Prenatal Genetic Diagnosis by Genomic Sequencing: A Prospective Evaluation
Conditions: Fetal Structural Anomalies
Sponsor: Columbia University

1 
Prenatal Genetic Diagnosis by Genomic Sequencing: 
A Prospective Evaluation 
(PrenatalSEQ) 
ClinicalTrials.gov ID: NCT03936101
14 January 2019 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
3 
Table of Contents 
 
1 
Introduction ........................................................................................................................................... 5 
1.1 
Study Abstract ............................................................................................................................... 5 
1.2 
Objectives ..................................................................................................................................... 5 
1.3 
Purpose of the Study Protocol ....................................................................................................... 6 
2 
Background ........................................................................................................................................... 7 
2.1 
Introduction ................................................................................................................................... 7 
2.2 
Preliminary Studies ....................................................................................................................... 8 
2.2.1 
Postnatal Studies ................................................................................................................... 8 
2.2.2 
Prenatal Studies ..................................................................................................................... 8 
2.2.3 
Healthcare Utilization and Cost ............................................................................................ 9 
2.2.4 
Educational & Psychosocial Needs ..................................................................................... 10 
2.3 
Rationale for the Study ............................................................................................................... 10 
3 
Study Design ....................................................................................................................................... 12 
3.1 
Design Summary ......................................................................................................................... 12 
3.2 
Eligibility Criteria ....................................................................................................................... 12 
3.2.1 
Prenatal sequencing group .................................................................................................. 12 
3.2.2 
Unsequenced prenatal group ............................................................................................... 13 
4 
Study Procedures ................................................................................................................................ 14 
4.1 
Screening for Eligibility and Consent ......................................................................................... 14 
4.1.1 
Informed consent................................................................................................................. 14 
4.2 
Enrollment ................................................................................................................................... 15 
4.2.1 
Prenatal sequencing group .................................................................................................. 15 
4.2.2 
Unsequenced prenatal group ............................................................................................... 16 
4.3 
Baseline Data Collection ............................................................................................................. 16 
4.4 
Sequencing Methods ................................................................................................................... 17 
4.5 
Variant Evaluation and Return of Results ................................................................................... 17 
4.6 
Study Procedures ........................................................................................................................ 18 
4.7 
Psycho-Social Surveys ................................................................................................................ 19 
4.7.1 
In-depth Qualitative Interviews .......................................................................................... 20 
4.8 
Healthcare Utilization and Change in Management ................................................................... 20 
4.9 
Adverse Event Reporting ............................................................................................................ 21 
4.10 
Study Outcome Measures and Ascertainment ............................................................................ 21 
4.10.1 
Primary Outcome ................................................................................................................ 21 
4.10.2 
Secondary Outcomes (Prenatal sequencing and unsequenced prenatal) ............................. 21 
4.10.3 
Secondary Outcomes (Prenatal sequencing only) ............................................................... 22 
5 
Statistical Considerations .................................................................................................................... 23 
5.1 
Power and sample size ................................................................................................................ 23 
5.2 
Prenatal Sequencing Group Only ................................................................................................ 23 
5.3 
Prenatal Sequencing and Prenatal Unsequenced Groups ............................................................ 24 
5.3.1 
Healthcare utilization and cost ............................................................................................ 24 
5.3.2 
Psychosocial outcomes ....................................................................................................... 25 
6 
Data Collection ................................................................................................................................... 26 
6.1 
Web Data Entry Systems ............................................................................................................ 26 
6.1.1  
Data Collection Forms ........................................................................................................ 26 
6.2 
Centralized Data Management .................................................................................................... 27 
6.3 
Performance Monitoring ............................................................................................................. 27 
7 
WGS Pipeline Development ............................................................................................................... 28 
7.1 
Evaluation of the incremental value of WGS compared to WES in understanding the etiology of 
birth defects ............................................................................................................................................. 28 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
4 
8 
Study Administration .......................................................................................................................... 29 
8.1 
Organization and Funding ........................................................................................................... 29 
8.1.1 
Clinical Coordinating Center .............................................................................................. 29 
8.1.2 
Data Coordinating Center ................................................................................................... 29 
8.2 
Committees ................................................................................................................................. 29 
8.2.1 
Steering Committee............................................................................................................. 29 
8.2.2 
Operations Committee ........................................................................................................ 30 
8.2.3 
Variant Adjudication Committee ........................................................................................ 30 
8.2.4 
Variant Oversight Committee ............................................................................................. 30 
8.2.5 
Clinical Adjudication Committee ....................................................................................... 30 
8.2.6 
WGS Pipeline Development Committee ............................................................................ 30 
8.2.7 
Data and Safety Monitoring Board (DSMB) ...................................................................... 30 
9 
Study Timetable .................................................................................................................................. 31 
9.1 
Training and Certification ........................................................................................................... 31 
9.2 
Recruitment and Data Collection Period..................................................................................... 31 
9.3 
Final Analysis ............................................................................................................................. 32 
9.4 
Data Archiving and Sample Storage ........................................................................................... 32 
9.4.1 
Data Archiving .................................................................................................................... 32 
9.4.2 
Data Sharing ........................................................................................................................ 32 
9.4.3 
Specimen Storage and Sharing ........................................................................................... 32 
Appendix A. Eligible Major Structural Anomalies..................................................................................... 33 
 
 
 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
5 
1 Introduction 
1.1 Study Abstract 
Whole exome and whole genome sequencing (WGS) have expanded the ability to determine the genetic 
etiology of previously undiagnosed disorders. This study is a multicenter prospective cohort study to 
evaluate the emerging technology of sequencing for the management of fetuses with structural anomalies. 
The hypothesis is that a significant subset of fetal structural anomalies has a genetic etiology identifiable 
by sequencing and that prenatal knowledge of this information will improve perinatal care, reduce 
unnecessary diagnostic testing, reduce the cost of care, and improve quality of life for both the child and 
the family. The aims of this study are to investigate these multiple aspects of prenatal sequencing in a 
single study with an innovative integrated prospective design, which will permit a robust evaluation of the 
benefits and risks of delivering diagnostic and prognostic genetic testing results in a prenatal setting. 
The study will determine, in a sequential population of pregnancies with selected fetal structural 
anomalies and a negative or non-causal chromosomal microarray (CMA), the frequency of pathogenic, 
likely pathogenic, and uncertain genomic variants identifiable by sequencing. To determine the impact of 
this information on clinical care, a control population of unsequenced pregnancies with similar structural 
anomalies will be prospectively recruited and the infants from both cohorts will be followed up to 1 year 
of age. This study component will evaluate differences in healthcare management and cost through 
discharge from hospital post-delivery, and perinatal and infant outcomes through 1 year of life. The 
educational, counseling and psychosocial impact of sequencing results during the prenatal period, in the 
nursery and through 1 year of life also will be evaluated. Since the analytical and clinical tools needed for 
the full translation of sequencing into care are still developing, optimization of bioinformatic tools to 
improve identification of pathogenic and likely pathogenic mutations associated with prenatal phenotypes 
of established disease genes will be investigated, as well as identification of new genes associated with 
presently undiagnosed fetal/neonatal phenotypes. This study will provide an in-depth evaluation of the 
prenatal diagnostic value of sequencing prior to its responsible introduction into practice and will provide 
independent data to guide its translation. 
1.2 Objectives 
The main objective of this multi-center collaborative study is to evaluate sequencing (both whole exome 
sequencing [WES] and WGS) as a prenatal diagnostic tool in pregnancies with a structural anomaly and a 
negative or only non-causal karyotype/ chromosome microarray analysis (CMA). Specifically, the aims 
are as follows: 
1. To determine in pregnancies with structural anomalies which are CMA negative (or have only non-
causal findings), the frequency of pathogenic, likely pathogenic, and uncertain genomic variants 
identifiable by sequencing. 
a. Examine the relative yield of pathogenic, likely pathogenic, and uncertain genomic variants 
identifiable by WGS compared with analysis of the coding sequence alone (simulating WES). 
2. To evaluate the impact of having sequencing results available prenatally on clinical management, 
including health care utilization and costs, comparing outcomes of pregnancies and neonates with in 
utero diagnosed structural anomalies that have undergone prenatal sequencing with those that have 
not. 
3. To evaluate the educational/counseling and psychosocial needs of pregnant couples having a fetus 
with a structural anomaly and assessing the psychological impact of sequencing and return of results 
on couples and determine the correlation with demographic factors, knowledge and attitudes about 
genetics, baseline parental attitudes and beliefs, and genetic test results. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
6 
4. To expand the diagnostic yield and clinical utility of fetal sequencing by developing and optimizing 
bioinformatic tools capable of efficiently and rapidly identifying  pathogenic and likely pathogenic 
variants with prenatal presentations.  
1.3 Purpose of the Study Protocol 
This protocol describes the background, design and organization of the study and may be viewed as a 
written agreement among the study investigators. It is approved by the funding agency (NICHD), the 
study Steering Committee, the Data Safety Monitoring Board (DSMB), the single Institutional Review 
Board (IRB) of the Clinical Coordinating Center and participating laboratories before recruitment begins. 
Any changes to the protocol during the study period require the approval of the Steering Committee and 
the IRB.   
A manual of operations supplements the protocol with detailed specifications of the study procedures. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
7 
2 Background 
2.1 Introduction 
With advances in prenatal imaging, increasingly there is the ability to detect birth defects prenatally and 
use this information to optimize perinatal and neonatal management. Simultaneously, molecular genetic 
diagnostics have facilitated more precise identification of the underlying cause of these birth defects and 
provided additional prognostic information to improve prenatal and postnatal management. The first 
major improvement in prenatal genomic diagnosis was chromosome microarray analysis (CMA) which 
identifies copy number variants (CNVs) of 100 kb to 10Mb that are below the resolution of most 
karyotypes. A multicenter study evaluating the clinical utility of prenatal CMA reported that CMA 
identified the same aneuploidies and unbalanced rearrangements as karyotyping but also identified 
additional, clinically significant cytogenetic information.1 Among women with a normal karyotype, CMA 
identified clinically relevant deletions or duplications in 6.0% with a fetal structural anomaly and in 1.7% 
of those with indications of advanced maternal age or positive aneuploidy screening results. The 
frequency of CNVs varied by anomaly, with the highest frequency (16%) occurring in fetuses with 
cardiac defects.   
Congenital anomalies affect 2-4% of all infants and are responsible for 20% of perinatal deaths.2 Prenatal 
genetic evaluation of fetal anomalies is limited to karyotype and CMA performed on amniocytes or 
chorionic villi. The karyotype is abnormal in 20-25% and CMA is abnormal in 5-10% of fetal anomalies, 
leaving 60-70% without a genetic diagnosis.3 WGS is the process of determining the complete DNA 
sequence of an organism's genome at a single time and includes both the coding and non-coding regions.  
Whole exome sequencing (WES) analyzes only the coding regions of the genome (exons; DNA code for 
making proteins). Until recently, researchers have elected to use WES because it costs less and because 
the functional consequences of positive findings occurring within coding regions of genes are much easier 
to interpret. Because of its high cost and incomplete knowledge about the role of non-coding variants in 
disease, WGS has primarily been used as a research tool. This may change as sequencing costs decrease 
and knowledge of the functional consequences of non-coding genetic variants increases. Numerous recent 
reports describe the speed and reliability of WGS in the context of rapid clinical sequencing since library 
preparation is not required4-6 making WGS an advantageous technology for rapid genomic testing in the 
future.  
Prenatal sequencing has the potential to alter the life course and reduce morbidity and mortality by 
guiding pregnancy management, delivery plans and/or the treatment of fetuses and newborns with some 
genetic abnormalities.7 One example is in utero fetal intervention now performed for certain anomalies 
including aortic stenosis to avoid hypoplastic left heart syndrome, urinary diversion for posterior urethral 
valves, drainage of pleural effusions and chylothorax, surgical repair for spina bifida, tracheal occlusion 
for congenital diaphragmatic hernia, and amnio-infusion for renal agenesis.8-14 For all of these, knowing 
their genetic etiology aids clinical management decisions by providing information about other associated 
medical features that affect the likelihood of success of the intervention. In addition, prenatal diagnosis of 
some metabolic conditions could guide early postnatal treatment and avoid metabolic crises that can lead 
to long term neurocognitive deficits. For example, pyruvate dehydrogenase deficiency can present 
prenatally with structural anomalies and is modifiable by a ketogenic diet and thiamine 
supplementation.15,16 Finally, mesenchymal stem cell transplantation for osteogenesis imperfecta has been 
reported to increase skeletal mineralization and growth velocity without adverse outcomes.17-20 With 
developments in gene and stem cell therapy, this type of treatment may increasingly become an option for 
other conditions. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
8 
2.2 Preliminary Studies 
2.2.1 Postnatal Studies 
WES, which analyzes the coding regions of the genome (exons) is a powerful diagnostic tool in adults 
and children with genetically heterogeneous conditions.21,22 Compared with a 10% diagnostic yield with 
karyotype and CMA, postnatal WES has diagnostic yields of >30% in adults and children with 
phenotypes suggestive of monogenic disorders.22 The mean and median overall diagnostic yields of a 
series of 16 studies was 36% and 38%, respectively, but was highly dependent on the a priori risk for a 
monogenic disorder. In addition to the diagnostic utility of WES in these populations, WES analysis has 
resulted in discovery of new disease genes with pleiotropic phenotypes. 
WGS is now gaining traction as a diagnostic and discovery strategy for adults and children with a 
suspected genetic disorder that remains undiagnosed after WES analysis, or as a first-line approach in lieu 
of WES. WES includes an exon capture step, which adds time and reagent costs, and biases against 
coverage in GC-rich regions. WGS does not include this selection step, providing more uniform coverage 
which allows a lower mean read depth, offers ability to detect copy number variants (CNVs) with higher 
resolution than CMA and more complex balanced re-arrangements. The lower read depth however will 
also make it more difficult to detect mosaic mutations which are an important source of mutations in 
neurodevelopmental disorders including autism and epilepsy.23,24 WGS was found to detect up to 3% of 
protein-coding variants missed by WES.25 Recent WGS studies have found more causative variants in 
coding and non-coding regions in autism.6 In addition, an increased burden of rare de novo variants in 
non-coding regions for congenital heart disease and diaphragmatic hernia has been seen (Chung, WC; 
unpublished data). 
2.2.2 Prenatal Studies 
Only a few case series of prenatal WES have been published to date with a wide range of indications, 
including pregnancy terminations, fetal demises with fetal anomalies, euploid fetuses with 
sonographically detected single or multiple fetal structural anomalies, and increased nuchal translucency 
(NT) > 3.5 mm have been reported26,27 with widely variable diagnostic yields of 6.2 - 80%. The 2 largest 
series of trio exomes on over 200 fetal anomalies each had overall diagnostic rates of 6.2 and 7.5%, but 
was higher (14.3% and 16.0%) for fetuses with multiple anomalies.27  
Table 1. This table describes the frequency of pathogenic variants by whole exome sequencing in 
various publications  
First 
Author, 
Year 
# 
Cases 
Cohort Criteria 
Method 
Pathogenic 
Variants* 
Normand, 
201828 
146 
Fetuses with ultrasound anomalies and a suspected 
Mendelian disorder 
62 Trio 
46/146 (32%) 
Aarabi, 
201829 
20 
1 or more major structural congenital anomaly 
detected by ultrasound 
Trio 
4/20 (20%) 
Fu, 
201730 
196  
Fetuses with structural abnormalities 
147 Proband-
only  
 
49 Trio 
34/147 (23.1%) 
 
13/49 (26.5%) 
Lei, 
201731 
30  
Fetuses with congenital anomalies of the kidney 
and urinary tract 
23 Proband-only 
3/23 (13%) 
 
7 Trio 
 
1/7 (14%) 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
9 
Total 
4/30 (13.3%)  
Vora, 
201732 
15 
Fetuses with multiple congenital anomalies highly 
suggestive of an underlying genetic disorder 
Trio 
7/15 (47%) 
Yates, 
201733 
84 
Fetuses with ultrasound abnormalities that resulted 
in fetal demise or pregnancy termination 
29 Proband-only  
4/29 (14%)  
 
45 Trios 
 
11/45 (24%) 
6 Quads/4 
Maternal Duos 
2/10 (20%) 
Total: 
17/84 (20%) 
 
Pangalos, 
201634 
14 
Prenatal ultrasound abnormalities or malformations 
Proband-only 
6/14 (43%) 
Alamillo, 
201535 
7 
Multiple congenital anomalies on prenatal 
ultrasound 
Trio 
4/7 (57%) 
Drury, 
201536 
24 
Fetuses with an increased NT (>3.5 mm) or other 
ultrasound abnormality 
14 Proband-only 
 
2/14 (14%) 
 
10 Trio 
3/10 (30%) 
Total 
5/24 (21%)  
 
Carss, 
201437 
30 
Structural abnormalities identified on prenatal 
ultrasound 
Trio 
3/30 (10%)  
 
Yang, 
201438 
11 
Terminated fetuses with anomalies  
Trio 
6/11 (54%) 
2.2.3 Healthcare Utilization and Cost 
Integration of WES or WGS into clinical care requires not only demonstration of the ability to identify the 
underlying etiology of a disorder but also requires evidence that care is improved, providing value to the 
health care system. While WGS is currently a relatively expensive test (≥$15,000 per trio), the 
information may lead to significant alterations in care utilization. Given that healthcare costs can be 
$250,000 or more in the first year of life for some complex congenital anomalies, the genetic diagnostic 
information may be net cost saving. Even if not cost-saving, if clinical outcomes are improved 
significantly, the incremental cost of WGS may be justifiable based on its cost effectiveness. Recent 
studies in critically ill neonates and infants have suggested that establishing a diagnosis leads to more 
focused management and reduction in healthcare utilization and reduced cost. Meng et al evaluated 
diagnostic WES for 278 critically ill infants within the first 100 days of life and found a genetic diagnosis 
in 36.7%.39 These new diagnoses led to care modification in 52%, including initiation of new 
subspecialist care, redirection of care, changes in medication/diet, or completion of major procedures 
(e.g., transplant). The greatest impact was in infants receiving rapid “critical trio sequencing”.39 A meta-
analysis found that change in clinical management by WGS results was 27% (4 studies with 136 children) 
compared with 17% by WES (12 studies and 992 children) and 6% by CMA (8 studies of 4,271 
children).40 Another study evaluating the cost-effectiveness of WES in a pediatric setting reported the 
mean duration of the diagnostic odyssey was 6 years, and that the diagnostic trajectory for WES 
performed at the initial tertiary presentation resulted in an incremental saving of $6800 per additional 
diagnosis compared with the standard diagnostic pathway.41  In summary, studies have suggested that 
sequencing early in the disease course may provide the maximal benefit by modifying clinical 
management, reducing the time to diagnosis and cost. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
10 
2.2.4 Educational & Psychosocial Needs 
The added prognostic information about health, treatability, life expectancy and neurodevelopmental, 
behavioral and cognitive function is far beyond what is learned from ultrasound findings and standard 
testing, and can have a powerful impact on parents. Identification of fetal genomic information has 
significant value to the management of the pregnancy. Ultrasound offers an anatomic phenotype but is 
incapable of evaluating long-term neurocognitive potential, limiting accurate prognostication and 
counseling. Karyotyping and CMA can offer disease specific counseling in a combined ~30-40% of 
detectable cases to provide couples with the information necessary to make informed reproductive 
decisions, leaving significant ambiguity in 60-70%. Equally important, knowledge of the genotype can 
direct additional fetal, neonatal and pediatric management. 
Efforts to understand the psychosocial and behavioral impact of integrating genomic technologies into 
adult and pediatric practice are ongoing42-44, but to date, little empirical work has been done to understand 
the unique challenges of applying genomic sequencing to a prenatal population.45 Attitudes towards 
prenatal screening and diagnosis are influenced by ethnicity, socioeconomic status, cultural and religious 
beliefs, and experiences with disability.45-49 
While WGS results may illuminate the situation when a well-known genetic condition is diagnosed, there 
are challenges in counseling for some of the newer genetic conditions for which less data are available. In 
CMA, variants of uncertain clinical significance (VUS) and those associated with variable expressivity 
occur in a proportion of cases with structural anomalies (5.3%) and require extensive genetic 
counseling.50  Other counseling issues include the identification of adult onset disorders of both the fetus 
and parent. To understand attitudes and unmet needs in the adoption of CMA testing semi-structured 
interviews with parents and counselors were performed to evaluate pre-test counseling, reporting of 
results, and assessing patient and provider experiences.47 From the patient perspective, 5 key themes were 
identified 1) accepting CMA testing was an easy decision to make as patients would obtain additional 
information on their baby’s health at no additional cost, 2) patients were blindsided by the results in cases 
where they initially received a normal karyotype result followed by an abnormal CMA, 3) abnormal 
results left patients shocked, anxious, confused and overwhelmed, 4) patients needed support to manage, 
understand and act on the microarray results, and 5) uncertain findings were felt to be toxic knowledge 
and patients wished they did not receive the results. It is anticipated that the needs for prenatal sequencing 
will be similar to those of prenatal CMA and that prior studies will inform the educational and support 
materials developed for prenatal sequencing.  
2.3 Rationale for the Study 
Sequencing itself is a transformative technology to identify comprehensively genetic variants accounting 
for a phenotype, but its application to prenatal diagnosis has not been investigated prospectively in a large 
cohort.22 This study population of unselected consecutive cases will be unique in that the majority of 
published series to date have only included select phenotypes felt to have a genetic etiology. It is possible 
that these studies have overestimated the frequency and underestimated the phenotypic variability of in 
utero genetic disease based upon the case ascertainment. A more comprehensive, less biased evaluation of 
the molecular etiology of birth defects should lead to the discovery of previously unknown genes and 
phenotypic associations. To date, only descriptive series of the diagnostic yield of selected prenatal WES 
or WGS have been reported on fetuses with a structural anomaly, but none have evaluated the benefit of a 
specific genetic diagnosis.36,37,51-53  
This study will be the first to perform a cost-effectiveness analysis to evaluate whether prenatal 
knowledge of the genetic cause of a fetal single-gene disorder will lead to altered fetal/neonatal 
management, costs, and outcomes. Also important are the pertinent psychosocial effects of introducing 
sequencing into a prenatal setting.  

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
11 
Before integration into standard care, the diagnostic capabilities of prenatal WGS must mature by 
developing new bioinformatic tools to specifically facilitate accurate and efficient prenatal interpretation 
and novel gene discovery. This can be accomplished initially by analyzing and reporting the variants from 
the coding regions of the genome and then subsequently analyzing the non-coding regions, allowing 
comparison of the differences in diagnostic yield. The current missing pieces are insufficient WGS data in 
fetuses with abnormalities, the lack of variant analysis algorithms tuned to the prenatal setting, and 
insufficient publicly deposited data to inform clinical care for prenatal genomic testing and rare 
developmental disorders. As more data become available (WGS data from adults, more WES/WGS from 
more diverse communities, Human Cell Atlas data, and ATAC seq data from appropriate times in 
development), it will increasingly be possible to interpret the coding and noncoding regions that are 
relevant to developmental disorders. 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
12 
3 Study Design 
3.1 Design Summary 
This multicenter, prospective observational cohort study will evaluate prenatal sequencing among 
pregnancies with fetal structural anomalies recruited at three university based medical centers and 
evaluated at three university genetic laboratories.  A total of 1,100 pregnancies with fetal structural 
anomalies and meeting eligibility criteria will be enrolled into the study. Of these, 750 will undergo 
prenatal genomic sequencing (prenatal sequencing group) and the remaining 350 pregnancies will not 
have any prenatal genomic sequencing (unsequenced prenatal group). Enrollment of pregnancies with an 
isolated nuchal translucency measurements ≥ 3.5 mm will be restricted to 5% within each group 
(sequenced and unsequenced) and isolated estimated fetal weight <5th %ile also will be restricted to 5% 
for each group (sequenced and unsequenced). 
The prenatal sequencing group will be used to determine the frequency of pathogenic, likely pathogenic, 
and uncertain genomic variants identifiable by sequencing and the relative yield of sequencing.  The 
prenatal sequencing group will be compared with the unsequenced prenatal group to evaluate health care 
management, health care utilization and cost, perinatal outcomes, and the psychosocial needs of pregnant 
couples.  Mothers, fathers and infants will be followed through 1year postpartum. 
3.2 Eligibility Criteria 
3.2.1 Prenatal sequencing group 
3.2.1.1 Inclusion Criteria 
1. 
Fetus identified by ultrasound and/or MRI with at least one of the following: 
a. One or more major structural anomalies (Appendix A)  
b. A nuchal translucency measurement of ≥ 3.5 mm  
c. A fetus less than 24 weeks 0 days gestation with normal anatomy and sonographically 
estimated fetal weight <5th %ile without maternal hypertension, type I diabetes, or other 
maternal disorders known to alter fetal growth.  
2. 
Negative prenatal CMA (or those with CMA findings not related to the ultrasound finding) 
3. 
Singleton gestation 
4. 
Gestational age less than 36 weeks, 0 days to allow for availability of sequencing results before 
delivery 
3.2.1.2 Exclusion Criteria 
1. 
Maternal or paternal age less than 18 years old 
2. 
Proven infectious or teratogenic cause of fetal anomaly 
3. 
Planned termination of the pregnancy 
4. 
Unavailable blood or saliva samples from both biologic parents prior to sequencing 
5. 
Parental unwillingness to participate in 1 year postnatal follow-up 
6. 
Language barrier (non-English or Spanish speaking) 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
13 
7. 
Delivery planned at a site other than one of the study centers or associated hospitals 
8. 
Previous consent to the unsequenced prenatal group or enrollment in a previous pregnancy 
 
3.2.2 Unsequenced prenatal group 
3.2.2.1 Inclusion Criteria 
1. Fetus identified by ultrasound and/or MRI with at least one of the following: 
a. One or more major structural anomalies (Appendix A)  
b. A nuchal translucency measurement of ≥ 3.5 mm 
c. A fetus less than 24 weeks 0 days gestation with normal anatomy and sonographically 
estimated fetal weight <5th %ile without maternal hypertension, type I diabetes, or other 
maternal disorders known to alter fetal growth 
2. 
Negative prenatal or postnatal CMA (or those with CMA findings not related to the ultrasound 
finding) 
3. 
Declined prenatal sequencing  
4. 
Singleton gestation 
3.2.2.2 Exclusion Criteria 
1. 
Maternal or paternal age less than 18 years old 
2. 
Proven infectious or teratogenic cause of fetal anomaly 
3. 
Positive prenatal NIPT screening for trisomy 21,18 or 13.  Positive 22q11.2 prenatal NIPT testing 
with consistent ultrasound findings is also an exclusion. 
4. 
Planned termination of the pregnancy 
5. 
Parental unwillingness to participate in 1 year postnatal follow-up 
6. 
Language barrier (non-English or Spanish speaking)  
7. 
Delivery planned at a site other than one of the study centers or associated hospitals 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
14 
4 Study Procedures  
4.1 Screening for Eligibility and Consent  
Eligible patients with specified fetal structural anomalies will be identified by prenatal ultrasound and/or 
fetal MRI. Center investigators and their staff in the outpatient ultrasound prenatal diagnostic units will be 
responsible for identifying eligible patients. To maximize recruitment, coordinators at each center will 
screen the prenatal diagnostic logs on a daily basis to identify eligible patients.  
Upon recognition of a specified fetal anomaly, the maternal fetal medicine physician (MFM)/ 
ultrasonographer and/or a genetic counselor will explain the ultrasound finding as per routine clinical 
care. At the conclusion of the initial evaluation/consult, women will be informed by the 
physician/counselor and/or coordinator about the study. Once the potential participant indicates 
willingness to hear more about the study, a study coordinator will meet with the parents, review 
eligibility, and explain the study.  Women who choose to undergo prenatal invasive testing will be 
approached for the prenatal sequencing group but will not be enrolled until a negative or non-causal 
karyotype/CMA is confirmed (with the exception of those enrolled into expedited prenatal sequencing, 
section 4.2.1). Women who choose not to have any diagnostic testing or decline sequencing will be 
approached for participation in the unsequenced prenatal group. All women with a specified fetal 
anomaly will be recorded on a screening log that collects screening ID, date screened, planned invasive 
prenatal testing, ultrasound anomaly, screening status code and reason for study ineligibility (if 
applicable).  A participant flowchart is shown in Figure 1. 
Figure 1. Screening flowchart 
Structural anomaly
 
Termination
 
Continued 
pregnancy
 
Not eligible
 
Invasive testing
 
No invasive testing
 
Consent to blood 
draw for fetal seq
 
Declines consent to 
blood draw
 
KT/CMA not 
causative
 
KT/CMA positive & 
causative
 
Offer sequencing
 
If accept, consent 
to Sequencing Grp
 
If decline, offer 
enrollment into 
Unsequenced Grp
 
Not eligible
 
KT/CMA not 
causative
 
KT/CMA positive & 
causative
 
Offer sequencing
 
If accept, enroll into 
Sequencing Grp
 
If decline, offer 
enrollment into 
Unsequenced Grp
 
Not eligible
 
Pre-/postnatal KT/
CMA not causative
 
Pre-/postnatal KT/
CMA positive & 
causative
 
Pre-/postnatal KT/
CMA not done
 
Continue in study
 
Exclude from 
analysis, stop study 
procedures
 
Exclude from 
analysis, stop study 
procedures
 
 
 
4.1.1 Informed consent 
Written informed consent must be obtained from participants and the biological father of the fetus prior to 
any study procedures.  Three consents may be employed for this study: 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
15 
• 
Short consent for potentially eligible women in the prenatal sequencing group that allows for 
collection of study samples prior to determination of the CMA and karyotype result.  If 
CMA/karyotype results come back negative, eligible participants will sign the full study informed 
consent form to proceed with sequencing. 
• 
Full study consent for the prenatal sequencing group that describes the specific group’s 
participation in the study and risks 
• 
Full study consent for the prenatal unsequenced group that describes the specific group’s 
participation in the study and risks 
Participants in both the sequenced and unsequenced groups will be assured that their participation in the 
study is purely voluntary and that patient care will not be affected if they decline participation in the 
study.  Patients who are not fluent in English will be enrolled by a person fluent in Spanish. Both verbal 
and written informed consent and authorization will be obtained in that language; if this is not possible 
the patient will be excluded. 
4.2 Enrollment 
4.2.1 Prenatal sequencing group 
For the prenatal sequencing group, chorionic villus sampling (CVS) or amniocentesis will be performed 
per standard of care. For CVS sampling, a portion of the villi, cultured villi or extracted DNA will be sent 
to the sequencing laboratory.  For amniocentesis, the usual draw of 40cc of fluid will be divided by the 
cytogenetics lab, with a portion used for clinical testing (minimum standard amount as per diagnostic 
laboratory practice) and from the remainder, DNA will be extracted for sequencing in a CLIA laboratory.  
Additional DNA for sequencing will be extracted from the cultured cells and the sequencing lab will 
determine which is the optimal source for sequencing, after quality and quantity verification. 
Potentially eligible participants will sign the screening consent form prior to collection of parental 
samples.  Thirty ml of maternal blood will be collected for sequencing and future research.  Paternal 
blood (up to 10 ml) or buccal swab/saliva will be collected for sequencing and future research. Every 
effort will be made to obtain samples from both parents prior to or at the time of the diagnostic procedure. 
If the father is absent, a sample will be obtained as soon as possible. If no paternal sample is obtained, the 
participant will be classified as a screen failure and will not be enrolled into the study. The parental 
samples will be sent for DNA extraction as soon as they become available. 
If the clinical karyotype, CMA or any other non-sequencing test result is known to be positive prior to 
sequencing, and felt to be causative of the anomaly, the karyotype/CMA results will be recorded, the 
participant will be classified as a screen failure and not enrolled, and parental samples/DNA will be 
discarded.  
If the clinical karyotype/CMA results are negative (or non-causative), eligibility criteria will be 
confirmed, the participant and biological father will sign the study consent form either in person or 
electronically, parental samples will be collected if needed, and genome sequencing will proceed.   
Parents declining participation at this time will be considered as having refused consent; these participants 
may be offered participation in the unsequenced prenatal group if eligibility criteria are met. Those 
choosing to terminate their pregnancy based on ultrasound/CMA findings are ineligible. 
“Expedited” prenatal sequencing 
An alternative to the prenatal diagnostic flow described above permits prenatal sequencing to be 
performed in parallel with the CMA, i.e. before the fetus is confirmed as CMA negative. This diagnostic 
workflow is at the discretion of the study research team and participants will be considered enrolled once 
they have signed the study consent and parental samples have been collected. If the CMA is negative or 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
16 
unrelated to the fetal anomaly, the case will continue to be part of the prenatal sequencing group and 
included in the analyses. In pregnancies in which the CMA result is found to be positive (and presumed 
causal of the anomaly), sequencing will be discontinued or if already completed the results will be 
recorded and reported to the family in the appropriate manner. The karyotype/CMA results will be 
recorded; however, follow-up of the pregnancy through delivery will not take place, these participants 
will not be included in the analyses and not contribute to the sample size.    
4.2.2 Unsequenced prenatal group 
The unsequenced prenatal group will include participants that decline invasive testing and those that plan 
to have invasive testing but decline sequencing (Figure 1).  For participants that plan to have invasive 
testing but decline sequencing, enrollment into the study will occur after the clinical karyotype/CMA 
results are confirmed negative and the participant signs the prenatal unsequenced consent. Participants 
that decline invasive testing will be enrolled at the time of diagnosis of the fetal anomaly and they sign 
the unsequenced prenatal group consent.  Women from this group who later elect invasive testing are not 
eligible to enroll in the prenatal sequencing group but will be included in the unsequenced prenatal 
analyses if the karyotype and CMA are confirmed negative or non-causal. It is anticipated that most 
patients that do not have invasive testing typically will have CMA performed after birth as part of clinical 
care. Continued follow-up of the pregnancy and 1-year postpartum period will not take place if a positive 
causal CMA is identified. 
Postpartum genomic sequencing may be performed at the discretion of the clinical care team as part of 
routine care.   
4.3 Baseline Data Collection 
In addition to data collected for eligibility and consent, the following information will be obtained at 
enrollment from either medical record review or participant interview:  
• 
Maternal and paternal: age, race, ethnicity, education, income, household composition, religion, 
religiosity, number of children, and family history 
• 
Maternal medical and psychiatric history including maternal history of disorders with teratogenic 
risk (e.g. diabetes, teratogen exposure including medications, diseases during pregnancy (e.g. 
CMV, Zika)  
• 
Obstetrical history including  
• 
Clinical estimated date of delivery and type of conception 
• 
Pre-procedure ultrasounds including date and detailed information on anomaly(ies) 
• 
Number of prior pregnancies and gestational age of delivery  
• 
Unexplained infertility  
• 
Family history of learning disabilities, genetic disorders, congenital anomalies, stillbirths or infant 
deaths, significant medical problems, fetal/child structural or growth findings, consanguinity, 
deafness and psychiatric illnesses. 
• 
Prenatal screening and diagnostic test results; carrier screening and results; blood type and screen; 
rubella immunity status 
A copy of de-identified ultrasound, karyotype and CMA reports will be transferred to the DCC for all 
enrolled patients. A study research chart should be kept, containing copies of the patient’s signed consent 
and ultrasound reports. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
17 
4.4 Sequencing Methods 
Prenatal sequencing will be performed for patients in one of the three laboratories, i.e. the Institute of 
Genomic Medicine (IGM) at Columbia University Medical Center, in the Human Genome Sequencing 
Center CLIA certified Clinical Laboratory (HGSC-CL) at Baylor College of Medicine, and at the High 
Throughput Sequencing Facility at the University of North Carolina Chapel Hill.  
Sequencing will be performed on an Illumina NovaSeq 6000 system. All positive sequencing results will 
be confirmed using Sanger sequencing and reported by a CLIA certified laboratory. It is possible that 
non-parentage (maternity or paternity) may be suspected through initial genomic analysis. If there is 
suspicion that the parent of the fetus is not the biological parent, further analysis of the genomic data will 
be suspended and the family will be informed they are not eligible for study participation. This approach 
will be addressed during the informed consent process. 
Data quality control, including alignment, variant calling, filtering and prioritization will be performed 
using best practices refined at the local institutions. 
4.5 Variant Evaluation and Return of Results   
All candidate variants will first be evaluated locally at each site by a team including the site PI who is a 
board certified clinical geneticist, a study genetic counselor, a variant curation scientist, and a board-
certified molecular geneticist with clinical genome sequencing experience to determine candidate variants 
relevant to the clinical phenotype. Factors used in this evaluation include the ACMG classification of the 
variant, whether its inheritance mode is consistent with the clinical condition, and the extent of 
phenotypic overlap with previously reported cases.  Initially, candidate variants will be reported in the 
coding regions only; however it is anticipated that over the course of the study this may change to also 
incorporate reporting in non-coding regions. 
Results that are considered pathogenic or likely pathogenic by the local team will be confirmed by Sanger 
sequencing and reported to genetic counselor and principal investigator who will relay the results to the 
provider and the family. In cases in which time is critical (e.g. preterm labor, impending fetal procedure, 
fetal compromise) and a causative variant is suspected, the genetic counselor/principal investigator may 
report results either verbally to the provider as a “research finding” or as a written preliminary result 
report prior to Sanger confirmation.  
Compelling variants in novel genes that are not yet disease associated will be classified as variants of 
uncertain significance (VUS) with the qualification that they are in “genes of uncertain clinical 
significance (GUS)”.  For compelling variants in known disease genes that have not yet been associated 
with this disease phenotype (also classified as VUS), in-house databases will be checked for similar 
variants, automated PubMed alerts will be set-up to be notified of new publications, and tools such as 
GeneMatcher will be used to help find other individuals with variants in the same gene. Uncertain 
variants will be Sanger confirmed and reported to the patient based on the judgment of the local team 
after consultation with the Oversight Committee. To build consensus in interpretation, the local Variant 
Interpretation teams will have video conferences on which uncertain results will be discussed.  
For all reportable results, a copy of the Sanger confirmation report and additional relevant information as 
applicable will be prepared by the local team and returned to the clinical study genetic counselor who will 
share the result with the ordering MFM/OB physician and place it in the electronic medical record 
(EMR). The ordering MFM/OB physicians and the study genetic counselor will report the results to the 
family and discuss the implications for clinical care. Study clinical geneticists will be available for 
consultation. Results will also be available to the neonatal care team for use after delivery.  A copy of the 
de-identified report will be transferred to the DCC for all patients. 
Local site requirements will dictate the reporting (if at all) of ACMG secondary or other incidental 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
18 
findings of the parents and fetus. Because of the stressful nature of receiving the fetal diagnosis, 
discussions of medically actionable secondary findings that will be reported will be deferred until a 
formal postpartum genetic counseling consultation, anticipated to occur within 6 weeks of infant 
discharge or death with the exception of findings in the fetus that may impact treatment decisions on the 
newborn which will be reported immediately. 
Carrier status for Mendelian disorders in either parent will be reported according to the local site 
requirements. 
Parents will be informed of negative fetal sequencing results by way of a “research” report since negative 
results will not be Sanger confirmed.  
4.6 Study Procedures   
Enrolled women will be followed from screening through 12 months postpartum unless a positive and 
casual CMA is reported prenatally or postnatally. Data will be collected at the following time points: 
Table 2. Summary of Data Collection 
One month after discussion of study results 
or 8 weeks after enrollment for the 
unsequenced prenatal group 
• Maternal and paternal psychosocial survey 
 
Delivery and neonatal care 
• Pregnancy complications & delivery outcomes 
• Neonatal phenotype and outcomes 
• Coordinator review of management changes done by 
chart review 
• Physician surveys assessing change in management 
(sequencing group only) 
• Results of clinical care ordered genetic testing for 
unsequenced group including karyotype/CMA and any 
subsequent sequencing Reports of healthcare utilization 
and charges (prenatal) from diagnosis of fetal anomaly 
to delivery 
• Healthcare utilization and charges from delivery until 
discharge or death (neonatal) 
One month post discharge  
• Maternal and paternal psychosocial survey 
12 months postpartum 
• Maternal and paternal psychosocial survey, including 
maternal brief survey of healthcare utilization 
• Participant phone interview to document infant weight 
and length by maternal self-report and child 
development measures using the Ages and Stages 
questionnaire (ASQ-3) 
 
Neonatal phenotypes will be re-evaluated within one week of birth to identify additional clinical features. 
If significant new clinical findings are discovered, the sequencing variants will be re-evaluated 
immediately by the local team and any new diagnoses confirmed and reported by the study staff  to the 
neonatologist or other appropriate care provider who will report this to the family in concert with the 
study coordinator.  A copy of de-identified genetic reports (CMA and/or sequencing) will be transferred 
to the DCC for all enrolled patients in the unsequenced group. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
19 
4.7 Psycho-Social Surveys   
Each participant (both members of the couple) will complete an online 20-minute survey at 3 time points 
and will receive a gift card after the last survey is completed. The three time points are: 
• 
One month after disclosure of study results or 8 weeks after enrollment for the unsequenced 
prenatal group  
• 
One month after discharge from the hospital or end of the pregnancy if there is a fetal demise or 
pregnancy termination  
• 
Twelve months postpartum 
The measures collected at each time period are detailed in Table 3.   
Table 3. Pyschosocial Survey Measures 
Variable 
Measure 
Time 
 
 
Post results/ 
enroll 
1mth   
pp 
12mth 
pp 
Anxiety 
Personal Health Questionnaire-8 
X 
X 
X 
Depression 
General Anxiety Disorder 
X 
X 
X 
Genetic Knowledge 
Adapt from measures used in 
CSER/eMERGE 
X 
 
 
Numeracy  
Adapt from measures used in 
CSER/eMERGE 
X 
 
 
General Optimism 
Life Orientation Test 
X 
 
 
Tolerance with 
Ambiguity 
Tolerance with Ambiguity 
X 
 
 
Perceived control over 
health 
Internal Health Locus of Control 
X 
 
 
Genetic Essentialism 
Parrott Genetic Essentialism 
X 
 
 
Consenting and 
Education Experience 
Modified Genetic Counseling Satisfaction 
Scale and Doctor-Patient perceptions of 
communication 
X 
 
 
Satisfaction w/ 
decision to Participate  
Decision Regret Scale 
X 
X 
X 
Satisfaction w/ 
decision to continue  
Decision Regret Scale 
X 
X 
X 
Parent-Infant 
relationship  
Parent-Infant Attachment Questionnaire 
(Reck 16 question) 
 
X 
X 
Parenting stress & 
anxiety for child 
Child Vulnerability scale 54 
 
X 
X 
Parents’ experience of 
parenting 
Parent Sense of Competence Scale 
 
 
X 
Therapeutic 
Optimism/Prognosis 
Brief Illness Perception Questionnaire 
X 
X 
X 
Marital Relationship 
Kansas Marital Satisfaction Scale  
X 
X 
X 
Quality of life 
ITQOL: 
https://www.healthactchq.com/surveys.php 
 
 
X 
Prenatal sequencing only 
Results Disclosure 
Disclosure and non-disclosure of results to 
X 
X 
X 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
20 
and Secrecy 
others 
Understanding of 
Results 
Adapt from measures used in CSER/ 
eMERGE 
X 
X 
X 
Emotional Response 
to Results 
FACToR-12 (12 items) 
X 
X 
X 
Perceptions of 
uncertainty of results 
PUGS perceptions of uncertainties in genetic 
sequencing 
X 
X 
X 
 
The end of the survey will have a free text box for participants to add any other details they wish to share 
and also ask if the participant is willing to speak by phone in a semi-structured interview (Section 4.7.1). 
4.7.1 In-depth Qualitative Interviews 
The impact of learning about the prenatal WGS test and return of prenatal WGS results will be evaluated 
by interviewing 45 sets of parents (90 participants) one month after result disclosure or an equivalent time 
for those who decline testing, after discharge from the hospital, and 12 months after delivery. Those 
willing to be interviewed will indicate this at the end of their psychosocial survey.  Depending on the 
number of couples who volunteer for an interview, research staff will purposefully sample a range of 
parental ages, ethnicities, educational level, site, and fetal anomaly.  Participants with positive or 
uncertain genetic test results and those who had difficulty adjusting to the information or were dissatisfied 
with participation in the study and have decision regret will be oversampled.  Interviews at each time 
point will focus on the following: 
• 
The interview after results disclosure will focus on educational needs for consenting to the study, 
how results are disclosed, how much information is provided and by whom, understanding of the 
results, and support needed during the pregnancy after results are provided.  
• 
The interview after discharge will focus on how the results of the genomic test influenced 
decisions about medical management including delivery, neonatal care, planning, connecting with 
other families with the same condition, disclosure of results to others, and impact on interpersonal 
relationships.  
• 
The interview 12 months after delivery will focus on impact of fetal genomic results on longer-
term management, perceptions of the child and potential vulnerability and impact on family 
relationships.  
Both parents will be interviewed since men are often not included in these studies and are an integral part 
of the decision-making process and long-term outcome of the family. The interview data will be used to 
develop recommendations and identify factors associated with decision satisfaction, and to develop 
recommendations for pre-and post-test genetic counseling that reflect the needs of women and their 
partners as they make decisions about undergoing prenatal sequencing, or continuing a pregnancy after 
learning about a known or uncertain fetal sequencing abnormality. 
4.8 
Healthcare Utilization and Change in Management 
Information on healthcare utilization and charges will be collected starting at the time of the diagnosis of 
the fetal anomaly through discharge from the hospital postpartum. Healthcare utilization will be 
abstracted from the prenatal and neonatal records and include both outpatient and inpatient visits, 
laboratory tests, imaging tests, invasive testing, prenatal and postnatal surgery, primary care visits, 
specialty care visits, diagnostic procedures, and medications. In addition, site coordinators will work with 
hospital billing to obtain detailed billing records from delivery admittance through discharge for each 
study participant, including the hospital charge ratio. In the 12-month participant psychosocial survey, 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
21 
information will also be recorded on healthcare utilization post neonatal discharge, including number of 
infant hospitalizations, surgeries, procedures and specialist visits. 
To identify how sequencing results changed the original care plan, the site coordinators will review all 
prenatal and postnatal medical records from diagnosis until discharge or death. Specifically, the clinical 
diagnosis, anticipated prognosis, procedures, medications, diet, surgery, non-routine testing and imaging 
will be documented.  Physician surveys evaluating change in clinical management (i.e., the reasons and 
timing for management decisions and whether they were influenced by a genetic test result) will be 
completed by both the MFM/OB for prenatal assessment and a designated neonatal fellow in consultation 
with the clinical care team at each institution. The survey will include questions focused on clinical 
utility, such as: 1) have there been any changes to the patient’s treatment plan based on the sequencing 
results, 2) have there been any changes to the way that you counsel the patient/family regarding the 
immediate medical management as a result of the sequencing results, and 3) have there been any changes 
to the way that you counsel the patient/family regarding the long term medical management as a result of 
the sequencing results. The care and the results of the surveys will be presented and reviewed by the 
Clinical Adjudication Committee which will determine whether an alteration in health care management 
occurred based on sequencing results. 
4.9 
Adverse Event Reporting 
Detailed information concerning adverse events assessed to be definitely, probably or possibly related to 
study procedures will be collected and evaluated throughout the conduct of the study.  Adverse events 
will be reported to the Data and Safety Monitoring Board.   
4.10 Study Outcome Measures and Ascertainment  
4.10.1 Primary Outcome 
1. Pathogenic, likely pathogenic, or VUS variants identified by sequencing and deemed reportable 
by the Variant Adjudication Committee   
2. Healthcare costs from time of diagnosis of anomaly to infant discharge between sequenced and 
unsequenced groups 
4.10.2 Secondary Outcomes (Prenatal sequencing and unsequenced prenatal) 
1. Perinatal outcomes including gestational age at delivery, major morbidities including length of 
ventilator support, sepsis, need for pressor support, need for ECMO,  metabolic abnormalities 
(e.g., acidosis, elevated uric acid, hypo-/hyperglycemia), intraventricular 
hemorrhage/periventricular leukomalacia, encephalopathy, and seizure  
2. Neonatal/infant death at time of discharge and at 12 months of age 
3. Length of initial NICU stay and number of days spent in the hospital between initial discharge 
and 12 months of age 
4. Infant weight and length at 12 months of age 
5. Developmental parameters (communication, gross motor, fine motor, problem solving and 
personal-social) at 12 months of age using ASQ-3 
6. Anxiety following result disclosure (or 8 weeks post enrollment for the unsequenced group), 
neonatal discharge and 12 months postpartum  
7. Depression following result disclosure (or 8 weeks post enrollment for the unsequenced group), 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
22 
neonatal discharge and 12 months postpartum 
8. Quality of life for the patient and family at 12 months postpartum  
9. Incremental cost per Quality Adjusted Life Year (QALY)  
4.10.3 Secondary Outcomes (Prenatal sequencing only) 
10. Apparent prenatal phenotypic expansion from currently defined pediatric phenotypes 
11. Variants of uncertain significance (VUS) that have not yet been associated with this disease 
phenotype  
12. VUS subclassified as compelling variants in novel genes that are not yet disease associated (genes 
of uncertain clinical significance; GUS)  
13. Pathogenic, likely pathogenic and VUS variants identified by sequencing (coding and non-coding 
regions) compared with coding regions only (digital WES) 
14. Pathogenic, likely pathogenic and VUS variants identified by analysis of a proband alone 
compared to a proband-parent trio  
15. Change in management decisions attributable to genomic results defined as changes to the 
patient’s treatment plan or changes to the counseling of the patient/family regarding the 
immediate or long-term medical management 
16. Accuracy of parental understanding of genetic test results 
17. Educational/counseling and social support needs of the mother and father 
18. Changes in classification of sequencing variants over time 
19. Turnaround time of sequencing components and how it changes over time. 
 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
23 
5 Statistical Considerations  
5.1 
Power and sample size 
The first primary outcome for this study is the frequency of pathogenic, likely pathogenic, or VUS 
genomic variants identified by sequencing among participants with a normal karyotype and CMA. The 
sample size is based on the incremental yield reported in a prospective study of WES performed at 
Columbia University in a similar population.55 Among 234 parent-fetus trios without CMA abnormalities, 
a genetic diagnosis was reported in 22 (9.4%). The precision of the estimate for a range of sample sizes 
and estimates is shown in Table 4. An estimate of 9.4% and sample size of 750 in the prenatal sequenced 
group will have a precision of 2.3% (confidence interval half-width) with a 93% probability.  
Table 4. Precision for range of estimates and sample sizes with at least 90% probability 
Estimate 
Sample size 
CI ½-width 
9.0% 
700 
2.4% 
 
750 
2.3% 
 
800 
2.2% 
9.4% 
700 
2.4% 
 
750 
2.3% 
 
800 
2.3% 
9.8% 
700 
2.5% 
 
750 
2.4% 
 
800 
2.3% 
 
The second primary outcome is healthcare costs per case from the diagnosis of an anomaly until neonatal 
discharge. The utilization of health care and cost only will include women that deliver at one of the study 
centers. Given that some of the recruiting centers include referrals in which the women will not deliver at 
the study center, power estimates assume cost will be available on 500 of the 750 in the prenatal 
sequencing group. Similarly, of the 350 unsequenced prenatal controls, 15% are assumed to have a 
positive pre-/postnatal CMA or deliver elsewhere resulting in approximately 300 unsequenced prenatal 
controls available for analysis. The total charges for deliveries at Columbia University that required a 
NICU admission in 2016 and 2017 (partial year) were used to provide cost estimates.  The charges follow 
a lognormal distribution approximately with a mean total charge of $277,902 and standard deviation of 
$389,484.  Assuming 500 participants in the prenatal WGS group, and 300 unsequenced prenatal control 
participants, the study will have 80% power to detect a mean ratio of 0.80 (20% reduction) with an 
alpha=0.05 two-sided.  
The secondary analyses focused on educational, counseling and psychosocial needs will compare the 
prenatal sequencing (those receiving a genetic result of any type) with the unsequenced prenatal group. 
Assuming an 80% completion rate will result in 600 prenatal sequencing and 280 unsequenced prenatal 
controls. Assuming a prevalence of 30% in the unsequenced prenatal control group for binary outcomes, 
the study will have more than 80% power to show a 30% reduction (30% to 21%) in the prenatal 
sequencing group with an alpha=0.05 two-sided. For a continuous outcome, the study will have 80% 
power to detect a small effect size given by Cohen’s d=0.2 with an alpha=0.05 two-sided.56 
5.2 
Prenatal Sequencing Group Only 
The primary and secondary outcomes for the sequencing variants are descriptive and will be reported as 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
24 
the observed proportion (frequency) with 95% confidence intervals. Exact confidence intervals will be 
reported as appropriate. Sequencing findings include variants or no variants (negative). Variants are 
further classified as pathogenic, likely pathogenic, or VUS. VUS is further categorized by whether the 
variants are in genes related to the fetal phenotype or known to cause severe childhood disease, or in a 
gene of uncertain significance (GUS). Sequencing findings will be reported overall, by single vs. multiple 
anomalies, by organ system (abdominal wall, CNS, face/ear, effusion, intrauterine fetal growth, GI tract, 
genitalia, heart, neck, renal tract, skeletal, spine, thorax), and by key demographics including maternal 
age, race/ethnicity and fetal sex. Pearson’s chi-square or Fisher’s Exact test for categorical variables and 
Mann-Whitney U test for continuous outcomes will be used to assess associations by variant 
classification.   
For each variant meeting the primary outcome definition (pathogenic, likely pathogenic, and uncertain 
genomic variants), the type of test that would identify the variant (WGS only, WES) will be determined 
centrally and used to report the incremental number of pathogenic, likely pathogenic, and VUS variants 
identified by WGS compared with WES.  
Change in management decisions attributable to sequencing results are descriptive and will be reported as 
the frequency with 95% confidence intervals overall and by variant classification.    
5.3 Prenatal Sequencing and Prenatal Unsequenced Groups 
Clinical management including health care utilization and cost, and pregnancy and neonatal outcomes 
will be compared among the prenatal sequencing group and the unsequenced prenatal group. Women 
whose fetus or neonate is found to have positive/causal findings on CMA will be excluded from these 
analyses. Initially, demographic data and severity of ultrasound findings will be compared to ensure there 
are no significant differences between the two study groups (prenatal sequencing and unsequenced 
prenatal group).  If differences are found, the covariates will be adjusted for in the analyses.  
Categorical variables will be reported as the number and frequency and associations assessed by the 
Pearson’s chi-square or Fisher’s Exact test. The distributions for continuous variables will be assessed for 
normality and transformed to fit a normal distribution if possible. Normal distributions will be reported as 
mean and standard deviation and compared with the t-test, and non-normal data will be reported as 
median and interquartile range and compared with the Mann-Whitney U test. 
For each outcome, if the study groups show a significant difference, interactions will be tested and 
subgroup analyses conducted if the interaction is significant (p<0.05). Pre-specified subgroup analyses 
include maternal age, race/ethnicity and fetal sex. 
The amount of missing data, missing data patterns, and identification of variables associated with 
missingness will be explored to inform the primary and sensitivity statistical analyses. Analytic 
techniques used to address missing data bias will be used as appropriate. A two-sided nominal p-value 
less than 0.05 will indicate statistical significance.  
5.3.1 Healthcare utilization and cost 
Direct medical costs including cost of prenatal/postnatal sequencing, medical care for the women and 
newborn (hospital costs, surgeries, emergency room visits, physician office visits, outpatient services, 
home health services, and medications) will be estimated using health care charges associated with each 
of these services. Unit costs for outpatient services, surgical procedures, laboratory tests, medications and 
consultations will be adjusted by health system specific cost-to-charge ratios. If cost-to-charge ratios are 
not available, published ratios or in rare cases Medicaid reimbursements will be used.   
The effect or outcome measure for cost effectiveness will be quality-adjusted life years (QALYs). 
Maternal and neonatal QALYs will be estimated by applying published utility weights (1=perfect health, 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
25 
0=death) from various health states to components of the conditions found by sequencing.  Cost-
effectiveness of prenatal sequencing will be evaluated as the incremental cost per QALY with a threshold 
of less than $100,000 per QALY as cost effective. Cost benefit analysis will also be performed and be 
evaluated by dividing the costs of care and outcomes from the intervention by the costs of usual care. A 
CBA < 1 indicates that the intervention is cost saving and thereby cost beneficial. 
All estimates of costs and outcomes will be reported as means with 95% confidence intervals.  Sensitivity 
analysis will be conducted to evaluate the impact of uncertainty on the results. Varying probabilities (e.g. 
baseline risk of sequencing findings, change in neonatal outcomes) and cost parameters (e.g. cost of 
sequencing, healthcare costs) will be used to take into account potential clinical scenarios that might 
deviate from the baseline estimates. One-way deterministic sensitivity analysis for individual factors will 
be employed and Monte Carlo simulation to assess the robustness of the findings by simultaneously 
sampling distributions around multiple parameters within the model and report 95% confidence ellipses 
and acceptability curves. 
5.3.2 Psychosocial outcomes 
The secondary psychosocial outcomes are descriptive and will be reported as the observed proportion 
(frequency) with 95% confidence intervals. Exact confidence intervals will be reported as appropriate. All 
outcomes will be assessed at three time periods (post disclosure, post discharge, and 12 months 
postpartum) and each time period will be analyzed separately. The initial analyses will compare prenatal 
sequencing (those receiving a genetic result of any type) with the unsequenced prenatal group. 
Exploratory analyses will include pairwise comparisons between each variant classification (unsequenced 
prenatal group, negative sequencing result, VUS result, and pathogenic/likely pathogenic sequencing 
results).  
Associations between the outcomes and key demographics also will be assessed. Further, association 
between outcomes and study groups will be tested in a multivariate setting adjusting for other variables 
hypothesized to affect outcome (e.g. demographics, genetic essentialism and genetic optimism). 
Continuous outcomes will be analyzed using a multivariate linear regression model and binary outcomes 
using a logistic regression model. Standard model selection methods and regression diagnostics will be 
performed to assess goodness of fit.  
A thematic analysis will be performed for the qualitative interview data to identify patterns or themes in 
these data.  Summary statistics will be reported for the themes.   

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
26 
6 Data Collection 
6.1 Web Data Entry Systems 
A web data entry system will be set up to present data screens for the entry of the data listed below.  Data 
will be collected on standardized forms on which most responses have been pre-coded. Data collection, 
including summary sequencing result data, will be either directly entered from source material and 
entered on the web interface or entered on case report forms for later keying on site.  For collection of 
pre- and postnatal phenotype data, use in MIDAS of the Human Phenotype Ontology (HPO) will be 
incorporated.  Documents such as the prenatal ultrasound and sequencing results reports will be 
transferred to the DCC via the MIDAS system once de-identified 
The forms will be set up in 2 web-based data entry systems as described below:  
• 
MIDAS (Multimodal Integrated Data Acquisition System) is a data entry and management 
system designed specifically for research studies. Data will be entered using a web interface into 
the MySQL database located at the Data Coordinating Center. The system allows extensive data 
auditing and reporting to assist users with data correction/verification as well as patient 
management.  
• 
REDCap is a web-based and database-backed platform.  The psychosocial survey instruments will 
be completed directly by study participants using REDCap software hosted by the Clinical 
Coordinating Center. Data will be exported regularly to the DCC.  
6.1.1  Data Collection Forms 
The following forms will be entered into MIDAS: 
PG01: 
Screening Log  
PG01A 
Lab sample tracking form  
PG02:  
Screening Results and Eligibility Form   
PG03: 
Prenatal Imaging Form  
PG04: 
Baseline Data Form (includes demographics, relevant maternal history including previous 
pregnancy data) 
PG06: 
Sequencing Results Summary Form 
PG07: 
Delivery/ Neonatal Clinical Outcome Form includes outcomes through discharge 
PG08: 
Health Management Report to be completed by attending provider(s) 
PG09: 
Clinical Cost Form includes prenatal and pre-discharge cost 
PG10: 
One Year Outcome Form 
PG11: 
Ages and Stages Questionnaire (ASQ-3) 
PG12: 
Adverse Event Form 
PG13: 
Patient Status Form includes withdrawal status 
 
 
The following form will be entered directly into REDCap by participants:  
PG14: 
Psychosocial survey 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
27 
6.2 Centralized Data Management  
The DCC will monitor on an ongoing basis the acquisition, completeness and quality of data. This will 
include review of laboratory compliance in timeliness of reporting prenatal diagnosis testing, including 
the evaluation and then the reporting of sequencing results to patients, where applicable.  These data will 
be edited for missing, out of range and inconsistent values, and queries forwarded to their point of origin 
for review and resolution. Reports including timelines for form completion will be generated prompting 
submission of outstanding data forms.  
Bioinformatics files generated during sequencing will be stored initially at the sequencing laboratories 
and finally submitted directly to NIH repository(ies). The DCC will prepare summary and clinical data for 
submission to the appropriate NIH data repository.  (See Data archiving) 
6.3 Performance Monitoring 
Site visits will be conducted by DCC staff to the recruitment centers and the sequencing laboratories, 
accompanied by a laboratory supervisor from another participating laboratory or a member of Variant 
Oversight Committee.  The purpose of each site visit is to review study procedures, assess compliance 
with the study protocol, and assess the quality of the study data and records.  A written report will be 
reviewed by the Steering Committee.   
The DCC will also present regular reports to the Steering Committee. These include: 
• 
Monthly recruitment reports - reports of the number of patients screened and enrolled by month and 
by recruitment site.  
• 
Quarterly Steering Committee reports - a report detailing recruitment, baseline patient characteristics, 
data quality, incidence of missing data and adherence to study protocol by recruitment site/ 
sequencing laboratory. 
 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
28 
7 WGS Pipeline Development 
 
7.1 Evaluation of the incremental value of WGS compared to WES in 
understanding the etiology of birth defects 
This study will provide important insights into the genetic basis of birth defects.  At present, the etiology 
of many structural anomalies can be determined by WES and as additional genes and variants in the 
coding regions are related to specific phenotypes, additional causes will become known.  However, 
studies to date strongly suggest that WES, while clinically valuable, will not identify all causes. By 
exploring the frequency of de novo variation in regulatory and other non-coding regions of the genome 
and evaluating their distribution in functional classes (SNVs, SVs and CNVs), additional understanding 
of genetic causes of developmental alterations will occur. Accordingly, this exploratory aim will 
comprehensively interrogate WGS data to identify the role of errors in noncoding regions in the etiology 
of birth defects.  The aim will also explore the development of pipelines and software to improve the 
integration of WGS into clinical care. This will require the development of multiple variant calling tools 
to catalog a comprehensive set of variants including SNVs/Indels, STRs, SVs, and CNVsI from WGS. 
The current variant assessment framework will be extended to include the interpretation of WGS data 
outside of protein-coding regions through the evaluation of: 1) Regions intolerant to variation as assessed 
from population genetics; 2) Cis-regulatory regions known to regulate genes that are intolerant to 
functional variation and to affect transcript levels or splicing; and 3) enhancer elements and other eQTLs 
deduced to impact expression of known intolerant genes. This will result in a comprehensive set of 
regulatory regions that would be especially useful in evaluating SVs and CNVs where a functional effect 
(such as loss of important enhancer) is easier to predict. Although similar tools have previously been used 
for specific research ends, this study will refine and adjust their heuristic filters to achieve optimal 
sensitivity and appropriateness for clinical care and expand the diagnostic yield of WGS. 
Furthermore, in partnership with Illumina Inc., these tools will be benchmarked to newer implementations 
of variant callers that have been specifically tuned for WGS data and optimized for speed and 
computational efficiency. To identify intolerant regions, previous experience using population allele 
frequencies of standing variation will form a basis on which to quantify purifying selection. The recently 
developed Orion, an intolerance metric based on the difference between an observed and expected site 
frequency spectrum under a neutral model, showed that it could accurately identify intolerant regions 
devoid of functional annotation. This method will be applied on a ten-fold larger WGS cohort (N=62,000, 
https://bravo.sph.umich.edu/freeze5) to calculate the observed variation and estimate the expected site 
frequency spectrum using mutation rates under hepta-nucleotide context (a tri-nucleotide context used 
originally) to refine a set of intolerant regions for assessment. It is anticipated that a hepta-nucleotide 
context will capture selection that is likely to occur at DNA-binding motifs more optimally and therefore 
allow a truer estimate of regional site frequency spectrum using the neutral model. Indeed, ~80% of 
GWAS non-coding variants map to putative cis-regulatory elements (CREs). Synonymous and non-
canonical intronic variants will be assessed using a recently developed TRaP score (shown to identify 
putative deleterious synonymous and intronic substitutions with >98% specificity). Data from the 
ENCyclopedia Of DNA Elements (ENCODE) (such as ChIP-seq, Hi-C, ChIA-Pet, and 5C-seq) will be 
incorporated to determine a set of regulatory elements of intolerant and haplo-insufficient genes. Finally, 
regulatory regions linked to intolerant genes will be incorporated using correlations between enhancer 
activity and gene expression across human tissues.  
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
29 
8 Study Administration 
8.1 Organization and Funding 
This study represents a collaboration of clinical geneticists, maternal fetal medicine physicians, genetic 
counselors, pediatric geneticists, biostatisticians, social sciences researchers, bioinformaticians and 
laboratory-based whole genome sequencing technical specialists. 
Table 5. Participating Organizations and Roles 
 
Organization 
Role 
Clinical Coordinating Center 
(CCC) 
Columbia University 
Project leadership, management, central IRB, 
overall coordination, central patient follow-up 
site 
Recruitment Sites and 
sequencing Laboratories 
Baylor, Columbia, UNC  
Recruiting for the study and performing whole 
genome sequencing, variant calling, 
interpretation, confirmation, and reporting of 
results   
Data Coordinating Center 
(DCC) 
George Washington University 
Biostatistics Center 
Protocol development, data collection, 
oversight and analysis 
Cost-effectiveness expertise 
Oregon Health Sciences 
University 
Oversight of cost analysis 
 Sequencing technology 
Illumina 
Support for sequencing 
8.1.1 Clinical Coordinating Center 
The Clinical Coordinating Center (CCC) is responsible for leading and overseeing all aspects of this 
study.  In this capacity the CCC will centrally coordinate study implementation, ongoing study 
management, patient follow-up, and publication of the study results.  The CCC will serve as the liaison 
between the participating study locations, the funding agency and the Data Coordinating Center.   
8.1.2 Data Coordinating Center 
The Data Coordinating Center (DCC) is responsible for all aspects of biostatistical design, data 
management, statistical analyses, and preparation of publications based on the study results.  
8.2 Committees 
8.2.1 Steering Committee 
The Steering Committee is the policy and decision-making group, and assumes overall responsibility for 
the management and conduct of the study including protocol development, oversight of the conduct of the 
protocol, analysis and interpretation of data, and reporting results in presentations and publications. The 
committee will ensure that there are synergies between the components of the project and will evaluate 
and provide overall direction. It will also ensure that the team makes measurable progress toward stated 
goals, operates within budget, follows federal policies, and submits required reports in a timely manner.   
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
30 
8.2.2 Operations Committee 
The Operations Committee is responsible for monitoring study implementation, recruitment, day to day 
management, quality control, coordination and efficient communications. The committee also oversees 
protocol training, study logistics, recruitment progress, and monitors study updates.  
8.2.3 Variant Adjudication Committee 
The Variant Adjudication Committee will include external experts who will assist with interpretation of 
variants for which consensus interpretation by the local lab directors and PIs cannot be reached.  The 
Committee’s determination will be considered the official study result. 
8.2.4 Variant Oversight Committee 
The Variant Oversight Committee will include study lab directors/study lead and relevant site lab 
members and genetic counselors from each site. The Committee will identify systematic differences 
between sequencing laboratories, if any, and will serve as a “learning lab” to standardize fetal sequencing 
results. Specific case examples will be used to illustrate processes and examine any differences.  
8.2.5 Clinical Adjudication Committee 
The Clinical Adjudication Committee will include experts in maternal fetal medicine, genetics and 
pediatrics. The Committee will review the health management reports to ascertain, without bias, whether 
an alteration in health care management occurred based on sequencing results.  
8.2.6 WGS Pipeline Development Committee 
The WGS Pipeline Development Committee which includes bioinformaticians from each site will 
develop a WGS pipeline for fetal diagnosis and identify new pathways. 
8.2.7 Data and Safety Monitoring Board (DSMB) 
The DSMB will be made up of the external members of the Variant and Clinical Adjudication 
committees. The committee will approve the study protocol prior to initiation of the study and meet every 
6 months in the course of the study. It will review interim and final reports on recruitment, and monitor 
logistical issues and adverse events. The committee may also recommend protocol modification or early 
termination of the study due to unexpected problems. 
 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
31 
9 Study Timetable 
 
The timetable for the study is depicted below. 
 
Figure 2. Timeline 
Finalize Study Documents / IRB / 
Training / Certification
 
Recruitment
 
Follow-up
 
Closeout/ Data Analysis / 
Data Archiving
 
2018
2024
2019
2020
2021
2022
2023
 
 
9.1 Training and Certification 
The DCC will collaborate with the Clinical Coordinating Center to organize a workshop to train the 
genetic counselors at recruitment sites and the follow-up staff at Columbia in study procedures for this 
study, including data form completion and web entry.  The manual of operations will serve as the text for 
the training.   
Additionally, DCC personnel will work with the Clinical Coordinating Center and Lab Practices 
Committee to clarify and standardize laboratory practices relating to sample handling and WGS analysis.  
This will include review of the electronic format for the recording of results in the MIDAS database. Data 
handling methods will be standardized across sites, and technical staff at the DCC will be responsible for 
preparing systems to achieve this.   
Before the study can be implemented, each recruiting site must be certified.  Certification will include 
successful completion of the training session.   
9.2 Recruitment and Data Collection Period 
Educational materials will be developed targeted to participants and referring physicians.   
There are 1,800 pregnancies/year with structural anomalies eligible for enrollment in the study at the three 
centers. Assuming that 40% of these will undergo diagnostic testing, and 25% will have an abnormal 
karyotype or CMA, and 50% of those eligible will consent to sequencing, it is estimated that 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
32 
approximately 270 patients per year or 23 patients will be enrolled per month, with overall recruitment of 
750 patients completed in ~33 months. The 350 controls would be recruited within the same time period.   
9.3 Final Analysis 
After a two-month period for completion of data entry and cleaning of the database for the recruitment 
portion of the main study, the recruitment/results sections of the data set will be locked and available for 
analysis. At the end of the 12 month postpartum follow-up period, approximately three months will be 
required to complete the final report to the Steering Committee and to submit the study’s primary report 
for publication. 
 
9.4 Data Archiving and Sample Storage 
9.4.1 Data Archiving 
At the conclusion of the study, the final dataset will be prepared by the DCC for archiving and sharing. 
Full de-identification of the data will be undertaken prior to submission to an NIH or public repository, 
including allocation of random record IDs replacing the Study ID used in the course of the study. Dates 
are converted to days and consideration is given to appropriate handling of unique aspects of clinical data 
which may serve to identify individuals. Genomic sequencing VCF files will be de-identified in a similar 
manner and the appropriate random ID to match the clinical record will be applied. Laboratories will be 
responsible for depositing any large sequencing files in the requisite repositories directly.  
9.4.2 Data Sharing 
In accordance with the NIH Genomic Data Sharing policy, sequencing data (including VCF files) and 
clinical data will be shared with other scientific investigators and through the controlled access dbGAP 
repository or comparable genomics commons, the Sequence Read Archive, and any NIH Birth Defects 
Commons that is established. A final dataset containing clinical and phenotypic data will be submitted to 
the NICHD data repository (DASH).  In addition, new algorithms and allele frequency data will be shared 
with the newly developed Precision FDA platform as applicable. 
9.4.3 Specimen Storage and Sharing 
Remaining DNA from the trios will be stored at the local sequencing sites for secondary analyses and 
ancillary studies. The Steering Committee will review and approve proposals for use of the remaining 
DNA.  The DNA will be made available to non-study investigators with appropriate IRB approval, Data 
Use Agreements and/or Material Transfer Agreements. Samples will be re-labeled using a random sample 
ID that is linked to the random record ID prior to distribution to non-study investigators.  

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
33 
Appendix A. Eligible Major Structural Anomalies 
Abdominal wall 
Heart 
 
Bladder exstrophy/Epispadia 
 
Anomalous pulmonary venous return 
 
Body-stalk anomaly 
 
Aortic stenosis/atresia 
 
Cloacal exstrophy 
 
Arrythmia 
 
Gastroschisis 
 
 
ASD 
 
Omphalocele 
 
AV canal defect 
CNS 
 
Coarctation 
 
Absent or hypoplastic cerebellar vermis 
 
Dextrocardia 
 
Agenesis of corpus collosum 
 
Double outlet right vertricle (DORV) 
 
Anencephaly/Acrania 
 
Ebsteins anomaly 
 
Arachnoid cyst  
 
Heart tumor 
 
Cerebellar hypoplasia 
 
Hypoplastic left heart 
 
Chiari malformation 
 
Hypoplastic right heart 
 
Dandy-Walker malformation 
 
Interrupted aortic arch 
 
Encephalocele 
 
Myxoma 
 
Heterotopia 
 
Pulmonary stenosis/atresia 
 
Holoprosencephaly 
 
Tetralogy of Fallot 
 
Hydranencephaly 
 
Transposition 
 
Iniencephaly 
 
Truncus Arteriosus 
 
Macrocephaly (relative to fetal size) 
 
VSD 
 
Megalencephaly 
 
 
Single ventricle 
 
Microcephaly 
 
Tricuspid atresia/stenosis 
 
Lissencephaly 
 
VSD 
 
Parenchymal defect (gyral anomaly) 
 
 
NS cardiac (abn 4-chamber view/outflow tracts, atrial/vent dilation) 
 
Posterior fossa cyst 
Abdomen, intestine, and liver anomalies 
 
Spina bifida 
 
Abnormal adrenal glands (tumor, uncertain) 
 
Tumor 
 
Bladder (dilated tense/floppy, ureterocele, duplex system) 
 
Vascular anomaly 
 
Bilateral congenital hydronephrosis 
 
Ventriculomegaly/Hydrocephaly 
 
Echogenic kidney 
Ear 
 
Horseshoe kidney 
 
Anotia  
 
Large kidney 
 
Outer ear malformation 
 
Multicystic kidney 
Neck 
 
Pelvic kidney 
 
Cystic hygroma 
 
Polycystic kidney 
 
Teratoma 
 
Small kidney 
Eye 
 
Multi-cystic renal dysplasia 
 
Anophthalmia/Micropthalmos 
 
Renal agenesis 
 
Congenital cataract 
 
Urethra (absent, dilated/valves) 
 
Cyclopia 
Skeletal 
 
Hypertelorism 
 
Skeletal dysplasia 
 
Hypotelorism 
 
Cloverleaf skull 
Face 
 
Hip dislocation/dysplasia 
 
Facial tumor 
 
Limb defect 
 
Lip – Cleft 
 
Foot (absent, oligo-/poly-/syn-dactyly, rocker bottom foot, split foot) 
Genitalia 
 
 
Hand (absent, brachy-/oligo-/ syn-dactyly, overlapping fingers,  
polydactyly (only if non-familial), split hand) 
 
Ambiguous genitalia 
 
Hypospadias 
 
Joints (fixed extended, fixed flexed)  
 
Micropenis 
 
Talipes 
 
 
 
Long bones (absent, bowed, short (<1st %ile), fracture) 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
34 
Nose  
Skin 
 
Depressed nasal bridge 
 
Congenital skin disorder 
 
Palate – Cleft 
 
Hemangioma 
 
Abnormal profile 
 
Tumor, unspecified 
 
Frontal bossing 
Spine 
 
Micrognathia/retrognathia 
 
Kyphosis 
Gastro-intestinal tract 
 
Sacral agenesis 
 
Ano-rectal atresia and stenosis 
 
Sacrococcygeal teratoma 
 
Large bowel obstruction 
 
Scoliosis 
 
Small bowel obstruction 
 
Sirenomelia 
 
Duodenal atresia/Stenosis 
Thorax/Respiratory 
 
Situs abnormality 
 
Congenital diaphragmatic hernia 
Head shape 
 
Choanal atresia 
 
Abnormal skull shape 
 
Congenital lung lesion/CCAM 
 
Craniosynostosis 
 
Hydrothorax 
 
Abnormal calcification 
 
Hypoplastic thorax 
Effusion 
 
Bell-shaped thorax 
 
Hydrops 
 
Short ribs 
 
 
Ascites 
Other (reviewed centrally prior to enrollment) 
 
Lymphangioma 
 
 
Pleural effusion 
 
 
 
Skin edema 
 
 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
35 
References 
 
1. 
Wapner RJ, Martin CL, Levy B, et al. Chromosomal microarray versus karyotyping for prenatal 
diagnosis. The New England journal of medicine 2012;367:2175-84. 
2. 
Osterman MJ, Kochanek KD, MacDorman MF, Strobino DM, Guyer B. Annual summary of vital 
statistics: 2012-2013. Pediatrics 2015;135:1115-25. 
3. 
Shaffer LG, Rosenfeld JA, Dabell MP, et al. Detection rates of clinically significant genomic 
alterations by microarray analysis for specific anomalies detected by ultrasound. Prenatal diagnosis 
2012;32:986-95. 
4. 
Miller NA, Farrow EG, Gibson M, et al. A 26-hour system of highly sensitive whole genome 
sequencing for emergency management of genetic diseases. Genome medicine 2015;7:100. 
5. 
Stavropoulos DJ, Merico D, Jobling R, et al. Whole Genome Sequencing Expands Diagnostic Utility 
and Improves Clinical Management in Pediatric Medicine. NPJ genomic medicine 2016;1. 
6. 
Turner TN, Coe BP, Dickel DE, et al. Genomic Patterns of De Novo Mutation in Simplex Autism. 
Cell 2017;171:710-22 e12. 
7. 
Illsinger S, Das AM. Impact of selected inborn errors of metabolism on prenatal and neonatal 
development. IUBMB life 2010;62:403-13. 
8. 
Matsui H, Gardiner H. Fetal intervention for cardiac disease: the cutting edge of perinatal care. 
Seminars in fetal & neonatal medicine 2007;12:482-9. 
9. 
Bienstock JL, Birsner ML, Coleman F, Hueppchen NA. Successful in utero intervention for bilateral 
renal agenesis. Obstetrics and gynecology 2014;124:413-5. 
10. Deprest J, Gratacos E, Nicolaides KH, Group FT. Fetoscopic tracheal occlusion (FETO) for severe 
congenital diaphragmatic hernia: evolution of a technique and preliminary results. Ultrasound in 
obstetrics & gynecology : the official journal of the International Society of Ultrasound in Obstetrics 
and Gynecology 2004;24:121-6. 
11. Jani J, Gratacos E, Greenough A, et al. Percutaneous fetal endoscopic tracheal occlusion (FETO) for 
severe left-sided congenital diaphragmatic hernia. Clinical obstetrics and gynecology 2005;48:910-
22. 
12. Knox EM, Kilby MD, Martin WL, Khan KS. In-utero pulmonary drainage in the management of 
primary hydrothorax and congenital cystic lung lesion: a systematic review. Ultrasound in obstetrics 
& gynecology : the official journal of the International Society of Ultrasound in Obstetrics and 
Gynecology 2006;28:726-34. 
13. Adzick NS, Thom EA, Spong CY, et al. A randomized trial of prenatal versus postnatal repair of 
myelomeningocele. The New England journal of medicine 2011;364:993-1004. 
14. Johnson MP, Bukowski TP, Reitleman C, Isada NB, Pryde PG, Evans MI. In utero surgical treatment 
of fetal obstructive uropathy: a new comprehensive approach to identify appropriate candidates for 
vesicoamniotic shunt therapy. Am J Obstet Gynecol 1994;170:1770-6; discussion 6-9. 
15. Sofou K, Dahlin M, Hallbook T, Lindefeldt M, Viggedal G, Darin N. Ketogenic diet in pyruvate 
dehydrogenase complex deficiency: short- and long-term outcomes. Journal of inherited metabolic 
disease 2017;40:237-45. 
16. Castiglioni C, Verrigni D, Okuma C, et al. Pyruvate dehydrogenase deficiency presenting as isolated 
paroxysmal exercise induced dystonia successfully reversed with thiamine supplementation. Case 
report and mini-review. European journal of paediatric neurology : EJPN : official journal of the 
European Paediatric Neurology Society 2015;19:497-503. 
17. Chitty LS DA, Gottschalk I, et al. EP21.04: BOOSTB4: a clinical study to determine safety and 
efficacy of pre- and/or postnatal stem cell transplantation for treatment of osteogenesis imperfecta. 
Ultrasound in obstetrics & gynecology : the official journal of the International Society of 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
36 
Ultrasound in Obstetrics and Gynecology 2016;48:356-. 
18. Horwitz EM, Prockop DJ, Gordon PL, et al. Clinical responses to bone marrow transplantation in 
children with severe osteogenesis imperfecta. Blood 2001;97:1227-31. 
19. Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone 
marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nature medicine 
1999;5:309-13. 
20. Gotherstrom C, Westgren M, Shaw SW, et al. Pre- and postnatal transplantation of fetal 
mesenchymal stem cells in osteogenesis imperfecta: a two-center experience. Stem cells translational 
medicine 2014;3:255-64. 
21. Cukier HN, Dueker ND, Slifer SH, et al. Exome sequencing of extended families with autism reveals 
genes shared across neurodevelopmental and neuropsychiatric disorders. Molecular autism 2014;5:1. 
22. Yang Y, Muzny DM, Reid JG, et al. Clinical whole-exome sequencing for the diagnosis of 
mendelian disorders. The New England journal of medicine 2013;369:1502-11. 
23. Krupp DR, Barnard RA, Duffourd Y, et al. Exonic Mosaic Mutations Contribute Risk for Autism 
Spectrum Disorder. American journal of human genetics 2017;101:369-90. 
24. Lim ET, Uddin M, De Rubeis S, et al. Rates, distribution and implications of postzygotic mosaic 
mutations in autism spectrum disorder. Nature neuroscience 2017;20:1217-24. 
25. Sanders SJ, Neale BM, Huang H, et al. Whole genome sequencing in psychiatric disorders: the 
WGSPD consortium. Nature neuroscience 2017;20:1661-8. 
26. Abou Tayoun AN, Spinner NB, Rehm HL, Green RC, Bianchi DW. Prenatal DNA Sequencing: 
Clinical, Counseling, and Diagnostic Laboratory Considerations. Prenatal diagnosis 2018;38:26-32. 
27. Best S, Wou K, Vora N, Van der Veyver IB, Wapner R, Chitty LS. Promises, pitfalls and 
practicalities of prenatal whole exome sequencing. Prenatal diagnosis 2018;38:10-9. 
28. Normand EA, Braxton A, Nassef S, et al. Clinical exome sequencing for fetuses with ultrasound 
abnormalities and a suspected Mendelian disorder. Genome medicine 2018;10:74. 
29. Aarabi M, Sniezek O, Jiang H, et al. Importance of complete phenotyping in prenatal whole exome 
sequencing. Human genetics 2018;137:175-81. 
30. Fu F, Li R, Li Y, et al. Whole exome sequencing as a diagnostic adjunct to clinical testing in fetuses 
with structural abnormalities. Ultrasound in obstetrics & gynecology : the official journal of the 
International Society of Ultrasound in Obstetrics and Gynecology 2018;51:493-502. 
31. Lei TY, Fu F, Li R, et al. Whole-exome sequencing for prenatal diagnosis of fetuses with congenital 
anomalies of the kidney and urinary tract. Nephrology, dialysis, transplantation : official publication 
of the European Dialysis and Transplant Association - European Renal Association 2017;32:1665-
75. 
32. Vora NL, Powell B, Brandt A, et al. Prenatal exome sequencing in anomalous fetuses: new 
opportunities and challenges. Genetics in medicine : official journal of the American College of 
Medical Genetics 2017;19:1207-16. 
33. Yates CL, Monaghan KG, Copenheaver D, et al. Whole-exome sequencing on deceased fetuses with 
ultrasound anomalies: expanding our knowledge of genetic disease during fetal development. 
Genetics in medicine : official journal of the American College of Medical Genetics 2017;19:1171-8. 
34. Pangalos C, Hagnefelt B, Lilakos K, Konialis C. First applications of a targeted exome sequencing 
approach in fetuses with ultrasound abnormalities reveals an important fraction of cases with 
associated gene defects. PeerJ 2016;4:e1955. 
35. Alamillo CL, Powis Z, Farwell K, et al. Exome sequencing positively identified relevant alterations 
in more than half of cases with an indication of prenatal ultrasound anomalies. Prenatal diagnosis 
2015;35:1073-8. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
37 
36. Drury S, Williams H, Trump N, et al. Exome sequencing for prenatal diagnosis of fetuses with 
sonographic abnormalities. Prenatal diagnosis 2015;35:1010-7. 
37. Carss KJ, Hillman SC, Parthiban V, et al. Exome sequencing improves genetic diagnosis of 
structural fetal abnormalities revealed by ultrasound. Human molecular genetics 2014;23:3269-77. 
38. Yang Y, Muzny DM, Xia F, et al. Molecular findings among patients referred for clinical whole-
exome sequencing. Jama 2014;312:1870-9. 
39. Meng L, Pammi M, Saronwala A, et al. Use of Exome Sequencing for Infants in Intensive Care 
Units: Ascertainment of Severe Single-Gene Disorders and Effect on Medical Management. JAMA 
pediatrics 2017;171:e173438. 
40. Clark MM, Stark Z, Farnaes L, et al. Meta-analysis of the diagnostic and clinical utility of genome 
and exome sequencing and chromosomal microarray in children with suspected genetic diseases. 
NPJ genomic medicine 2018;3:16. 
41. Tan TY, Dillon OJ, Stark Z, et al. Diagnostic Impact and Cost-effectiveness of Whole-Exome 
Sequencing for Ambulant Children With Suspected Monogenic Conditions. JAMA pediatrics 
2017;171:855-62. 
42. Biesecker BB, Klein W, Lewis KL, et al. How do research participants perceive "uncertainty" in 
genome sequencing? Genetics in medicine : official journal of the American College of Medical 
Genetics 2014;16:977-80. 
43. Gray SW, Martins Y, Feuerman LZ, et al. Social and behavioral research in genomic sequencing: 
approaches from the Clinical Sequencing Exploratory Research Consortium Outcomes and Measures 
Working Group. Genetics in medicine : official journal of the American College of Medical Genetics 
2014;16:727-35. 
44. Kaphingst KA, Ivanovich J, Biesecker BB, et al. Preferences for return of incidental findings from 
genome sequencing among women diagnosed with breast cancer at a young age. Clinical genetics 
2016;89:378-84. 
45. Kalynchuk EJ, Althouse A, Parker LS, Saller DN, Jr., Rajkovic A. Prenatal whole-exome 
sequencing: parental attitudes. Prenatal diagnosis 2015;35:1030-6. 
46. Kuppermann M, Nakagawa S, Cohen SR, Dominguez-Pareto I, Shaffer BL, Holloway SD. Attitudes 
toward prenatal testing and pregnancy termination among a diverse population of parents of children 
with intellectual disabilities. Prenatal diagnosis 2011;31:1251-8. 
47. Bernhardt BA, Soucier D, Hanson K, Savage MS, Jackson L, Wapner RJ. Women's experiences 
receiving abnormal prenatal chromosomal microarray testing results. Genetics in medicine : official 
journal of the American College of Medical Genetics 2013;15:139-45. 
48. Case AP, Ramadhani TA, Canfield MA, Wicklund CA. Awareness and attitudes regarding prenatal 
testing among Texas women of childbearing age. Journal of genetic counseling 2007;16:655-61. 
49. Kuppermann M, Gates E, Washington AE. Racial-ethnic differences in prenatal diagnostic test use 
and outcomes: preferences, socioeconomics, or patient knowledge? Obstetrics and gynecology 
1996;87:675-82. 
50. Wapner RJ, Driscoll DA, Simpson JL. Integration of microarray technology into prenatal diagnosis: 
counselling issues generated during the NICHD clinical trial. Prenatal diagnosis 2012;32:396-400. 
51. Hillman SC, Willams D, Carss KJ, McMullan DJ, Hurles ME, Kilby MD. Prenatal exome 
sequencing for fetuses with structural abnormalities: the next step. Ultrasound in obstetrics & 
gynecology : the official journal of the International Society of Ultrasound in Obstetrics and 
Gynecology 2015;45:4-9. 
52. Economides DL, Whitlow BJ, Kadir R, Lazanakis M, Verdin SM. First trimester sonographic 
detection of chromosomal abnormalities in an unselected population. British journal of obstetrics and 
gynaecology 1998;105:58-62. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
Version 1.0 
Protocol # 
 
 
 
 
 
 
 
 
 
14 Jan 2019 
38 
53. Whitlow BJ, Chatzipapas IK, Lazanakis ML, Kadir RA, Economides DL. The value of sonography 
in early pregnancy for the detection of fetal abnormalities in an unselected population. British journal 
of obstetrics and gynaecology 1999;106:929-36. 
54. Forsyth BW, Horwitz SM, Leventhal JM, Burger J, Leaf PJ. The child vulnerability scale: an 
instrument to measure parental perceptions of child vulnerability. Journal of pediatric psychology 
1996;21:89-101. 
55. Wapner R PS, Brennan K, Bier L, Wou K, Goldstein D. Whole exome sequencing in the evaluation 
of fetal structural anomalies: A prospective study of sequential patients. Am J Obstet 
Gynecol;216(1):S5-S6. 
56. J C. Statistical Power Analysis for the Behavioral Sciences (second ed.). Hillsdale, NJ: Lawrence 
Erlbaum Associates; 1988. 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
1 
Prenatal Genetic Diagnosis by Genomic Sequencing 
(PrenatalSEQ) 
  
 
  
 
Grant Title: Prenatal Genetic Diagnosis by Genomic Sequencing: A 
Prospective Evaluation 
 
Grant Number: R01HD055651 
 
 
Funded by: The National Institute of Child Health and Human Development 
 
 
 
 
Version Number: v.1.8 
 
27 March 2023 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
2 
Protocol Amendment History (Cumulative): 
Date 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
27 Mar 2023 
1.1, 3.1, 
3.2.3, 4.1, 
4.1.1, 
4.2.3, 4.6 
Added enrollment of retrospective 
unsequenced prenatal group 
Prospective enrollment of 
unsequenced prenatal group was 
not feasible. To meet sample 
size target, added enrollment of 
retrospective unsequenced 
prenatal group participants. 
 
4.7 
Added additional questions to the 
psychosocial survey measures on 
COVID-19, reasons for joining the 
sequenced or unsequenced group, and 
the impact of sequencing results  
To gain better understanding of 
the impact of these events. 
 
4.10 
Clarified neonatal hospital charges are 
between delivery and infant discharge 
or death 
Fetal deaths will not have 
neonatal charges 
 
4.10.2 
Clarified secondary outcomes that will 
be available only on those enrolled 
prospectively 
Retrospective unsequenced 
prenatal group will not have 
outcomes assessed at 12 months 
postpartum 
 
5.1 
Removed power calculations for the 
secondary analysis focused on 
educational, counseling and 
psychosocial needs. 
Study will not have sufficient 
power to evaluate since the 
retrospective unsequenced 
prenatal group will not collect 
these outcomes 
 
5.3 
Clarified twelve month postpartum 
outcomes will be compared only 
among those individuals enrolled 
prospectively 
Retrospective unsequenced 
participants will not collect 
these outcomes 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
3 
22 Dec 2021 
3.1, 4.4, 
8.1 
Clarified that there are now four labs 
performing sequencing for the study 
Columbia has switched to using 
the New York Genome Center 
(NYGC) as their sequencing lab 
 
3.2.1, 
3.2.2 
Added the inclusion of dichorionic 
diamniotic twin gestations in which 
only one fetus has an eligible anomaly.  
Monochorionic diamniotic (MCDA) 
and monochorionic monoamniotic 
(MCMA) twins and a gestation in 
which both fetuses have an anomaly 
are excluded. 
Improve generalizability of the 
results by including twin 
gestation.  MCDA and MCMA 
are excluded due to the 
increased risk of adverse 
pregnancy outcomes. 
 
3.2.1.2, 
3.2.2.2 
Updated exclusion criteria to eliminate 
the need for delivery at a study center 
or associated hospital 
This will allow enrollment of 
individuals who deliver at 
outside hospitals  
 
4.2.1 
Clarified that either the twin with the 
anomaly or both twins will be sampled 
according to local clinical practice 
The study only requires the twin 
with the anomaly to be sampled, 
but some sites sample both as 
part of clinical practice 
 
4.3 
Clarified that data collection will 
include twin gestation 
This will be collected in order to 
account for twin gestations 
 
4.5 
Clarified that the reporting of 
secondary fetal findings will be per 
local site guidelines 
This will allow sites to report 
secondary fetal findings during 
pregnancy 
 
4,6, 4.8 
Clarified that for twin gestations, 
neonatal and cost data will only be 
collected for the twin with the anomaly 
Outcomes will only be analyzed 
for the fetus with the anomaly 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
4 
Date 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
18 May 2021 
3.2.1.2 
Clarified exclusion criteria for the 
sequenced group so that only those 
with positive gene panels are excluded 
This will allow those patients 
with negative gene panels to be 
included which could benefit 
from whole genome sequencing 
 
3.2.2.2 
Clarified that CMA findings that are 
pathogenic or likely pathogenic as well 
as causal are excluded 
Pathogenic or likely pathogenic 
variants that are related to the 
prenatal ultrasound finding will 
be excluded 
 
4.1 
Updated eligibility flow chart to reflect 
changes to sample collection 
Maternal buccal swab/saliva or 
blood may be collected 
 
4.2.1 
Added that maternal buccal 
swab/saliva may be collected instead 
of blood 
There are times when collecting 
blood is not feasible, and DNA 
can be extracted from either 
blood or saliva 
 
4.7.1 
Clarified that there will be one 
interview between 2 weeks after 
results disclosure or 4 weeks after 
enrollment for the unsequenced group 
and 15 months after delivery 
Each parent will be interviewed 
once, and the interview will be 
scheduled at some point during 
study follow-up 
13 Nov 2020 
3.1, 
3.2.1.1, 
3.2.2.1 
Clarified definition of isolated nuchal 
translucency to be between 3.5 mm 
and 4.5 mm without evidence of cystic 
hygroma  
This will distinguish between 
elevated nuchal translucency 
and cystic hygroma 
 
3.2.2.2, 
4.2.2, 4.6, 
5.1, 5.3 
Clarified that those with a CMA with 
pathogenic variants that are related to 
the prenatal ultrasound finding will 
only be excluded if the CMA was done 
prior to neonatal discharge 
Results obtained prior to 
discharge could impact cost and 
outcomes that are collected 
through discharge 
 
4.1 
Updated eligibility flow chart to reflect 
changes to exclusion criteria in the 
unsequenced group 
Only those that receive a CMA 
with pathogenic variants that are 
related to the prenatal ultrasound 
finding prior to neonatal 
discharge will be excluded 
 
4.2.1, 
4.2.2, 4.6, 
5.1, 5.3 
Clarified that CMA findings must be 
pathogenic or likely pathogenic as well 
as causal 
Only pathogenic or likely 
pathogenic variants that are 
related to the prenatal ultrasound 
finding will be excluded 
 
4.3 
Deleted carrier screening and results, 
blood type, and screen from baseline 
data collection 
No longer collected 
 
4.7 
Clarified that the couple will receive 
one gift card 
A gift card will be given to the 
couple if at least one parent 
completes the surveys 
 
6.1.1 
Added the PG07B Infant 
Dysmorphology Form to the list of 
forms 
Document any changes in infant 
phenotype assessed at birth and 
12 months 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
5 
Date 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
13 Nov 2020 
8.1 
Deleted recruitment site information 
and clarified that Baylor, Columbia, 
and UNC are the sequencing labs 
Baylor, Columbia, and UNC are 
the only sequencing labs, but 
additional sites may be added to 
improve recruitment 
 
9.2 
Deleted three when specifying the 
number of centers  
Additional sites may be added to 
improve recruitment 
15 Jun 2020 
3.2.1, 
3.2.2 
Clarified inclusion criteria for both 
groups to include suspected major 
anomalies 
Some anomalies are only 
suspected in utero and not 
confirmed until after birth 
 
3.2.1 
Added imminent delivery planned to 
the sequencing group exclusion criteria 
Sequencing results would not be 
available prior to delivery if an 
imminent delivery is planned 
 
3.2.1, 
3.2.2 
Added intrauterine fetal demise to the 
exclusion criteria for both groups 
Fetal death prior to enrollment 
was always an exclusion but not 
explicitly stated. 
 
3.2.2 
Deleted negative prenatal or postnatal 
CMA from the unsequenced group 
inclusion criteria and reworded as an 
exclusion to exclude prenatal or 
postnatal CMA that are related to the 
prenatal ultrasound finding. 
Not all infants will have 
postnatal CMA.  Therefore, only 
those infants that are tested and 
found to have a positive will be 
excluded 
 
3.2.2 
Added an inclusion to the unsequenced 
group to only include participants with 
a gestational age less than 36 weeks 
Align with similar criteria in the 
sequenced group and to enable 
the psychosocial survey to be 
completed prior to delivery 
 
3.2.2 
Clarified the exclusion criteria in the 
unsequenced group to exclude other 
genetic causes of the anomaly 
Align with similar criteria in the 
sequenced group 
 
3.2.1, 
3.2.2, 
4.1.1, 
4.2.1, 4.4 
Changed biologic to genetic 
Corrected terminology 
 
4.1.1 
Clarified informed consent will be 
signed rather than written  
Allows the use of electronic 
consenting 
 
4.2.2 
Clarified that postpartum genetic 
testing is at the discretion of the 
clinical care team. 
Not all infants in the 
unsequenced group will have a 
prenatal or postnatal CMA. 
 
4.3 
Deleted that a research chart will be 
kept with copies of the consent and 
ultrasound reports. 
All documentation is stored 
electronically. 
 
4.5 
Clarified that only sequencing findings 
reported to the patient will be 
confirmed with Sanger sequencing 
Some findings, if they are not 
causal, will not be reported to 
the patient.  Any findings that 
are reported will be Sanger 
confirmed. 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
6 
Date 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
15 Jun 2020 
4.6, 4.7, 
4.10 
Changed the timing of the first 
psychosocial survey to two weeks 
after discussion of study results or 
4 weeks after enrollment for the 
unsequenced group 
To improve the rate of survey 
completion and to enable the survey 
to be completed prior to delivery 
 
4.6, 4.8, 
4.10, 5.1, 
5.3.1 
Changed the time period for 
maternal healthcare utilization and 
cost to presentation to hospital for 
delivery to discharge 
Charge data will be collected 
directly from the billing data which 
is only available for the hospital 
admission. 
 
4.7 
Added that if the patient delivers 
before completion of the first 
psychosocial survey, a survey that 
combines elements of the first two 
surveys will be administered. 
Allows for completion of questions 
that would otherwise be missed. 
 
4.8 
Clarified that the healthcare charge 
data that will be collected includes 
current procedural terminology 
medical code, description, total 
units and total hospital charge. 
Charge data will be collected 
directly from the billing data and 
not from quantifying tests, 
procedures and medications. 
 
5.3 
Added type of anomaly (lethal vs 
non-lethal) to the list of planned 
subgroup analyses 
To evaluate if there is an interaction 
between group and type of anomaly  
 
6.1.1 
Deleted PG09A Medication 
Utilization Log 
Medication use will be collected 
directly from the billing records 
 
8.2.7 
Changed the DSMB review to at 
least annually 
The risk for this study is low and 
does not require more frequent 
review 
 
9.2 
Added if recruitment falls below 
targets, additional sites will be 
added 
To improve recruitment if targets 
are not met 
 
9.4.3 
Clarified that remaining DNA will 
be stored at Columbia at the 
completion of the study 
Samples will all be sent once 
follow-up is complete. 
 
Appendix 
A 
Clarified typos in the list of 
anomalies and that only moderate 
to severe bilateral hydronephrosis 
meets eligibility 
Only moderate to severe bilateral 
hydronephrosis would qualify as a 
major structural anomaly 
18 Nov 2019 
3.2.1 
Updated inclusion criteria for 
prenatal sequencing group to 
include twin gestation reduced to 
singleton, either spontaneously or 
therapeutically, if the reduction 
occurred by 13 weeks, 6 days.  
Early reductions are treated as a 
singleton pregnancy. 
 
3.2.1 
Updated exclusion criteria for 
prenatal sequencing group to 
include other genetic causes of the 
fetal anomaly. 
Sequencing should only be 
performed on participants that do 
not have a known cause of the 
anomaly. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
7 
Date 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
18 Nov 2019 
3.2.2 
Updated inclusion criteria for 
prenatal unsequenced group to 
include twin gestation reduced to 
singleton, either spontaneously or 
therapeutically, if the reduction 
occurred by 13 weeks, 6 days. 
Early reductions are treated as a 
singleton pregnancy. 
 
4.1 
Screening flowchart was clarified 
to start with eligible anomalies. 
The flowchart for women that do 
not have invasive testing was 
clarified to include consent to the 
unsequenced group. 
Only eligible anomalies enter the 
screening process.  
Women must consent to the 
unsequenced group before they 
enter the study flow. 
 
4.2.1 
Added that amniotic supernatant 
will be stored for future research. 
These samples allow for exploration 
of other prenatal diagnosis 
applications. 
 
4.2.1 
Clarified that either the short 
consent for sample collection or 
full study consent may be signed 
at the screening visit. 
Participants may sign either consent 
when they are initially approached. 
 
4.5 
Added that any VUS that is 
reported to the participant will be 
Sanger confirmed. 
All results reported to a participant 
must be Sanger confirmed. 
 
4.8 
Clarified that healthcare utilization 
in the prenatal period will be 
collected from billing records 
when possible. 
Abstraction from billing records is 
more efficient if they are available. 
 
6.1.1 
Updated the name of the Adverse 
Event Form to include 
Unanticipated Problems. 
Unanticipated problems must be 
reported to the IRB. 
19 Apr 2019 
3.2.1 
Added prenatal sequencing or 
planned prenatal sequencing, 
including gene panels as an 
exclusion to the prenatal 
sequencing group 
Women included in this assessment 
of prenatal sequencing should not 
be getting sequencing as part of 
clinical care as interpretation of 
results and outcomes may vary 
 
3.2.2 
Added unable to consent both 
biologic parents as an exclusion to 
the prenatal unsequenced group 
To be consistent with the sequenced 
group, as it is important to keep 
cohorts as comparable as possible. 
 
4.5 
Clarified that formal notification 
will occur for negative fetal 
sequencing results only if testing is 
performed in a CLIA approved 
laboratory. 
Formal reports may be given if a 
CLIA approved laboratory is used 
in testing. 
 
4.6 
Clarified wording in Table 2 and 
added assessment of post-
discharge genetic testing 
Important to capture genetic testing 
that happens after discharge and up 
through the 12 month follow-up 
 
4.6 
Added assessment of infant 
phenotype at 12 months 
To ensure the results are 
reevaluated if new phenotypic 
information becomes available. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
8 
 
Date 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
19 Apr 2019 4.8 
Clarified change in management 
with be assessed by the physician in 
conjunction with the site 
coordinator 
Change in management will require 
coordination between the site 
coordinator and the physician 
 
4.10.2 
Changed length of ventilator 
support to mechanical ventilation 
More clinically relevant outcome 
 
6.1.1 
Added Infant Clinical Structural 
Anomalies Form PG07A 
Document any changes in infant 
phenotype assessed at 12 months 
4 Feb 2019 
4.2.1 
Added  investigation of non-
invasive prenatal testing (NIPT) to 
future research of maternal samples 
There is an existing plan to evaluate 
NIPT as part of future research in 
the maternal samples that is detailed 
in the consent. 
 
4.5 
Deleted reporting of any findings to 
participants prior to Sanger 
confirmation. 
Findings must be confirmed prior to 
reporting unless exemption 
requested by IRB.   
 
4.5 
Deleted reporting of negative 
findings to participants 
Findings must be confirmed prior to 
reporting and negative findings will 
not be confirmed. 
 
6.1.1 
Updated data collection forms list 
New forms added 
 
9.4.3 
Clarified sample storage includes 
samples for future research and that 
storage of all samples will be at 
Columbia University 
A single repository is most efficient. 
 
4.2.1 
Added  investigation of non-
invasive prenatal testing (NIPT) to 
future research of maternal samples 
There is an existing plan to evaluate 
NIPT as part of future research in 
the maternal samples that is detailed 
in the consent. 
 
4.5 
Deleted reporting of any findings to 
participants prior to Sanger 
confirmation. 
Findings must be confirmed prior to 
reporting unless exemption 
requested by IRB.   
 
4.5 
Deleted reporting of negative 
findings to participants 
Findings must be confirmed prior to 
reporting and negative findings will 
not be confirmed. 
 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
9 
Table of Contents 
 
1 
Introduction ......................................................................................................................................... 11 
1.1 
Study Abstract ............................................................................................................................. 11 
1.2 
Objectives ................................................................................................................................... 11 
1.3 
Purpose of the Study Protocol ..................................................................................................... 12 
2 
Background ......................................................................................................................................... 13 
2.1 
Introduction ................................................................................................................................. 13 
2.2 
Preliminary Studies ..................................................................................................................... 14 
2.2.1 
Postnatal Studies ................................................................................................................. 14 
2.2.2 
Prenatal Studies ................................................................................................................... 14 
2.2.3 
Healthcare Utilization and Cost .......................................................................................... 15 
2.2.4 
Educational & Psychosocial Needs ..................................................................................... 16 
2.3 
Rationale for the Study ............................................................................................................... 16 
3 
Study Design ....................................................................................................................................... 18 
3.1 
Design Summary ......................................................................................................................... 18 
3.2 
Eligibility Criteria ....................................................................................................................... 18 
3.2.1 
Prenatal sequencing group .................................................................................................. 18 
3.2.2 
Prospective unsequenced prenatal group ............................................................................ 19 
3.2.3 
Retrospective unsequenced prenatal group ......................................................................... 20 
4 
Study Procedures ................................................................................................................................ 21 
4.1 
Screening for Eligibility and Consent ......................................................................................... 21 
4.1.1 
Informed consent................................................................................................................. 22 
4.2 
Enrollment ................................................................................................................................... 22 
4.2.1 
Prenatal sequencing group .................................................................................................. 22 
4.2.2 
Unsequenced prenatal group ............................................................................................... 23 
4.2.3 
Retrospective unsequenced prenatal group ......................................................................... 23 
4.3 
Baseline Data Collection ............................................................................................................. 23 
4.4 
Sequencing Methods ................................................................................................................... 24 
4.5 
Variant Evaluation and Return of Results ................................................................................... 24 
4.6 
Study Procedures ........................................................................................................................ 25 
4.7 
Psycho-Social Surveys ................................................................................................................ 26 
4.7.1 
In-depth Qualitative Interviews .......................................................................................... 27 
4.8 
Healthcare Utilization and Change in Management ................................................................... 28 
4.9 
Adverse Event Reporting ............................................................................................................ 28 
4.10 
Study Outcome Measures and Ascertainment ............................................................................ 28 
4.10.1 
Primary Outcome ................................................................................................................ 28 
4.10.2 
Secondary Outcomes (Prenatal sequencing and unsequenced prenatal) ............................. 28 
4.10.3 
Secondary Outcomes (Prenatal sequencing only) ............................................................... 29 
5 
Statistical Considerations .................................................................................................................... 30 
5.1 
Power and sample size ................................................................................................................ 30 
5.2 
Prenatal Sequencing Group Only ................................................................................................ 30 
5.3 
Prenatal Sequencing and Prenatal Unsequenced Groups ............................................................ 31 
5.3.1 
Healthcare utilization and cost ............................................................................................ 31 
5.3.2 
Psychosocial outcomes ....................................................................................................... 32 
6 
Data Collection ................................................................................................................................... 33 
6.1 
Web Data Entry Systems ............................................................................................................ 33 
6.1.1  
Data Collection Forms ........................................................................................................ 33 
6.2 
Centralized Data Management .................................................................................................... 34 
6.3 
Performance Monitoring ............................................................................................................. 34 
7 
WGS Pipeline Development ............................................................................................................... 35 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
10 
7.1 
Evaluation of the incremental value of WGS compared to WES in understanding the etiology of 
birth defects ............................................................................................................................................. 35 
8 
Study Administration .......................................................................................................................... 36 
8.1 
Organization and Funding ........................................................................................................... 36 
8.1.1 
Clinical Coordinating Center .............................................................................................. 36 
8.1.2 
Data Coordinating Center ................................................................................................... 36 
8.2 
Committees ................................................................................................................................. 36 
8.2.1 
Steering Committee ............................................................................................................. 36 
8.2.2 
Operations Committee ........................................................................................................ 36 
8.2.3 
Variant Adjudication Committee ........................................................................................ 37 
8.2.4 
Variant Oversight Committee ............................................................................................. 37 
8.2.5 
Clinical Adjudication Committee ....................................................................................... 37 
8.2.6 
WGS Pipeline Development Committee ............................................................................ 37 
8.2.7 
Data and Safety Monitoring Board (DSMB) ...................................................................... 37 
9 
Study Timetable .................................................................................................................................. 38 
9.1 
Training and Certification ........................................................................................................... 38 
9.2 
Recruitment and Data Collection Period ..................................................................................... 38 
9.3 
Final Analysis ............................................................................................................................. 39 
9.4 
Data Archiving and Sample Storage ........................................................................................... 39 
9.4.1 
Data Archiving .................................................................................................................... 39 
9.4.2 
Data Sharing ........................................................................................................................ 39 
9.4.3 
Specimen Storage and Sharing ........................................................................................... 39 
Appendix A. Eligible Major Structural Anomalies..................................................................................... 40 
 
 
Tables 
Table 1. This table describes the frequency of pathogenic variants by whole exome sequencing in various 
publications .......................................................................................................................................... 14 
Table 2. Summary of Data Collection ........................................................................................................ 25 
Table 3. Psychosocial Survey Measures ..................................................................................................... 26 
Table 4. Precision for range of estimates and sample sizes with at least 90% probability ......................... 30 
Table 5. Participating Organizations and Roles .......................................................................................... 36 
 
 
Figures 
Figure 1. Prospective screening flowchart .................................................................................................. 21 
Figure 2. Timeline ....................................................................................................................................... 38 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
11 
1 Introduction 
1.1 Study Abstract 
Whole exome and whole genome sequencing (WGS) have expanded the ability to determine the genetic 
etiology of previously undiagnosed disorders. This study is a multicenter cohort study to evaluate the 
emerging technology of sequencing for the management of fetuses with structural anomalies. The 
hypothesis is that a significant subset of fetal structural anomalies has a genetic etiology identifiable by 
sequencing and that prenatal knowledge of this information will improve perinatal care, reduce 
unnecessary diagnostic testing, reduce the cost of care, and improve quality of life for both the child and 
the family. The aims of this study are to investigate these multiple aspects of prenatal sequencing in a 
single study with an innovative integrated design, which will permit a robust evaluation of the benefits 
and risks of delivering diagnostic and prognostic genetic testing results in a prenatal setting. 
The study will determine, in a sequential population of pregnancies with selected fetal structural 
anomalies and a negative or non-causal chromosomal microarray (CMA), the frequency of pathogenic, 
likely pathogenic, and uncertain genomic variants identifiable by sequencing. To determine the impact of 
this information on clinical care, a control population of unsequenced pregnancies with similar structural 
anomalies will be prospectively recruited and the infants from both cohorts will be followed up to 1 year 
of age. Due to limited prospective enrollment of the controls, a portion of the unsequenced pregnancies 
will be enrolled retrospectively through medical record review. This study component will evaluate 
differences in healthcare management and cost through discharge from hospital post-delivery, and 
perinatal and infant outcomes through 1 year of life. The educational, counseling and psychosocial impact 
of sequencing results during the prenatal period, in the nursery and through 1 year of life also will be 
evaluated. The retrospective unsequenced pregnancies will not be included in 1-year follow-up, as they 
will be enrolled under a waiver of consent. Since the analytical and clinical tools needed for the full 
translation of sequencing into care are still developing, optimization of bioinformatic tools to improve 
identification of pathogenic and likely pathogenic mutations associated with prenatal phenotypes of 
established disease genes will be investigated, as well as identification of new genes associated with 
presently undiagnosed fetal/neonatal phenotypes. This study will provide an in-depth evaluation of the 
prenatal diagnostic value of sequencing prior to its responsible introduction into practice and will provide 
independent data to guide its translation. 
1.2 Objectives 
The main objective of this multi-center collaborative study is to evaluate sequencing (both whole exome 
sequencing [WES] and WGS) as a prenatal diagnostic tool in pregnancies with a structural anomaly and a 
negative or only non-causal karyotype/ chromosome microarray analysis (CMA). Specifically, the aims 
are as follows: 
1. To determine in pregnancies with structural anomalies which are CMA negative (or have only non-
causal findings), the frequency of pathogenic, likely pathogenic, and uncertain genomic variants 
identifiable by sequencing. 
a. Examine the relative yield of pathogenic, likely pathogenic, and uncertain genomic variants 
identifiable by WGS compared with analysis of the coding sequence alone (simulating WES). 
2. To evaluate the impact of having sequencing results available prenatally on clinical management, 
including health care utilization and costs, comparing outcomes of pregnancies and neonates with in 
utero diagnosed structural anomalies that have undergone prenatal sequencing with those that have 
not. 
3. To evaluate the educational/counseling and psychosocial needs of pregnant couples having a fetus 
with a structural anomaly and assessing the psychological impact of sequencing and return of results 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
12 
on couples and determine the correlation with demographic factors, knowledge and attitudes about 
genetics, baseline parental attitudes and beliefs, and genetic test results. 
4. To expand the diagnostic yield and clinical utility of fetal sequencing by developing and optimizing 
bioinformatic tools capable of efficiently and rapidly identifying  pathogenic and likely pathogenic 
variants with prenatal presentations.  
1.3 Purpose of the Study Protocol 
This protocol describes the background, design and organization of the study and may be viewed as a 
written agreement among the study investigators. It is approved by the funding agency (NICHD), the 
study Steering Committee, the Data Safety Monitoring Board (DSMB), the single Institutional Review 
Board (IRB) of the Clinical Coordinating Center and participating laboratories before recruitment begins. 
Any changes to the protocol during the study period require the approval of the Steering Committee and 
the IRB.   
A manual of operations supplements the protocol with detailed specifications of the study procedures. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
13 
2 Background 
2.1 Introduction 
With advances in prenatal imaging, increasingly there is the ability to detect birth defects prenatally and 
use this information to optimize perinatal and neonatal management. Simultaneously, molecular genetic 
diagnostics have facilitated more precise identification of the underlying cause of these birth defects and 
provided additional prognostic information to improve prenatal and postnatal management. The first 
major improvement in prenatal genomic diagnosis was chromosome microarray analysis (CMA) which 
identifies copy number variants (CNVs) of 100 kb to 10Mb that are below the resolution of most 
karyotypes. A multicenter study evaluating the clinical utility of prenatal CMA reported that CMA 
identified the same aneuploidies and unbalanced rearrangements as karyotyping but also identified 
additional, clinically significant cytogenetic information.1 Among women with a normal karyotype, CMA 
identified clinically relevant deletions or duplications in 6.0% with a fetal structural anomaly and in 1.7% 
of those with indications of advanced maternal age or positive aneuploidy screening results. The 
frequency of CNVs varied by anomaly, with the highest frequency (16%) occurring in fetuses with 
cardiac defects.   
Congenital anomalies affect 2-4% of all infants and are responsible for 20% of perinatal deaths.2 Prenatal 
genetic evaluation of fetal anomalies is limited to karyotype and CMA performed on amniocytes or 
chorionic villi. The karyotype is abnormal in 20-25% and CMA is abnormal in 5-10% of fetal anomalies, 
leaving 60-70% without a genetic diagnosis.3 WGS is the process of determining the complete DNA 
sequence of an organism's genome at a single time and includes both the coding and non-coding regions.  
Whole exome sequencing (WES) analyzes only the coding regions of the genome (exons; DNA code for 
making proteins). Until recently, researchers have elected to use WES because it costs less and because 
the functional consequences of positive findings occurring within coding regions of genes are much easier 
to interpret. Because of its high cost and incomplete knowledge about the role of non-coding variants in 
disease, WGS has primarily been used as a research tool. This may change as sequencing costs decrease 
and knowledge of the functional consequences of non-coding genetic variants increases. Numerous recent 
reports describe the speed and reliability of WGS in the context of rapid clinical sequencing since library 
preparation is not required4-6 making WGS an advantageous technology for rapid genomic testing in the 
future.  
Prenatal sequencing has the potential to alter the life course and reduce morbidity and mortality by 
guiding pregnancy management, delivery plans and/or the treatment of fetuses and newborns with some 
genetic abnormalities.7 One example is in utero fetal intervention now performed for certain anomalies 
including aortic stenosis to avoid hypoplastic left heart syndrome, urinary diversion for posterior urethral 
valves, drainage of pleural effusions and chylothorax, surgical repair for spina bifida, tracheal occlusion 
for congenital diaphragmatic hernia, and amnio-infusion for renal agenesis.8-14 For all of these, knowing 
their genetic etiology aids clinical management decisions by providing information about other associated 
medical features that affect the likelihood of success of the intervention. In addition, prenatal diagnosis of 
some metabolic conditions could guide early postnatal treatment and avoid metabolic crises that can lead 
to long term neurocognitive deficits. For example, pyruvate dehydrogenase deficiency can present 
prenatally with structural anomalies and is modifiable by a ketogenic diet and thiamine 
supplementation.15,16 Finally, mesenchymal stem cell transplantation for osteogenesis imperfecta has been 
reported to increase skeletal mineralization and growth velocity without adverse outcomes.17-20 With 
developments in gene and stem cell therapy, this type of treatment may increasingly become an option for 
other conditions. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
14 
2.2 Preliminary Studies 
2.2.1 Postnatal Studies 
WES, which analyzes the coding regions of the genome (exons) is a powerful diagnostic tool in adults 
and children with genetically heterogeneous conditions.21,22 Compared with a 10% diagnostic yield with 
karyotype and CMA, postnatal WES has diagnostic yields of >30% in adults and children with 
phenotypes suggestive of monogenic disorders.22 The mean and median overall diagnostic yields of a 
series of 16 studies was 36% and 38%, respectively, but was highly dependent on the a priori risk for a 
monogenic disorder. In addition to the diagnostic utility of WES in these populations, WES analysis has 
resulted in discovery of new disease genes with pleiotropic phenotypes. 
WGS is now gaining traction as a diagnostic and discovery strategy for adults and children with a 
suspected genetic disorder that remains undiagnosed after WES analysis, or as a first-line approach in lieu 
of WES. WES includes an exon capture step, which adds time and reagent costs, and biases against 
coverage in GC-rich regions. WGS does not include this selection step, providing more uniform coverage 
which allows a lower mean read depth, offers ability to detect copy number variants (CNVs) with higher 
resolution than CMA and more complex balanced re-arrangements. The lower read depth however will 
also make it more difficult to detect mosaic mutations which are an important source of mutations in 
neurodevelopmental disorders including autism and epilepsy.23,24 WGS was found to detect up to 3% of 
protein-coding variants missed by WES.25 Recent WGS studies have found more causative variants in 
coding and non-coding regions in autism.6 In addition, an increased burden of rare de novo variants in 
non-coding regions for congenital heart disease and diaphragmatic hernia has been seen (Chung, WC; 
unpublished data). 
2.2.2 Prenatal Studies 
Only a few case series of prenatal WES have been published to date with a wide range of indications, 
including pregnancy terminations, fetal demises with fetal anomalies, euploid fetuses with 
sonographically detected single or multiple fetal structural anomalies, and increased nuchal translucency 
(NT) > 3.5 mm have been reported26,27 with widely variable diagnostic yields of 6.2 - 80%. The 2 largest 
series of trio exomes on over 200 fetal anomalies each had overall diagnostic rates of 6.2 and 7.5%, but 
was higher (14.3% and 16.0%) for fetuses with multiple anomalies.27  
Table 1. This table describes the frequency of pathogenic variants by whole exome sequencing in 
various publications  
First 
Author, 
Year 
# 
Cases 
Cohort Criteria 
Method 
Pathogenic 
Variants* 
Normand, 
201828 
146 
Fetuses with ultrasound anomalies and a suspected 
Mendelian disorder 
62 Trio 
46/146 (32%) 
Aarabi, 
201829 
20 
1 or more major structural congenital anomaly 
detected by ultrasound 
Trio 
4/20 (20%) 
Fu, 
201730 
196  
Fetuses with structural abnormalities 
147 Proband-
only  
 
49 Trio 
34/147 (23.1%) 
 
13/49 (26.5%) 
Lei, 
201731 
30  
Fetuses with congenital anomalies of the kidney 
and urinary tract 
23 Proband-only 
3/23 (13%) 
 
7 Trio 
 
1/7 (14%) 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
15 
Total 
4/30 (13.3%)  
Vora, 
201732 
15 
Fetuses with multiple congenital anomalies highly 
suggestive of an underlying genetic disorder 
Trio 
7/15 (47%) 
Yates, 
201733 
84 
Fetuses with ultrasound abnormalities that resulted 
in fetal demise or pregnancy termination 
29 Proband-only  
4/29 (14%)  
 
45 Trios 
 
11/45 (24%) 
6 Quads/4 
Maternal Duos 
2/10 (20%) 
Total: 
17/84 (20%) 
 
Pangalos, 
201634 
14 
Prenatal ultrasound abnormalities or malformations 
Proband-only 
6/14 (43%) 
Alamillo, 
201535 
7 
Multiple congenital anomalies on prenatal 
ultrasound 
Trio 
4/7 (57%) 
Drury, 
201536 
24 
Fetuses with an increased NT (>3.5 mm) or other 
ultrasound abnormality 
14 Proband-only 
 
2/14 (14%) 
 
10 Trio 
3/10 (30%) 
Total 
5/24 (21%)  
 
Carss, 
201437 
30 
Structural abnormalities identified on prenatal 
ultrasound 
Trio 
3/30 (10%)  
 
Yang, 
201438 
11 
Terminated fetuses with anomalies  
Trio 
6/11 (54%) 
2.2.3 Healthcare Utilization and Cost 
Integration of WES or WGS into clinical care requires not only demonstration of the ability to identify the 
underlying etiology of a disorder but also requires evidence that care is improved, providing value to the 
health care system. While WGS is currently a relatively expensive test (≥$15,000 per trio), the 
information may lead to significant alterations in care utilization. Given that healthcare costs can be 
$250,000 or more in the first year of life for some complex congenital anomalies, the genetic diagnostic 
information may be net cost saving. Even if not cost-saving, if clinical outcomes are improved 
significantly, the incremental cost of WGS may be justifiable based on its cost effectiveness. Recent 
studies in critically ill neonates and infants have suggested that establishing a diagnosis leads to more 
focused management and reduction in healthcare utilization and reduced cost. Meng et al evaluated 
diagnostic WES for 278 critically ill infants within the first 100 days of life and found a genetic diagnosis 
in 36.7%.39 These new diagnoses led to care modification in 52%, including initiation of new 
subspecialist care, redirection of care, changes in medication/diet, or completion of major procedures 
(e.g., transplant). The greatest impact was in infants receiving rapid “critical trio sequencing”.39 A meta-
analysis found that change in clinical management by WGS results was 27% (4 studies with 136 children) 
compared with 17% by WES (12 studies and 992 children) and 6% by CMA (8 studies of 4,271 
children).40 Another study evaluating the cost-effectiveness of WES in a pediatric setting reported the 
mean duration of the diagnostic odyssey was 6 years, and that the diagnostic trajectory for WES 
performed at the initial tertiary presentation resulted in an incremental saving of $6800 per additional 
diagnosis compared with the standard diagnostic pathway.41  In summary, studies have suggested that 
sequencing early in the disease course may provide the maximal benefit by modifying clinical 
management, reducing the time to diagnosis and cost. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
16 
2.2.4 Educational & Psychosocial Needs 
The added prognostic information about health, treatability, life expectancy and neurodevelopmental, 
behavioral and cognitive function is far beyond what is learned from ultrasound findings and standard 
testing, and can have a powerful impact on parents. Identification of fetal genomic information has 
significant value to the management of the pregnancy. Ultrasound offers an anatomic phenotype but is 
incapable of evaluating long-term neurocognitive potential, limiting accurate prognostication and 
counseling. Karyotyping and CMA can offer disease specific counseling in a combined ~30-40% of 
detectable cases to provide couples with the information necessary to make informed reproductive 
decisions, leaving significant ambiguity in 60-70%. Equally important, knowledge of the genotype can 
direct additional fetal, neonatal and pediatric management. 
Efforts to understand the psychosocial and behavioral impact of integrating genomic technologies into 
adult and pediatric practice are ongoing42-44, but to date, little empirical work has been done to understand 
the unique challenges of applying genomic sequencing to a prenatal population.45 Attitudes towards 
prenatal screening and diagnosis are influenced by ethnicity, socioeconomic status, cultural and religious 
beliefs, and experiences with disability.45-49 
While WGS results may illuminate the situation when a well-known genetic condition is diagnosed, there 
are challenges in counseling for some of the newer genetic conditions for which less data are available. In 
CMA, variants of uncertain clinical significance (VUS) and those associated with variable expressivity 
occur in a proportion of cases with structural anomalies (5.3%) and require extensive genetic 
counseling.50  Other counseling issues include the identification of adult onset disorders of both the fetus 
and parent. To understand attitudes and unmet needs in the adoption of CMA testing semi-structured 
interviews with parents and counselors were performed to evaluate pre-test counseling, reporting of 
results, and assessing patient and provider experiences.47 From the patient perspective, 5 key themes were 
identified 1) accepting CMA testing was an easy decision to make as patients would obtain additional 
information on their baby’s health at no additional cost, 2) patients were blindsided by the results in cases 
where they initially received a normal karyotype result followed by an abnormal CMA, 3) abnormal 
results left patients shocked, anxious, confused and overwhelmed, 4) patients needed support to manage, 
understand and act on the microarray results, and 5) uncertain findings were felt to be toxic knowledge 
and patients wished they did not receive the results. It is anticipated that the needs for prenatal sequencing 
will be similar to those of prenatal CMA and that prior studies will inform the educational and support 
materials developed for prenatal sequencing.  
2.3 Rationale for the Study 
Sequencing itself is a transformative technology to identify comprehensively genetic variants accounting 
for a phenotype, but its application to prenatal diagnosis has not been investigated prospectively in a large 
cohort.22 This study population of unselected consecutive cases will be unique in that the majority of 
published series to date have only included select phenotypes felt to have a genetic etiology. It is possible 
that these studies have overestimated the frequency and underestimated the phenotypic variability of in 
utero genetic disease based upon the case ascertainment. A more comprehensive, less biased evaluation of 
the molecular etiology of birth defects should lead to the discovery of previously unknown genes and 
phenotypic associations. To date, only descriptive series of the diagnostic yield of selected prenatal WES 
or WGS have been reported on fetuses with a structural anomaly, but none have evaluated the benefit of a 
specific genetic diagnosis.36,37,51-53  
This study will be the first to perform a cost-effectiveness analysis to evaluate whether prenatal 
knowledge of the genetic cause of a fetal single-gene disorder will lead to altered fetal/neonatal 
management, costs, and outcomes. Also important are the pertinent psychosocial effects of introducing 
sequencing into a prenatal setting.  

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
17 
Before integration into standard care, the diagnostic capabilities of prenatal WGS must mature by 
developing new bioinformatic tools to specifically facilitate accurate and efficient prenatal interpretation 
and novel gene discovery. This can be accomplished initially by analyzing and reporting the variants from 
the coding regions of the genome and then subsequently analyzing the non-coding regions, allowing 
comparison of the differences in diagnostic yield. The current missing pieces are insufficient WGS data in 
fetuses with abnormalities, the lack of variant analysis algorithms tuned to the prenatal setting, and 
insufficient publicly deposited data to inform clinical care for prenatal genomic testing and rare 
developmental disorders. As more data become available (WGS data from adults, more WES/WGS from 
more diverse communities, Human Cell Atlas data, and ATAC seq data from appropriate times in 
development), it will increasingly be possible to interpret the coding and noncoding regions that are 
relevant to developmental disorders. 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
18 
3 Study Design 
3.1 Design Summary 
This multicenter, observational cohort study will evaluate prenatal sequencing among pregnancies with 
fetal structural anomalies recruited at university based medical centers and evaluated at four genetic 
laboratories.  A total of 1,100 pregnancies with fetal structural anomalies and meeting eligibility criteria 
will be enrolled into the study. Of these, 750 will undergo prenatal genomic sequencing (prenatal 
sequencing group) and the remaining 350 pregnancies will not have any prenatal genomic sequencing 
(unsequenced prenatal group). Enrollment of pregnancies with an isolated nuchal translucency 
measurements ≥ 3.5 mm and < 4.5 mm without evidence of cystic hygroma will be restricted to 5% 
within each group (sequenced and unsequenced) and isolated estimated fetal weight <5th %ile also will 
be restricted to 5% for each group (sequenced and unsequenced). 
The prenatal sequencing group will be used to determine the frequency of pathogenic, likely pathogenic, 
and uncertain genomic variants identifiable by sequencing and the relative yield of sequencing.  The 
prenatal sequencing group will be compared with the unsequenced prenatal group (prospective and 
retrospective) to evaluate health care management, health care utilization and cost, and perinatal 
outcomes. The prenatal sequencing group will be compared with the prospective unsequenced prenatal 
group to evaluate psychosocial needs of pregnant couples.  Mothers, fathers and infants will be followed 
through 1 year postpartum for participants enrolled prospectively. 
3.2 Eligibility Criteria 
3.2.1 Prenatal sequencing group 
3.2.1.1 Inclusion Criteria 
1. 
Fetus identified by ultrasound and/or MRI with at least one of the following: 
a. One or more suspected major structural anomalies (Appendix A)  
b. A nuchal translucency measurement of ≥ 3.5 mm and < 4.5 mm without evidence of 
cystic hygroma 
c. A fetus less than 24 weeks 0 days gestation with normal anatomy and sonographically 
estimated fetal weight <5th %ile without maternal hypertension, type I diabetes, or other 
maternal disorders known to alter fetal growth.  
2. 
Negative prenatal CMA (or those with CMA findings not related to the ultrasound finding) 
3. 
Singleton gestation or dichorionic diamniotic (DCDA) twin gestation.  
4. 
Gestational age less than 36 weeks, 0 days to allow for availability of sequencing results before 
delivery 
3.2.1.2 Exclusion Criteria 
1. 
Imminent delivery planned (within 3 weeks of enrollment) as sequencing results would not be 
available prior to delivery 
2. 
Prenatal sequencing or planned prenatal sequencing performed outside of the study.  Gene panels 
may be performed prior to enrollment but the results must be known and negative to be eligible 
(positive or unknown results are an exclusion).  

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
19 
3. 
Maternal or paternal age less than 18 years old 
4. 
Proven infectious, teratogenic, or other genetic cause of fetal anomaly 
5. 
For twin gestation, anomalies in both fetuses (NT, IUGR, or structural anomaly) 
6. 
Monochorionic diamniotic (MCDA) and monochorionic monoamniotic (MCMA) twins as well as 
higher-order multifetal gestations 
7. 
Planned termination of the fetus or intrauterine fetal demise (IUFD) 
8. 
Unavailable blood or saliva samples from both genetic parents prior to sequencing 
9. 
Parental unwillingness to participate in 1 year postnatal follow-up 
10. Language barrier (non-English or Spanish speaking) 
11. Previous consent to the unsequenced prenatal group or enrollment in a previous pregnancy 
3.2.2 Prospective unsequenced prenatal group 
3.2.2.1 Inclusion Criteria 
1. Fetus identified by ultrasound and/or MRI with at least one of the following: 
a. One or more suspected major structural anomalies (Appendix A)  
b. A nuchal translucency measurement of ≥ 3.5 mm and < 4.5 mm without evidence of cystic 
hygroma 
c. A fetus less than 24 weeks 0 days gestation with normal anatomy and sonographically 
estimated fetal weight <5th %ile without maternal hypertension, type I diabetes, or other 
maternal disorders known to alter fetal growth 
2. Declined prenatal sequencing  
3. Singleton gestation or dichorionic diamniotic (DCDA) twin gestation.  
4. Gestational age less than 36 weeks, 0 days 
3.2.2.2 Exclusion Criteria 
1. 
Prenatal or postnatal (prior to neonatal discharge) CMA with pathogenic or likely pathogenic 
variants that are related to the prenatal ultrasound finding  
2. 
Maternal or paternal age less than 18 years old 
3. 
Proven infectious, teratogenic, or other genetic cause of fetal anomaly 
4. 
Positive prenatal NIPT screening for trisomy 21, 18 or 13.  Positive 22q11.2 prenatal NIPT testing 
with consistent ultrasound findings is also an exclusion. 
5. For twin gestation, anomalies in both fetuses (NT, IUGR, or structural anomaly) 
6. Monochorionic diamniotic (MCDA) and monochorionic monoamniotic (MCMA) twins as well as 
higher-order multifetal gestations. 
7. 
Planned termination of the fetus or intrauterine fetal demise (IUFD) 
8. 
Unable to consent both genetic parents 
9. 
Parental unwillingness to participate in 1 year postnatal follow-up 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
20 
10. Language barrier (non-English or Spanish speaking)  
3.2.3 Retrospective unsequenced prenatal group 
Any changes to the prospective unsequenced prenatal group criteria are italicized below. 
3.2.3.1 Inclusion Criteria 
1. Fetus identified by ultrasound and/or MRI with at least one of the following: 
a. One or more suspected major structural anomalies (Appendix A)  
b. A nuchal translucency measurement of ≥ 3.5 mm and < 4.5 mm without evidence of cystic 
hygroma 
c. A fetus less than 24 weeks 0 days gestation with normal anatomy and sonographically 
estimated fetal weight <5th %ile without maternal hypertension, type I diabetes, or other 
maternal disorders known to alter fetal growth 
2. Did not pursue prenatal sequencing  
3. Singleton gestation or dichorionic diamniotic (DCDA) twin gestation.  
4. Gestational age less than 36 weeks, 0 days at date study-specified anomalies first diagnosed 
3.2.3.2 Exclusion Criteria 
1. Prenatal or postnatal (prior to neonatal discharge) CMA with pathogenic or likely pathogenic variants 
that are related to the prenatal ultrasound finding  
2. Maternal or paternal age less than 18 years old 
3. Proven infectious, teratogenic, or other genetic cause of fetal anomaly 
4. Positive prenatal NIPT screening for trisomy 21, 18 or 13.  Positive 22q11.2 prenatal NIPT testing 
with consistent ultrasound findings is also an exclusion. 
5. For twin gestation, anomalies in both fetuses (NT, IUGR, or structural anomaly) 
6. Monochorionic diamniotic (MCDA) and monochorionic monoamniotic (MCMA) twins as well as 
higher-order multifetal gestations. 
7. Termination of the fetus or intrauterine fetal demise (IUFD) within 3 weeks of date of initial anomaly 
diagnosis 
8. Language barrier (non-English or Spanish speaking)  
9. Delivery at a site other than one of the study centers or associated hospitals 
10. Unable to access prenatal or neonatal  records 
 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
21 
 
4 Study Procedures  
4.1 Screening for Eligibility and Consent  
Eligible patients with specified fetal structural anomalies will be identified by prenatal ultrasound and/or 
fetal MRI. Center investigators and their staff in the outpatient ultrasound prenatal diagnostic units will be 
responsible for identifying eligible patients. To maximize recruitment, coordinators at each center will 
screen the prenatal diagnostic logs on a daily basis to identify eligible patients.  
Upon recognition of a specified fetal anomaly, the maternal fetal medicine physician (MFM)/ 
ultrasonographer and/or a genetic counselor will explain the ultrasound finding as per routine clinical 
care. At the conclusion of the initial evaluation/consult, women will be informed by the 
physician/counselor and/or coordinator about the study. Once the potential participant indicates 
willingness to hear more about the study, a study coordinator will meet with the parents, review 
eligibility, and explain the study.  Women who choose to undergo prenatal invasive testing will be 
approached for the prenatal sequencing group but will not be enrolled until a negative or non-causal 
karyotype/CMA is confirmed (with the exception of those enrolled into expedited prenatal sequencing, 
section 4.2.1). Women who choose not to have any diagnostic testing or decline sequencing will be 
approached for participation in the unsequenced prenatal group. A portion of the unsequenced group will 
be enrolled through medical record review under a waiver of consent. All women with a specified fetal 
anomaly will be recorded on a screening log that collects screening ID, date screened, planned invasive 
prenatal testing, ultrasound anomaly, screening status code and reason for study ineligibility (if 
applicable).  A participant flowchart is shown in Figure 1. 
Figure 1. Prospective screening flowchart 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
22 
4.1.1 Informed consent 
Signed informed consent must be obtained from participants and the genetic father of the fetus prior to 
any study procedures for prospective enrollment.  Three consents may be employed for this study: 
• 
Short consent for potentially eligible women in the prenatal sequencing group that allows for 
collection of study samples prior to determination of the CMA and karyotype result.  If 
CMA/karyotype results come back negative, eligible participants will sign the full study informed 
consent form to proceed with sequencing. 
• 
Full study consent for the prenatal sequencing group that describes the specific group’s 
participation in the study and risks 
• 
Full study consent for the prenatal unsequenced group that describes the specific group’s 
participation in the study and risks 
Participants in both the sequenced and unsequenced groups will be assured that their participation in the 
study is purely voluntary and that patient care will not be affected if they decline participation in the 
study.  Patients who are not fluent in English will be enrolled by a person fluent in Spanish. Both verbal 
and written informed consent and authorization will be obtained in that language; if this is not possible 
the patient will be excluded. 
Enrollment of participants in the retrospective unsequenced prenatal group will occur under a waiver of 
consent. 
4.2 Enrollment 
4.2.1 Prenatal sequencing group 
For the prenatal sequencing group, chorionic villus sampling (CVS) or amniocentesis will be performed 
per standard of care. For twin gestations, either the twin with the anomaly or both twins will be sampled 
according to local clinical practice.  For CVS sampling, a portion of the villi, cultured villi or extracted 
DNA will be sent to the sequencing laboratory.  For amniocentesis, the usual draw of 40cc of fluid will be 
divided by the cytogenetics lab, with a portion used for clinical testing (minimum standard amount as per 
diagnostic laboratory practice) and from the remainder, DNA will be extracted for sequencing in a CLIA 
laboratory.  Additional DNA for sequencing will be extracted from the cultured cells and the sequencing 
lab will determine which is the optimal source for sequencing, after quality and quantity verification. 
Amniotic fluid supernatant will be kept for future research. 
Potentially eligible participants will sign either the screening consent form or study consent form prior to 
collection of parental samples.  Thirty ml of maternal blood or buccal swab/saliva will be collected for 
sequencing and future research; future research will include investigating non-invasive prenatal testing 
(NIPT). Paternal blood (up to 10 ml) or buccal swab/saliva will be collected for sequencing and future 
research. Every effort will be made to obtain samples from both parents prior to or at the time of the 
diagnostic procedure. If the father is absent, a sample will be obtained as soon as possible. If no paternal 
sample is obtained, the participant will be classified as a screen failure and will not be enrolled into the 
study. The parental samples will be sent for DNA extraction as soon as they become available. 
If the clinical karyotype, CMA or any other non-sequencing test result is known to be positive prior to 
sequencing, and felt to be causative of the anomaly, the karyotype/CMA results will be recorded, the 
participant will be classified as a screen failure and not enrolled, and parental samples/DNA will be 
discarded.  
If the clinical karyotype/CMA results are negative (or non-causative), eligibility criteria will be 
confirmed, the participant and genetic father will sign the study consent form (if not already signed), 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
23 
either in person or electronically, parental samples will be collected if needed, and genome sequencing 
will proceed.  This defines the time enrolled in the study.  Parents declining participation at this time will 
be considered as having refused consent; these participants may be offered participation in the 
unsequenced prenatal group if eligibility criteria are met. Those choosing to terminate their pregnancy 
based on ultrasound/CMA findings are ineligible. 
“Expedited” prenatal sequencing 
An alternative to the prenatal diagnostic flow described above permits prenatal sequencing to be 
performed in parallel with the CMA, i.e. before the fetus is confirmed as CMA negative. This diagnostic 
workflow is at the discretion of the study research team and participants will be considered enrolled once 
they have signed the study consent and parental samples have been collected. If the CMA is negative or 
unrelated to the fetal anomaly, the case will continue to be part of the prenatal sequencing group and 
included in the analyses. In pregnancies in which the CMA result is found to be pathogenic or likely 
pathogenic (and presumed causal of the anomaly), sequencing will be discontinued or if already 
completed the results will be recorded and reported to the family in the appropriate manner. The 
karyotype/CMA results will be recorded; however, follow-up of the pregnancy through delivery will not 
take place, these participants will not be included in the analyses and not contribute to the sample size.    
4.2.2 Unsequenced prenatal group 
The prospective unsequenced prenatal group will include participants that decline invasive testing and 
those that plan to have invasive testing but decline sequencing (Figure 1).  For participants that plan to 
have invasive testing but decline sequencing, enrollment into the study will occur after the clinical 
karyotype/CMA results are confirmed negative and the participant signs the prenatal unsequenced 
consent. Participants that decline invasive testing will be enrolled at the time of diagnosis of the fetal 
anomaly and they sign the unsequenced prenatal group consent.  Women from this group who later elect 
invasive testing are not eligible to enroll in the prenatal sequencing group but will be included in the 
unsequenced prenatal analyses if the karyotype and CMA are confirmed negative or non-causal.  
Postpartum genetic testing (karyotype and CMA) and genomic sequencing may be performed at the 
discretion of the clinical care team as part of routine care.  Continued follow-up of the pregnancy and 1-
year postpartum period will not take place if a pathogenic or likely pathogenic causal CMA is identified 
prior to neonatal discharge. 
4.2.3 Retrospective unsequenced prenatal group 
The retrospective unsequenced prenatal group will include participants from the screening log who did 
not actively decline enrollment and are otherwise eligible.  Participants will be considered enrolled at the 
time of diagnosis of the fetal anomaly.   
4.3 Baseline Data Collection 
In addition to data collected for eligibility and consent, the following information will be obtained at 
enrollment from either medical record review or participant interview:  
• 
Maternal and paternal: age, race, ethnicity, education, income, household composition, religion, 
religiosity, number of children, and family history 
• 
Maternal medical and psychiatric history including maternal history of disorders with teratogenic 
risk (e.g. diabetes, teratogen exposure including medications, diseases during pregnancy (e.g. 
CMV, Zika)  
• 
Obstetrical history including  

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
24 
• 
Clinical estimated date of delivery and type of conception 
• 
Pre-procedure ultrasounds including date and detailed information on anomaly(ies) 
• 
Number of prior pregnancies and gestational age of delivery  
• 
Unexplained infertility  
• 
Twin gestation 
• 
Family history of learning disabilities, genetic disorders, congenital anomalies, stillbirths or infant 
deaths, significant medical problems, fetal/child structural or growth findings, consanguinity, 
deafness and psychiatric illnesses. 
• 
Prenatal screening and diagnostic test results; rubella immunity status 
A copy of de-identified imaging, karyotype and CMA reports will be transferred to the DCC for all 
enrolled patients.  
4.4 Sequencing Methods 
Prenatal sequencing will be performed for patients in one of the four laboratories, i.e. the Institute of 
Genomic Medicine (IGM) at Columbia University Medical Center, New York Genome Center (NYGC), 
in the Human Genome Sequencing Center CLIA certified Clinical Laboratory (HGSC-CL) at Baylor 
College of Medicine, and at the High Throughput Sequencing Facility at the University of North Carolina 
Chapel Hill.  
Sequencing will be performed on an Illumina NovaSeq 6000 system. All positive sequencing results will 
be confirmed using Sanger sequencing and reported by a CLIA certified laboratory. It is possible that 
non-parentage (maternity or paternity) may be suspected through initial genomic analysis. If there is 
suspicion that the parent of the fetus is not the genetic parent, further analysis of the genomic data will be 
suspended and the family will be informed they are not eligible for study participation. This approach will 
be addressed during the informed consent process. 
Data quality control, including alignment, variant calling, filtering and prioritization will be performed 
using best practices refined at the local institutions. 
4.5 Variant Evaluation and Return of Results   
All candidate variants will first be evaluated locally at each site by a team including the site PI who is a 
board certified clinical geneticist, a study genetic counselor, a variant curation scientist, and a board-
certified molecular geneticist with clinical genome sequencing experience to determine candidate variants 
relevant to the clinical phenotype. Factors used in this evaluation include the ACMG classification of the 
variant, whether its inheritance mode is consistent with the clinical condition, and the extent of 
phenotypic overlap with previously reported cases.  Initially, candidate variants will be reported in the 
coding regions only; however it is anticipated that over the course of the study this may change to also 
incorporate reporting in non-coding regions. 
Compelling variants in novel genes that are not yet disease associated will be classified as variants of 
uncertain significance (VUS) with the qualification that they are in “genes of uncertain clinical 
significance (GUS)”.  For compelling variants in known disease genes that have not yet been associated 
with this disease phenotype (also classified as VUS), in-house databases will be checked for similar 
variants, automated PubMed alerts will be set-up to be notified of new publications, and tools such as 
GeneMatcher will be used to help find other individuals with variants in the same gene. To build 
consensus in interpretation, the local Variant Interpretation teams will have video conferences on which 
uncertain results will be discussed.  

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
25 
Any pathogenic, likely pathogenic or VUS finding that are reported to the participant will be confirmed 
by Sanger sequencing. For all reportable results, a copy of the Sanger confirmation report and additional 
relevant information as applicable will be prepared by the local team and returned to the clinical study 
genetic counselor who will share the result with the ordering MFM/OB physician and place it in the 
electronic medical record (EMR). The ordering MFM/OB physicians and the study genetic counselor will 
report the results to the family and discuss the implications for clinical care. Study clinical geneticists will 
be available for consultation. Results will also be available to the neonatal care team for use after 
delivery.  A copy of the de-identified report will be transferred to the DCC for all patients. 
Local site requirements will dictate the reporting (if at all) of Sanger-confirmed ACMG secondary or 
other incidental findings of the parents and fetus.   Findings in the fetus that may impact treatment 
decisions on the newborn will be reported immediately. 
Carrier status for Mendelian disorders in either parent will be reported according to the local site 
requirements. 
Parents will be formally informed of negative fetal sequencing results only if testing is performed in a 
CLIA approved laboratory.  
4.6 Study Procedures   
Women who are enrolled prospectively will be followed from screening through 12 months postpartum 
unless a pathogenic or likely pathogenic causal CMA is reported prenatally or postnatally prior to 
neonatal discharge. Women who are enrolled retrospectively will be followed from screening through 
delivery discharge.  For twin gestations, neonatal data will only be collected for the fetus with the 
anomaly. Data will be collected at the following time points: 
Table 2. Summary of Data Collection 
Two weeks after discussion of study results 
or 4 weeks after enrollment for the 
unsequenced prenatal group 
• Maternal and paternal psychosocial survey 
 
Delivery and neonatal care 
• Pregnancy complications & delivery outcomes 
• Neonatal phenotype and outcomes 
• Coordinator and physician review of medical records 
to assess change in management (sequencing group 
only) 
• Results of clinical care ordered genetic testing for 
unsequenced group including karyotype/CMA and 
any subsequent sequencing  
• Reports of healthcare utilization and charges from 
admission for delivery to discharge (maternal) 
• Healthcare utilization and charges from delivery until 
discharge or death (neonatal) 
One month post discharge (prospective 
only) 
• Maternal and paternal psychosocial survey 
12 months postpartum (prospective only) 
• Maternal and paternal psychosocial survey 
• Infant weight and length, genetic testing post 
discharge, and healthcare utilization by maternal self-
report  

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
26 
• Ages and Stages questionnaire (ASQ-3) 
 
Neonatal phenotypes will be re-evaluated within one week of birth and infant phenotypes at 12 months to 
identify additional clinical features. If significant new clinical findings are discovered, the sequencing 
variants will be re-evaluated immediately by the local team and any new diagnoses confirmed and 
reported by the study staff to the neonatologist, pediatrician, or other appropriate care provider who will 
report this to the family in concert with the study coordinator.  A copy of de-identified genetic reports 
(CMA and/or sequencing) will be transferred to the DCC for all enrolled patients in the unsequenced 
group. 
4.7 Psycho-Social Surveys   
Each participant enrolled prospectively (both members of the couple) will complete an online 20-minute 
survey at 3 time points and the couple will receive a gift card after the last survey is completed. The three 
time points are: 
• 
Two weeks after disclosure of study sequencing results or 4 weeks after enrollment for the 
unsequenced prenatal group  
• 
One month after discharge from the hospital or end of the pregnancy if there is a fetal demise or 
pregnancy termination  
• 
Twelve months postpartum 
If the patient delivers before completion of the first survey, a survey that combines elements of the first 
two surveys will be administered. The measures collected at each time period are detailed in Table 3.   
Table 3. Psychosocial Survey Measures 
Variable 
Measure 
Time 
 
 
Post 
results/ 
enroll 
1mth   
pp 
12mth 
pp 
Anxiety 
Personal Health Questionnaire-8 
X 
X 
X 
Depression 
General Anxiety Disorder 
X 
X 
X 
Genetic Knowledge 
Adapted from measures used in 
CSER/eMERGE 
X 
 
 
Numeracy  
Adapted from measures used in 
CSER/eMERGE 
X 
 
 
General Optimism 
Life Orientation Test 
X 
 
 
Tolerance with 
Ambiguity 
Tolerance with Ambiguity 
X 
 
 
Perceived control 
over health 
Internal Health Locus of Control 
X 
 
 
Genetic Essentialism 
Parrott Genetic Essentialism 
X 
 
 
Consenting and 
Education Experience 
Modified Genetic Counseling Satisfaction 
Scale and Doctor-Patient perceptions of 
communication 
X 
 
 
Satisfaction w/ 
decision to Participate  
Decision Regret Scale 
X 
X 
X 
Satisfaction w/ 
decision to continue  
Decision Regret Scale 
X 
X 
X 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
27 
Parent-Infant 
relationship  
Parent-Infant Attachment Questionnaire 
(Reck 16 question) 
 
X 
X 
Parenting stress & 
anxiety for child 
Child Vulnerability scale 54 
 
X 
X 
Parents’ experience of 
parenting 
Parent Sense of Competence Scale 
 
 
X 
Therapeutic 
Optimism/Prognosis 
Brief Illness Perception Questionnaire 
X 
X 
X 
Marital Relationship 
Kansas Marital Satisfaction Scale  
X 
X 
X 
Quality of life 
ITQOL: 
https://www.healthactchq.com/surveys.php 
 
 
X 
COVID-19 
History of maternal and infant COVID-19 
infection 
X 
X 
X 
Study group 
Reasons for joining the sequenced or 
unsequenced group 
X 
 
 
Prenatal sequencing only 
Results Disclosure 
and Secrecy 
Disclosure and non-disclosure of results to 
others 
X 
X 
X 
Understanding of 
Results 
Adapt from measures used in CSER/ 
eMERGE 
X 
X 
X 
Emotional Response 
to Results 
FACToR-12 (12 items) 
X 
X 
X 
Perceptions of 
uncertainty of results 
PUGS perceptions of uncertainties in genetic 
sequencing 
X 
X 
X 
Impact of sequencing 
results 
Impact of sequencing results on pregnancy 
management 
 
X 
X 
 
The end of the survey will have a free text box for participants to add any other details they wish to share 
and also ask if the participant is willing to speak by phone in a semi-structured interview (Section 4.7.1). 
4.7.1 In-depth Qualitative Interviews 
The impact of learning about the prenatal WGS test and return of prenatal WGS results will be evaluated 
by interviewing 45 sets of parents who were enrolled prospectively (90 participants) once between two 
weeks after result disclosure or four weeks after enrollment for participants in the unsequenced group and 
15 months after delivery. Those willing to be interviewed will indicate this at the end of their 
psychosocial survey.  Depending on the number of couples who volunteer for an interview, research staff 
will purposefully sample a range of parental ages, ethnicities, educational level, site, and fetal anomaly.  
Participants with positive or uncertain genetic test results and those who had difficulty adjusting to the 
information or were dissatisfied with participation in the study and have decision regret will be 
oversampled.   
Both parents will be interviewed since men are often not included in these studies and are an integral part 
of the decision-making process and long-term outcome of the family. The interview data will be used to 
develop recommendations and identify factors associated with decision satisfaction, and to develop 
recommendations for pre- and post-test genetic counseling that reflect the needs of women and their 
partners as they make decisions about undergoing prenatal sequencing, or continuing a pregnancy after 
learning about a known or uncertain fetal sequencing abnormality. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
28 
4.8 
Healthcare Utilization and Change in Management 
Information on healthcare utilization and charges will be collected starting at the time of presentation to 
the hospital for delivery through discharge. Site coordinators will work with hospital billing to obtain 
detailed billing records from delivery admittance through discharge for each study participant, including 
the hospital charge ratio. Current Procedural Terminology (CPT) medical code, description, total units 
and total hospital charge will be collected. At the 12-month follow-up phone call for participants who 
were enrolled prospectively, information will also be recorded on healthcare utilization post neonatal 
discharge, including number of infant hospitalizations, surgeries, procedures and specialist visits. For 
twin gestations, this information will only be collected for the twin with the anomaly. 
To identify if sequencing results changed the original care plan, the site coordinators will review all 
prenatal and postnatal medical records in conjunction with the designated physician from diagnosis until 
discharge or death to document change in healthcare management. Change in healthcare management will 
be completed by both the MFM/OB for prenatal assessment and a designated neonatal fellow in 
consultation with the clinical care team at each institution. The survey will include questions focused on 
clinical utility, such as: 1) have there been any changes to the patient’s treatment plan based on the 
sequencing results, 2) have there been any changes to the way that you counsel the patient/family 
regarding the immediate medical management as a result of the sequencing results, and 3) have there been 
any changes to the way that you counsel the patient/family regarding the long term medical management 
as a result of the sequencing results. The changes in healthcare management will be presented and 
reviewed by the Clinical Adjudication Committee which will confirm whether an alteration in health care 
management occurred based on sequencing results. 
4.9 
Adverse Event Reporting 
Detailed information concerning adverse events assessed to be definitely, probably or possibly related to 
study procedures will be collected and evaluated throughout the conduct of the study.  Adverse events 
will be reported to the Data and Safety Monitoring Board.   
4.10 Study Outcome Measures and Ascertainment  
4.10.1 Primary Outcome 
1. Pathogenic, likely pathogenic, or VUS variants identified by sequencing and deemed reportable 
by the Variant Adjudication Committee   
2. Maternal hospital charges between time of presentation to hospital for delivery to discharge, and 
neonatal hospital charges between delivery and infant discharge or death 
4.10.2 Secondary Outcomes (Prenatal sequencing and unsequenced prenatal) 
1. Perinatal outcomes including gestational age at delivery, major morbidities including mechanical 
ventilation, sepsis, pressor support, ECMO,  metabolic abnormalities (e.g., acidosis, elevated uric 
acid, hypo-/hyperglycemia), intraventricular hemorrhage, periventricular leukomalacia, 
encephalopathy, and seizure  
2. Neonatal/infant death at time of discharge and at 12 months of age* 
3. Length of initial NICU stay and number of days spent in the hospital between initial discharge 
and 12 months of age* 
4. Infant weight and length at 12 months of age* 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
29 
5. Developmental parameters (communication, gross motor, fine motor, problem solving and 
personal-social) at 12 months of age using ASQ-3* 
6. Anxiety following result disclosure (or 4 weeks post enrollment for the unsequenced group), 
neonatal discharge and 12 months postpartum*  
7. Depression following result disclosure (or 4 weeks post enrollment for the unsequenced group), 
neonatal discharge and 12 months postpartum* 
8. Quality of life for the patient and family at 12 months postpartum*  
9. Incremental cost per Quality Adjusted Life Year (QALY) 
* Only available on participants enrolled prospectively and followed through 12 months postpartum 
4.10.3 Secondary Outcomes (Prenatal sequencing only) 
10. Apparent prenatal phenotypic expansion from currently defined pediatric phenotypes 
11. Variants of uncertain significance (VUS) that have not yet been associated with this disease 
phenotype  
12. VUS subclassified as compelling variants in novel genes that are not yet disease associated (genes 
of uncertain clinical significance; GUS)  
13. Pathogenic, likely pathogenic and VUS variants identified by sequencing (coding and non-coding 
regions) compared with coding regions only (digital WES) 
14. Pathogenic, likely pathogenic and VUS variants identified by analysis of a proband alone 
compared to a proband-parent trio  
15. Change in management decisions attributable to genomic results defined as changes to the 
patient’s treatment plan or changes to the counseling of the patient/family regarding the 
immediate or long-term medical management 
16. Accuracy of parental understanding of genetic test results 
17. Educational/counseling and social support needs of the mother and father 
18. Changes in classification of sequencing variants over time 
19. Turnaround time of sequencing components and how it changes over time. 
 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
30 
5 Statistical Considerations  
5.1 
Power and sample size 
The first primary outcome for this study is the frequency of pathogenic, likely pathogenic, or VUS 
genomic variants identified by sequencing among participants with a normal karyotype and CMA. The 
sample size is based on the incremental yield reported in a prospective study of WES performed at 
Columbia University in a similar population.55 Among 234 parent-fetus trios without CMA abnormalities, 
a genetic diagnosis was reported in 22 (9.4%). The precision of the estimate for a range of sample sizes 
and estimates is shown in Table 4. An estimate of 9.4% and sample size of 750 in the prenatal sequenced 
group will have a precision of 2.3% (confidence interval half-width) with a 93% probability.  
Table 4. Precision for range of estimates and sample sizes with at least 90% probability 
Estimate 
Sample size 
CI ½-width 
9.0% 
700 
2.4% 
 
750 
2.3% 
 
800 
2.2% 
9.4% 
700 
2.4% 
 
750 
2.3% 
 
800 
2.3% 
9.8% 
700 
2.5% 
 
750 
2.4% 
 
800 
2.3% 
 
The second primary outcome is maternal and neonatal hospital charges. The utilization of health care and 
cost only will include women that deliver at one of the study centers. Given that some of the recruiting 
centers include referrals in which the women will not deliver at the study center, power estimates assume 
cost will be available on 500 of the 750 in the prenatal sequencing group. Similarly, of the 350 
unsequenced prenatal controls, 15% are assumed to have a pathogenic or likely pathogenic causal pre-
/postnatal CMA prior to neonatal discharge or deliver elsewhere resulting in approximately 300 
unsequenced prenatal controls available for analysis. The total charges for deliveries at Columbia 
University that required a NICU admission in 2016 and 2017 (partial year) were used to provide cost 
estimates.  The charges follow a lognormal distribution approximately with a mean total charge of 
$277,902 and standard deviation of $389,484.  Assuming 500 participants in the prenatal WGS group, 
and 300 unsequenced prenatal control participants, the study will have 80% power to detect a mean ratio 
of 0.80 (20% reduction) with an alpha=0.05 two-sided.  
5.2 
Prenatal Sequencing Group Only 
The primary and secondary outcomes for the sequencing variants are descriptive and will be reported as 
the observed proportion (frequency) with 95% confidence intervals. Exact confidence intervals will be 
reported as appropriate. Sequencing findings include variants or no variants (negative). Variants are 
further classified as pathogenic, likely pathogenic, or VUS. VUS is further categorized by whether the 
variants are in genes related to the fetal phenotype or known to cause severe childhood disease, or in a 
gene of uncertain significance (GUS). Sequencing findings will be reported overall, by single vs. multiple 
anomalies, by organ system (abdominal wall, CNS, face/ear, effusion, intrauterine fetal growth, GI tract, 
genitalia, heart, neck, renal tract, skeletal, spine, thorax), and by key demographics including maternal 
age, race/ethnicity and fetal sex. Pearson’s chi-square or Fisher’s Exact test for categorical variables and 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
31 
Mann-Whitney U test for continuous outcomes will be used to assess associations by variant 
classification.   
For each variant meeting the primary outcome definition (pathogenic, likely pathogenic, and uncertain 
genomic variants), the type of test that would identify the variant (WGS only, WES) will be determined 
centrally and used to report the incremental number of pathogenic, likely pathogenic, and VUS variants 
identified by WGS compared with WES.  
Change in management decisions attributable to sequencing results are descriptive and will be reported as 
the frequency with 95% confidence intervals overall and by variant classification.    
5.3 Prenatal Sequencing and Prenatal Unsequenced Groups 
Clinical management including health care utilization and cost, and pregnancy and neonatal outcomes 
will be compared among the prenatal sequencing group and the unsequenced prenatal group (prospective 
and retrospective). Women whose fetus or neonate is found to have pathogenic or likely pathogenic 
causal findings on CMA prior to neonatal discharge will be excluded from these analyses. Twelve month 
postpartum outcomes will be compared only among those individuals enrolled prospectively. Initially, 
demographic data and severity of ultrasound findings will be compared to ensure there are no significant 
differences between the two study groups (prenatal sequencing and unsequenced prenatal group).  If 
differences are found, the covariates will be adjusted for in the analyses.  
Categorical variables will be reported as the number and frequency and associations assessed by the 
Pearson’s chi-square or Fisher’s Exact test. The distributions for continuous variables will be assessed for 
normality and transformed to fit a normal distribution if possible. Normal distributions will be reported as 
mean and standard deviation and compared with the t-test, and non-normal data will be reported as 
median and interquartile range and compared with the Mann-Whitney U test. 
For each outcome, if the study groups show a significant difference, interactions will be tested and 
subgroup analyses conducted if the interaction is significant (p<0.05). Pre-specified subgroup analyses 
include maternal age, race/ethnicity, fetal sex, and type of anomaly (lethal vs non-lethal). 
The amount of missing data, missing data patterns, and identification of variables associated with 
missingness will be explored to inform the primary and sensitivity statistical analyses. Analytic 
techniques used to address missing data bias will be used as appropriate. A two-sided nominal p-value 
less than 0.05 will indicate statistical significance.  
5.3.1 Healthcare utilization and cost 
Direct medical costs include cost of all medical care for the woman and newborn during the delivery 
hospitalization. These costs will be estimated using health care charges associated with each of these 
services. Unit costs will be adjusted by health system specific cost-to-charge ratios. If cost-to-charge 
ratios are not available, published ratios or in rare cases Medicaid reimbursements will be used.   
The effect or outcome measure for cost effectiveness will be quality-adjusted life years (QALYs). 
Maternal and neonatal QALYs will be estimated by applying published utility weights (1=perfect health, 
0=death) from various health states to components of the conditions found by sequencing.  Cost-
effectiveness of prenatal sequencing will be evaluated as the incremental cost per QALY with a threshold 
of less than $100,000 per QALY as cost effective. Cost benefit analysis will also be performed and be 
evaluated by dividing the costs of care and outcomes from the intervention by the costs of usual care. A 
CBA < 1 indicates that the intervention is cost saving and thereby cost beneficial. 
All estimates of costs and outcomes will be reported as means with 95% confidence intervals.  Sensitivity 
analysis will be conducted to evaluate the impact of uncertainty on the results. Varying probabilities (e.g. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
32 
baseline risk of sequencing findings, change in neonatal outcomes) and cost parameters (e.g. cost of 
sequencing, healthcare costs) will be used to take into account potential clinical scenarios that might 
deviate from the baseline estimates. One-way deterministic sensitivity analysis for individual factors will 
be employed and Monte Carlo simulation to assess the robustness of the findings by simultaneously 
sampling distributions around multiple parameters within the model and report 95% confidence ellipses 
and acceptability curves. 
5.3.2 Psychosocial outcomes 
The secondary psychosocial outcomes are descriptive and will be reported as the observed proportion 
(frequency) with 95% confidence intervals. Exact confidence intervals will be reported as appropriate. All 
outcomes will be assessed at three time periods (post disclosure/enrollment, post discharge, and 12 
months postpartum) and each time period will be analyzed separately. The initial analyses will compare 
prenatal sequencing (those receiving a genetic result of any type) with the prospective unsequenced 
prenatal group. Exploratory analyses will include pairwise comparisons between each variant 
classification (unsequenced prenatal group, negative sequencing result, VUS result, and pathogenic/likely 
pathogenic sequencing results).  
Associations between the outcomes and key demographics also will be assessed. Further, association 
between outcomes and study groups will be tested in a multivariate setting adjusting for other variables 
hypothesized to affect outcome (e.g. demographics, genetic essentialism and genetic optimism). 
Continuous outcomes will be analyzed using a multivariate linear regression model and binary outcomes 
using a logistic regression model. Standard model selection methods and regression diagnostics will be 
performed to assess goodness of fit.  
A thematic analysis will be performed for the qualitative interview data to identify patterns or themes in 
these data.  Summary statistics will be reported for the themes.   

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
33 
6 Data Collection 
6.1 Web Data Entry Systems 
A web data entry system will be set up to present data screens for the entry of the data listed below.  Data 
will be collected on standardized forms on which most responses have been pre-coded. Data collection, 
including summary sequencing result data, will be either directly entered from source material and 
entered on the web interface or entered on case report forms for later keying on site.  For collection of 
pre- and postnatal phenotype data, use in MIDAS of the Human Phenotype Ontology (HPO) will be 
incorporated.  Documents such as the prenatal ultrasound and sequencing results reports will be 
transferred to the DCC via the MIDAS system once de-identified 
The forms will be set up in 2 web-based data entry systems as described below:  
• 
MIDAS (Multimodal Integrated Data Acquisition System) is a data entry and management 
system designed specifically for research studies. Data will be entered using a web interface into 
the MySQL database located at the Data Coordinating Center. The system allows extensive data 
auditing and reporting to assist users with data correction/verification as well as patient 
management.  
• 
REDCap is a web-based and database-backed platform.  The psychosocial survey instruments will 
be completed directly by study participants using REDCap software hosted by the Clinical 
Coordinating Center. Data will be exported regularly to the DCC.  
6.1.1  Data Collection Forms 
The following forms will be entered into MIDAS: 
PG01: 
Screening Log  
PG01A 
Lab sample tracking form  
PG02:  
Screening Results and Eligibility Form   
PG03: 
Prenatal Imaging Form  
PG04: 
Baseline Data Form (includes demographics, relevant maternal history including previous 
pregnancy data) 
PG06: 
Sequencing Results Summary Form 
PG06A: Variant Data Log (includes mutation type, description and inheritance) 
PG07: 
Delivery/ Neonatal Clinical Outcome Form includes outcomes through discharge 
PG07A: Infant Clinical Structural Anomalies Form 
PG07B: Infant Dysmorphology Form 
PG08: 
Healthcare Management Form 
PG09: 
Healthcare Utilization Form 
PG10: 
One Year Outcome Form 
PG11: 
Ages and Stages Questionnaire (ASQ-3) 
PG12: 
Adverse Event and Unanticipated Problem Form 
PG13: 
Patient Status Form includes withdrawal status 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
34 
 
 
The following form will be entered directly into REDCap by participants:  
PG14: 
Psychosocial survey 
6.2 Centralized Data Management  
The DCC will monitor on an ongoing basis the acquisition, completeness and quality of data. This will 
include review of laboratory compliance in timeliness of reporting prenatal diagnosis testing, including 
the evaluation and then the reporting of sequencing results to patients, where applicable.  These data will 
be edited for missing, out of range and inconsistent values, and queries forwarded to their point of origin 
for review and resolution. Reports including timelines for form completion will be generated prompting 
submission of outstanding data forms.  
Bioinformatics files generated during sequencing will be stored initially at the sequencing laboratories 
and finally submitted directly to NIH repository(ies). The DCC will prepare summary and clinical data for 
submission to the appropriate NIH data repository.  (See Data archiving) 
6.3 Performance Monitoring 
Site visits will be conducted by DCC staff to the recruitment centers and the sequencing laboratories, 
accompanied by a laboratory supervisor from another participating laboratory or a member of the Variant 
Oversight Committee.  The purpose of each site visit is to review study procedures, assess compliance 
with the study protocol, and assess the quality of the study data and records.  A written report will be 
reviewed by the Steering Committee.   
The DCC will also present regular reports to the Steering Committee. These include: 
• 
Monthly recruitment reports - reports of the number of patients screened and enrolled by month and 
by recruitment site.  
• 
Quarterly Steering Committee reports - a report detailing recruitment, baseline patient characteristics, 
data quality, incidence of missing data and adherence to study protocol by recruitment site/ 
sequencing laboratory. 
 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
35 
7 WGS Pipeline Development 
 
7.1 Evaluation of the incremental value of WGS compared to WES in 
understanding the etiology of birth defects 
This study will provide important insights into the genetic basis of birth defects.  At present, the etiology 
of many structural anomalies can be determined by WES and as additional genes and variants in the 
coding regions are related to specific phenotypes, additional causes will become known.  However, 
studies to date strongly suggest that WES, while clinically valuable, will not identify all causes. By 
exploring the frequency of de novo variation in regulatory and other non-coding regions of the genome 
and evaluating their distribution in functional classes (SNVs, SVs and CNVs), additional understanding 
of genetic causes of developmental alterations will occur. Accordingly, this exploratory aim will 
comprehensively interrogate WGS data to identify the role of errors in noncoding regions in the etiology 
of birth defects.  The aim will also explore the development of pipelines and software to improve the 
integration of WGS into clinical care. This will require the development of multiple variant calling tools 
to catalog a comprehensive set of variants including SNVs/Indels, STRs, SVs, and CNVs from WGS. 
The current variant assessment framework will be extended to include the interpretation of WGS data 
outside of protein-coding regions through the evaluation of: 1) Regions intolerant to variation as assessed 
from population genetics; 2) Cis-regulatory regions known to regulate genes that are intolerant to 
functional variation and to affect transcript levels or splicing; and 3) enhancer elements and other eQTLs 
deduced to impact expression of known intolerant genes. This will result in a comprehensive set of 
regulatory regions that would be especially useful in evaluating SVs and CNVs where a functional effect 
(such as loss of important enhancer) is easier to predict. Although similar tools have previously been used 
for specific research ends, this study will refine and adjust their heuristic filters to achieve optimal 
sensitivity and appropriateness for clinical care and expand the diagnostic yield of WGS. 
Furthermore, in partnership with Illumina Inc., these tools will be benchmarked to newer implementations 
of variant callers that have been specifically tuned for WGS data and optimized for speed and 
computational efficiency. To identify intolerant regions, previous experience using population allele 
frequencies of standing variation will form a basis on which to quantify purifying selection. The recently 
developed Orion, an intolerance metric based on the difference between an observed and expected site 
frequency spectrum under a neutral model, showed that it could accurately identify intolerant regions 
devoid of functional annotation. This method will be applied on a ten-fold larger WGS cohort (N=62,000, 
https://bravo.sph.umich.edu/freeze5) to calculate the observed variation and estimate the expected site 
frequency spectrum using mutation rates under hepta-nucleotide context (a tri-nucleotide context used 
originally) to refine a set of intolerant regions for assessment. It is anticipated that a hepta-nucleotide 
context will capture selection that is likely to occur at DNA-binding motifs more optimally and therefore 
allow a truer estimate of regional site frequency spectrum using the neutral model. Indeed, ~80% of 
GWAS non-coding variants map to putative cis-regulatory elements (CREs). Synonymous and non-
canonical intronic variants will be assessed using a recently developed TRaP score (shown to identify 
putative deleterious synonymous and intronic substitutions with >98% specificity). Data from the 
ENCyclopedia Of DNA Elements (ENCODE) (such as ChIP-seq, Hi-C, ChIA-Pet, and 5C-seq) will be 
incorporated to determine a set of regulatory elements of intolerant and haplo-insufficient genes. Finally, 
regulatory regions linked to intolerant genes will be incorporated using correlations between enhancer 
activity and gene expression across human tissues.  
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
36 
8 Study Administration 
8.1 Organization and Funding 
This study represents a collaboration of clinical geneticists, maternal fetal medicine physicians, genetic 
counselors, pediatric geneticists, biostatisticians, social sciences researchers, bioinformaticians and 
laboratory-based whole genome sequencing technical specialists. 
Table 5. Participating Organizations and Roles 
 
Organization 
Role 
Clinical Coordinating Center 
(CCC) 
Columbia University 
Project leadership, management, central IRB, 
overall coordination, central patient follow-up 
site 
Sequencing Laboratories 
Baylor, Columbia, UNC, 
NYGC 
Performing whole genome sequencing, variant 
calling, interpretation, confirmation, and 
reporting of results   
Data Coordinating Center 
(DCC) 
George Washington University 
Biostatistics Center 
Protocol development, data collection, 
oversight and analysis 
Cost-effectiveness expertise 
Oregon Health Sciences 
University 
Oversight of cost analysis 
 Sequencing technology 
Illumina 
Support for sequencing 
8.1.1 Clinical Coordinating Center 
The Clinical Coordinating Center (CCC) is responsible for leading and overseeing all aspects of this 
study.  In this capacity the CCC will centrally coordinate study implementation, ongoing study 
management, patient follow-up, and publication of the study results.  The CCC will serve as the liaison 
between the participating study locations, the funding agency and the Data Coordinating Center.   
8.1.2 Data Coordinating Center 
The Data Coordinating Center (DCC) is responsible for all aspects of biostatistical design, data 
management, statistical analyses, and preparation of publications based on the study results.  
8.2 Committees 
8.2.1 Steering Committee 
The Steering Committee is the policy and decision-making group, and assumes overall responsibility for 
the management and conduct of the study including protocol development, oversight of the conduct of the 
protocol, analysis and interpretation of data, and reporting results in presentations and publications. The 
committee will ensure that there are synergies between the components of the project and will evaluate 
and provide overall direction. It will also ensure that the team makes measurable progress toward stated 
goals, operates within budget, follows federal policies, and submits required reports in a timely manner.   
 
8.2.2 Operations Committee 
The Operations Committee is responsible for monitoring study implementation, recruitment, day to day 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
37 
management, quality control, coordination and efficient communications. The committee also oversees 
protocol training, study logistics, recruitment progress, and monitors study updates.  
8.2.3 Variant Adjudication Committee 
The Variant Adjudication Committee will include external experts who will assist with interpretation of 
variants for which consensus interpretation by the local lab directors and PIs cannot be reached.  The 
Committee’s determination will be considered the official study result. 
8.2.4 Variant Oversight Committee 
The Variant Oversight Committee will include study lab directors/study lead and relevant site lab 
members and genetic counselors from each site. The Committee will identify systematic differences 
between sequencing laboratories, if any, and will serve as a “learning lab” to standardize fetal sequencing 
results. Specific case examples will be used to illustrate processes and examine any differences.  
8.2.5 Clinical Adjudication Committee 
The Clinical Adjudication Committee will include experts in maternal fetal medicine, genetics and 
pediatrics. The Committee will review the health management reports to ascertain, without bias, whether 
an alteration in health care management occurred based on sequencing results.  
8.2.6 WGS Pipeline Development Committee 
The WGS Pipeline Development Committee which includes bioinformaticians from each site will 
develop a WGS pipeline for fetal diagnosis and identify new pathways. 
8.2.7 Data and Safety Monitoring Board (DSMB) 
The DSMB will be made up of the external members of the Variant and Clinical Adjudication 
committees. The committee will approve the study protocol prior to initiation of the study and meet at 
least annually in the course of the study. It will review interim and final reports on recruitment, and 
monitor logistical issues and adverse events. The committee may also recommend protocol modification 
or early termination of the study due to unexpected problems. 
 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
38 
9 Study Timetable 
 
The timetable for the study is depicted below. 
 
Figure 2. Timeline 
Finalize Study Documents / IRB / 
Training / Certification
 
Recruitment
 
Follow-up
 
Closeout/ Data Analysis / 
Data Archiving
 
2018
2024
2019
2020
2021
2022
2023
 
 
9.1 Training and Certification 
The DCC will collaborate with the Clinical Coordinating Center to organize a workshop to train the 
genetic counselors at recruitment sites and the follow-up staff at Columbia in study procedures for this 
study, including data form completion and web entry.  The manual of operations will serve as the text for 
the training.   
Additionally, DCC personnel will work with the Clinical Coordinating Center and Lab Practices 
Committee to clarify and standardize laboratory practices relating to sample handling and WGS analysis.  
This will include review of the electronic format for the recording of results in the MIDAS database. Data 
handling methods will be standardized across sites, and technical staff at the DCC will be responsible for 
preparing systems to achieve this.   
Before the study can be implemented, each recruiting site must be certified.  Certification will include 
successful completion of the training session.   
9.2 Recruitment and Data Collection Period 
Educational materials will be developed targeted to participants and referring physicians.   
There are 1,800 pregnancies/year with structural anomalies eligible for enrollment in the study at the 
participating centers. Assuming that 40% of these will undergo diagnostic testing, and 25% will have an 
abnormal karyotype or CMA, and 50% of those eligible will consent to sequencing, it is estimated that 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
39 
approximately 270 patients per year or 23 patients will be enrolled per month, with overall recruitment of 
750 patients completed in ~33 months. The 350 controls would be recruited within the same time period. 
If recruitment falls below targets, additional sites may be added.  
9.3 Final Analysis 
After a two-month period for completion of data entry and cleaning of the database for the recruitment 
portion of the main study, the recruitment/results sections of the data set will be locked and available for 
analysis. At the end of the 12 month postpartum follow-up period, approximately three months will be 
required to complete the final report to the Steering Committee and to submit the study’s primary report 
for publication. 
 
9.4 Data Archiving and Sample Storage 
9.4.1 Data Archiving 
At the conclusion of the study, the final dataset will be prepared by the DCC for archiving and sharing. 
Full de-identification of the data will be undertaken prior to submission to an NIH or public repository, 
including allocation of random record IDs replacing the Study ID used in the course of the study. Dates 
are converted to days and consideration is given to appropriate handling of unique aspects of clinical data 
which may serve to identify individuals. Genomic sequencing VCF files will be de-identified in a similar 
manner and the appropriate random ID to match the clinical record will be applied. Laboratories will be 
responsible for depositing any large sequencing files in the requisite repositories directly.  
9.4.2 Data Sharing 
In accordance with the NIH Genomic Data Sharing policy, sequencing data (including VCF files) and 
clinical data will be shared with other scientific investigators and through the controlled access dbGAP 
repository or comparable genomics commons, the Sequence Read Archive, and any NIH Birth Defects 
Commons that is established. A final dataset containing clinical and phenotypic data will be submitted to 
the NICHD data repository (DASH).  In addition, new algorithms and allele frequency data will be shared 
with the newly developed Precision FDA platform as applicable. 
9.4.3 Specimen Storage and Sharing 
At the completion of the study, remaining DNA from the trios and additional samples collected for future 
research will be stored at Columbia University for secondary analyses and ancillary studies. The Steering 
Committee will review and approve proposals for use of the remaining DNA and samples.  The DNA and 
samples will be made available to non-study investigators with appropriate IRB approval, Data Use 
Agreements and/or Material Transfer Agreements. Samples will be re-labeled using a random sample ID 
that is linked to the random record ID prior to distribution to non-study investigators.  
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
40 
Appendix A. Eligible Major Structural Anomalies 
Abdominal wall 
Heart 
 
Bladder exstrophy  
 
Anomalous pulmonary venous return 
 
Body-stalk anomaly  
 
Aortic stenosis/atresia 
 
Cloacal exstrophy 
 
Arrhythmia 
 
Gastroschisis 
 
 
ASD 
 
Omphalocele 
 
AV canal defect 
CNS 
 
Coarctation 
 
Absent or hypoplastic cerebellar vermis 
 
Dextrocardia 
 
Agenesis of corpus collosum 
 
Double outlet right ventricle (DORV) 
 
Anencephaly/Acrania 
 
Ebsteins anomaly 
 
Arachnoid cyst  
 
Heart tumor 
 
Cerebellar hypoplasia 
 
Hypoplastic left heart 
 
Chiari malformation 
 
Hypoplastic right heart 
 
Dandy-Walker malformation 
 
Interrupted aortic arch 
 
Encephalocele 
 
Myxoma 
 
Heterotopia 
 
Pulmonary stenosis/atresia 
 
Holoprosencephaly 
 
Tetralogy of Fallot 
 
Hydranencephaly 
 
Transposition 
 
Iniencephaly 
 
Truncus Arteriosus 
 
Macrocephaly (relative to fetal size) 
 
VSD 
 
Megalencephaly 
 
 
Single ventricle 
 
Microcephaly 
 
Tricuspid atresia/stenosis 
 
Lissencephaly 
 
NS cardiac (abn 4-chamber view/outflow tracts, atrial/vent dilation) 
 
Parenchymal defect (gyral anomaly) 
 
 
 
 
Posterior fossa cyst 
Abdomen, intestine, and liver anomalies 
 
Spina bifida 
 
Abnormal adrenal glands (tumor, uncertain) 
 
Tumor 
 
Bladder (dilated tense/floppy, ureterocele, duplex system) 
 
Vascular anomaly 
 
Moderate/severe bilateral hydronephrosis/urinary tract dilation 
 
Ventriculomegaly/Hydrocephaly 
 
Echogenic kidney 
Ear 
 
Horseshoe kidney 
 
Anotia  
 
Large kidney 
 
Outer ear malformation 
 
Multicystic kidney 
Neck 
 
Pelvic kidney 
 
Cystic hygroma 
 
Polycystic kidney 
 
Teratoma 
 
Small kidney 
Eye 
 
Multi-cystic renal dysplasia 
 
Anophthalmia/Micropthalmos 
 
Renal agenesis 
 
Congenital cataract 
 
Urethra (absent, dilated/valves) 
 
Cyclopia 
Skeletal 
 
Hypertelorism 
 
Skeletal dysplasia 
 
Hypotelorism 
 
Cloverleaf skull 
Face 
 
Hip dislocation/dysplasia 
 
Facial tumor 
 
Limb defect 
 
Lip – Cleft 
 
Foot (absent, oligo-/poly-/syn-dactyly, rocker bottom foot, split foot) 
Genitalia 
 
 
Hand (absent, brachy-/oligo-/ syn-dactyly, overlapping fingers,  
polydactyly (only if non-familial), split hand) 
 
Ambiguous genitalia 
 
Epispadias 
 
Joints (fixed extended, fixed flexed)  
 
Hypospadias 
 
Talipes 
 
Micropenis 
 
Long bones (absent, bowed, short (<1st %ile), fracture) 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
41 
Nose  
Skin 
 
Depressed nasal bridge 
 
Congenital skin disorder 
 
Palate – Cleft 
 
Hemangioma 
 
Abnormal profile 
 
Tumor, unspecified 
 
Frontal bossing 
Spine 
 
Micrognathia/retrognathia 
 
Kyphosis 
Gastro-intestinal tract 
 
Sacral agenesis 
 
Ano-rectal atresia and stenosis  
 
Sacrococcygeal teratoma 
 
Large bowel obstruction 
 
Scoliosis 
 
Small bowel obstruction 
 
Sirenomelia 
 
Duodenal atresia/Stenosis 
Thorax/Respiratory 
 
Situs abnormality  
 
Congenital diaphragmatic hernia 
Head shape 
 
Choanal atresia 
 
Abnormal skull shape 
 
Congenital lung lesion/CCAM 
 
Craniosynostosis 
 
Hydrothorax 
 
Abnormal calcification 
 
Hypoplastic thorax 
Effusion 
 
Bell-shaped thorax 
 
Hydrops 
 
Short ribs 
 
 
Ascites 
Other (reviewed centrally prior to enrollment) 
 
Lymphangioma 
 
 
Pleural effusion 
 
 
 
Skin edema 
 
 
 
 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
42 
References 
 
1. 
Wapner RJ, Martin CL, Levy B, et al. Chromosomal microarray versus karyotyping for prenatal 
diagnosis. The New England journal of medicine 2012;367:2175-84. 
2. 
Osterman MJ, Kochanek KD, MacDorman MF, Strobino DM, Guyer B. Annual summary of vital 
statistics: 2012-2013. Pediatrics 2015;135:1115-25. 
3. 
Shaffer LG, Rosenfeld JA, Dabell MP, et al. Detection rates of clinically significant genomic 
alterations by microarray analysis for specific anomalies detected by ultrasound. Prenatal diagnosis 
2012;32:986-95. 
4. 
Miller NA, Farrow EG, Gibson M, et al. A 26-hour system of highly sensitive whole genome 
sequencing for emergency management of genetic diseases. Genome medicine 2015;7:100. 
5. 
Stavropoulos DJ, Merico D, Jobling R, et al. Whole Genome Sequencing Expands Diagnostic Utility 
and Improves Clinical Management in Pediatric Medicine. NPJ genomic medicine 2016;1. 
6. 
Turner TN, Coe BP, Dickel DE, et al. Genomic Patterns of De Novo Mutation in Simplex Autism. 
Cell 2017;171:710-22 e12. 
7. 
Illsinger S, Das AM. Impact of selected inborn errors of metabolism on prenatal and neonatal 
development. IUBMB life 2010;62:403-13. 
8. 
Matsui H, Gardiner H. Fetal intervention for cardiac disease: the cutting edge of perinatal care. 
Seminars in fetal & neonatal medicine 2007;12:482-9. 
9. 
Bienstock JL, Birsner ML, Coleman F, Hueppchen NA. Successful in utero intervention for bilateral 
renal agenesis. Obstetrics and gynecology 2014;124:413-5. 
10. Deprest J, Gratacos E, Nicolaides KH, Group FT. Fetoscopic tracheal occlusion (FETO) for severe 
congenital diaphragmatic hernia: evolution of a technique and preliminary results. Ultrasound in 
obstetrics & gynecology : the official journal of the International Society of Ultrasound in Obstetrics 
and Gynecology 2004;24:121-6. 
11. Jani J, Gratacos E, Greenough A, et al. Percutaneous fetal endoscopic tracheal occlusion (FETO) for 
severe left-sided congenital diaphragmatic hernia. Clinical obstetrics and gynecology 2005;48:910-
22. 
12. Knox EM, Kilby MD, Martin WL, Khan KS. In-utero pulmonary drainage in the management of 
primary hydrothorax and congenital cystic lung lesion: a systematic review. Ultrasound in obstetrics 
& gynecology : the official journal of the International Society of Ultrasound in Obstetrics and 
Gynecology 2006;28:726-34. 
13. Adzick NS, Thom EA, Spong CY, et al. A randomized trial of prenatal versus postnatal repair of 
myelomeningocele. The New England journal of medicine 2011;364:993-1004. 
14. Johnson MP, Bukowski TP, Reitleman C, Isada NB, Pryde PG, Evans MI. In utero surgical treatment 
of fetal obstructive uropathy: a new comprehensive approach to identify appropriate candidates for 
vesicoamniotic shunt therapy. Am J Obstet Gynecol 1994;170:1770-6; discussion 6-9. 
15. Sofou K, Dahlin M, Hallbook T, Lindefeldt M, Viggedal G, Darin N. Ketogenic diet in pyruvate 
dehydrogenase complex deficiency: short- and long-term outcomes. Journal of inherited metabolic 
disease 2017;40:237-45. 
16. Castiglioni C, Verrigni D, Okuma C, et al. Pyruvate dehydrogenase deficiency presenting as isolated 
paroxysmal exercise induced dystonia successfully reversed with thiamine supplementation. Case 
report and mini-review. European journal of paediatric neurology : EJPN : official journal of the 
European Paediatric Neurology Society 2015;19:497-503. 
17. Chitty LS DA, Gottschalk I, et al. EP21.04: BOOSTB4: a clinical study to determine safety and 
efficacy of pre- and/or postnatal stem cell transplantation for treatment of osteogenesis imperfecta. 
Ultrasound in obstetrics & gynecology : the official journal of the International Society of 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
43 
Ultrasound in Obstetrics and Gynecology 2016;48:356-. 
18. Horwitz EM, Prockop DJ, Gordon PL, et al. Clinical responses to bone marrow transplantation in 
children with severe osteogenesis imperfecta. Blood 2001;97:1227-31. 
19. Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone 
marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nature medicine 
1999;5:309-13. 
20. Gotherstrom C, Westgren M, Shaw SW, et al. Pre- and postnatal transplantation of fetal 
mesenchymal stem cells in osteogenesis imperfecta: a two-center experience. Stem cells translational 
medicine 2014;3:255-64. 
21. Cukier HN, Dueker ND, Slifer SH, et al. Exome sequencing of extended families with autism reveals 
genes shared across neurodevelopmental and neuropsychiatric disorders. Molecular autism 2014;5:1. 
22. Yang Y, Muzny DM, Reid JG, et al. Clinical whole-exome sequencing for the diagnosis of 
mendelian disorders. The New England journal of medicine 2013;369:1502-11. 
23. Krupp DR, Barnard RA, Duffourd Y, et al. Exonic Mosaic Mutations Contribute Risk for Autism 
Spectrum Disorder. American journal of human genetics 2017;101:369-90. 
24. Lim ET, Uddin M, De Rubeis S, et al. Rates, distribution and implications of postzygotic mosaic 
mutations in autism spectrum disorder. Nature neuroscience 2017;20:1217-24. 
25. Sanders SJ, Neale BM, Huang H, et al. Whole genome sequencing in psychiatric disorders: the 
WGSPD consortium. Nature neuroscience 2017;20:1661-8. 
26. Abou Tayoun AN, Spinner NB, Rehm HL, Green RC, Bianchi DW. Prenatal DNA Sequencing: 
Clinical, Counseling, and Diagnostic Laboratory Considerations. Prenatal diagnosis 2018;38:26-32. 
27. Best S, Wou K, Vora N, Van der Veyver IB, Wapner R, Chitty LS. Promises, pitfalls and 
practicalities of prenatal whole exome sequencing. Prenatal diagnosis 2018;38:10-9. 
28. Normand EA, Braxton A, Nassef S, et al. Clinical exome sequencing for fetuses with ultrasound 
abnormalities and a suspected Mendelian disorder. Genome medicine 2018;10:74. 
29. Aarabi M, Sniezek O, Jiang H, et al. Importance of complete phenotyping in prenatal whole exome 
sequencing. Human genetics 2018;137:175-81. 
30. Fu F, Li R, Li Y, et al. Whole exome sequencing as a diagnostic adjunct to clinical testing in fetuses 
with structural abnormalities. Ultrasound in obstetrics & gynecology : the official journal of the 
International Society of Ultrasound in Obstetrics and Gynecology 2018;51:493-502. 
31. Lei TY, Fu F, Li R, et al. Whole-exome sequencing for prenatal diagnosis of fetuses with congenital 
anomalies of the kidney and urinary tract. Nephrology, dialysis, transplantation : official publication 
of the European Dialysis and Transplant Association - European Renal Association 2017;32:1665-
75. 
32. Vora NL, Powell B, Brandt A, et al. Prenatal exome sequencing in anomalous fetuses: new 
opportunities and challenges. Genetics in medicine : official journal of the American College of 
Medical Genetics 2017;19:1207-16. 
33. Yates CL, Monaghan KG, Copenheaver D, et al. Whole-exome sequencing on deceased fetuses with 
ultrasound anomalies: expanding our knowledge of genetic disease during fetal development. 
Genetics in medicine : official journal of the American College of Medical Genetics 2017;19:1171-8. 
34. Pangalos C, Hagnefelt B, Lilakos K, Konialis C. First applications of a targeted exome sequencing 
approach in fetuses with ultrasound abnormalities reveals an important fraction of cases with 
associated gene defects. PeerJ 2016;4:e1955. 
35. Alamillo CL, Powis Z, Farwell K, et al. Exome sequencing positively identified relevant alterations 
in more than half of cases with an indication of prenatal ultrasound anomalies. Prenatal diagnosis 
2015;35:1073-8. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
44 
36. Drury S, Williams H, Trump N, et al. Exome sequencing for prenatal diagnosis of fetuses with 
sonographic abnormalities. Prenatal diagnosis 2015;35:1010-7. 
37. Carss KJ, Hillman SC, Parthiban V, et al. Exome sequencing improves genetic diagnosis of 
structural fetal abnormalities revealed by ultrasound. Human molecular genetics 2014;23:3269-77. 
38. Yang Y, Muzny DM, Xia F, et al. Molecular findings among patients referred for clinical whole-
exome sequencing. Jama 2014;312:1870-9. 
39. Meng L, Pammi M, Saronwala A, et al. Use of Exome Sequencing for Infants in Intensive Care 
Units: Ascertainment of Severe Single-Gene Disorders and Effect on Medical Management. JAMA 
pediatrics 2017;171:e173438. 
40. Clark MM, Stark Z, Farnaes L, et al. Meta-analysis of the diagnostic and clinical utility of genome 
and exome sequencing and chromosomal microarray in children with suspected genetic diseases. 
NPJ genomic medicine 2018;3:16. 
41. Tan TY, Dillon OJ, Stark Z, et al. Diagnostic Impact and Cost-effectiveness of Whole-Exome 
Sequencing for Ambulant Children With Suspected Monogenic Conditions. JAMA pediatrics 
2017;171:855-62. 
42. Biesecker BB, Klein W, Lewis KL, et al. How do research participants perceive "uncertainty" in 
genome sequencing? Genetics in medicine : official journal of the American College of Medical 
Genetics 2014;16:977-80. 
43. Gray SW, Martins Y, Feuerman LZ, et al. Social and behavioral research in genomic sequencing: 
approaches from the Clinical Sequencing Exploratory Research Consortium Outcomes and Measures 
Working Group. Genetics in medicine : official journal of the American College of Medical Genetics 
2014;16:727-35. 
44. Kaphingst KA, Ivanovich J, Biesecker BB, et al. Preferences for return of incidental findings from 
genome sequencing among women diagnosed with breast cancer at a young age. Clinical genetics 
2016;89:378-84. 
45. Kalynchuk EJ, Althouse A, Parker LS, Saller DN, Jr., Rajkovic A. Prenatal whole-exome 
sequencing: parental attitudes. Prenatal diagnosis 2015;35:1030-6. 
46. Kuppermann M, Nakagawa S, Cohen SR, Dominguez-Pareto I, Shaffer BL, Holloway SD. Attitudes 
toward prenatal testing and pregnancy termination among a diverse population of parents of children 
with intellectual disabilities. Prenatal diagnosis 2011;31:1251-8. 
47. Bernhardt BA, Soucier D, Hanson K, Savage MS, Jackson L, Wapner RJ. Women's experiences 
receiving abnormal prenatal chromosomal microarray testing results. Genetics in medicine : official 
journal of the American College of Medical Genetics 2013;15:139-45. 
48. Case AP, Ramadhani TA, Canfield MA, Wicklund CA. Awareness and attitudes regarding prenatal 
testing among Texas women of childbearing age. Journal of genetic counseling 2007;16:655-61. 
49. Kuppermann M, Gates E, Washington AE. Racial-ethnic differences in prenatal diagnostic test use 
and outcomes: preferences, socioeconomics, or patient knowledge? Obstetrics and gynecology 
1996;87:675-82. 
50. Wapner RJ, Driscoll DA, Simpson JL. Integration of microarray technology into prenatal diagnosis: 
counselling issues generated during the NICHD clinical trial. Prenatal diagnosis 2012;32:396-400. 
51. Hillman SC, Willams D, Carss KJ, McMullan DJ, Hurles ME, Kilby MD. Prenatal exome 
sequencing for fetuses with structural abnormalities: the next step. Ultrasound in obstetrics & 
gynecology : the official journal of the International Society of Ultrasound in Obstetrics and 
Gynecology 2015;45:4-9. 
52. Economides DL, Whitlow BJ, Kadir R, Lazanakis M, Verdin SM. First trimester sonographic 
detection of chromosomal abnormalities in an unselected population. British journal of obstetrics and 
gynaecology 1998;105:58-62. 

Prenatal Genetic Diagnosis By Genomic Sequencing   
 
 
 
 
 Version 1.8 
 
 
 
 
 
 
 
 
 
 
     27 March 2023 
45 
53. Whitlow BJ, Chatzipapas IK, Lazanakis ML, Kadir RA, Economides DL. The value of sonography 
in early pregnancy for the detection of fetal abnormalities in an unselected population. British journal 
of obstetrics and gynaecology 1999;106:929-36. 
54. Forsyth BW, Horwitz SM, Leventhal JM, Burger J, Leaf PJ. The child vulnerability scale: an 
instrument to measure parental perceptions of child vulnerability. Journal of pediatric psychology 
1996;21:89-101. 
55. Wapner R PS, Brennan K, Bier L, Wou K, Goldstein D. Whole exome sequencing in the evaluation 
of fetal structural anomalies: A prospective study of sequential patients. Am J Obstet 
Gynecol;216(1):S5-S6. 
56. J C. Statistical Power Analysis for the Behavioral Sciences (second ed.). Hillsdale, NJ: Lawrence 
Erlbaum Associates; 1988.