Genomic Medicine for Ill Neonates and Infants (The GEMINI Study)
Phase: NA
Conditions: Pediatric: Genetic Syndrome
Sponsor: Tufts Medical Center

  
 
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Study Title: 
 
Genomic Medicine for Ill Neonates and Infants  
(The GEMINI Study) 
Grant Title: 
Precision Medicine in the Diagnosis of Genetic 
Disorders in Neonates (U01TR002271) 
NCT Identified 
Number: 
NCT03890679 
Co-Principal 
Investigators:   
 
Jonathan M. Davis, MD 
The Floating Hospital for Children at Tufts Medical 
Center 
Tel: 617-636-5322 
E-mail: jdavis@tuftsmedicalcenter.org 
 
Jill Maron, MD, MPH 
The Floating Hospital for Children at Tufts Medical 
Center 
Tel: 617-636-0766 
E-mail: jmaron@tuftsmedicalcenter.org 
Pager: 617-647-2971 
 
Project 
Manager: 
Anne Kurfiss, MPH 
The Floating Hospital for Children at Tufts Medical 
Center 
Tel: 617-636-7134 
E-mail: akurfiss@tuftsmedicalcenter.org 
Funded by: 
National Center for Advancing Translational Sciences 
(NCATS) 
Protocol 
Version: 
2.0, 05DEC2019 
 
 

  
 
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Table of Contents 
STATEMENT OF COMPLIANCE ............................................................................................................................. 4 
TERMINOLOGY .......................................................................................................................................................... 5 
1 
PROTOCOL SUMMARY ............................................................................................................................... 6 
1.1 
Synopsis ................................................................................................................................................ 6 
1.2 
Schema ................................................................................................................................................. 8 
2 
INTRODUCTION .......................................................................................................................................... 22 
2.1 
Study Rationale .................................................................................................................................. 22 
2.2 
Background......................................................................................................................................... 24 
2.3 
Risk/Benefit Assessment .................................................................................................................. 27 
2.3.1 
Known Potential Risks ................................................................................................... 27 
2.3.2 
Known Potential Benefits .............................................................................................. 30 
2.3.3 
Assessment of Potential Risks ..................................................................................... 31 
3 
OBJECTIVES AND ENDPOINTS .............................................................................................................. 32 
4 
STUDY DESIGN ........................................................................................................................................... 34 
4.1 
Overall Design .................................................................................................................................... 34 
4.2 
Scientific Rationale for Study Design.............................................................................................. 35 
4.3 
End of Study Definition ..................................................................................................................... 35 
5 
STUDY POPULATION ................................................................................................................................ 36 
5.1 
Inclusion Criteria ................................................................................................................................ 36 
5.2 
Exclusion Criteria ............................................................................................................................... 36 
5.3 
Screen Failures .................................................................................................................................. 37 
5.4 
Strategies for Recruitment and Retention ...................................................................................... 37 
6 
STUDY INTERVENTION ............................................................................................................................ 38 
6.1 
Study Intervention(s) Administration ............................................................................................... 38 
6.1.1 
Study Intervention Description ..................................................................................... 38 
6.2 
Study Intervention Compliance ........................................................................................................ 46 
6.3 
Concomitant Therapy ........................................................................................................................ 46 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT DISCONTINUATION/ 
WITHDRAWAL .......................................................................................................................................................... 47 
7.1 
Discontinuation of Study Intervention ............................................................................................. 47 
7.2 
Participant Discontinuation/Withdrawal from the Study ............................................................... 47 
7.3 
Lost to Follow-Up ............................................................................................................................... 48 
8 
STUDY ASSESSMENTS AND PROCEDURES ..................................................................................... 49 
8.1 
Screening & Informed Consent ....................................................................................................... 49 
8.2 
Enrollment ........................................................................................................................................... 51 
8.3 
Safety and Other Assessments ....................................................................................................... 64 
8.4 
Adverse Events and Serious Adverse Events ............................................................................... 64 
8.4.1 
Definition of Adverse Events (AE) ............................................................................... 64 
8.4.2 
Definition of Serious Adverse Events (SAE) .............................................................. 65 
8.4.3 
Classification of an Adverse Event .............................................................................. 65 
8.4.4 
Time Period and Frequency for Event Assessment and Follow-Up ....................... 67 
8.4.5 
Adverse Event Reporting .............................................................................................. 67 
8.4.6 
Serious Adverse Event Reporting ............................................................................... 67 
8.4.7 
Reporting Events to Participants ................................................................................. 68 
8.5 
Unanticipated Problems .................................................................................................................... 68 
8.5.1 
Definition of Unanticipated Problems (UP) ................................................................. 68 

  
 
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8.5.2 
Unanticipated Problem Reporting ................................................................................ 69 
8.5.3 
Reporting Unanticipated Problems to Participants ................................................... 70 
9 
STATISTICAL CONSIDERATIONS .......................................................................................................... 71 
9.1 
Statistical Hypotheses ....................................................................................................................... 71 
9.2 
Sample Size Determination .............................................................................................................. 71 
9.3 
Populations for Analyses .................................................................................................................. 71 
9.4 
Statistical Analyses............................................................................................................................ 72 
9.4.1 
General Approach .......................................................................................................... 72 
9.4.2 
Analysis of the Primary Efficacy Endpoint(s) ............................................................. 73 
9.4.3 
Analysis of the Secondary Endpoint(s) ....................................................................... 75 
9.4.4 
Safety Analyses .............................................................................................................. 77 
9.4.5 
Baseline Descriptive Statistics ..................................................................................... 77 
9.4.6 
Planned Interim Analyses ............................................................................................. 77 
9.4.7 
Sub-Group Analyses ..................................................................................................... 77 
9.4.8 
Tabulation of Individual participant Data .................................................................... 78 
9.4.9 
Exploratory Analyses ..................................................................................................... 78 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS .............................. 79 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ........................................................... 79 
10.1.1 
Informed Consent Process ........................................................................................... 79 
10.1.2 
Study Discontinuation and Closure ............................................................................. 83 
10.1.3 
Confidentiality and Privacy ........................................................................................... 83 
10.1.4 
Future Use of Stored Specimens and Data ............................................................... 86 
10.1.5 
Key Roles and Study Governance .............................................................................. 87 
10.1.6 
Safety Oversight ............................................................................................................. 87 
10.1.7 
Clinical Monitoring .......................................................................................................... 88 
10.1.8 
Quality Assurance and Quality Control ....................................................................... 89 
10.1.9 
Data Handling and Record Keeping ............................................................................ 89 
10.1.10 
Protocol Deviations ........................................................................................................ 90 
10.1.11 
Publication and data Sharing Policy ............................................................................ 90 
10.1.12 
Conflict of Interest Policy .............................................................................................. 91 
11 
ABBREVIATIONS ........................................................................................................................................ 92 
12 
PROTOCOL AMENDMENT HISTORY ..................................................................................................... 95 
13 
REFERENCES ............................................................................................................................................. 99 
14 
APPENDICES ............................................................................................................................................. 104 
 

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STATEMENT OF COMPLIANCE 
 
The trial will be carried out in accordance with the United States (US) Code of Federal 
Regulations (CFR) applicable to clinical studies (45 CFR Part 46). 
  
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who 
are responsible for the conduct, management, or oversight of NIH-funded clinical trials 
have completed Human Subjects Protection and GCP Training. 
 
The protocol, informed consent form(s), recruitment materials, and all participant 
materials will be submitted to the Institutional Review Board (IRB) for review and 
approval. Approval of the protocol, consent form, and Data Safety Monitoring Plan 
(DSMP) must be obtained before any participant is enrolled.  Any amendment to the 
protocol will require review and approval by the IRB before the changes are 
implemented.  
 
______________________________________  
    
Printed Name 
_______________________________________   
__ __ / __ __ __ / __ __ 
Signature  
 
 
 
                                 Date  
 

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TERMINOLOGY 
Term 
Definition as used in this protocol 
Infant 
The research subject and proband with signs/symptoms 
consistent with a possible genetic disorder; in most cases the 
infant will likely be a neonate, but not necessarily   
Guardian/parent 
The person who has authority to legally consent for the infant to 
participate in this research, this person may or may not be a 
biological parent. 
Biological 
parent(s), 
biological mother 
or biological father 
Who is thought to be the true parent of the infant and may 
provide a blood sample for genetic sequencing and trio analysis; 
biological parents participating in this study are also considered 
research subjects and may withdraw their consent at any time.  
Enrolling Site 
The hospital at which the infant is enrolled into the study. 
Laboratories 
Refers to Athena Diagnostics, a wholly-owned subsidiary of 
Quest Diagnostics; Rady Children’s Institute for Genomic 
Medicine – Clinical Genome Center (RCIGM-CGC); and Sema4 
which are conducting the genetic sequencing and/or 
confirmatory tests. 
Positive Result 
Diagnostic findings related to phenotype - pathogenic variant(s) 
in genes interpreted to be responsible for, or contributing to, the 
infant’s phenotype (variant classification of pathogenic or likely 
pathogenic). 
Negative Result 
The result that pathogenic variants associated with the infant’s 
clinical phenotype were not detected. 
Variant of 
Unknown 
Significance (VUS) 
A variant for which the clinical significance is unknown. The 
variant detected may or may not explain the infant’s current 
clinical symptoms. 
Suspicious VUS  
A VUS where the phenotypic fit is very good or functional 
confirmatory tests are readily available or the results are 
actionable. 
Medically 
Actionable 
There is a preventative measure or a treatment available to cure 
or ameliorate symptoms and improve outcome. 
Secondary / 
Incidental Findings 
Pathogenic or likely pathogenic mutations listed in the ACMG 59 
or in other genes unrelated to the phenotype. Secondary 
findings are not intentionally sought in this protocol. The RCIGM 
laboratory report will use the term incidental findings in order to 
emphasize that a targeted analysis for secondary findings is not 
done. The ICF will use the term additional results. 
Provisional result 
A positive result that has not been confirmed by another method.  
Final Result 
Either a positive result or VUS that is confirmed by another 
method, often Sanger sequencing/qPCR, or a negative result. 

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1 
 
PROTOCOL SUMMARY 
1.1 SYNOPSIS  
Title: 
 
Genomic Medicine for Ill Neonates and Infants  
(The GEMINI Study) 
Study Intervention: NewbornDx Sequencing Evaluation (NewbornDx), Rapid 
Whole Genome Sequencing (rWGS) 
Study Description: 
This multicenter, prospective clinical trial will examine the 
diagnostic yield and clinical utility of NewbornDx and rWGS 
testing in high-risk infants with signs/symptoms consistent with 
a possible genetic disorder. Infants will undergo both 
NewbornDx and rWGS testing in parallel. Genetic sequences 
via NewbornDx and rWGS will be generated for the infant 
(proband) and when available, the biological parents, and 
analyzed. NewbornDx and rWGS test results will be returned 
to the infant’s clinician, medical record and parent/guardian. 
Those infants analyzed as a duo or trio will have a 
retrospective analysis done of only the infant’s data to 
determine if the same result would have been obtained with 
and without the parent samples. Clinical utility will be 
measured by clinician opinion and changes in the infant’s care 
as a result of the genetic sequencing test results. Quality of 
life (QoL) and medical resource utilization by parent survey 
will be collected until the infant is one year corrected 
gestational age. These data, along with a retrospective chart 
review of infants with suspected genetic disorders, will be 
used to understand 1-year cost and health outcomes that 
would have been incurred in the absence of the advanced 
testing. The resulting data from the trial will be used in the 
economic evaluation comparing NewbornDx, rWGS, and SOC 
over a 1-year period and used as basis to simulate the lifetime 
cost-effectiveness of these testing strategies. A web-based 
clinical reference database to provide references, clinical 
management guidelines, opportunities for clinical trial 
participation, and support groups for each condition will be 
developed with separate interfaces for the parent/guardian(s) 
and medical provider. The clinical reference database will be 
qualitatively assessed by a survey of medical providers. 
Objectives: 
 To estimate the diagnostic yield of NewbornDx and rWGS 
testing in identifying genetic disorders of unknown etiology  
 To assess the clinical utility of genomic sequencing 

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 To examine the economic impact and health outcomes of 
NewbornDx and rWGS in infants compared with SOC 
diagnostic testing, over a one-year and lifetime horizon  
 To develop and qualitatively assess the web-based clinical 
reference database of supporting information for medical 
providers and parents 
Endpoints: 
Primary Endpoints:  
 A confirmed genetic disorder detected by NewbornDx  
 A confirmed genetic disorder detected by rWGS  
 Time from sample collection to positive test result  
 Clinical utility of genomic sequencing (care changes, 
time to initiation of appropriate treatment or redirection 
of care including the withholding or withdrawing of life-
sustaining treatment) 
Secondary Endpoints:  
 One-year cost-effectiveness of SOC, NewbornDx and 
rWGS testing  
 Lifetime cost-effectiveness of SOC, NewbornDx and 
rWGS testing 
 User satisfaction with the clinical reference database 
Study Population: 
400 infants less than one year corrected gestational age 
(CGA) admitted to a hospital (NICU/PICU/ CICU/inpatient 
floor) participating in this study with signs/symptoms 
consistent with a possible genetic disorder  
Phase: 
N/A  
Enrolling Sites: 
The Floating Hospital for Children at Tufts Medical Center, 
Cincinnati Children's Hospital Medical Center, Mount Sinai - 
Kravis Children’s Hospital, Children’s Hospital of Pittsburgh, 
Rady Children's Hospital - San Diego, and North Carolina 
Children's Hospital 
Study Duration: 
54 months 
Participant 
Duration: 
 
From enrollment until 1 year CGA 
 
 

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1.2 SCHEMA 
 
 
Enroll in REDCap system 
Urgent Testing Determination 
Part 1: Screening and Enrollment 
Informed consent 
Screening 
Upload Redacted ICF to REDCap 
Blue = Enrolling site 

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Add Sample ID#s and ship 
to Athena and RCIGM 
Part 2: Sample Collection and HPO 
Obtain blood samples and label 
with Name and DOB 
Mount 
Sinai 
Subject?  
Yes 
No 
Order rWGS in the  
RCIGM Portal 
Sema4 extracts DNA, 
ships to RCIGM 
Add Sample ID#s and ship 
to Athena and Sema4 
Complete Athena 
NewbornDx Paper 
Requisition 
Site extracts phenotype, 
uploads HPO terms to 
RCIGM ordering portal 
Blue = Site 
Green = Sema4 

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NewbornDx 
Primary 
pathogenic, likely 
pathogenic or 
suspicious VUS? 
No 
Yes 
Send primary 
provisional result 
for confirmation 
testing 
Provide provisional 
result with 
management 
guidance 
Part 3: NewbornDx/rWGS Tests 
Enrolling site 
(except Mount 
Sinai) returns 
provisional 
result to 
clinician/parents
/MR 
Athena 
RCIGM 
Blue = Enrolling site 
Orange = Athena 
Purple = RCIGM 
Provisional 
reporting? 
No 
Yes 
rWGS  
Primary 
pathogenic, likely 
pathogenic or 
suspicious VUS? 
Yes 
No 
Send primary 
provisional result 
for confirmation 
testing 
Provide provisional 
result with 
management 
guidance 
Provisional 
reporting? 
Yes 
No 

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Secondary Infant 
Finding? 
Yes
No 
Part 4: Secondary Findings for the Infant 
Yes
No 
Yes
No 
Opt-in for infant 
secondary finding? 
Disorder onset in 
childhood? 
Medically actionable 
or ACMG59? 
Yes
No
Don’t Report 
Secondary Infant 
Finding? 
No 
Yes
No
Yes
No
Yes
Opt-in for infant 
secondary finding? 
Disorder onset in 
childhood? 
Medically actionable 
or ACMG59? 
No 
Yes
RCIGM 
Athena 
Send infant secondary 
finding for confirmation 
testing 
Send infant secondary 
finding for 
confirmation testing 

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Athena will not 
report secondary 
findings for a 
parent 
Don’t Report 
Part 5: Secondary Findings for a Biological Parent 
Athena 
Secondary finding for a parent? 
Opt-in parent secondary findings? 
Send parent secondary finding for 
confirmation testing 
On the ACMG59 List? 
No 
Yes 
No 
Yes 
No 
Yes 
RCIGM 

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Part 6: Confirmation Testing & Return of Results 
Athena 
RCIGM 
Orange = Quest 
Purple = RCIGM 
Athena conducts confirmation 
testing 
RCIGM conducts confirmation 
testing 
Findings to 
confirm? 
No 
Yes 
Findings to 
confirm? 
Yes 
No 
Athena sends CLIA-
certified final report for 
infant to enrolling site and 
Tufts 
RCIGM sends CLIA-
certified final report(s) for 
infant and if applicable, 
parent, to site and Tufts; 
(exception: RCIGM 
workflow for Mount Sinai 
subjects shown in Part 7) 

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Luci dchart
Part 7: RCIGM Workflow for Subjects from Mount Sinai Only. All other sites proceed to Part 8. 
This workflow will be eliminated when rWGS becomes New York State approved 

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Concordant 
results? 
Adjudication 
Procedures  
Enrolling site and Tufts MC co-PIs and Project Manager 
receive confirmed results 
No 
Study Workflow Part 8 - Reconciliation of Results 
Enrolling site informs clinician and guardian of final 
results, documents how results will be used in clinical care 
(all CLIA certified lab reports, even if discordant, remain in 
infant’s medical record); 
Site returns any secondary findings to biological parent 
after confirming opt-in; refers to adult genetics 
Blue = Enrolling site 
Gray = Tufts 
Yes 

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 Study Workflow Part 9 – Adjudication Procedures 
 

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Final parent interview and QoL instruments at 1 
Year CGA 
After discharge home, monthly parent survey and 
QoL instruments every 3 months 
Physician clinical utility survey 
Chart abstraction of clinical actions 
Part 9: Post Result through 1 Year CGA 
Blue = Enrolling site 
At transfer or discharge home, obtain follow-up 
contact information, complete QoL instruments, 
upload redacted source documents to REDcap 

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Schedule of Activities (SoA) and Data Case Report Forms 
A= Athena (Quest), 
R=RCIGM, S4=Sema4 
Procedures 
Screening 
Enrollment 
Genetic Sequencing 
Return of final result 
Transfer to facility 
Discharge home 
Follow-up #1-11 
1 year CGA 
Site 
Evaluate inclusion/exclusion criteria  
X  
 
 
 
 
 
 
Site 
Consult clinical genetics or specialty service per SOC 
X  
 
 
 
 
 
 
Site 
Informed consent process  
X  
 
 
 
 
 
 
Site 
Complete screening log 
X  
 
 
 
 
 
 
Consented and Enrolled Infants 
Site 
Enroll infant in REDCap 
 
X  
 
 
 
 
 
Site 
Determine if the infant requires urgent testing   
 
X  
 
 
 
 
 
Site 
Obtain blood samples for infant and biological parent(s) 
 
X  
 
 
 
 
 
Site 
Order rWGS (or Ultra Rapid for Urgent Cases) in the RCIGM 
portal; indicate secondary finding elections for infant and each 
biological parent  
 
X  
 
 
 
 
 
Site 
Complete Athena Laboratory requisition for NewbornDx, 
indicate infant secondary finding election  
 
X  
 
 
 
 
 
Site 
Notify Athena of need for urgent testing, if applicable 
 
X  
 
 
 
 
 
Site 
Ship samples to Athena Diagnostics and RCIGM (or Sema4*)  
X  
 
 
 
 
 
Site 
Scan redacted ICF signature pages into REDCap  
 
X  
 
 
X  
X 
Site 
Provide clinical information and HPO terms to RCIGM portal 
and Athena 
 
 
X  
 
 
 
 
A 
Complete NewbornDx [duo/trio if parent sample(s) are 
available] 
 
 
X  
 
 
 
 
R 
Complete rWGS [duo/trio if parent sample(s) are available]  
 
 
X  
 
 
 
 
A, R 
Return verbal provisional result, if applicable,  with 
management guidance to enrolling site clinical staff*, site PI, 
study-wide Co-PI   
 
 
X  
 
 
 
 
Site 
Enter provisional result, if applicable, with management 
guidance as a research note to infant’s medical record and 
notify the infant’s parent/guardians 
 
 
X  
 
 
 
 
A, R, S4 Complete confirmation testing for pathogenic, likely 
pathogenic and suspicious VUS findings* 
 
 
X  
 
 
 
 
A, R, S4 Return final infant result to enrolling site research team and 
study-wide co-PIs/project manager   
 
 
 
X  
 
 
 

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A= Athena (Quest), 
R=RCIGM, S4=Sema4 
Procedures 
Screening 
Enrollment 
Genetic Sequencing 
Return of final result 
Transfer to facility 
Discharge home 
Follow-up #1-11 
1 year CGA 
R, S4 
Return confirmed parental secondary findings to the enrolling 
site if applicable and the parent opted in on the ICF* 
 
 
 
X  
 
 
 
Site 
Return the final result for the infant to the clinical team, 
infant’s medical record and the infant’s parent/guardians 
 
 
 
X  
 
 
 
Site 
Notify the biological parent of confirmed secondary findings, if 
applicable and that parent opted in on ICF 
 
 
 
X  
 
 
 
Tufts, 
Site 
Follow adjudication procedures if the final results from 
NewbornDx and rWGS are discordant or if Sema4 does not 
confirm the rWGS result 
 
 
 
X  
 
 
 
Site 
Administer clinical utility survey   
 
 
 
X  
 
 
 
Site 
Obtain hospital bill(s) for infant hospitalization during which 
the research testing occurred 
 
 
 
 
X X  
 
Site 
Obtain the parent/guardian contact information for follow-up  
 
 
 
 
X X  
 
Site 
Administer parent SF-12 QoL and child visual analog scale 
 
 
 
 
 
X X X 
Site 
Administer or if completed by email, review the 
parent/guardian Resource Utilization monthly interview 
 
 
 
 
X X X X 
Site 
Obtain medical records as necessary (i.e. a new diagnosis or 
adverse event) 
 
 
 
 
 
X X X 
A, R, S4 Monitor for non-conforming events or errors 
 
 
X X  
 
 
 
Site 
Monitor for adverse events 
 
X X X X X X X 
Site 
Monitor for protocol deviations 
  
X X X X X X X 
*Mount Sinai will send blood samples for the infant and each biological parent to Sema4 instead of RCIGM. 
Sema4 will extract the DNA, save one aliquot for future confirmation testing and ship one aliquot to RCIGM. 
Test results that are reported by RCIGM rWGS will be treated as research results for subjects from Mount 
Sinai. Results from RCIGM rWGS will be placed in the infant’s research file and not the infant’s medical 
record. Positive and VUS results reported by RCIGM that are not also reported by Athena Diagnostics will 
be confirmed by Sema4 and a Sema4 laboratory report will be placed in the infant’s medical record. The 
protocol allows for the return of a provisional result when a treatment is available and waiting for 
confirmation testing puts the infant at irreversible harm. Provisional results will not be given for subjects from 
Mount Sinai when the provisional result is found only by RCIGM rWGS. A provisional result may be given 
for results from Athena Diagnostics NewbornDx. 
 
 

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Electronic Case Report Form Submission Schedule  
[X] =Data reported as applicable 
Data Case Report Forms 
Time point 
Screening 
Enrollment 
Return of 
Result 
Transfer 
and/or 
Discharge 
Home 
Follow-up 
#1-11 
1 Year 
CGA 
  
Forms Required for All Subjects Approached 
Demographics 
X 
 
 
 
 
 
System(s)  
X 
 
 
 
 
 
Consent 
X 
 
 
 
 
 
 
Forms Required for All Subjects Enrolled 
Enrollment 
 
X 
 
 
 
 
Urgent Testing 
 
X 
 
 
 
 
Source Documents 
 
X 
X 
[X] 
[X] 
[X] 
Prenatal History 
 
X 
 
 
 
 
Birth History 
 
X 
 
 
 
 
HPO 
 
X 
 
 
 
 
Specimens/Results 
 
X 
X 
 
 
 
Results Concordance 
 
 
X 
 
 
 
Clinical Utility Survey 
 
 
X 
 
 
 
Clinical Actions 
 
 
X 
 
 
 
Transfer/Discharge 
 
 
 
X 
 
 
Child-Visual Analog Scale 
 
 
 
X 
X1 
X 
SF-12 
 
 
 
X 
X1 
X 
Hospital Bill 
 
 
 
X 
 
 
CPT 
 
 
 
X 
 
 
Resource Utilization Survey 
 
 
 
 
X 
X 
Completion 
 
[X] 
[X] 
[X] 
[X] 
X 
 
Forms Required for All Subjects Enrolled as applicable 
Clinical Genetic Tests 
 
[X] 
[X] 
[X] 
[X] 
[X] 
Newborn Screening 
 
[X] 
[X] 
[X] 
[X] 
[X] 
Medications 
 
[X] 
[X] 
[X] 
 
 
Surgeries 
 
[X] 
[X] 
[X] 
 
 
Death 
 
[X] 
[X] 
[X] 
[X] 
[X] 
Adverse Event 
 
[X] 
[X] 
[X] 
[X] 
[X] 
Protocol Deviation 
 
[X] 
[X] 
[X] 
[X] 
[X] 
1The Child-Visual Analog Scale and SF-12 are completed at discharge home, then every 3 months and at 1-year 
CGA. 

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Other forms utilized by the enrolling site   
Screening 
Enrollment 
Return of result 
Transfer and/or 
Discharge home 
Follow-up #1-11 
1-year CGA 
Site Screening and Enrollment Log 
X 
 
 
 
 
 
Demographics Source Document* 
X 
 
 
 
 
 
Documentation of Informed Consent* 
 
X 
 
 
 
 
Eligibility Checklist*  
 
X 
 
 
 
 
Informed Consent Checklist*   
 
X 
 
 
 
 
Documentation of Informed Consent for the Medical Record* 
 
X 
 
 
 
 
Blood Specimen Collection Source Document* 
 
X 
 
 
 
 
HPO Source Document* 
 
X 
 
 
 
 
Athena Diagnostics Laboratory Requisition  
 
X 
 
 
 
 
RCIGM Laboratory Requisition (printed from RCIGM portal)  
 
X 
 
 
 
 
Sema4 Laboratory Requisition (Mount Sinai only) 
 
X 
 
 
 
 
Documentation of Verbal Result*   
 
 
X 
 
 
 
Parent/Guardian Contact Information Form   
 
 
 
X 
 
 
Documentation of Follow-up * 
 
 
 
 
X 
X 
Study Workflow Checklist * 
 
X 
X 
X 
X 
X 
 *Optional use by site if other source documentation or site-specific forms exists 
 
Data sent from each laboratory; data stratified by infant case ID (RCIGM), accession number (Athena) 
or sample ID.  
Preliminary result [gene(s) and variant(s)] prior to confirmation testing  
Date and time of sample receipt 
Date and time the preliminary result was determined prior to confirmation testing 
If the preliminary result was returned provisionally, the date/time the verbal result was called to the site 
If the preliminary result was returned provisionally, the date/time the provisional written report was sent 
to the site 
Whether the preliminary result was determined using proband-only, duo or trio data 
Whether or not the confirmation testing confirmed the preliminary result 
Infant-only analysis for subjects who were initially analyzed as a duo/trio   
 
 

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2 
INTRODUCTION 
 
2.1 STUDY RATIONALE  
 
Congenital abnormalities and genetic disorders are a leading cause of infant mortality in 
the US1. While newborn screening (NBS) has dramatically reduced infant morbidity and 
mortality for some genetic disorders, these improvements have not had a significant 
impact in Neonatal Intensive Care Units (NICU) where 10-25% of all NICU admissions 
are the result of a genetic disorder. Infants with a genetic disorder remain in the hospital 
approximately 40% longer than those without genetic disorders. Due to the non-specific 
presentation of many of these genetic disorders, many infants do not receive a definitive 
diagnosis in a timely fashion, if at all.  
 
Undiagnosed genetic disorders contribute significantly to infant mortality and morbidity. 
Each year, an estimated 14.4% of neonates are admitted to the NICU for treatment of 
an acute condition, with average medical costs of $76,164 per patient. Overall mortality 
rates in the NICU vary from 0.8% to 6.2%, increasing to as high as 57% when there are 
significant delays in appropriate diagnosis3-5. Genetic abnormalities (e.g. structural and 
metabolic disorders) are a leading cause of death in these infants with over 8,500 
known genetic disorders as major contributors5-15. While we do not yet know the genetic 
basis of many disorders in neonates, it has been estimated that more than 1,900 genes 
appear to be relevant in the neonatal period with symptom onset generally occurring 
prior to 2 years of age. Until the recent development of rWGS, timely molecular 
diagnosis of many suspected genetic disorders did not occur. In fact, preliminary studies 
demonstrate that neonates with a genetic disorder risk going undetected or being 
misdiagnosed, with 45% of diagnoses made via rWGS not included in the clinician's 
initial differential diagnosis16. The result can be a 5-year gap between disorder onset 
and diagnosis due to: 1) profound clinical and genetic heterogeneity; 2) limited standard 
genetic testing currently available (e.g. sequential and single gene sequencing); 3) 
prolonged length of time required to obtain the results of standard genetic tests; and 4) 
deferral of testing to post discharge since most payers do not reimburse hospitals for 
genetic testing while a neonate remains hospitalized17-22.  
 
When performed early in the clinical course, rWGS triples the diagnostic rate compared 
to SOC23, 24. This provides preliminary evidence that rWGS or NewbornDx could be 
more cost effective and clinically useful than current standard of care (SOC)25. 
Furthermore, data from the NSIGHT study (NCT02225522) indicates a diagnosis is 
made only 15% of the time in the neonatal period using SOC testing compared to 44% 
for rWGS70. The total cost for clinical rWGS trio is ~ $19,500,25,26 suggesting that 
NewbornDx panel followed by a reflex to rWGS (if necessary) may be a more optimal 
use of resources25,28-31. Here we propose to examine the diagnostic yield of NewbornDx 
and rWGS, assess the clinical utility of genomic testing, and evaluate each technique’s 
relative cost using data collected from the trial along with simulation modeling. The NIH, 
the National Academy of Medicine (NAM), and several European agencies have 
consistently identified scientific and operational barriers to implementation of genomic 
medicine. Specifically, the “burden to clinicians in obtaining, interpreting, and managing 

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results”, including “integrating effective clinical decision support tools into the EMR” are 
some of the biggest challenges of scaling genomic medicine32-35.  
NewbornDx offers many of the benefits of rWGS by: 1) detecting some non-exonic 
regions, sequence variants, chromosomal level abnormalities and high homology 
regions; 2) covering multiple phenotypes; 3) improving interpretability; 4) lowering costs; 
5) obtaining relatively rapid results and 6) expanding utilization worldwide (potentially 
conducting the panel on every neonate as the next generation of NBS). NewbornDx and 
rWGS also offer future opportunities to incorporate other features such as the ability to 
detect variants that alter protein abundance, initiation codons, stop codons and triplet 
expansion repeats. These future enhancements will assist neonates who currently 
cannot be confirmed as positive when there is a single heterozygous pathogenic variant 
in an autosomal recessive disorder and a second variant cannot be detected. However, 
unlike   rWGS, NewbornDx will not include newly discovered disorder genes (although 
they can be added in the future). rWGS is more comprehensive and covers larger areas 
of the genome allowing examination of intronic regulatory variants compared to 
NewbornDx. It is therefore very timely to understand the diagnostic yield of rWGS and 
NewbornDx, the clinical utility of rapid genomic testing, and the cost-effectiveness of the 
approaches compared to each other and the current standard of care. 
Since disorder progression can be extremely rapid in neonates, a molecular diagnosis 
must be made as soon as possible in order to impact outcome19,20,24,26,33,34. Stark, et.al 
performed a study supporting the utilization of molecular diagnostic platforms to improve 
outcomes, reporting a 58% diagnostic rate in neonates who underwent WES compared 
to a 17% diagnostic rate in neonates who underwent standard genetic investigations23. 
Despite these high-yield diagnostic rates, the average time-to-molecular diagnosis using 
conventional WGS and WES is prolonged (>3-4 weeks), limiting their clinical utility. In 
particular, neonates receiving an early molecular diagnosis (37%) did better than those 
diagnosed later (20%). Initiation of palliative care and avoidance of major morbidity 
were higher among those with an early diagnosis (17%) compared to those with a later 
diagnosis (9%). Overall, these studies suggest that outcomes can be significantly 
improved with earlier diagnosis (similar to NBS). NBS identifies 12,500 affected babies 
per year at <10 days of life or about 0.3%35. Although rapid diagnosis through NBS is 
known to save thousands of lives per year, it represents just a small fraction of the 
genetic disorder burden in neonates and is limited to detection of protein levels using 
Tandem Mass Spectroscopy. In addition, these protein levels are often adversely 
affected by the treatments delivered in the NICU such as total parenteral nutrition. By 
enhancing diagnostic yield, cost-effectiveness, and time to diagnosis, NewbornDx or 
rWGS could facilitate more rapid initiation of appropriate therapeutic interventions or 
redirection of the goal of care from cure to comfort and/or the withdrawing of life-
sustaining treatment5,24,36-41. The goal of a precision medicine approach in the NICU is 
to apply rapid molecular diagnostics in order to supplement empiric, phenotype-driven 
management with genotype differentiated treatments. 
 
Innovation 
 
The NewbornDx panel covers 98.4% of the genes in which sequence variants 
may cause known neonatal onset phenotypes.  

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 
The NewbornDx panel, like rWGS, offers opportunities for future improvements 
through the addition of newly discovered genes as well as the development of 
features that allow genome enrichment on the protein level that are 
complementary to NBS. 
 
Simple clinical reporting formats have been developed where results can be 
delivered electronically to the point of care through the EMR and easily 
referenced for future use. 
 
Our approach will establish interoperability and scalability across all healthcare 
institutions even where genetics expertise may be limited. This critical step 
towards implementation is vital since the vast majority of neonates are admitted 
to NICUs where there may be limited access to experts in Medical Genetics. 
 
The incorporation of NewbornDx or rWGS into neonatal care does not have to 
impact the infant’s future with information regarding possible later adult onset 
diagnoses. 
 
This prospective, multicenter trial will provide the first prospective comparison of 
NewbornDx and rWGS in NICUs with long-term follow-up to elucidate risks vs. 
rewards, costs vs. benefits, and impact on morbidity and mortality. 
 
Neonates at the six CTSI sites are ethnically and racially diverse, which will 
address ongoing criticism of existing rWGS trials that lack sufficient ethnic/racial 
diversity to insure generalizability. 
 
2.2 BACKGROUND  
 
In-silico comparison of 
diagnostics rates demonstrate 
that a rapid neonatal 
NewbornDx (aka TNGS) panel 
may detect up to 90% of the 
same disorders as WGS at 
substantially lower cost (Figure 
1).  
 
We performed a retrospective in 
silico comparison of the 1,722 
gene NewbornDx panel to 
already published rWGS and 
rWES clinical data from three 
studies representing 67 patients 
and 75 variants19,20,33. 
Compared to WES/WGS, the 
NewbornDx panel would have detected 85-94% of the same variants. A rapid 
NewbornDx panel using DBS has already been clinically validated under an SBIR 
Phase I grant through NICHD42. The panel is available as a laboratory developed test 
and is composed of 1,722 genes representing disorders that affect all systems (Figure 
2), including all coding regions and clinically relevant non-coding regions.  

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Figure 2. NewbornDx panel representing all 16 systems 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The phenotypes for most of these disorders typically present before 2 years of age. 
Specifically, of the 2,300 genes with neonatal onset phenotypes in OMIM, 1,750 are the 
result of sequence variants (76%) and 150 are the result of copy number variants 
(CNVs) (6%). There are an additional 150 tumor cell line genes (7%) and 250 genes 
(11%) with benign phenotypes. Eliminating the latter two groups leaves 1,900 genes 
with neonatal onset phenotypes, from which we have curated the final list of 1,722 
genes while eliminating portions of the genome that are challenging to sequence on any 
platform. The result is a comprehensive NewbornDx panel that represents 98.4% of the 
known genes in which sequence variants result in disorders presenting in the neonatal 
period (Figure 3).  
Audiological 
Hearing loss (syndromic): 127 genes 
Structural abnormalities: 24 genes 
 
Cancer Predisposition 
Neuroendocrine/CNS: 22 genes 
Hematologic: 50 genes 
 
Metabolic 
Mitochondrial: 132 genes 
Carbohydrate metabolism disorder: 23 genes 
Lysosomal storage disease: 38 genes 
 
Neurological 
Brain malformations: 196 genes 
Seizures: 304 genes 
Microcephaly: 266 genes 
 
Hematologic 
Anemia: 82 genes 
Bleeding disorder: 29 genes 
Platelet disorder: 35 genes 
 
Gastrointestinal 
Large and small intestine: 10 genes 
Liver disease: 42 genes 
Failure to thrive: 18 genes 
 
Skeletal 
Short stature: 84 genes 
Contractures: 31 genes 
Bone fragility: 29 genes 
 
Respiratory 
Respiratory distress: 39 genes 
Abnormal breathing: 12 genes 
Muscle 
Muscle atrophy/dystrophy: 44 genes 
Hypotonia: 81 genes 
 
Skin 
Abnormal pigmentation: 25 genes 
Abnormal hair and hair growth: 18 genes 
Blistering: 18 genes 
 
Endocrine 
Adrenal dysfunction: 15 genes 
Hypo-/hyper-glycemia: 44 genes 
Hyper-/hypo-parathyroidism: 32 genes 
 
Immune 
Recurrent fever/recurrent infections: 18 genes 
Immunodeficiency: 56 genes 
 
Cardiac 
Cardiomyopathy: 106 genes 
Structural defects: 108 genes 
Conduction defects: 107 genes 
 
Genitourinary 
Renal disease: 115 genes 
Genital abnormalities: 37 genes 
 
Ophthalmologic 
Abnormal eye movements: 46 genes 
Anterior chamber dysgenesis: 27 genes 
Optic nerve abnormalities: 42 genes 
 

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The ability of NewbornDx to detect CNVs increases this number to 98.5% of total genes 
known to cause neonatal disorders. The panel shows analytic sensitivity of 98% for 
single nucleotide variants with an average depth of coverage of 139x (>97% of bases 
are covered at 20x). Extraction of DNA from DBS has also been validated and can be 
performed using an existing sample collection kit. 
 
In preliminary studies, NewbornDx compared favorably to rWGS, and SOC. We 
performed NewbornDx on 10 trios from Pittsburgh's Children’s Hospital (our collaborator 
Dr. Vockley) and 6 patients from Rady Children’s Hospital (Dr. Kingsmore). Neonates 
were chosen based on the clinical suspicion of an underlying genetic condition with 
symptom onset early in life. Results were returned within an average of 13 days. We 
found 7 patients with causative variants for a diagnostic yield of 47%, in line with that 
observed in previous publications using WES and WGS. One of the diagnoses was a 
condition not included in the pre-test differential diagnosis, consistent with the findings 
in Petrikin et al.16. Results from Dr. Kingsmore’s study (NCT02225522) suggest 
NewbornDx may have detected 100% of diagnostic findings generated using WGS33. 
 
There is a strong need for prospective studies examining the clinical utility of 
NewbornDx   and rWGS in neonates. While studies have compared WES/WGS in the 
NICU, they lacked generalizability to a larger population since: 1) they were 
underpowered or were not prospective, 2) patients were selected based on a positive 
family history, or 3) the variants were detected in previously characterized genes and 
therefore potentially detectable on the NewbornDx panel, 4) they did not utilize recently 
Figure 3: Contribution of variant types to conditions with neonatal-onset 
presentations.  

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developed rapid methods for rWES or rWGS24,25,27,33,43. As such, no prospective 
assessment of the clinical utility, cost-effectiveness or outcomes associated with a 
NewbornDx-based diagnosis of genetic disorders has been conducted. Such evidence 
is critical to justify the use of the methodology given the cost limitations imposed for 
hospitalized neonates (inpatient genetic testing is expensive, often bundled, and usually 
not reimbursed independently). In particular, no study has examined the temporal 
dynamics of genetic disorder progression in the most vulnerable neonates and defined 
specific metrics to evaluate how NewbornDx and/or rWGS will best guide treatment and 
reduce morbidity and mortality. Finally, there are limited studies defining best practices 
related to NewbornDx or rWGS with regard to communicating information to the medical 
team and family. This includes: 1) providing timely results with appropriate genetic 
counseling, 2) reviewing information about available clinical trials and/or approved drugs 
to treat the condition, or 3) offering redirection of care from cure to comfort and/or 
withdrawing of life-sustaining treatment. We propose a prospective study of the 
effectiveness of NewbornDx and rWGS in infants admitted to the hospital with a 
suspected genetic disorder. This transformative study represents one of the first times 
key stakeholders with expertise in neonatal clinical trials, medical genetics, and 
molecular diagnostics are participating in a prospective study of this size and scope. 
 
2.3 RISK/BENEFIT ASSESSMENT   
 
2.3.1 KNOWN POTENTIAL RISKS  
 
Blood Sampling: Blood samples should pose minimal health risks to participants. Every 
attempt will be made to obtain blood from the enrolled infant at the time that routine 
samples are collected for clinical indications. For the infant, a minimum of two and a 
maximum of five dried blood spots are needed for NewbornDx, a total blood volume of 
1mL for rWGS. This one-time blood sample from the infant should not cause undue 
trauma or excessive blood loss. If the clinical team does not feel it is safe to obtain the 
sample from the infant, then we will obtain the blood just prior to a packed red blood cell 
transfusion. The 3ml of blood from each biological parent is considered minimal for an 
adult. Risks associated with blood draws are usually minor and include pain, anxiety, 
bleeding, bruising, fainting, and rarely infection at the site. All samples will be collected 
by qualified staff following hospital guidelines and standard precautions.   
 
Loss of confidentiality: Since we are storing identifying information about the infant and 
parents, there is a small risk that this information may be compromised. All study related 
material will be stored securely. Each enrolled infant will be assigned a unique study 
identification number which does not include any identifiers. Infant information entered 
into the password-protected REDCap System and documents used for remote 
monitoring will be identified by the study ID number. Each site will keep the link between 
the study ID number and identifiers for all subjects enrolled at their site in a secure 
password-protected spreadsheet. Blood samples, medical information provided on the 
laboratory requisition form, sequencing data, and results from the laboratory will be 
identifiable by name and date of birth. This is to ensure that: 1) the correct infant can be 
identified immediately 24/7 in the case of a time sensitive result that could influence 

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survival and outcome; and 2) to eliminate any errors with matching of study ID to infant 
at the enrolling site. The ICF clearly indicates that the labeling of specimens, medical 
information, and reports to/from the laboratories will contain identifying information. All 
genomic sequencing data will be electronically stored at the laboratory that conducted 
the test in password-protected databases accessible only to research personnel. In 
addition, this study is automatically issued a COC. PHI related to a genetic diagnosis 
may need to be shared with government agencies or other organizations involved in the 
research. WGS may uniquely identify any person. Since a COC may not apply in all 
circumstances and WGS can identify any person, it is essential that this be included in 
the ICF. 
 
Secondary findings for the infant and biological parent(s): NewbornDx and rWGS may 
detect secondary findings. These include diagnoses for the infant or a biological parent 
not related to the infant’s symptoms, misattributed parentage, and suspected incest. 
Parents/guardians of infants will be counseled prior to enrollment about the information 
available from each platform.    
 
The infant’s parent/guardian will choose whether or not to receive secondary diagnoses 
for the infant. This will be by an opt-in or opt-out (express choice of one or the other) 
included in the ICF. A secondary diagnosis for the infant will only be reported if both 
criteria 1 and 2 are met and at least one of 3a or 3b:  
1. The parent/guardian opted to receive secondary findings for the infant, AND 
2. The disorder onset may occur in childhood, AND 
3. At least one of the following:  
a) The disorder is medically actionable  AND/OR 
b) The disorder involves a gene on the “American College of Medical Genetics 
and Genomics (ACMG) list” of genes recommended for reporting of 
secondary findings at the time of testing46  
 
It is possible a secondary diagnosis will eventually become a primary diagnosis (in the 
case where the infant has not yet grown into the phenotype, an atypical presentation, or 
the field advances) and the parent/guardian will not have been given the relevant 
information because the parent/guardian elected not to receive secondary findings for 
the infant. Information about conditions with an onset occurring only in adulthood will not 
be given for the infant.  
 
If the parent/guardian opted-in for infant secondary findings, a biological parent will have 
the choice to opt-in on the ICF to receive secondary findings. A secondary diagnosis for 
a biological parent will only be reported if the biological parent opted in to receive 
secondary findings and the disorder involves a gene on the ACMG list recommended to 
report as secondary findings at the time of testing. There are some circumstances in 
which a biological parent of an infant could learn about a current condition or an 
increased risk for an adult-onset disorder even if they do not opt-in to receive secondary 
findings by the inheritance and penetrance pattern of the infant’s disorder.  
 

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Misattributed parentage (mistaken paternity or an egg donor) will not be reported by the 
laboratory or the enrolling site. The ICF indicates that misattributed parentage could be 
inferred from the results. For example, the infant is found to have an autosomal 
recessive condition and one parent is found not to carry this condition and de novo 
mutations are known to be rare. Additionally, the ICF states that there are privacy risks 
if the parent/guardian and/or biological parent receives the raw sequencing data and 
gives it to another person for analysis. Privacy risks which include misattributed 
parentage will be explained if a parent/guardian requests to receive raw sequencing 
data. 
 
This testing could detect incest, defined as “a sexual relationship between people too 
closely related to marry each other (such as a person having a sexual relationship with 
a parent, grandparent, brother, or sister).” If incest is suspected, this information will be 
reported to the site. The site will notify child protective services. Risks of discovering 
and reporting incest may include possible criminal charges depending upon state law. 
Risks to the infant include behavioral problems and removal from the home. The 
capability of the sequencing tests to detect incest, the legal requirement to report it to 
child protective services and associated risks is in the informed consent form.  
 
The ICF states that secondary findings in the infant or biological parents is not the 
primary purpose of this research study and just because a secondary finding isn’t 
reported doesn’t mean any disease-causing genetic changes don’t exist. 
 
Misinterpretation of results: We expect to miss a small number of structural variants 
(SVs) using short read rWGS (NewbornDx does not detect structural variants) that are 
causal. We will monitor the percentage of identified SVs and be careful not to label 
cases with a negative result as not genetic in origin. Experienced personnel will perform 
the genomic testing according to well-established protocols. Although we may not find a 
result, this does not mean the infant will not continue to have symptoms or even have a 
genetic disorder. Similarly, the infant’s health may not improve during this study; it may 
get worse or stay the same. All infants will continue to receive routine care as directed 
by the clinical team, including routine NBS and other genetic testing. 
 
Risk of discrimination associated with genetic testing: Federal and State laws generally 
make it illegal for health insurance companies, group health plans, and most employers 
to discriminate against a person based on their genetic information. The Genetic 
Information Non-discrimination Act (GINA) indicates that genetic information cannot be 
used by certain health insurance companies when making decisions about eligibility for 
health insurance or how much insurance costs. These insurance companies can still 
use any genetic information to help them decide whether or not they will pay health 
insurance claims. This law also indicates that employers with fifteen or more employees 
may not use genetic information when making decisions to hire, promote, or fire an 
individual or when setting the terms of employment. Protections by this law may not 
apply to certain military, veterans and federal employees. These laws do not protect 
against genetic discrimination by companies that sell life, disability, or long-term care 

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insurance. Information regarding the protections and limitations of Genetic Information 
Nondiscrimination Act (GINA) will be included in the ICF.  
 
Emotional distress: Genetic testing may cause anxiety or emotional distress due to 
feelings of responsibility for passing genetic variants to children. If parents/guardians 
and/or biological parents opt-in to receive secondary findings for their infant and/or 
themselves, this information may improve their overall health, but could provoke anxiety 
by knowing the information. Genetic counselors will be available to all 
parent/guardian(s) and/or biological parents at all times while enrolled in the study to 
answer questions or addressing concerns related to testing or results. 
 
2.3.2 KNOWN POTENTIAL BENEFITS  
This study may or may not benefit the infant. An infant may receive a diagnosis or 
additional information regarding his or her medical condition. The main benefits to the 
infant include a faster time to diagnosis, a more accurate diagnosis, and initiation of 
appropriate interventions faster than possible with standard diagnostic testing. These 
benefits have the potential to significantly reduce morbidity, mortality, other diagnostic 
testing, overall hospital costs, and time to initiate appropriate treatment and time to 
discharge. There will be no direct benefit to participation for unaffected family members 
except where a diagnosis provides information regarding reproductive decisions and 
recurrence risks. The targeted nature of the NewbornDx testing enables reporting of 
results without providing secondary findings that might burden families.  
The NewbornDx panel and rWGS may each have specific benefits including: 
 NewbornDx only detects variants in genes known to be actionable in the 
neonatal period.   
 NewbornDx avoids the financial burden of rWGS.  
 NewbornDx decreases the ethical considerations of rWGS related to secondary 
findings or findings related to parents; however, these findings can be associated 
with improved health for patients and families.  
 Rapid return of results (either by rWGS and/or NewbornDx) 
 rWGS provides coverage of CNVs and other genetic disorders 
Importance of the knowledge to be gained:  
rWGS is slowly becoming integrated into neonatal care in an attempt to broaden 
diagnostic coverage while reducing time to diagnosis. However, each platform is 
expensive, may identify disorders with limited therapeutic options, and uncover adult 
onset disorders. NewbornDx adopts the philosophy of NBS by providing assessment of 
1,722 genes matched to phenotypes presenting in the neonatal period, including those 
where a timelier intervention can improve outcome. Our preliminary data indicate that 
NewbornDx may be superior to traditional diagnostic testing and genome scale 
sequencing by transforming empiric, phenotype-driven management into genotype-
informed treatment plans that improve outcome without the ethical, financial and 
technical burdens associated with WGS. If NewbornDx has a comparable diagnostic 
yield as rWGS, with favorable cost effectiveness compared with rWGS and standard of 
care, it could easily be adopted worldwide. This study has the potential to advance the 

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expanding field of molecular diagnosis in high-risk neonates and even enhance 
traditional NBS. 
2.3.3 ASSESSMENT OF POTENTIAL RISKS 
 
 Drawing of blood can be associated with pain, bruising, anemia, and possible 
infection. 
 A genetic diagnosis could be missed, with clinicians thinking it is unlikely to be 
present since the genetic testing is negative. 
 If parents/guardians and/or biological parents opt-in to receive secondary findings for 
their child and/or themselves, this information may improve their overall health, but 
also may provoke anxiety in learning about the information. 
 If parents/guardians and/or biological parents opt-out of receiving secondary 
findings, they will not receive information that could potentially impact their health or 
their child’s health. 
 Despite the presence of a COC, PHI related to a genetic diagnosis may need to be 
revealed to government agencies or other organizations involved in the research. 
 For a critically ill infant, a preliminary diagnosis may be disclosed to the clinical team 
without time for confirmatory testing. Potential therapy may be initiated that may not 
be necessary. 
 If a diagnosis is made, the information may not be provided to the clinical team in 
sufficient time to improve outcome. 
 It is not clear how reporting of variants of unknown significance will impact care and 
outcome. 

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3 
OBJECTIVES AND ENDPOINTS 
Primary Objectives 
Endpoints 
Endpoint 
Justification  
To estimate the 
diagnostic yield of 
NewbornDx and 
rWGS in identifying 
genetic disorders of 
unknown etiology 
Primary  
A confirmed genetic disorder 
detected by NewbornDx  
A confirmed genetic disorder 
detected by rWGS 
Assess diagnostic 
capability of the 
platforms 
Secondary 
For diagnoses by rWGS, if 
trio testing was required to 
confirm a diagnosis 
Assess need for 
including 
biological parents 
To assess the clinical 
utility of genomic 
sequencing   
Primary  
Time from sample collection 
to positive test result  
Assess the 
timeliness of an 
accurate result  
Primary  
Clinical utility of genomic 
sequencing  
Assess impact on 
care 
Secondary  
If the infant does not receive 
a diagnosis, calculated at 
discharge and at 1year CGA 
Assess the 
clinical impact of 
platform(s) 
Secondary Objectives 
Endpoints 
Endpoint 
Justification  
To examine the 
economic impact and 
health outcomes of 
NewbornDx and 
rWGS in infants 
compared with SOC 
diagnostic testing, 
over a one-year and 
lifetime horizon 
Secondary  
One-year cost-effectiveness 
of NewbornDx and rWGS 
compared with SOC testing  
Assess economic 
value of 
alternative testing 
strategies 
Secondary  
Lifetime cost-effectiveness of 
NewbornDx and rWGS, 
compared with SOC testing 
Assess economic 
value of 
alternative testing 
strategies 
To develop and 
qualitatively assess 
the web-based clinical 
reference database of 
supporting information 
for medical providers 
and parents 
Secondary  
Satisfaction of the web-
based clinical reference 
database  
Assess ease of 
use and 
usefulness of 
information 
among medical 
providers   
Exploratory 
Objectives 
Endpoints 
Endpoint 
Justification  
To inform the 
NewbornDx panel 
with findings from 
rWGS that were not 
identified with 
NewbornDx 
Exploratory  
Conditions detected by 
rWGS that were not detected 
by NewbornDx 
Expand and 
improve the 
diagnostic 
accuracy of the 
NewbornDx panel   

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How endpoint(s) will be adjudicated  
1. 
Confirmed genetic disorders detected by NewbornDx or rWGS: the laboratories 
conducting testing could report descrepant results due to differences in 
confirmatory methodology, variant types or methods of interpretation. Methods 
used for confirmation testing at each laboratory will be documented. If results are 
discordant due to a variant classification, the laboratories will each provide the 
evidence supporting their result and variant classification. This summary will be 
given to the enrolling site PI to share with the clinical team; a subsequent phone 
call with the labs may be arranged upon request from the site. The clinical team 
who is caring for the infant is in the best position to determine if one result aligns 
best with the infant’s clinical presentation and their view will be captured in the 
study data. Additionally, a subset of the study geneticists on the steering 
committee who are independent of the labs will review the redacted lab reports 
and lab summaries and provide what they think is the most accurate result for an 
additional study endpoint. The labs are available for questions by the steering 
committee. The opinion of the steering committee members may be shared with 
the enrolling site upon the site’s request. Detailed procedures are outlined in the 
MOP. 
2. 
Clinical utility of genomic sequencing testing: it is possible that the clinician 
completing the clinical utility survey (refer to section 8.1) might indicate an 
opinion or action that differs from the medical record data abstraction reported by 
the study coordinator on the Clinical Actions after Genomic Sequencing form. In 
such a case, the research team at the site will review the relevant information 
with the clinical team to find out and document the reason for the discrepancy 
and if applicable, make any corrections (i.e. a survey completed for the wrong 
infant). 

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4 
STUDY DESIGN  
 
4.1 OVERALL DESIGN 
 
This multicenter, prospective, clinical trial will estimate the diagnostic yield of 
NewbornDx and rWGS testing and assess the clinical utility of genomic sequencing 
among high-risk infants with signs/symptoms consistent with a possible genetic 
disorder. The relative cost effectiveness of each testing method will be evaluated 
through simulation.   
There are three components to this study: 
1. Infants will undergo SOC diagnostic testing as well as NewbornDx and rWGS in 
parallel. Genetic sequences via NewbornDx and rWGS will be generated for the 
infant (proband) and when available, the biological parents, and analyzed 
together. NewbornDx and rWGS test results will be returned to the infant’s 
clinician, medical record and parent/guardian. Those infants analyzed as a duo 
or trio will have a retrospective analysis done of only the infant’s data to 
determine if the same result would have been obtained with and without parent 
samples. Clinical utility will be measured by clinician opinion and changes in the 
infant’s care as a result of the genetic sequencing test results. QoL and medical 
resource utilization will be collected by parent survey monthly, until the infant is 
one year CGA.  
2. A retrospective chart review of neonates with suspected genetic disorders 
completed at 3 sites, will be used to elucidate 1-year cost and health outcomes 
that would have been incurred in the absence of available NewbornDx and rWGS 
testing. The retrospective review will include 300 neonates born 12-18 months 
prior to the start of the study and performed under a separately submitted 
amendment to the central IRB. The cost and health outcome data will be used in 
an economic evaluation comparing NewbornDx, rWGS, and SOC over 1-year 
and a child’s lifetime time trajectory. 
3. A user-friendly clinical reference database will be developed to provide medical 
providers and families with information about genetic disorders, treatments, 
clinical trials and advocacy groups. A qualitative assessment of the clinical 
reference database will be conducted through surveys of clinicians involved in 
the infants’ care. Once the database is completed and prior to implementation of 
the clinical reference database into this study, an amendment will be submitted to 
the IRB along with the survey.  
 
Hypothesis #1: NewbornDx will detect at least 60% of the diagnoses identified by rWGS 
 
Hypothesis #2: NewbornDx will be substantially less costly than rWGS or SOC testing 
over a one year and lifetime horizon  
 
Hypothesis #3: Medical providers will be satisfied with the web-based clinical reference 
database of supporting information. 
 
Interim analyses are not planned.  

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4.2 SCIENTIFIC RATIONALE FOR STUDY DESIGN 
 
There is a strong need for prospective, comparative studies comparing the applicability 
of NewbornDx and rWGS in the neonatal population. While studies have been done 
comparing WES/WGS in the NICU, they lacked generalizability to a larger population 
since: 1) they were underpowered or were not prospective, 2) patients were selected 
based on a positive family history, or 3) the variants were detected in previously 
characterized genes and therefore potentially detectable on the NewbornDx panel. As 
such, no prospective assessment of the clinical utility, cost- effectiveness, or outcomes 
associated with a NewbornDx-based diagnosis of genetic diseases has been 
conducted. The proposed study design will maintain equipoise with direct comparative 
analysis between two comprehensive genetic sequencing approaches for use in infants 
in an attempt to determine their cost-effectiveness and diagnostic yield for use in this 
vulnerable population. 
 
4.3 END OF STUDY DEFINITION 
 
An infant is considered to have completed the study if he or she has undergone 
NewbornDx and rWGS and completed the last interview at 1 year CGA or died prior to1 
year CGA.  
 
 

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5 
STUDY POPULATION 
 
5.1 INCLUSION CRITERIA 
 
In order to be eligible to participate in this study, an infant must meet all of the following 
criteria: 
1. Documented informed consent from the parent/guardian  
2. Signs/symptoms consistent with a possible genetic disorder including, but not 
limited to dysmorphic features, congenital anomalies, hypotonia, seizures, 
clotting, poor oral feeding skills 
3. Admitted to a hospital (NICU, PICU, CVICU, inpatient floor) participating in this 
study at the time of enrollment (enrollment is not limited to the initial 
hospitalization after birth, it may occur during a readmission)   
4. Less than one year CGA  
 
There is not a requirement for clinical genetic testing prior to enrollment. However, 
enrollment in this study is not intended to replace targeted available genetic testing for 
specific suspected disorders that match the phenotype of the infant and make rWGS 
testing unnecessary. Examples include trisomies or a high likelihood of DiGeorge 
syndrome. In these cases, targeted genetic testing for a suspected disorder should be 
done prior to enrollment (e.g. DiGeorge syndrome and FISH for 22q11.2 deletion). 
Clinical genetic testing does not need to be completed prior to enrollment if there is 
more than one diagnosis on the differential diagnosis list. Other than these few 
situations, our goal should be to understand the role of utilizing rWGS and NewbornDx 
as a first line choice of testing. 
 
5.2 EXCLUSION CRITERIA 
 
An individual who meets any of the following criteria will be excluded from participation 
in this study: 
1. A known genetic diagnosis (e.g. prenatal testing, biochemical testing and/or 
enzyme analysis)  
2. Major congenital anomaly (renal, cardiac, hepatic, neurologic, or pulmonary 
malformations) associated with a chromosomal anomaly detected on prenatal 
testing (e.g. ultrasound, genetic testing) 
3. Presence of documented congenital infection (e.g. congenital cytomegalovirus) 
4. Infants considered non-viable due to prematurity (< 23 0/7 weeks GA) 
5. Infants who are not expected to receive medical care in the US healthcare 
system from time of discharge home until 1 year CGA  
 
Lack of participation by one or both biological parents in the trio analysis does not 
preclude the infant from participating in this study. This situation could occur if a 
biological parent does not provide consent for his/her own blood sample to be used for 
a trio analysis, if a blood sample cannot be obtained, or in the case of an egg or sperm 
donor. 

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5.3 SCREEN FAILURES 
 
Screen failures are defined as infants whose parent(s) consent to participation in the 
clinical trial but who do not have NewbornDx or rWGS performed. This could occur if a 
clinical diagnosis was determined after consent was obtained but prior to the start of 
any study procedures (section 7 addresses the procedure for subject withdrawal if a 
clinical diagnosis is determined after study procedures commence but before the 
completion of genomic sequencing). A minimal set of screen failure information will be 
collected including demography, screen failure details and eligibility criteria.  
 
5.4 STRATEGIES FOR RECRUITMENT AND RETENTION 
 
The anticipated number of infants to be screened is approximately 1200 in order for 400 
infants to complete the study. Six US sites will recruit for this study. The 
parent/guardian(s) of all eligible infants will be approached. Enrollment is expected to 
include about 50% of each sex and proportions of each race that are represented in the 
general population in the multiple sites where the studies will be performed. The entire 
ICF will be translated into Spanish and short forms in other non-English languages will 
be utilized. 
 
This study involves monthly follow-up from discharge home until 1 year CGA. In order to 
reduce the risk of lost to follow-up, study staff will have the parent/guardian(s) fill out the 
Follow-up Contact Information form at the time of transfer or discharge home. This form 
will document several methods of contact for the parent/guardian(s) and a family 
member or friend who would know how to reach the parent/guardian(s) if their contact 
information changes or they are unreachable. Then, the study coordinators will contact 
the family monthly until 1 year CGA to complete the parent surveys/interviews. 
 
 

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6 
STUDY INTERVENTION 
 
6.1 STUDY INTERVENTION(S) ADMINISTRATION 
 
6.1.1 STUDY INTERVENTION DESCRIPTION 
 
NewbornDx and rWGS are CLIA/CAP certified and will be completed for all infants. The 
Athena (Quest) and RCIGM laboratories will be blinded to each other’s result until all 
final results are returned.  
 
Phenotype Extraction and Provision of Clinical Information to the Laboratories 
1. A sub-group of clinicians from each site will manually review the medical record, 
extract relevant clinical information and determine the Human Phenotype 
Ontology (HPO) terms (refer to MOP). The sub-group of clinicians will consist of 
a minimum of 3 clinicians with at least one a geneticist or genetic counselor.   
2. A small portion of the medical records (genetics consult note, other sub-specialty 
note (neurology, hematology, etc.) and NICU notes will be selected. 
3. The HPO terms and medical records will be provided by the sites to the 
laboratories via upload to the RCIGM ordering portal. Athena will receive this 
data to its systems through a secure FTP site or encrypted email. Athena will not 
have access to the rWGS results through this mechanism. 
4. Once HPO terms are submitted by the site, the laboratories may either add 
additional terms based on the last consult/progress notes submitted by the site or 
contact the site research team for clarification of symptoms. If this happens by 
either Athena (Quest) or RCIGM, the additional or revised HPO terms needs to 
be redirected to the site for upload to the portal. This ensures any new 
information is shared with both laboratories.   
 
Athena Diagnostics (Quest) 
NewbornDx Methods and Workflow  
Rapid NewbornDx will be performed at Athena (Quest) Diagnostics to CLIA/CAP 
requirements. DBS will be collected on filter paper and sequenced. Custom 
oligonucleotide probe libraries (Agilent SureSelectQXT) will be utilized to capture 
genomic DNA regions and targeted libraries created as described earlier42. The content 
of the assay (regions of interest) will remain constant for participants in the study. 
Sequencing of the targeted libraries will be performed on a NextSeq 500 (Illumina, San 
Diego, CA). Sequencing reads will be aligned to the reference genome (hg19/GRCH 
build 37) using Burrows-Wheeler Aligner for short alignments (BWA-MEM) followed by a 
Genome Analysis Toolkit v3.6 (GATK) haplotype variant caller running on the Edico 
Dragen™ Platform, and annotated with Enterprise genome interpretation software 
(Fabric Genomics, Oakland, CA). All subject FASTQ, Variant Call File (VCF) and other 
data files will be securely stored within the Athena (Quest) Diagnostics Isilon storage.  
 
 

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Athena (Quest) Genomic Data Analysis 
Data analysis will be completed by a custom bioinformatics pipeline utilizing Burrows-
Wheeler Aligner & Genome Analysis Toolkit v3.6. All reported DNA variants will be 
reviewed by at least one trained genomic variant scientist, one genomic science 
specialist, and one board-certified laboratory director prior to the final report being 
issued. 
 
Athena (Quest) Variant Pathogenicity Determination   
Variant pathogenicity is assessed following a standardized evaluation protocol71 
consistent with the ACMG standards and guidelines for the interpretation of sequence 
variants49. Data is gathered from multiple sources, including but not limited to, internal 
datasets, published literature, public databases, and in-silico prediction tools. Evidence 
is reviewed and assessed by a genomic variant scientist using a standard operation 
procedure. The proposed pathogenicity classifications derived from the data are 
reviewed and approved by a board-certified laboratory director.   
 
Athena (Quest) Variant Causality Determination  
Variant causality will be determined by the infant's clinical presentation, family history, 
and any other laboratory results provided. Relevant HPO terms (see phenotype 
extraction above) will be used with Phenomizer or Phenolyzer to create a differential 
diagnosis based on the neonate’s clinical presentation. Phevor (Fabric Genomics) will 
be used to prioritize DNA variants based on the genes’ associated phenotypes 
compared to the HPO terms provided by the site. Fabric Genomics will also be used to 
filter variants based on variables such as inheritance pattern, frequency of the DNA 
variant in the general population, variant type (missense, nonsense, etc.), and presence 
in a variant database (i.e. ClinVar or HGMD). Variant filtering will be based on a 
standardized set of criteria used by genomic variant scientists and board-certified 
laboratory directors. Sample reports will be drafted and reviewed by a team composed 
of genomic variant scientists, genetic counselors, and a board-certified director prior to 
being released to the client. 
 
RCIGM-CGC  
rWGS Methods and Workflow  
rWGS will be performed at the CLIA-certified, CAP accredited Rady Children’s Institute 
for Genomic Medicine – Clinical Genome Center (RCIGM-CGC). Sequencing is 
performed in accordance with the RCIGM- CGC standard operating procedures and 
validations. Briefly, genomic DNA is extracted from whole blood, PCR-free DNA libraries 
are made, and quality checks performed for library size and concentration. DNA 
libraries are then loaded on a NovaSeq 6000, and 2 x 100 nt sequencing to an average 
genome wide coverage of 40x. Short Tandem Repeat (STR) genotypes will be used to 
assure sample identity is maintained throughout the laboratory process. For each infant 
or biological parent sample, STR genotypes are generated by a commercial PCR 
amplification kit and are compared with those called by WGS process. Bcl files are 
automatically aligned to hg19/GRCH build 37 and nucleotide variants and small 
insertions and deletions are called with DRAGEN (Edico Genome) in ~1 hour. Files are 
uploaded to DNAnexus, where structural variants (SVs) are called using the union of 

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two algorithms (CNVnator and Manta). Redundant SVs are removed from the superset 
of SV calls. SVs will be annotated to determine affected genes and regulatory regions 
and compared to public and internal databases to determine MAF and filter common, or 
false positive SVs. A combined VCF (SNVs/indels and SVs) are uploaded to a rapid 
instance of Opal (Fabric Genomics), allowing annotation and display within 1 hour.  
 
rWGS Trio Testing 
When samples for the infant and parent(s) are received at the same time, RCIGM will 
generate infant and parent sequences simultaneously and the data from the infant and 
parent(s) will be analyzed together as a duo/trio. When infant samples are sent without 
the parent samples, RCIGM will sequence the infant sample and analyze the infant’s 
data. If a diagnosis is not determined or a VUS is identified after analyzing the infant 
alone, the parents’ samples, if subsequently become available, will be sequenced and 
data from the duo or trio analyzed together. The infant’s report of results will indicate 
whether or not trio testing was done. For all positive diagnoses made when only 
sequencing and analyzing the infant, the lab will subsequently run testing on the parent 
samples (if available) to determine inheritance (for example, targeted Sanger 
sequencing). 
 
RCIGM-CGC Genomic Data Analysis 
Data analysis will follow the standards set by the clinical diagnostic laboratory and in 
accordance with recommendations from the American College of Medical Genetics and 
Genomics (ACMG/AMP). A minimum of two expert data analysts will be responsible for 
analyzing the DNA variants following genomic sequencing. Analysts will be rotated 
among qualified staff, including but not limited to laboratory directors, genetic 
counseling staff, and trained medical and research staff. One of the analysts will then 
compile a report to be reviewed and reported by an ABMGG board-certified laboratory 
director. 
 
RCIGM-CGC Variant Pathogenicity Determination   
The laboratory directors as well as Medical Geneticists and Genetic Counselors at 
RCIGM and at each site will assess pathogenicity of variants according to ACMG/AMP 
recommendations for classifying sequence. New statistical approaches for variant 
classification are currently being actively developed by several groups and may be 
introduced upon verification of their utility. Knowledge of the pathogenicity of individual 
SNVs and SVs is rapidly evolving through community curation via HGMD, ClinVar and 
DECIPHER50 and through exhaustive mutant generation and functional testing for 
individual disorder genes. Variant classification will be performed using expert analysis 
of all data regarding each variant, including review of available literature and functional 
models.  
 
RCIGM-CGC Variant Causality Determination:  
Variant causality will be determined on a case by case basis depending on the infant's 
clinical presentation, family history, and any other laboratory results provided. Relevant 
HPO terms will be used with software tools, such as Phenomizer or Phenolyzer or 
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heterotaxy), to create a differential diagnosis list based on the neonate’s clinical 
presentation. Inputs for causality determination will be the annotated genomic variant 
file and the comprehensive differential diagnosis gene list. Alternatively, the Phevor tool 
in Opal52, 53 will be used to rank genes with relationship to the observed phenotype 
based on their known or suspected association(s). By allowing dynamic filtering of 
variants based on variables such as individual clinical features, disorders, genes, 
genotype, and inheritance pattern, Opal will assist in identification of a molecular 
differential diagnosis. Custom Opal settings can be saved, which allows configuration in 
a manner that can enable a provisional diagnosis to be determined rapidly. Opal also 
allows data mark-up, user tracking and data basing of variants and their classifications 
and export of fields in formats suitable for inclusion in diagnostic reports. In a typical 
interpretation session, variants will be filtered by allele frequency and presence in 
variant databases and prioritized based on phase, parent-of-origin, clinical features, and 
other characteristics unique to the infant or to the biological parents. All potential genetic 
inheritance patterns will be examined, including de novo, autosomal recessive, 
autosomal dominant, X-linked, mitochondrial, and, where possible, somatic variation 
(where trios may be needed). Where a single likely causative heterozygous variant for a 
recessive disorder is identified, the entire coding domain will be manually inspected 
using the Integrated Genome Viewer (IGV) for coverage and to screen for additional 
variants that may not have been called. Expert interpretation will be performed for all 
likely causative variants with regard to evidence for pathogenicity and causality. 
Likewise, the causality of structural variants in rWGS is assessed based on frequency in 
the RCIGM-CGC database and location with respect to the comprehensive differential 
diagnosis gene list54. This is an evolving area of interpretation. SV and nucleotide 
variant causality is examined together in order to identify compound heterozygosity in 
recessive disorders. 
 
Secondary/Incidental Findings for the Infant 
Secondary/incidental findings for an infant will not be actively sought. A dedicated 
analysis of the ACMG 59 secondary gene list will not be done. If a secondary/incidental 
finding is found during the infant analysis, the laboratory will only report it if both criteria 
1 and 2 are met and either 3a or 3b:  
1. The parent/guardian opted to receive secondary findings for the infant, AND 
2. The disorder onset may occur in childhood, AND 
3. At least one of the following:  
a) The disorder is medically actionable AND/OR 
b) The disorder involves a gene on the “American College of Medical Genetics 
and Genomics (ACMG) list” of genes recommended for reporting of 
secondary findings at the time of testing 
 
Secondary findings of conditions with an onset only in adulthood will not be returned for 
the infant. Only pathogenic or likely pathogenic variants are reported as a secondary/ 
incidental finding if they have the potential to impact the infant during childhood. 
 
The “ACMG list” refers to the current list of genes recommended to report as secondary 
findings. WGS covers all 59 genes currently recommended by the ACMG to return as 

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secondary findings, NewbornDx covers 30 of those genes. There are 52 genes on the 
ACMG list that may occur in childhood. In order to account for how the ACMG list might 
evolve during the study period, the ACMG list at the time testing is done will be used. 
Therefore, the list used for the first infant enrolled may be different than the list used for 
the last infant enrolled. If the ACMG list evolves during the study period, we will not 
retrospectively change what was initially reported; this aligns with current clinical 
practice.  
 
Primary and secondary findings for the infant, if reportable, will be included in one report 
from each laboratory.  
 
Secondary Findings for a Biological Parent 
A secondary finding for a biological parent may be identified through WGS when 
conducting the infant’s phenotypically driven analysis. Secondary findings for a parent 
will not be actively sought and a dedicated analysis of the ACMG 59 secondary gene list 
will not be done. There may be cases in which a biological parent elects to receive 
secondary findings and the parent’s sample does not undergo WGS. This could occur if 
the infant’s samples have been processed prior to the receipt of the parent samples. 
Secondary findings for a biological parent will not be reported by Athena Diagnostics.  
 
The laboratory will only report a secondary diagnosis for a biological parent if all of the 
following criteria are met: 
1. The biological parent opted in to receive secondary findings for him or herself. A 
parent may opt-in to receive secondary findings only if the parent/guardian opted-
in for infant secondary findings. 
2. The disorder is of a gene on the ACMG list of genes recommended to report as 
secondary findings at the time of testing.  
 
A separate report must be issued for any secondary findings for a biological parent.   
 
When Incidental/Secondary Findings Are Reported 
Secondary/Incidental finding found in Infant and 
Parent 
Did Infant 
Opt-In? 
Report for 
Infant? 
Did 
Parent 
Opt-in? 
Report for 
Parent? 
Included in ACMG59, adult onset 
Yes 
No 
Yes 
Yes, RCIGM* 
Included in ACMG59, adult onset 
Yes 
No 
No 
No 
Included in ACMG59, adult onset 
No 
No 
No 
No 
Included in ACMG59, childhood onset 
Yes 
Yes 
Yes 
Yes, RCIGM* 
Included in ACMG59, childhood onset 
Yes 
Yes 
No 
No 
Included in ACMG59, childhood onset 
No 
No 
No 
No 
Not on ACMG59, childhood onset, medically actionable  
Yes 
Yes 
Yes 
No 
Not on ACMG59, childhood onset, medically actionable  
Yes 
Yes 
No 
No 
Not on ACMG59, childhood onset, medically actionable  
No 
No 
No 
No 
*Athena/Quest will not report secondary/incidental findings for parents. 
 

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Confirmatory Testing 
With the exception of a provisional result, pathogenic and likely pathogenic variants (as 
determined by ACMG variant classification criteria) that relate to the neonate’s current 
phenotype as well as suspicious VUS will be clinically confirmed at RCIGM or Athena 
prior to the return of written results. Additional confirmation testing may be done by 
Sema4 for subjects enrolled at Mount Sinai (refer to Sema4 section below). Segregation 
analysis on biological parents will be performed when warranted. A provisional result 
will be given in cases in which delaying the return of results until confirmatory testing 
occurs places the infant at an increased risk of morbidity or mortality (a treatment 
modality is available whose delay in administration could cause irreversible harm). Prior 
to confirmation testing, the laboratory will return provisional results and management 
guidance via verbal report with read back followed by a written provisional result report 
to the enrolling site. Refer to the section, Sema4 and Subjects Enrolled at Mount Sinai, 
for when provisional results may be given for subjects from Mount Sinai.   
 
Confirmatory testing will subsequently be performed for all provisional results and a 
written report of the final result issued. The laboratory will make the decision about 
whether a finding meets the criteria to be given provisionally; if there is a question, the 
lab will consult with the study-wide PIs. A lethal condition will not be released as a 
provisional result; it will be confirmed prior to this result reported. 
 
Variant Classification Is confirmation testing needed? 
Pathogenic 
Yes 
Likely Pathogenic 
Yes 
Variant of Unknown 
Significance (VUS) 
Only if suspicious (where the phenotypic fit is very good 
and/or functional confirmatory tests are readily available 
and/or the results are actionable)  
Likely Benign 
No 
Benign 
No 
 
Secondary diagnoses 
Is confirmation 
testing needed? 
A secondary or incidental finding that will be reported 
Yes 
 
Category 
Definition 
Detected  
By NewbornDx 
Detected  
by rWGS Method(s) 
SNP 
variation in a single 
nucleotide 
Yes 
Yes 
Confirmation 
testing methods 
are at the 
discretion of the 
laboratory director. 
Common methods 
include MLPA, 
microarray, 
Sanger and qPCR. 
Indel  
an insertion or 
deletion of less than 
1 kb in length, in-
frame and frame shift 
Yes 
Yes 
Structural 
Variant  
Inversions, balanced 
translocations, 
CNVs; ~ 1 kb and 
larger  
No 
Yes 
 

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Sema4 and Subjects Enrolled at Mount Sinai 
Results returned to the medical record for subjects from Mount Sinai must be from a 
New York State CLIA certified lab and of a New York State conditionally approved test. 
Athena Diagnostics is a New York State CLIA certified lab and NewbornDx is a New 
York State conditionally approved test. Therefore, NewbornDx results for subjects from 
Mount Sinai will be returned to the research team, clinician(s) caring for the subject, the 
subject’s parent/guardian(s) and placed in the subjects’ medical record. Provisional 
results from NewbornDx may be acted upon prior to Athena’s own confirmation testing. 
 
Until the time RCIGM becomes a New York State CLIA certified laboratory and rWGS a 
New York State conditionally approved or approved test, subjects from Mount Sinai may 
require additional confirmation testing by Sema4. Sema4 is a New York State CLIA 
certified and Mount Sinai wholly owned laboratory. Procedures to be followed: 
1. The research team at Mount Sinai will send samples to Sema4. Sema4 will 
extract DNA from the infant and biological parent blood samples and send two 
aliquots to RCIGM-CGC while retaining an aliquot for possible future confirmation 
testing. DNA requirements: > 3 ug DNA (concentration ranges from 20 ng/ul to 
200 ng/ul) with Picogreen measurement. If the site used non-Picogreen 
measurement, 4-5 ug DNA (concentration ranges from 20-200 ng/ul). 
2. RCIGM will issue a research grade report of its results to the research team at 
Mount Sinai. This report will have the heading “research report” and also include 
disclaimers in the Regulatory Disclosures section. The research report will only 
be placed in the research file and not in the medical record. 
3. A provisional result will be given to the research team in order for them to alert 
Sema4 to start confirmation testing if required (i.e. RCIGM reports a provisional 
result which is not also reported by Athena). A RCIGM provisional result will not 
be shared with the clinical team, parent/guardian nor put in the medical record. 
4. Pathogenic, likely pathogenic or VUS results returned only by RCIGM will have 
additional confirmation testing and interpretation done at the Sema4 laboratory. If 
RCIGM and Athena report the same result, Sema4 confirmation testing is not 
required. At Sema4, confirmation testing will be done by targeted analysis with 
Sanger, or for CNVs with qPCR, MLPA, or ultra-high resolution exon array. 
5. RCIGM will send final results as a research report to the Mount Sinai research 
team. The Mount Sinai research team will compare the RCIGM research report 
with Athena’s report and alert Sema4 of confirmation testing as necessary.  
 
RCIGM Result  
Agreement with NewbornDx 
Is testing 
by Sema4 
required? 
What reports 
are put in the 
medical 
record? 
Pathogenic, likely 
pathogenic, VUS 
or secondary/ 
incidental result 
The findings reported by RCIGM are 
also reported and interpreted within the 
similar variant classification categories 
(P/LP v. VUS) by Quest/Athena. 
No 
NewbornDx  
RCIGM reports findings that Quest/ 
Athena did not report. 
Yes 
NewbornDx,  
Sema4  
Negative Result 
Yes 
No 
NewbornDx  
No 
No 
NewbornDx  

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If during the course of this study, the RCIGM rWGS test becomes CLIA certified in New 
York State and the rWGS test is New York State approved or conditionally approved, 
the processes involving Sema4 will be eliminated. 
 
Discordant Results  
It is possible that the final results of NewbornDx and rWGS may be discordant. It is also 
possible that the confirmatory testing done by Sema4 for infants enrolled at Mount Sinai 
will be discordant to the confirmed NewbornDx or rWGS results. This could be a result 
of both technical and interpretation differences:  
 technical differences of the platforms where a specific variant may be identified 
on one platform but not another 
 different genotyping and variant filter thresholds when both platforms return the 
same variant 
 different classification of the same variant as benign, likely benign, VUS, likely 
pathogenic or pathogenic  
Which methods are used for confirmation testing at each laboratory will be documented.  
 
It will be determined if the discordance is caused by the technical performance of the 
platform or variant filtering or classification. In cases of discordance due to variant 
classification, each lab will use an agreed upon format to provide a written document 
summarizing the evidence supporting their variant classification. Refer to section 8.1 
and the MOP for adjudication procedures. 
 
Inconclusive Results 
Under certain circumstances, a test result may not be conclusive. These situations will 
be reviewed on an individual basis and testing may be repeated or another sample 
requested. The reasons for inconclusive testing are explained in the result report. 
 
Requests for Re-analysis of Sequencing Data 
Requests for reanalysis can only be made after a significant change in phenotype 
occurs in the infant (e.g. new onset seizures) when results have already been returned. 
This may be permitted on a case-by-case basis only after discussion between the 
enrolling site PI and study-wide PIs. 
 
Retrospective Singleton (Proband) Analyses for Duo/Trios 
After the final result is returned for an infant using a duo or trio analysis, Athena 
analysts will conduct a retrospective analysis of only the infant’s NewbornDx data. The 
purpose of this analysis is to determine if the same result obtained with a duo/trio could 
have been obtained with only the infant’s sample. The analysts who conduct the 
retrospective analysis will not know the result that was returned for the subject. The 
retrospective infant-only analyses will occur on a periodic basis throughout the study.  
 
RCIGM will also perform a retrospective infant-only analysis on all subjects utilizing 
either an Artificial Intelligence platform or by performing a manual analysis similar to 
Athena at the end of the study. 
 

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6.2 STUDY INTERVENTION COMPLIANCE 
 
Adherence to the protocol regarding the study intervention will be assessed through the 
monitoring of critical processes as detailed in the Data and Safety Monitoring Plan 
(DSMP). 
 Site procedure for maintaining accurate link of infant with study ID number 
 Site procedure to ensure correct labeling of blood/DNA sample(s) 
 Accurate and relevant Human Phenotype Ontology terms and clinical information 
submitted by the enrolling site to the laboratory for use in analysis of variants with 
phenotype 
 Laboratory protocols adhering to CLIA/CAP/state guidelines  
 Laboratory procedures for accurate matching of a result with the infant 
 Laboratory procedures to ensure any secondary findings for the infant are only 
reported if the criteria pertaining to the infant as outlined in section 6.1.1 are met 
 Laboratory procedures to ensure secondary findings for a biological parent are only 
reported if the criteria pertaining to a parent as outlined in section 6.1.1 are met 
 Confirmation testing completed prior to reporting of pathogenic, likely pathogenic, or 
suspicious VUS results or the documentation of a provisional result exception 
 Accurate dissemination of the report of results from the laboratories to the infant’s 
EMR 
 
6.3 CONCOMITANT THERAPY 
  
There are no restrictions on concomitant medications, treatments or procedures allowed 
during the study. Medications and procedures will be captured during data collection. 
 
 
 

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7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/ WITHDRAWAL 
 
7.1 DISCONTINUATION OF STUDY INTERVENTION 
 
The study intervention is diagnostic tests. Discontinuation of the study intervention 
might occur if a clinical diagnosis is determined prior to the completion of NewbornDx or 
rWGS. In this case, the infant will be withdrawn from the study per section 7.2. 
 
7.2 PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
 
An infant’s parent/guardian or a biological parent can withdraw their own participation at 
any time and is not obligated to state the reason for withdrawal. However, the 
investigator should make a reasonable effort to ascertain the reason for withdrawal 
while fully respecting the parent/guardian’s or biological parents’ rights. 
 
An investigator may discontinue or withdraw an infant from the study for the following 
reasons: 
 A clinical diagnosis was determined prior to completing the genomic testing for 
the infant  
 Withdrawal is requested by the NIH or DSMB  
 If the infant meets an exclusion criterion (either newly developed or not 
previously recognized) that precludes further study participation (i.e. CMV testing 
that came back positive after enrollment) 
 If the blood samples cannot be safely obtained from the infant 
 The parent/guardian withdraws consent for the infant 
 
The reason for discontinuation of the infant or biological parent will be recorded in the 
study database. An infant for whom the parent/guardian provided consent but who does 
not receive the study intervention may be replaced. An infant for whom the 
parent/guardian provided consent and who received the study intervention and was 
subsequently withdrawn from the study will not be replaced. 
 
In the event of an infant death prior to the completion of genetic testing, the testing will 
continue to be done unless the parent/guardian indicates otherwise. There is a potential 
to provide information regarding a hereditary disorder for future children. 
 
If an infant is withdrawn from the study, the data already collected on the infant may be 
used unless the parent/guardian indicates otherwise in writing.   
 
The following actions will be taken at the local study site and laboratories as applicable 
after receiving a request to withdraw: 
 Upon receipt of request to withdraw, the enrolling site will notify RCIGM and 
Athena (Quest) of withdrawal of the infant and/or the biological parent(s). Mount 
Sinai will also notify Sema4.  

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 The enrolling site or laboratories that are in possession of unused blood from the 
infant and/or the biological parent(s) will ensure blood samples are destroyed.  
 Isolated DNA will be destroyed. The laboratories will permanently break any link 
between personal identifying information and sequencing data files from the 
infant and/or the biological parent(s).   
 The RCIGM laboratory will remove the REDCap Infant Study ID from its ordering 
portal. 
 If a back-up of the inventory database/informatics system is ever restored, then 
the laboratory should ensure that the relevant identifying records stored on the 
discarded samples log are again deleted from the records. 
 The enrolling site must document reasons for withdrawal and record it in the 
REDCap database. 
 The enrolling site will permanently break any link between personal identifying 
information and the anonymized records and data in REDCap by replacing 
protected health information (PHI) in the Master List of enrolled subject and 
REDCap Study Infant ID with a note that the infant has withdrawn.  
 The enrolling site and laboratories must shred any hard copies of associated 
identifying information not required to be maintained for regulatory purposes. 
 The enrolling site must retain the executed consent form and request for 
withdrawal in a separate, secure file of discontinued infants and redact any 
identifiers that link the infant to the data (such as infant’s study ID number). 
 The laboratories will certify that identifying links have been broken and as 
applicable, that biological material has been destroyed. 
 Only anonymous data may be used for future research after consent has been 
revoked. 
7.3 LOST TO FOLLOW-UP 
 
An infant will be considered lost to follow-up if the enrolling site is unable to contact a 
parent/guardian at the time the infant turns 1 year CGA plus 2 months. In order to 
reduce the risk of lost to follow-up, study staff will have the parent/guardian(s) provide 
several methods of contact for themselves as well as a family member or friend who 
would know how to reach them if their contact information changed or are unreachable 
(refer to Follow-up Contact Information form). The enrolling site will attempt to contact 
the parent/guardian(s) monthly. Before an infant is deemed lost to follow-up, the 
enrolling site will make every effort to regain contact with the infant (multiple contacts 
and, if necessary, a certified letter to the infant’s last known mailing address or local 
equivalent methods). These contact attempts should be documented in the infant’s 
study file. If the infant continues to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. It is permissible to 
contact the infant’s primary care provider to obtain relevant information on the condition 
of the infant if the parent/guardian(s) are not available and they have given permission 
by signing the ICF and/or medical record release form. 
 
 

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8 
STUDY ASSESSMENTS AND PROCEDURES 
 
8.1 SCREENING & INFORMED CONSENT 
 
Screening 
Infants born with a suspected genetic disorder will be screened for all inclusion/ 
exclusion criteria. Potential infants will be identified by study personnel reviewing the 
daily census of applicable hospital locations and relevant medical records or by the 
clinical team. Study or clinical personnel will identify potential candidates based on the 
primary diagnosis, signs/symptoms, and CGA. The study team will discuss eligibility of 
the infant with the attending physician of record and if in agreement, the family will be 
approached for the study.  
 
There is not a limit to how many infants can be enrolled at any one site. Infants who are 
already an inpatient at the time a site opens for enrollment may be approached for this 
study if they fulfill the inclusion/exclusion criteria. Those infants identified through the 
screening process will have the following data recorded on the Screening Log and/or 
entered into REDCap The Infant Study ID will be used to code screening data and is 
generated by REDCap consisting of a 4-digit number (1-digit site number and a 3-digit 
infant number). 
 
Category 
Information from the medical 
record which may be recorded 
on a paper or electronic 
screening log kept securely at 
the enrolling site 
Screening data that will be 
entered into REDCap if the 
infant is approached for study 
participation regardless of 
consent status 
Identifying 
Information 
name, date of birth, medical 
record number 
N/A 
Screening 
Process 
screening ID, date screened, 
location in hospital, whether or 
not the infant is eligible, if the 
family was approached for 
consent, date of approach, 
reason why an eligible infant was 
not approached 
screening ID  
Clinical 
Information 
gestational age at birth, age at 
time of screening, clinical 
signs/symptoms and primary 
system involvement, clinical 
differential diagnosis, prenatal 
and postnatal clinical genetic 
testing and newborn screening 
results, list of genetic tests 
gestational age at birth, age at 
time of screening, primary 
system(s) involvement related to 
patient nomination, sex of infant 

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pending, newborn screening test 
results, sex of infant  
Consent 
Process 
languages used during the 
consent process; if telephone 
consent was used; if clinical 
genetics was involved prior to the 
consent process; enrollment 
status; reason for declining study 
if voluntarily given 
languages used during the 
consent process; if telephone 
consent was used; if clinical 
genetics was involved prior to the 
consent process; enrollment 
status; reason for declining study 
if voluntarily given 
Demographics 
type of health insurance, race 
and ethnicity of the biological 
mother and biological father (in 
order to represent the infant)  
type of health insurance, race 
and ethnicity of the biological 
mother and biological father, 5-
digit zip code   
 
Identifying information from the screening log will be destroyed at the end of the study.  
Note: for screened patients that have already been entered into REDCap under the 
current HIPAA authorization waiver by the IRB, we will first delete the primary 
signs/symptoms already captured in the database and then add the primary system 
involvement and zip code. The purpose of removing primary signs/symptoms and 
replacing it with system involvement is to reduce the risk of identification due to unique 
features once zip code is added. 
 
Informed Consent 
Consent by one parent/guardian is required for participation by the infant. Each 
biological parent will give permission to obtain and use his or her own blood sample and 
elect parental secondary findings. Written documentation of informed consent from the 
parent/guardian and/or biological parent(s) must be documented prior to the conduct of 
any study procedures. Refer to section 10.1.1 Process of Informed Consent. 
 
If the parent/guardian provides consent for the infant to participate, study personnel at 
the enrolling site will: 
 Review the ICF and ensure that it is properly completed  
 Review whether or not the parent/guardian opted-in to receive secondary findings for 
the infant  
 Review whether or not the biological parent(s) opted-in to receive secondary findings 
for themselves  
 Provide the parent/guardian and the biological parents, as applicable, with a copy of 
the signed ICF  
 Notify the attending physician of record of the parent/guardian’s decision to 
participate 
 Record that consent was obtained in the infant’s medical record  
 Place a copy of the signed ICF into the infant’s medical record  
 File the original signed ICF in the research file 

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 Complete screening log and the Demographics, Symptoms and Consent eCRFs in 
REDCap  
 
If the parent/guardian refuses participation for the infant, study personnel will: 
 Communicate refusal to relevant study or clinical personnel 
 Complete screening log and the Demographics, Symptoms and Consent eCRFs 
in REDCap  
 
8.2 ENROLLMENT  
 
PI Assessment of whether or not an infant requires urgent testing 
This protocol will prioritize testing of infants who are severely ill. Infants with the 
following conditions may qualify for the most rapid testing possible: 
 Infant requires mechanical ventilation 
 Infant is exhibiting severe neurological complications (i.e. uncontrolled seizures) 
 Infant is hemodynamically unstable 
 Site PI selects this option at his/her discretion 
There is not a specific timeframe in which urgent testing must be returned; rather, the 
laboratory does its best to expedite testing. Refer to the “MOP about how to notify 
laboratories of urgent testing.  
 
Enrollment in REDCap System 
Complete the enrollment and urgent testing eCRFs in REDCap. The Infant Study ID and 
linking infant identifiers will be added to the secure site master enrollment log. The 
Infant Study ID will be used for all data entered into REDCap and in study files. 
 
Ordering of Laboratory Tests, Collection and Shipment of Blood Samples 
Three laboratories are involved in this protocol: Athena (Quest), RCIGM and Sema4.  
1. Athena Diagnostics (a subsidiary of Quest Diagnostics) is performing the 
NewbornDx panel (infant and when available, the biological parents) and 
confirmation testing for pathogenic, likely pathogenic and suspicious VUS 
findings. 
2. RCIGM is performing rWGS (infant and when available, the biological parents) 
and confirmation testing for pathogenic, likely pathogenic and suspicious VUS. 
3. In some circumstances, Sema4 will be involved with DNA extraction and 
confirmation testing only for infants enrolled at Mount Sinai (refer to section 
6.1.1).  
 
 
 

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Blood Sample Collection  
Blood samples are collected as follows: 
Who 
Samples to Collect 
Infant 
 A minimum of two and maximum of five fully saturated dried blood 
spots (DBS) using study-provided Perkin Elmer 226 filter paper*  
 Two 0.5ml blood samples in study-provided EDTA tubes (one 0.5ml 
sample is acceptable, but two are preferred) 
Mother 
 Two 0.5ml blood samples in study-provided EDTA tubes (one 0.5ml 
sample is acceptable, but two are preferred) 
 One 2mL blood sample in a study-provided EDTA tube   
Father 
 Two 0.5ml blood samples in study-provided EDTA tubes (one 0.5ml 
sample is acceptable, but two are preferred) 
 One 2mL blood sample in a study-provided EDTA tube 
*In circumstances in which an infant meets urgent criteria and where allowing the required 3-hour 
timeframe for a blood spot card to dry prior to shipping may adversely affect time to diagnosis and 
potential life-saving treatment, 1ml of whole blood in an EDTA tube may be obtained in lieu of the blood 
spot card and sent to Athena. 
 
Refer to the MOP for detailed instructions about the timing and source of blood 
samples, collection procedures and documentation of the date/time of blood collection. 
If possible, collect blood from the infant at the time of routine clinical testing to minimize 
discomfort. If the clinical team does not feel it is safe to withdraw 1ml of blood from the 
infant, wait until just prior to the time a packed red blood cell transfusion is administered. 
 
All blood samples will be obtained by qualified staff following the hospital guidelines and 
standard precautions. DNA extracted and held prior to enrollment may be used for this 
study (i.e. the clinical team obtained a sample prior to putting an infant on ECMO 
knowing an untainted sample may be needed later for clinical purposes). 
 
Obtain whole blood samples from each biological parent near the time the samples are 
being collected from the infant. In the case of an assay failure or a damaged sample, an 
additional blood sample for the infant or a biological parent may be obtained and 
shipped to the laboratory.  
 
Labeling of Blood Samples 
Each sample should be labeled with name, date of birth and/or MRN, and sample ID 
number using permanent ink. Label each sample with name and date of birth and/or 
MRN at the time of sample collection. Add the sample ID number for each sample after 
you complete the laboratory requisition forms. The sample ID numbers for rWGS are 
generated by the RCIGM ordering portal at the time the online requisition is completed. 
The sample ID numbers for NewbornDx will be generated manually by the site as 
instructed in the MOP. 
 
 
 

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Blood Sample Storage Conditions until Shipment 
Keep the blood samples refrigerated at 4◦C until the time of shipment (up to 5 days). Do 
not freeze. The DBS may be kept at room temperature until shipment. 
 
Order NewbornDx and rWGS 
Complete the requisitions for each laboratory as follows and according to the MOP:  
The site 
shipping 
samples 
Requisitions 
to complete 
Process 
All sites 
(except 
Mount 
Sinai) 
Athena 
Diagnostics 
Complete the paper Athena requisition according to the 
MOP, keep a copy in the infant’s research file and 
include it when shipping samples to Athena. 
RCIGM 
Login to the HIPAA-compliant RCIGM ordering portal and 
complete the online test requisition form according to the 
instructions in the MOP. The RCIGM ordering portal will 
1) assign a RCIGM Portal Case ID and 2) generate a 
RCIGM sample ID code for each sample. Print the 
requisition form, keep a copy in the infant’s research file 
and include it when shipping samples to RCIGM.  
Mount 
Sinai 
Athena 
Diagnostics  
Complete the paper Athena requisition according to the 
MOP, keep a copy in the infant’s research file and 
include it when shipping samples to Athena. 
RCIGM 
Login to the HIPAA-compliant RCIGM ordering portal and 
complete the online test requisition form according to the 
instructions in the MOP. The RCIGM ordering portal will 
1) assign a RCIGM case ID and 2) generate a RCIGM 
sample ID code for each sample. Print the requisition 
form, keep a copy in the infant’s research file and include 
it when providing samples to Sema4 (don’t ship whole 
blood samples to RCIGM).  
Sema4  
Complete the paper-based Sema4 requisition, keep a 
copy in the infant’s research file and include both the 
Sema4 requisition and the RCIGM requisition when 
providing samples to Sema4. 
Sema4  
RCIGM  
Include the RCIGM requisition completed by and 
received from Mount Sinai when shipping the DNA 
samples to RCIGM. 
 
Be sure to indicate in the appropriate fields on all requisitions whether or not the 
parent/guardian opted-in to receive secondary findings for the infant and if each 
biological parent opted-in for their own secondary findings.  
 
 

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Provision of Clinical Information to the Laboratories 
HPO Terms will be provided by the site to the laboratories to inform the data 
interpretation of the genomic sequencing (refer to section 6.1.1, Phenotype Extraction 
and instructions in the MOP). It is the responsibility of the site to ensure the infant’s 
clinical condition is monitored while NewbornDx and rWGS are in process. Refer to the 
MOP about how to communicate any changes (either positive or negative) in the 
phenotype of the infant after the HPO terms are sent. 
 
Laboratory Notification of Urgent Testing 
If the site PI determines an infant requires urgent testing (refer to urgent testing criteria 
above), the site should order an “ultra-rapid” WGS test in the RCIGM portal and contact 
Athena at the time of sample shipment to inform them of urgent testing. Mount Sinai 
should contact Sema4 to extract the DNA and ship it to RCIGM as quickly as possible. 
How to notify the laboratories of urgent testing is detailed in the MOP. 
 
Blood Sample Shipment 
The shipping address, shipping conditions and when to delay shipment of the infant’s 
sample to obtain parent samples is described in the MOP. Only laboratory personnel 
with International Air Transport Association (IATA) certification should package/ship 
specimens. Ship samples overnight delivery. RCIGM must receive the blood samples 
by 10AM PST to ensure it is on that day’s sequencing run. Athena Diagnostics does not 
accept samples on Sundays. RCIGM will receive samples on Sunday if the ordering 
provider determines that the patient is critically ill and requires results as fast as 
possible; contact RCIGM prior to shipping samples. 
 
Receiving 
Lab 
Enrolling 
Site 
What the Enrolling Sites Ship 
Athena  
All Sites 
o Infant: minimum of 2 Dried Blood Spots on one DBS Card 
o Mother: 2ml of whole blood in one EDTA tube 
o Father: 2ml of whole blood in one EDTA tube 
o Athena Requisition Form 
RCIGM 
All sites 
except 
Mount 
Sinai 
o Infant: 0.5ml whole blood in each of two EDTA tubes  
o Mother: 0.5ml whole blood in each of two EDTA tubes 
o Father: 0.5ml whole blood in each of two EDTA tubes 
o RCIGM Requisition Form  
Sema4* 
Only Mount 
Sinai 
o Infant: 0.5ml whole blood in each of two EDTA tubes 
o Mother: 0.5ml whole blood in each of two EDTA tubes 
o Father: 0.5ml whole blood in each of two EDTA tubes 
o Sema4 Requisition Form 
o RCIGM Requisition Form 
*Sema4 will isolate DNA from the blood samples, ship DNA aliquots for the infant and each 
biological parent to RCIGM and keep an aliquot at Mount Sinai for possible confirmation testing.  
 
 
 

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Tracking of Samples 
When the samples are shipped, the site research team should retain the tracking 
number for each shipment to confirm the package arrived at the applicable laboratory. 
Sites should send the courier name and tracking number applicable to Athena (Quest) 
and RCIGM via the email addresses provided in the MOP.  
 
Data Collection and Entry into the REDCap System 
The following information from the infant’s medical record entered into REDCap by staff 
at each enrolling site. Enrolling sites will have access to data entered into REDCap for 
infants at their own site. The study-wide co-PIs (Davis/Maron) and monitoring staff will 
have access to all data in REDCap.  
 
Refer to the detailed grid of Data eCRFs in section 1.3 Schedule of Activities and the 
eCRF Instructions. Information collected and entered into the REDCap database for 
enrolled infants will include: 
 identifiers (date of birth, date of hospital admission, dates of tests/procedures, 
parent email address if the parent wishes to complete follow-up surveys online – 
this email is not released or used outside of this study and will be removed when 
de-identifying the dataset) 
 birth information (mode of delivery, birth weight, APGAR score, newborn 
screening results, pregnancy complications) 
 phenotypic information (clinical symptoms, clinical presentations, HPO terms)  
 clinical information (diagnoses, laboratory and imaging test results including 
clinical genetic testing results) 
 clinical genetic tests that would have been done if this research study was not 
available 
 genetic testing results as a result of study participation 
 any actions because of the research genetic test results 
 treatments (surgical procedures and medications) 
 hospital billing information (CPT codes, billing data) 
 medical visit, emergency room, hospitalization, procedures, treatments post 
discharge home  
 quality-of-life assessments 
 date and cause of death, if applicable 
 adverse events 
 
Data for this study will be collected at the following time points: at enrollment, following 
return of results, at time of an adverse event, at transfer, discharge home, withdrawal, 
or death and then monthly after discharge until the infant turns one-year CGA.   
 
Upload of Source Documents to the REDCap System 
Scanned source documents will be uploaded to the REDCap system for remote 
monitoring. Copies of medical records or source documents should be redacted of 
name and MRN or other account numbers, coded with the Infant Study ID, scanned to a 
PDF file and uploaded to REDCap.  
 

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Genomic Sequencing Results 
Please refer to Section 6.1 Description of Study Intervention for details regarding the 
genomic sequencing methods that will be completed. Results of the infant’s genomic 
testing will be shared with the research team, clinical team, the parent/guardian(s), and 
placed in the infant’s medical record as follows: 
Type of 
result 
Result reported by laboratory 
Action at site 
Provisional 
Positive 
Result 
Diagnostic findings related to 
phenotype - pathogenic variant(s) 
in genes interpreted to be 
responsible for, or contributing to, 
the infant’s phenotype will be 
reported (classification of 
pathogenic or likely pathogenic). A 
provisional result means the result 
has not been confirmed. A 
provisional result is only given if 
waiting for confirmation testing 
would put the infant at risk for 
significant morbidity or mortality (a 
treatment is available and a delay 
would cause irreversible harm). 
For all provisional results, 
confirmation testing will 
subsequently be performed. 
The enrolling site will receive 
management guidance by the 
laboratory and consult with their 
clinical genetics team. Provisional 
results will be entered in the 
infant’s medical record under a 
research note. The research note 
will outline what provisional results 
were reported. Provisional results 
may guide the implementation of 
an intervention for the infant. 
Following clinical confirmation of 
results, a clinical report of the final 
result will be placed in the medical 
record. In the event that the 
positive finding cannot be 
confirmed, a research note will be 
added to the medical record to 
facilitate communication among 
multiple specialty services.  
Confirmed 
Positive 
Result 
Diagnostic findings related to 
phenotype - pathogenic variant(s) 
in genes interpreted to be 
responsible for, or contributing to, 
the infant’s phenotype will be 
reported (classification of 
pathogenic or likely pathogenic). 
The result has been confirmed (for 
example with Sanger or qPCR). 
All positive results will be 
confirmed prior to return to the 
enrolling site with the exception of 
a provisional result. 
Confirmed positive results will 
yield a clinical diagnosis, a referral 
to clinical genetics and reported in 
the medical record. Results may 
guide the implementation of an 
intervention for the infant. 
Confirmed 
Variant of 
Unknown 
Significance 
(VUS) 
A variant for which the clinical 
significance is unknown. The 
variant detected may or may not 
explain the infant’s current clinical 
symptoms. The variant has been 
Suspicious VUS reported by the 
laboratories will be reported in the 
medical record. In the future, a 
variant may become known to be 
significant, and the parent/ 

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Type of 
result 
Result reported by laboratory 
Action at site 
confirmed. Only suspicious VUS 
(where the phenotypic fit is very 
good and/or functional 
confirmatory tests are readily 
available and/or the results are 
actionable) will be reported by the 
laboratory. 
guardian(s) of the infant will have 
this information to provide to their 
personal physician.   
Negative 
Result 
The result that pathogenic variants 
associated with the infant’s clinical 
phenotype were not detected 
(classification likely benign or 
benign). 
Negative results will be reported to 
the medical record in accordance 
with standard care for clinical 
laboratories to reflect the clinical 
testing ordered. 
Secondary/ 
Incidental 
Finding for 
the Infant 
Variant(s) not thought to be 
related to the infant’s current 
symptoms but still important for 
the person’s health will be 
reported if 1) the parent/guardian 
opted-in to receive additional 
results for the infant, 2) the onset 
of the disorder may occur in 
childhood, and 3) at least one of 
the following: a) the disorder is 
medically actionable (available 
treatment to cure or ameliorate 
symptoms or preventative 
measure), or b) The disorder is of 
a gene on the “ACMG list” of 
genes recommended for reporting 
of secondary findings at the time 
of testing. 
Upon receipt of a report with a 
secondary/incidental finding for 
the infant, the site will verify on the 
signed ICF that the 
parent/guardian(s) opted-in to 
receive additional results for the 
infant. The result will be given at 
the same time as a primary finding 
for the infant. 
Secondary/ 
Incidental 
Finding for 
a Biological 
Parent 
If a duo or trio is done, variant(s) 
not thought to be related to the 
infant’s current symptoms but still 
important for the biological 
parent’s health will be reported if 
1) the biological parent opted-in 
during the informed consent 
process to receive additional 
results and 2) the variant is of one 
of the 59 genes recommended by 
the ACMG to report back as 
secondary findings. Secondary 
findings for a biological parent will 
Upon receipt of a report with a 
secondary/incidental finding for 
the biological parent, the site will 
verify on the signed ICF that the 
biological parents opted-in to 
receive additional results. The 
result will be given to the biological 
parents by the enrolling site PI, 
study geneticist or genetics 
counselor and a referral to clinical 
genetics will be made. 

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Type of 
result 
Result reported by laboratory 
Action at site 
be returned in a separate report 
from the report issued for the 
infant and the biological parent 
report will not be put in the infant’s 
medical record. The report of a 
secondary finding for a biological 
parent will be issued to the 
enrolling site PI. 
 
Clinical Laboratory Report 
Standard clinical laboratory reports of results will be issued from Athena Diagnostics 
and RCIGM. The report of results will include: test result [positive (diagnosis), negative, 
variant of unknown significance], sequence variants detected (gene and transcript, 
chromosome, genomic coordinates, variant, confirmation status), variant interpretation, 
gene information, references, recommendations, test methodology, test limitations, 
regulatory disclosures, laboratory contact information, CLIA #, CAP #. Exception: Until 
conditional New York State CLIA certification and rWGS approval is obtained, RCIGM 
will issue a research report for subjects from Mount Sinai. For results that are confirmed 
by Sema4, Sema4 will issue a standard clinical laboratory report. 
 
Raw Sequencing Data 
Under California law (Assembly Bill No. 375), subjects have the right to obtain their own 
sequencing data from for-profit companies. Athena (Quest) Diagnostics is required to 
make sequencing data available upon request if paid a data recovery fee by the subject. 
This information is included in the informed consent form. While not mandated per law, 
raw data can be downloaded from the RCIGM portal for 60 days after the results are 
returned. After this time, sequencing data can be obtained by request with a turn-around 
time of approximately 1 week.  
 
At the end of the study, VUS may be re-analyzed to see if gains in the understanding of 
genotype and phenotype correlation have occurred over the length of the study which 
changes the infant’s results (i.e. a VUS becomes positive). In such a case, the site will 
attempt to notify the parent/guardian(s).   
 
Future Use of Specimens and Sequencing Data 
DNA may be stored for further testing if necessary for the management of the infant. 
Stored DNA will be destroyed at the conclusion of the study per section 10.1.4, Future 
Use of Stored Specimens and Data. 
 
 
 
 
 

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Process for the laboratory to return results to the research team and the medical record 
 
Notification of a Provisional Result: 
If any of the laboratories have a positive result requiring provisional reporting, the 
laboratory must: 
1. Call three parties: 1) the attending physician taking care of the infant, 2) the site 
PI and 3) study-wide co-PI Dr. Jill Maron. The lab will use the contact information 
provided by the site on the requisition to reach the attending physician of record. 
Therefore, the site will determine the best number (e.g. the clinical unit phone 
number where the infant is admitted or the beeper/phone that the NICU fellow 
holds 24/7). The phone numbers for each site PI and Dr. Maron are listed in the 
MOP. Provide verbal provisional result and management guidance with read 
back. The notifying laboratory will document the person to whom the result is 
reported and the date and time of notification (refer to the form, “Documentation 
of Verbal Result Returned”).  
2. Send an encrypted email of a PDF of the provisional result report to 1) the 
physician of record, 2) the site PI/research team, 3) the study-wide Co-PIs and 
project manager. The email addresses to reach the physician of record should be 
obtained during the verbal report. The RCIGM portal will be configured to 
automatically trigger a copy of the email alert to the site study team and the 
study-wide Co-PIs. Athena (Quest) will refer to the email addresses for each site 
PI and Dr. Maron listed in the MOP but these should also be included on the 
requisitions.  Refer to the MOP for contact information. The provisional result will 
not be digitally faxed directly into the EMR. 
 
The site will enter provisional results in the infant’s medical record under a research 
note. The research note will outline what provisional results were reported. Following 
clinical confirmation of results, a final clinical report will be placed in the medical 
record. In the event that the positive finding cannot be confirmed, a research note 
will be added to the medical record to facilitate communication among multiple 
specialty services.  
 
Notification by the Laboratory of a Confirmed Positive Result, Negative Result or 
Suspicious VUS: 
The laboratory must: 
1. Send an encrypted email of a PDF of the report to 1) the site research team and 2) 
the study-wide Co-PIs and project manager.  Refer to the MOP for contact 
information. 
2. In the case of 1) a confirmed positive result or 2) a final result that has the potential 
for immediate implications for the infant’s management (i.e. provisional result that is 
not confirmed), call the attending physician of record caring for the infant. Provide 
verbal results with read back. The notifying laboratory will document the person to 
whom the result was reported, and the date and time of notification (refer to the 
form, “Documentation of Verbal Result Returned”). After the site is contacted, the 
laboratory must call the site PI and Dr. Maron.  

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3. During the course of this study, we intend to move to a process in which the 
laboratory issues the report directly to the infant’s EMR by digital fax. For infants 
from Mount Sinai, the RCIGM report will be sent to the Mount Sinai PI, not the 
infant’s EMR, for input into the subject’s research file. After completion of 
confirmation testing by Sema4, if applicable, the Sema4 CLIA-certified report will be 
sent to the infant’s EMR. 
 
Notification by the Laboratory of Secondary Findings 
1. If the parent/guardian(s) opted-in to receive secondary findings for the infant, the 
laboratory will report secondary findings as described in section 6.1.1 in the 
return of results for the infant.  
2. If a biological parent opted-in to receive secondary findings for him or herself, the 
laboratory will return secondary findings for the individual biological parent as 
described in section 6.1.1 in a separate report from the test result for the infant. 
This report will not be sent to the infant’s EMR. It will be sent to the site PI. 
 
Process for returning results to the medical record 
It is intended for the report of results to be sent Athena and RCIGM directly to the 
infant’s EMR via digital fax; however, the enrolling site may need to manually deposit 
the report in the medical record until that process is completed. The enrolling site is 
responsible for ensuring that the clinical laboratory reports appear in the infant’s medical 
record. If the final results are discordant, all reports will still be placed in the medical 
record and a note should document how the results will be used in clinical care. In the 
case of infants enrolled at Mount Sinai, only results by a New York State CLIA certified 
lab of a New York State approved or conditionally approved test will be put in the 
infant’s medical record.  
 
Clinical genetics and related sub-specialty notes will be placed in the medical record per 
SOC. 
 
Adjudication of Discordant Results 
If the final results of NewbornDx and rWGS or the confirmation test results done at 
Sema4 are discordant, adjudication processes, as outlined in the MOP, will be followed. 
If results are discordant due to variant classification, the laboratories will each provide 
the evidence supporting their result and variant classification. This summary will be 
given to the enrolling site PI to share with the clinical team; a subsequent phone call 
with the labs may be arranged upon request from the site. The clinical team caring for 
the infant is in the best position to determine if one result best explains the phenotype of 
the infant and their view will be captured in the study data. Additionally, a subset of the 
study geneticists on the steering committee who are independent of the labs will review 
the redacted lab reports and lab summaries and provide what they think is the most 
accurate result for an additional study endpoint. Senior lab personnel are available for 
questions by the steering committee. The opinion of the steering committee members 
may be shared with the enrolling site upon the site’s request. Regardless of whether or 
not results between the tests are concordant, both the NewbornDx report and the rWGS 

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report will be put in the infant’s medical record. A note should be placed in the medical 
record to document how the results will be used in clinical care. 
 
Communication with the Physician of Record and the Infant’s Parent/Guardian(s) 
The site should wait until results from both NewbornDx and rWGS are returned before 
speaking with the parent/guardian about the results as long as waiting would not harm 
the infant (i.e. the result is not provisional or the infant does not require urgent testing). 
 

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Coordination Between Study 
Team and Physicians of 
Record re: Results 
Qualifications of  Personnel 
providing Genetic Results 
to Parents/Guardians 
Qualifications of  Personnel 
providing ongoing related 
information to 
parents/guardians 
Providing Results to 
Parents/Guardians when an 
Infant is Discharged Home 
Prior to Return of Results 
The site PI/sub-I will speak 
with the physician of record 
and suitable subspecialists 
(for example, genetics, 
neurology, cardiology, etc.) at 
the time each test result is 
returned in order to review 
results and formulate a plan 
prior to returning a result to 
the parent/guardian.  
The PI and/or sub-I at all 
sites are neonatologists or 
geneticists. 
If the patient is discharged 
home prior to the return of 
results, the primary 
pediatrician should be 
contacted along with hospital 
clinical and research 
personnel. 
Results will be disclosed to 
the family with as many 
members of the clinical 
team caring for the patient 
present as possible. At a 
minimum, each site will 
include one or more of the 
following: attending clinical 
geneticist, attending 
physician of record, 
attending neonatologist or 
geneticist who is a member 
of the study team. 
Additional attending sub-
specialists relevant to the 
particular genetic condition 
and genetic counselors 
may also participate in 
providing results to families. 
If clinical practice at a 
particular site is for licensed 
genetic counselors to return 
results without an attending 
physician as described 
above, the licensed genetic 
counselor may do so. 
Recommendations to the 
family about changes in 
management or further 
genetic testing will be 
addressed by the physician 
of record as well as clinical 
genetics and other sub-
specialties per standard-of-
care. 
If a genetic problem exists 
that can be treated but does 
not need to be treated in the 
neonatal period, a referral to 
outpatient clinical genetics 
will be made. 
It is strongly recommended 
that positive results or a VUS 
be disclosed in-person 
followed by a referral to a 
clinical geneticist. Depending 
on the need for timely action 
based on the results, it may 
be appropriate to return the 
result at the time of the next 
outpatient clinical genetics or 
related subspecialist 
appointment or to ask the 
family to come into the site to 
meet with the site PI to hear 
about the study results. 
Under extraordinary 
circumstances (where the 
family lives hours away 
and/or has moved out of 
state), diagnostic or VUS 
results may be given by 
phone along with a clinical 
genetics referral. It is 
acceptable to convey 
negative/no finding results by 
phone with an offer for follow 
up genetic counseling visit. 

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Communication with a Biological Parent about a Parent Secondary Finding 
Upon the receipt of a report with a secondary finding for a biological parent by the site 
PI, the site will verify on the signed ICF that the biological parent opted-in to receive 
secondary findings. The result will be given to the biological parent by the enrolling site 
PI, study geneticist or genetics counselor and a referral to clinical genetics will be made. 
Some sites may opt to create a medical record for each biological parent and scan the 
consent and individual parental secondary findings to that medical record.  
 
Genomic Sequencing Clinical Utility Survey 
At discharge home or within one month after the return of results for infants who 
discharge home prior to the return of results, the physician of record at the time the 
results are returned will be asked to complete a brief survey regarding the clinical utility 
of the genomic testing. The one physician under whom the infant is admitted at the time 
results are received will receive the survey (i.e. neonatologist if the infant is in the NICU, 
pediatric critical care attending if the infant is in the PICU). The survey will be sent via 
email or a paper copy handed to the physician. The goal of the survey is to identify what 
changes were made to the immediate management of the infant as a result of testing as 
well as the general utility of the diagnosis. If the infant is discharged (home or death) 
prior to return of results, the Clinical Utility Survey should be completed by the clinical 
geneticist, most relevant subspecialist or by the site PI after speaking with the 
pediatrician (if baby was discharged home). 
 
Parent/Guardian Contact Information, Medical Record Release and Diaries at 
Transfer/Discharge Home 
The parent/guardian(s) will fill out a contact information form with multiple methods of 
contact to ensure we are able to reach the parent/guardian or primary care provider until 
the infant is 1 year CGA. A quality of life (QoL) instrument (The Optum™ SF-12v2® 
Health Survey) and the Child Visual Analog Scale will be administered. 
 
Billing Information 
At transfer to another facility and/or discharge home, the enrolling site research staff will 
obtain the final hospital costs and charges, and all physician CPT codes, for the infant’s 
hospitalization.  
 
Parent/Guardian Interview and Medical Record Acquisition 
Following transfer of facility or discharge home, study staff will contact one 
parent/guardian monthly to assess how the infant is progressing until 1 year CGA. The 
parent/guardian will be asked about any new testing, diagnosis, medications, medical 
visits or hospitalizations, and any missed days of work due to their child’s disorder. 
Ideally, the parent will receive a link via email to complete the monthly parent survey 
online in the REDCap system; however, the parent/guardian may be contacted by e-
mail or phone per the parent/guardian’s preference. Self-reported QoL information will 
be collected from a parent/guardian at 3-month intervals to infer the impact on the family 
of any prolonged illness. The follow-up surveys will be translated into Spanish. In the 
case of a parent/guardian who does not read English or Spanish, the follow-up contact 
will need to be made with an interpreter by phone. Upon request by the health 

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economics analysts and as permitted in the ICF, medical records and billing information 
from any hospitalizations and any outpatient health care utilization may be obtained 
from the provider institutions to complement parent/guardian self-report.   
 
8.3 SAFETY AND OTHER ASSESSMENTS 
 
Enrolling sites must actively monitor, identify and assess adverse events (AE) in the 
study. Identification of AEs will be based on review of the medical record, observation 
by study staff and by report during the parent survey/interviews. For each AE, the 
investigator will determination whether the adverse event is serious, provide the 
duration (start and end dates), the severity grade (mild, moderate, severe), the 
relationship to the study testing (unlikely, possibly, and probably), action(s) taken, and 
the outcome. The AE assessment must be documented and retained with study files. 
The Investigator’s signature and date on the source document that supports the 
causality noted on the AE form ensures that a medically qualified assessment of 
causality was done. 
 
The laboratories conducting genetic sequencing for this study fall under the CLIA/CAP 
regulations. They continually monitor for errors and non-conforming events and will 
notify the study-wide PIs via phone/email of an error/event that affected a result for the 
infant. They will also notify the study-wide PIs if a data breach were to occur. In these 
instances, the study-wide PI would file an adverse event, and notify the DSMB, IRB, 
and NCATS as required.  The laboratories are not expected to report adverse events as 
described in sections 8.3 and 8.4. 
 
8.4 ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
 
8.4.1 DEFINITION OF ADVERSE EVENTS (AE) 
 
An Adverse Event for this study will only include the following: 
 An untoward medical occurrence related to obtaining the study blood samples 
 If findings for the infant thought to be secondary at the time results are reported 
are given to the research team, clinician or parent/guardian when the 
parent/guardian did not opt-in to receive secondary findings for their infant  
 If secondary findings for a parent are reported to the research team, clinician or 
parent when the parent did not opt-in to receive parental secondary findings  
 Intervention based on inaccurate result returned (testing error; provisional result 
not confirmed) 
 Intervention based on wrong result returned (human error, result not accurately 
matched up with the correct infant) 
 Untoward event related to an intervention initiated due to the genomic testing 
results (intervention that would not have been initiated without the genomic 
testing results) 
 Loss of confidentiality related to study participation 

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 Any new untoward medical occurrence related to study participation or the 
genetic sequencing results   
 A pre-existing condition that worsens in terms of frequency or intensity and the 
worsening is due to study participation/genetic sequencing results  
 The infant dies due to error because the infant was enrolled in this study 
(erroneous result or misidentification) 
 
Events not reported as Adverse Events for this study: 
 A pre-existing condition at the time of enrollment 
 A pre-existing condition that worsens in terms of frequency or intensity but the 
worsening is not related to study participation/genetic sequencing 
 A new untoward medical occurrence that is not related to study 
participation/genetic sequencing results and does not result in death 
 Death due to natural causes before genetic testing results are returned (i.e. 
prematurity) 
 Death due to parent/guardian wishes to withdraw life sustaining treatment if an 
infant is determined to have a lethal condition by genetic testing  
 
8.4.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)  
 
An adverse event (AE) or suspected adverse reaction is considered "serious" if in the 
view of either the investigator or sponsor, it results in any of the following outcomes: 
death, a life-threatening adverse event, prolongation of existing hospitalization, a 
persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions, or a congenital anomaly/birth defect. Important medical events that 
may not result in death, be life-threatening, or require more prolonged hospitalization 
may be considered serious when they may jeopardize the infant and require medical or 
surgical intervention to prevent one of the outcomes listed in this definition.  
 
Events that are not related to study participation or genomic sequencing will not be 
captured as an SAE since the definition of an adverse event for this study requires a 
relation to genetic testing (rather than the underlying disorder).  
 
8.4.3 CLASSIFICATION OF AN ADVERSE EVENT 
The PI or another licensed clinician who is a study member authorized on the 
delegation log will classify adverse events. 
8.4.3.1 SEVERITY OF EVENT 
  
The investigator should use the following definitions when assessing intensity of an 
adverse event: 
 Mild: Infant or a biological parent has minor findings but tolerates them well, and 
no or minimal intervention required 

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 Moderate: Infant or a biological parent experiences enough symptoms or findings 
to require intervention  
 Severe: Infant or a biological parent experiences symptoms or findings that 
require significant intervention 
 
8.4.3.2 RELATIONSHIP TO STUDY INTERVENTION 
 
All adverse events (AEs) must have their relationship to the genomic sequencing 
assessed. The investigator will examine and evaluate the participant based on temporal 
relationship and his/her clinical judgment.  
 
The criteria below are intended as guidelines to assist the Investigator in assessing the 
likelihood of a relationship between the genomic sequencing testing and the adverse 
event. The greater the correlation with the components and their respective elements, 
the more likely the genomic sequencing testing caused the adverse event. 
 Exposure: Is there evidence that the infant or biological parent had the testing 
performed such as: reliable history, patient records, assays run in applicable 
laboratories and results exist? 
 Time Course: Did the AE follow in a reasonable temporal sequence from 
performance of the testing? Is the time of onset of the AE compatible with the 
testing being performed? 
 Likely Cause: Is the AE not reasonably explained by another etiology such as an 
underlying disorder or other host or environmental factors? 
 
The following scale of criteria may be used as a guidance (not all criteria must be 
present in order to be indicative of a relationship to study genomic sequencing). 
 
Probably related to 
study genomic 
sequencing 
 There is evidence the infant or a biological parent had the 
genomic testing performed 
 The temporal sequence of the AE onset relative to genomic 
testing is reasonable 
 The AE is more likely explained by the genomic testing than 
by another cause 
Possibly related to 
study genomic 
sequencing 
 There is evidence the infant or a biological parent had the 
genomic testing performed 
 The temporal sequence of the AE onset relative to genomic 
testing is reasonable 
 The AE could have been due to another equally likely cause 
Unlikely related to 
study genomic 
sequencing 
 There is evidence the infant or a biological parent had the 
genomic testing performed 
 There is no temporal relationship to genomic testing 
 There is another more likely cause of the AE 
 

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8.4.3.3 EXPECTEDNESS  
 
The investigator will be responsible for determining whether an AE is expected or 
unexpected. An AE will be considered unexpected if the nature, severity, or frequency 
of the event is not consistent with the risk information previously described for the study 
intervention. An AE is considered unexpected if it is not consistent with the risk 
information described in the protocol. 
8.4.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND 
FOLLOW-UP 
 
AEs must be actively identified from the signing of the ICF until the infant reaches one 
year CGA. AEs must be followed until an outcome is known. All events that cannot be 
resolved by 30 days after the final assessment at 1 year CGA will be considered 
resolved by convention and entered in the REDCap system.    
 
8.4.5 ADVERSE EVENT REPORTING  
 
Reporting of non-serious AEs must occur using the Adverse Event eCRF in the 
REDCap system according to the following: 
Event 
Timeframe for Site to Report 
Timeframe for Study PI to Report 
AE, not serious 
Site enters AE into EDC within 7 
business days of identification.     
The study PI reports to the DSMB 
every 6 months and in the annual 
progress report to NCATS. 
 
8.4.6 SERIOUS ADVERSE EVENT REPORTING  
 
Reporting of SAEs must occur using the adverse event eCRF in the REDCap system 
according to the following: 
Event 
Timeframe for Site to Report 
Timeframe for Study-wide PI to 
Report 
SAE  
Site reports to the study-wide PI 
within 24 hours of learning 
about the event. 
The study-wide PI will alert the 
DSMB chair within 24 hours of 
learning about the event. If the SAE 
is also an unanticipated problem, 
the study-wide PI will also report to 
the central IRB and NCATS within 
24 hours of learning about the 
event. 
  
To ensure accurate clinical trial reporting and correct interpretation of safety signals, 
each death will be examined to determine cause. This may be difficult in the preterm 
infant population as there are often multiple events which contribute to death. For the 

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purposes of this clinical trial, the investigator will attribute the cause of death to the AE 
which had the greatest medical contribution to death, regardless of whether or not the 
event was the most proximal. For example, if an infant has a large intraventricular 
hemorrhage that leads to seizures and death, the death should be attributed to 
intraventricular hemorrhage, not seizures. The investigator should utilize all available 
information when attributing cause of death. Autopsy information should be utilized 
where available to amend the adverse event report if needed. It is recognized that the 
death certificate may not reflect clinical trial procedures and thus may not match the 
cause of death attributed by the investigator. In the rare situation for deaths in which no 
single AE represents the greatest medical contribution to death, the adverse event is 
listed as “Death not associated with an AE term” and one of the following causes must 
be selected: 1) death not otherwise specified (i.e. failure to thrive), 2) extreme 
prematurity, 3) multi-organ failure, and 4) sudden death (no documentation of cause, 
includes Sudden Infant Death Syndrome). For cases in which the cause of death is 
unclear, the final determination as to which AE or organ system the death should be 
attributed will be determined by the DSMB. 
 
8.4.7 REPORTING EVENTS TO PARTICIPANTS  
 
The parent/guardian of an infant will be told of AEs/SAEs in the course of the infant’s 
clinical care (i.e. untoward event related to treatment initiated from sequencing results) 
or in the instance of an inaccurate or wrong result returned. If the risk of participating in 
the study changes, re-consent of the parent/guardian or of a biological parent will occur 
at the discretion of the IRB and DSMB.   
 
 
8.5 UNANTICIPATED PROBLEMS 
 
8.5.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP) 
 
 “Unanticipated Problems Involving Risks to Subjects or Others” (“UPIRSO”). An event 
is considered an UPIRSO when it meets all of the following criteria: 
  
(1) It is unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB-
approved research protocol and informed consent document; and (b) the characteristics 
of the population being studied; 
 
Unexpected events could be either medical or non-medical events. 
 
(2)  It is related or possibly related to participation in the research (i.e. there is a 
reasonable possibility that the incident, experience, or outcome may have been caused 
by the procedures involved in the research); 
 
and, 
 

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(3) It places subjects or others [e.g. study team members or relatives of a subject] at a 
greater risk of harm (including physical, psychological, economic, or social harm) than 
was previously known or recognized 
 
8.5.2 UNANTICIPATED PROBLEM REPORTING  
 
Certain types of events (as defined below) and including unanticipated problems 
involving risks to subjects or others must be promptly reported to the IRB:  
Event 
Timeframe for Site to Report 
Timeframe for Study PI to Report 
Certain types of 
events (as 
defined below) 
Sites much report the event to 
the study-wide co-PIs as soon 
as possible but no later than 8 
working days after discovery or 
in the case of a death within 24 
hours of learning about the 
event. 
The study PI will alert the central 
IRB, DSMB Chair, and NIH   as 
soon as possible after the event is 
discovered by the site, but in all 
cases within 10 working days after 
discovery of the event by the site. 
Reportable deaths, as described in 
this section, must be reported as 
soon as possible but in all cases 
within 72 hours after discovery by 
the site. 
 
The following “certain types of events” must be promptly reported to the IRB: 
 
A. UPIRSO (refer to definition in section 8.4.1) 
 
B. POTENTIAL SERIOUS OR CONTINUING NON-COMPLIANCE:     
 
Non-Compliance is defined by the Organization as the failure to follow the research 
protocol, federal, state, or local laws or regulations governing human subjects research, 
institutional policies, or the requirements or determinations of the IRB.  Only incidents 
that may qualify as serious or continuing non-compliance must be promptly reported: 
 
(1) Serious Non-compliance is defined by the Organization as non-compliance that 
either (a) significantly harms or poses an increased risk of significant harm to subjects 
or others, or (b) significantly compromises the rights and welfare of the subjects or the 
integrity of the Organization’s human research protection program. 
 
(2) Continuing Non-compliance is defined by the Organization as a pattern of non-
compliance that significantly compromises the scientific integrity of the study or the 
rights and welfare of the subjects or the integrity of the Organization’s human research 
protection program.  When applying this definition, particular consideration may be 
given by the IRB to activity that recurs after a previous report has been evaluated by the 
IRB and corrective action has been instituted. 
 
C. OTHER EVENTS THAT REQUIRE PROMPT REPORTING: 

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In addition to the above, investigators must also promptly report the following:. 
 
 Potential Breaches of Confidentiality:  Any unauthorized disclosure of subject’s 
personally identifiable information. Please Note: Potential breaches of 
confidentiality that involve protected health information (PHI) must also be 
reported promptly to the HIPAA Privacy Officer. Please see guidance for further 
detail. 
 Incarceration of a parent participating in this study 
 Unresolved Subject Complaints:  Complaints of subjects when the complaint 
indicates unexpected risks or cannot be resolved by the research team. 
 Other events: There may be other events that should be promptly reportable to 
the IRB.   
 
Events that do not meet the above criteria should be summarized and reported to the 
IRB at the time of continuing review.  
 
8.5.3 REPORTING UNANTICIPATED PROBLEMS TO PARTICIPANTS  
Refer to section 8.3.7 
 
 

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9 
STATISTICAL CONSIDERATIONS  
 
9.1 STATISTICAL HYPOTHESES 
 
The analysis plan is descriptive. There will be no statistical hypothesis testing. 
 
9.2 SAMPLE SIZE DETERMINATION 
 
The sample proportion and 95% CI for the binary outcomes of NewbornDx and rWGS 
will be calculated. The table below shows the 95% confidence limits of the estimated 
proportion for a range of proportions, assuming the sample size is 400 completed 
infants. For example, if the sample proportion is 0.05 for disorders detected by rWGS 
but not NewbornDx, the 95% CI will be 0.03 to 0.07.  Since the distribution of time to 
diagnosis is unknown, it is difficult to speculate as to the width of the confidence interval 
for the mean or median. However, 40% to 60% (160 to 240 infants) are expected to 
have a genetic diagnosis which is a sufficient number for a precise estimate. 
 
Table: Confidence Interval Limits with 400 infants 
Proportion Lower 
Limit 
Upper 
Limit 
0.05  
0.03  
0.07  
0.10  
0.07  
0.13  
0.20  
0.16  
0.24  
0.30  
0.25  
0.35  
0.40  
0.35  
0.45  
0.50  
0.45  
0.55  
 
9.3 POPULATIONS FOR ANALYSES 
 
 ICF Dataset: All families who were approached for study participation  
 Intention-to-Treat (ITT) Dataset: all consented infants 
 Modified Intention-to-Treat Dataset: all consented infants for whom both 
NewbornDx and rWGS genomic sequencing results were returned 
 Per-Protocol Dataset: all consented infants who received both NewbornDx and 
rWGS (modified intention-to-treat dataset) and completed the study period for the 
analysis of the primary and secondary objectives without clinically important 
protocol deviations. Clinically important protocol deviations will be identified and 
documented prior to the database lock.  
 Time to Molecular Diagnosis Dataset: The Modified Intention-to-Treat Analysis 
Dataset excluding the infants who did not have a confirmed positive result  
 Urgent Testing Dataset: All infants who required urgent testing per section 8.1 

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 Safety Analysis Dataset: All infants for whom blood specimens were obtained or 
data collected 
 
9.4 STATISTICAL ANALYSES 
 
9.4.1 GENERAL APPROACH 
 All analyses will be descriptive. We will calculate the proportion and 95% CI among 
enrolled infants with confirmed genetic disorders detected by NewbornDx, by rWGS, by 
NewbornDx but not rWGS, and by rWGS but not NewbornDx. We will also calculate the 
95% CI for the ratio of the proportion detected by NewbornDx relative to the proportion 
detected by rWGS69. Although detection by SOC is an exclusion criterion, some infants 
will receive a diagnosis by SOC after enrollment. We will therefore also calculate the 
proportion and 95% CI of those receiving a diagnosis by SOC prior to the time that 
results from NewbornDx or rWGS become available. We will calculate the proportions 
and CIs for enrollees with no confirmed diagnosis of a genetic disorder. These will be 
calculated at discharge and at 1 year CGA. We will calculate the proportion of infants 
with conditions detected by NewbornDx that were not listed as part of the differential 
diagnosis at enrollment. The same calculation will be performed for rWGS. We will 
calculate the positive predictive value (PPV) of NewbornDx and rWGS (i.e. the 
proportion of positives that are confirmed by Sanger or PCR).  
 
Because a “gold standard” does not currently exist, it will not be possible to calculate 
the negative predictive value (NPV), sensitivity, or specificity of NewbornDx or rWGS. 
We will calculate the proportion for which there was clinical utility from genomic testing 
(change in drug treatment, surgical treatment, imaging, subspecialty consult, diet, other 
treatment, and redirection of care and/or withdrawal of life sustaining treatment). We will 
also calculate the proportion of testing where trios were ultimately required to confirm a 
diagnosis for rWGS) and if this approach increased the number with a confirmed 
diagnosis. Kaplan-Meier (K-M) curves will be used to estimate the time from enrollment 
to molecular diagnosis (separately for NewbornDx and rWGS) including all enrolled 
infants and excluding infants without a diagnosis of a genetic disorder and those 
diagnosed by SOC before genomic results are returned. The analysis will count as 
positive only those diagnoses that are eventually confirmed. We will also use K-M to 
estimate age at diagnosis and survival. A model will be developed to predict, using 
information available at the start of symptoms, whether the NewbornDx ultimately yields 
a confirmed and actionable diagnosis. Such a model could help to further target 
NewbornDx to the population that would benefit most from it, especially if the model 
discrimination is high (c-statistic greater than .90). The model will be validated using 
bootstrap validation. 
 
 

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9.4.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT(S) 
 
Primary Endpoints 
Definition 
Scale and 
measure  
Populations for 
which the 
analysis will be 
conducted 
A confirmed 
positive result 
detected by 
NewbornDx  
If NewbornDx and the 
confirmatory test have the 
same positive finding 
Binary: Yes/No; 
single endpoint 
Modified 
Intention-to-
Treat Dataset 
A confirmed 
positive result 
detected by rWGS 
If rWGS and the confirmatory 
test have the same positive 
finding 
Binary: Yes/No; 
single endpoint 
Modified 
Intention-to-
Treat Dataset 
Time to a positive 
result by 
NewbornDx *  
Among those who have a 
confirmed positive result at one 
year CGA by NewbornDx, 
rWGS, or another genetic test, 
comparison of date/time of 
sample collection to date/time 
of completion of NewbornDx 
prior to confirmation testing 
Interval: Hours; 
single endpoint 
Time to 
Molecular 
Diagnosis 
Dataset 
Time to a positive 
result by rWGS*  
Among those who have a 
confirmed positive result at one 
year CGA by NewbornDx, 
rWGS, or another genetic test, 
comparison of date/time of 
sample collection to date/time 
of completion of rWGS prior to 
confirmation testing 
Interval: Hours; 
single endpoint 
Time to 
Molecular 
Diagnosis 
Dataset 
Time to a result by 
NewbornDx* 
 
Among all babies tested, 
comparison of date/time of 
sample collection to date/time 
of completion of NewbornDx 
prior to confirmation testing 
Interval: Hours; 
single endpoint 
Modified 
Intention-to-
Treat Dataset 
Time to aresult by 
rWGS*   
Among all babies tested, 
comparison of date/time of 
sample collection to date/time 
of completion of rWGS prior to 
confirmation testing 
Interval: Hours; 
single endpoint 
Modified 
Intention-to-
Treat Dataset 
Time from 
enrollment to a 
positive result 
Among those who have a 
confirmed positive results at 
one year CGA by NewbornDx, 
rWGS or another genetic test, 
Interval: Hours; 
single endpoint 
Time to 
Molecular 

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Primary Endpoints 
Definition 
Scale and 
measure  
Populations for 
which the 
analysis will be 
conducted 
detected by 
NewbornDx* 
comparison of date/time of 
informed consent to date/time 
of completion of NewbornDx 
prior to confirmation testing 
Diagnosis 
Dataset 
 
Time from 
enrollment to a 
positive result 
detected by rWGS* 
Among those who have a 
confirmed positive results at 
one year CGA by  NewbornDx,  
rWGS, or another genetic test, 
comparison of date/time of 
informed consent to date/time 
of completion of rWGS prior to 
confirmation testing 
Interval: Hours; 
single endpoint 
Time to 
Molecular 
Diagnosis 
Dataset 
Clinical utility of 
genomic 
sequencing 
If clinical action was taken due 
to return of results  
 
Time from result to action 
taken 
 
Types of clinical actions taken 
(i.e. surgery, medication) 
 
Clinician opinion of utility 
Binary: Yes/No 
 
 
Interval 
 
 
Categorical  
 
Ordinal 
Modified 
Intention-to-
Treat Analysis 
Dataset 
If trio testing was 
required to obtain a 
diagnosis for 
rWGS 
If Trios were done  
 
 
If Trios increased diagnostic 
yield 
Binary: Yes/No; 
single endpoint 
 
Binary: Yes/No; 
single endpoint  
Modified 
Intention-to-
Treat Analysis 
Dataset 
If enrollees did not 
receive a diagnosis 
No confirmed diagnosis of a 
genetic disorder identified by 
any of the genomic sequencing 
tests  
Binary: Yes/No; 
single endpoint 
Modified 
Intention-to-
Treat Analysis 
Dataset 
*The infants who met the criteria for urgent testing may be analyzed separately. 
 
 

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9.4.3 ANALYSIS OF THE SECONDARY ENDPOINT(S) 
 
Secondary Endpoints 
1. One-year cost-effectiveness of NewbornDx and rWGS compared with SOC 
testing, from a societal perspective 
2. Lifetime year cost-effectiveness of NewbornDx and rWGS compared with SOC 
testing, from a societal perspective  
 
We will conduct an economic evaluation that will compare costs and health outcomes 
for infants with suspected genetic disorders who undergo: 1) traditional standard of care 
diagnostic testing, 2) NewbornDx, or 3) rWGS. One-year post-identification outcomes 
for NewbornDx and rWGS will be estimated from trial data, while outcomes for standard 
of care will rely on retrospective chart review.  We will use simulation modeling to 
extend our projections beyond 1-year post identification to a lifetime time horizon.  
 
In Trial Data Collection for NewbornDx and rWGS: 
This clinical trial provides a unique opportunity to examine observed health outcomes, 
care utilization, and family resource use for infants being evaluated for a suspected 
genetic disorder. During the trial, charge and cost data will be collected from the total 
hospital bill at discharge home from the index hospitalization in which the infant is 
enrolled in the study. Following discharge, the parent will be contacted every 30-days to 
collect resource utilization related to medical visits, hospitalizations, diagnostic tests, 
labs, procedures, and medications administered after discharge home until one year of 
age corrected for prematurity. Data will also be collected on: 1) mother and father lost 
work days; and 2) observed health outcomes, including death, diagnoses, and the 
timing of diagnosis. We will use a quality of life instrument to measure parental quality of 
life at monthly follow-up and to infer the value parents place on avoiding adverse health 
conditions. We will also collect parent-reported quality of life of the infant using a visual 
analog scale. Health economics practice guidelines call for inclusion of such outcomes, 
particularly in child health intervention evaluations59,60. 
 
Assessment of SOC testing 
As no infant in our trial will only receive SOC testing only (i.e., without NewbornDx and 
rWGS), we will estimate standard of care testing outcomes (costs, diagnostic findings, 
health outcomes) by using a retrospective chart review.   
In the retrospective chart review, we will collect resource utilization and health care 
outcome data from 300 infants born 12-18 months prior to the start of the study who had 
a genetic evaluation, but did not receive NewbornDx and rWGS. We chose infants born 
in this timeframe to minimize differences between contemporary clinical practice and 
clinical practice received by our retrospective cohort, hence maximizing the 
generalizability of our retrospective data to contemporary settings.   
 
 
 

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Simulation: Extending outcome estimates to a lifetime time horizon  
Following data collection, we will use simulation modeling to estimate outcomes (cost, 
and health outcomes) for all strategies: NewbornDx, rWGS and standard of care 
diagnostic testing over a 1-year and lifetime horizon. For each strategy, the simulation 
model will project disorder progression based on estimated health state transition 
probabilities. By associating each health condition (or “health state”) with a “utility 
weight” (QALYs gained per year in that state) and annual cost, the simulation will track 
hypothetical infants and sum QALY gains and incurred costs. Repeating this process 
with a large number of simulated infants will yield estimated population health and cost 
outcomes. Costs and utility weights will be estimated from trial data, and previously 
published estimates, making the assumption that these values depend only on the 
infant’s health state and not on the diagnostic testing technology. Thus, we assume that 
the costs and utility weight estimates apply to infants undergoing any of the three 
diagnostic testing approaches. 
 
Since NewbornDx and rWGS will identify a large number of disorders, disorders will be 
grouped based on estimated life expectancy and long-term severity. We will identify 
“disorder groups” by reviewing available data and literature on the expected outcomes 
of each identifiable disorder in conjunction with expert clinical judgment. We will also 
identify health states that appropriately represent the possible outcomes for each 
“disorder group” following review and synthesis of available evidence with input from our 
Steering Committee and DSMB.  
 
Our lifetime simulation model will require: 1) classification of health states that describe 
health outcomes associated with each study strategy (e.g. disorder, disability, death); 2) 
health state transition probabilities, which represent the likelihood that simulated 
patients will develop specific disorder and disability outcomes, conditional on their 
assigned strategy; 3) health state costs, which include both health care associated with 
a specific health state and other societal costs; and 4) health state utilities, which 
represent health state preferences for avoiding adverse health outcomes. For the time 
period extending beyond the 1-year CGA trial duration, we will identify model transition 
probabilities, health state costs, and health utilities from existing primary datasets and 
the literature. For many genetic disorders, the literature reports limited long-term follow 
up data that appropriately account for expected differences in health outcomes due to 
differences in time to diagnosis. Available data will guide short-term extrapolation 
beyond our trial timeframe. For long-term time periods for which there are no 
informative data, we will draw on expert opinion, and conduct appropriate sensitivity 
analyses to characterize the impact of the attendant uncertainty. Health state costs will 
include the health care and societal costs (e.g. family costs, special education). We will 
estimate health care utilization, family time and other resources use for each health 
state from available data sources and then multiply these utilization estimates by 
national unit prices. We will also estimate child health state utilities using available data 
and will include utility values for parental quality of life when possible. 
 
Cost-effectiveness analysis: With simulation findings corresponding to the 1-year CGA 
trial follow-up period and for extrapolation to a lifetime horizon, we will: 1) calculate 

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aggregate population costs and health outcomes (QALYs) for each trial strategy; 2) rank 
the strategies from least to greatest aggregate population health (total QALYs); and 3) 
calculate incremental QALYs and costs for the second strategy (compared to the first) 
and for the third strategy (compared to the second). Incremental QALYs are, by our 
sequencing of these comparisons, non-negative. If corresponding incremental costs are 
likewise non-negative, an incremental cost-effectiveness ratio (ICER) (incremental costs 
divided by incremental QALYs) will be calculated in a similar fashion. If incremental 
costs are negative in either case – that is, if either strategy reduces costs while 
improving health relative to its comparator – we refer to this strategy as “cost-saving”. 
 
Secondary 
Endpoint  
Definition 
Scale  
Populations for which 
the analysis will be 
conducted 
Satisfaction of the 
web-based clinical 
reference database  
Usefulness of information 
 
Ease of use of the clinical 
reference database 
Categorical 
  
Medical providers who 
use the clinical 
reference database 
after it is built 
 
In order to qualitatively assess the impact of the intervention, we will collect surveys 
from medical providers across all sites. This survey will be submitted to the IRB prior to 
implementation. 
 
9.4.4 SAFETY ANALYSES 
 
All-cause mortality will be evaluated at discharge home from the hospitalization during 
which enrollment occurs and at one-year CGA. Data will be reviewed to determine if 
diagnoses are made in a timely fashion and if the diagnoses are reported accurately.  
 
9.4.5 BASELINE DESCRIPTIVE STATISTICS 
 
Population description: Gestational age, birth weight, day of life at admission, and 
length of stay in the hospital will be summarized with means, medians, standard 
deviations, inter-quartile ranges, and 95% confidence intervals (CI). Prenatal 
evaluations, complications, and mode of delivery will be summarized with frequencies 
and percentages. Signs/ symptoms that warranted the testing will be summarized with 
frequencies and percentages.  
 
9.4.6 PLANNED INTERIM ANALYSES  
 
Interim analyses are not planned.  
 
9.4.7 SUB-GROUP ANALYSES 
 
The primary or secondary endpoints will not be analyzed by demographic sub-groups; 
we do not expect different results according to sex or race/ethnicity.  

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9.4.8 TABULATION OF INDIVIDUAL PARTICIPANT DATA 
 
N/A 
 
9.4.9 EXPLORATORY ANALYSES 
 
A review of the conditions detected by rWGS but not NewbornDx will be conducted and 
this review may potentially improve the NewbornDx panel. 
 
 

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10 SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS 
 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT 
CONSIDERATIONS 
 
10.1.1 
INFORMED CONSENT PROCESS 
 
10.1.1.1 
CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS 
PROVIDED TO PARTICIPANTS 
  
Consent materials for this protocol include and the Informed Consent Form and a 
Secondary Findings Parent Handout, both of which will be IRB-approved prior to use. 
The ICF will describe in detail study procedures and risks and will include check boxes 
to elect to receive secondary findings for the infant and each biological parent. The 
same informed consent forms and when applicable, assent forms, will be used at all 
sites. The ICF will be translated into Spanish. Short-forms will be used for all languages 
other than English or Spanish.  
 
10.1.1.2 
CONSENT PROCEDURES AND DOCUMENTATION 
 
 
Documentation of Consent 
Written documentation of informed consent is required prior to enrolling in the study or 
conduct of any study procedures with the exception of IRB-approved recruitment efforts 
and the unusual circumstance described in #7 of the telephone consent procedures. 
Written documentation of consent is required for the participation by the infant and each 
biological parent providing a blood sample.  
 
Consent when the Parent/Guardian of the Infant or a Biological Parent is > 18 Years 
Participation of the infant requires written documentation of consent by one parent/ 
guardian.  Participation of a biological parent requires written documentation of consent 
by that biological parent. 
 
Consent when the Parent/Guardian of the Infant is < 18 Years Old 
State 
Who provides consent for the 
Infant’s participation? 
Must a minor parent provide 
assent for the infant’s 
participation? 
California 
Minor parent 
N/A 
Massachusetts 
Minor parent 
N/A 
New York 
Minor parent 
N/A 
North Carolina 
One guardian of the minor parent No 
Ohio 
Minor parent 
N/A 
Pennsylvania 
Minor parent 
N/A 

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Consent when a Biological Parent is < 18 Years Old 
State 
Who provides consent for 
participation by the minor 
parent? 
Must a minor parent provide 
assent for his or her own 
participation? 
California 
One guardian of the minor 
parent 
Yes  
Massachusetts 
Minor parent  
N/A 
New York 
Minor parent  
N/A 
North Carolina 
One guardian of the minor 
parent 
Yes, assent signature line on 
ICF 
Ohio 
One guardian of the minor 
parent 
Yes, assent signature line on 
ICF 
Pennsylvania 
One guardian of the minor 
parent 
Yes, assent signature line on 
ICF 
 
Assent for a minor parent’s participation is required by 4 sites. North Carolina Children’s 
Hospital, Children’s Hospital of Pittsburgh and Cincinnati Children’s Hospital Medical 
Center will use an additional assent signature line on the ICF. A separate assent form 
for participation by a minor parent will only be used by Rady Children's Hospital - San 
Diego and submitted to the central IRB at the time of their site IRB application. 
 
By signing the informed consent form, the infant’s parent/guardian agrees that the infant 
will complete all evaluations required by the study, unless the infant’s parent/guardian 
withdraws the infant voluntarily or the infant is withdrawn from the trial for any reason.  
 
 The original signed ICF and if applicable, assent documents, will be put in the infant’s 
research file. A copy of the signed informed consent/assent documents will be: 
• provided to the parent/guardian(s) and biological parent(s) as applicable 
• put into the infant’s medical record and if applicable, each parent’s medical 
record 
An entry into the infant’s medical record will indicate informed consent was obtained 
(refer to the Documentation of Informed Consent template).  
 
Process of Informed Consent 
 
Informed consent is a process initiated prior to the individual’s agreeing to participate in 
the study and continuing throughout the individual’s study participation.  
 
1. Research staff at the enrolling site will communicate with the clinical team prior to 
approaching a parent/guardian. The clinical genetics team will be consulted as 
dictated by SOC at each site. The clinicians may be asked to speak with the 
parent/guardian(s) prior to their approach by the research team depending on 
family and clinical circumstances.  

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2. The parent/guardian(s) of the infant and as applicable, the biological parents of 
the infant, will be approached directly for consent. If the parent/guardian(s) are 
not directly available, they may be contacted via telephone in extenuating 
circumstances (see telephone consent below).  
3. Research staff will provide a comprehensive summary of the study including 
contact information for the research team, and if requested, consent/assent 
documents.  
4. The site-PI, co-investigator or a study coordinator who is experienced in genetic 
studies will obtain consent. This person will verbally explain the research study 
and answer any questions. The information available on each genetic 
sequencing platform including primary findings, secondary findings and the risk 
of finding out about mistaken paternity or suspected incest will be described 
(refer to section 2.3 Risks/Benefits). The following will be emphasized: 
participation is voluntary; the infant may be withdrawn from the study at any time 
without prejudice, and the decision not to participate will in no way adversely 
affect the quality of their infant’s medical care.   
5. The parent/guardian(s) and biological parents will have the opportunity to review 
the written consent form and ask questions prior to signing. Parent/guardian(s) 
will be given as much time to consider the study and read the consent 
documents as necessary. 
 
Non-English Speakers 
1. For non-English speaking families, a fully translated standard consent or an oral 
presentation accompanied by a short form may be used to obtain informed 
consent. The fully translated consent and the short form will be approved by the 
central IRB prior to its use.  
2. A Spanish translation of the informed consent form will be available; a short form 
will be used for all other languages. If a short form is not available for the family’s 
preferred language, consent will be obtained through a certified interpreter and 
documented on the English consent form. Short forms approved by the Johns 
Hopkins IRB can be found here: 
https://www.hopkinsmedicine.org/institutional_review_board/forms/short_form_tr
anslation.html 
3. An interpreter from the hospital will be called to assist with the consent process if 
needed. When an interpreter is not available in-person, a professional phone 
interpreter service may be used. Family members should not serve as an 
interpreter due to the complex nature of genetic testing.  
4. Interpreters may also serve as a witness to the short form consent process; their 
signature as an interpreter (or an interpreter reference ID number from a 
professional telephone interpreter service) will serve as a witness signature. The 
non-English speaking participant signs the short form consent in their language. 
The person obtaining consent signs the English version full consent. The 
interpreter/witness understands both languages, so signs both. 
5. The parent/guardian(s) and biological parent(s) who sign the consent documents 
will be given a copy of these documents. 
 

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Telephone consent 
It is the intention of the study team to obtain written consent from the parent/guardian 
for the infant and each biological parent if providing a sample whenever possible. It is 
anticipated that in certain circumstances, it may be necessary for the consent process 
to be conducted via telephone with written documentation of consent to follow. 
Examples of when this might be needed include: when the biological mother remains at 
the birthing hospital after her infant is transferred to the enrolling site and a 
parent/guardian or biological parent cannot physically visit because the family lives too 
far away. If the consent process is not able to be conducted in-person, telephone 
consent may be used with written documentation of consent to follow. 
   
The PI will ensure the procedures for securing telephone consent are followed: 
1. The absent party will be contacted by phone. The research staff will explain the 
entire research study and the process required for consent by telephone.  
An email address or fax number will be obtained in order to forward the consent 
document(s). A time will be scheduled for a full informed consent when both parties 
will be able to have the consent physically in front of them to sign.  For non-English 
speaking persons, refer to procedures for Non-English speaking persons above. 
2. The consent document(s) will be sent to the absent party. The absent party will be 
advised that they are welcome to read this document, but they will be asked not to 
sign the document until the time of the scheduled consent. 
3. At the time of the scheduled consent, the research staff will have a witness present 
to confirm that the telephone consent was conducted. All study information will be 
discussed, and any questions answered.  
4. Following the explanation of the study, the absent party will be asked to sign and 
date the document. The research staff and witness will also sign and date their copy. 
5. The absent party will be asked to return the signed document to the research team, 
either electronically or if necessary, by mail.  
6. Copies of both signed documents will be made and returned to the absent party. 
7. Written documentation of consent will be obtained prior to any study activities with 
one exception. In the circumstance where testing is urgent (clinical situation is life-
threatening) and written documentation of consent is not readily feasible (i.e. no 
access to a fax machine or scanner), verbal consent as documented by the research 
staff will be sufficient to obtain, ship and process blood samples. However, written 
documentation of consent by the parent/guardian must be documented prior to the 
return of any results.  
8. If necessary, the research staff will attempt to arrange for a blood collection kit to be 
delivered to the absent party. In the case where the biological mother is at another 
hospital and wishes to provide a blood sample, we will work with the birthing hospital 
to collect the sample and return the consent documentation.  
9. The consent will be documented per hospital and IRB policies. 
 
New Information 
Any new information that may affect willingness to continue in the study will be 
communicated by the investigator to the parent/guardian(s) or biological parent(s) in 

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accordance with IRB requirements. The informed consent document will be updated 
and re-consent will be obtained if necessary.    
 
Waiver of Consent 
Waiver of consent is requested for the clinician survey. After the genomic sequencing 
result is returned for an infant, the physician of record for the infant will complete a 
survey about changes in care from study results and perceived utility of testing. 
10.1.2 
STUDY DISCONTINUATION AND CLOSURE 
 
This study may be temporarily suspended or prematurely terminated if there is 
reasonable cause. The trial will be stopped for a safety review by the DSMB if at any 
point the act of conducting the trial is deemed detrimental or unsafe to infants. Reasons 
for stopping the trial may include the reporting of inaccurate test results due to either 
problems with the technology or the medical information interface. The trial may also be 
stopped if a data breech has occurred that puts an infant’s personal genomic data at 
risk. Formal stopping rules will be developed with the DSMB prior to the trial beginning. 
 
If the study is prematurely terminated or suspended, written notification documenting 
the reason for study suspension or termination will be provided by the study-wide 
Principal Investigator (PI) to site investigators, NCATS, the DSMB and the IRB. The site 
investigators will promptly inform the parent/guardian(s) of enrolled infants who have not 
yet completed the study and inform them of any changes to study procedures. 
  
Circumstances that may warrant termination or suspension include, but are not limited 
to: 
 Determination of unexpected, significant, or unacceptable risk to infants 
 Insufficient compliance to protocol requirements 
 Data that are not sufficiently complete and/or evaluable 
 
The study may resume once concerns about safety, protocol compliance, and data 
quality are addressed, and satisfy the DSMB, NCATS and the IRB. 
 
10.1.3 
CONFIDENTIALITY AND PRIVACY  
 
Confidentiality will be maintained by the participating investigators, their staff, and the 
sponsor(s). This confidentiality covers the clinical information relating to participating 
infants, testing of biological samples and genetic tests.  
 
All study related material will be stored securely by the site. Each enrolled infant will be 
assigned a unique study identification number which does not include any identifiers. 
The information entered into the password-protected REDCap System and the 
documents used for remote monitoring will be identified by the Infant Study ID number. 
Each site will keep the link between the Infant Study ID number and infant identifiers for 

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all subjects enrolled at their site in a secure manner (password-protected spreadsheet 
or hand-written log securely locked).  
 
The ICF will clearly indicate that the labeling of specimens, medical information and 
reports to/from the laboratories will contain identifying information. Confirmed genomic 
testing results will be returned from the laboratory to the site and the infant’s EMR and 
will contain patient identifiers. All genomic sequencing data will be electronically stored 
in password-protected databases at the laboratory that conducted the test. 
 
Paper-based study files, including the original ICF/assent documents and contact 
information related to the infant will be stored in a secure office of the research staff for 
internal use during the study. A copy of the signed ICF will be placed in the infant’s 
medical record. At the end of the study, all records will continue to be kept in a secure 
location (i.e. a secure storage facility) for as long a period as dictated by the reviewing 
IRB, Institutional policies, or sponsor requirements. 
 
Study research data about the infant, which is for purposes of statistical analysis and 
scientific reporting, will be transmitted to and stored at the Tufts Medical Center Data 
Coordinating Center. The REDCap and study management systems used by clinical 
sites and by Tufts Medical Center research staff will be secured and password 
protected. At the end of the study, all study databases will be de-identified and archived 
at Tufts Medical Center. 
 
Per Section 2012 of the 21st Century Cures Act as implemented in the 2017 NIH 
Certificates of Confidentiality Policy, this study is automatically issued a COC. This 
certificate protects identifiable research information from forced disclosure. It allows the 
investigator and others who have access to research records to refuse to disclose 
identifying information on research participation in any civil, criminal, administrative, 
legislative, or other proceeding, whether at the federal, state, or local level. By 
protecting researchers and institutions from being compelled to disclose information that 
would identify research participants, Certificates of Confidentiality help achieve the 
research objectives and promote participation in studies by helping assure 
confidentiality and privacy to participants. The fact that a COC may not apply in all 
circumstances is disclosed in the ICF. 
 
Clinical information will not be released without written permission of the infant’s 
parent/guardian, except as necessary for monitoring by the research staff for this study, 
IRB, DSMB, NIH, or OHRP. Senior project leadership or other authorized 
representatives may inspect all documents and records required to be maintained by 
the PIs. This documentation includes, but is not limited to medical records (office, clinic, 
or hospital) for the infants in this study. Clinical study sites will permit access to such 
records.  
 
Protection against a data security breach 
 Paper-based study files, including original signed ICFs, will be stored in a locked 
office of the study team members or a secure storage facility. A copy of the 

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signed ICF will be placed in the subject’s medical record (paper or scanned in to 
the EMR as applicable). All genomic sequencing data will be electronically stored 
accessible only to research personnel.   
 The RCIGM ordering portal to which each infant’s medical record is uploaded is 
HIPAA-compliant and requires a login and password 
 Any PHI distributed over email (return of results) must be encrypted and 
distributed via secure institutional email systems. Results will be returned to the 
electronic medical record consistent with each site’s HIPAA-compliant 
institutional policies.  
 Electronic study files containing PHI (i.e. a screening log) at each site must be 
kept secure (i.e. password-protected file in a restricted access folder maintained 
on a secure internal institutional server) 
 The REDCap system is password protected; limited staff at each site will be 
granted password login ID. All personnel must provide an active organizational 
email address before being issued an ID. The REDCap system requires complex 
passwords, and the frequency of forced password changes is every 90 days. 
Enrolling sites will only have access to data entered into the REDCap system for 
infants enrolled at their own site with the exception of the study-wide PIs, DCC 
and monitoring personnel who will have access to all sites’ data. Enrolling sites 
will not have access to export study data.  
 The data center in which the REDCap servers are housed has strict access 
control; only authorized core personnel may access the facility unescorted. Only 
authorized users are allowed to connect to the network and the security of the 
network is actively monitored. Power and environmental controls have several 
layers of backups. The institution actively logs and monitors all communication to 
the server (multiple firewall layers prevent direct external communication to the 
server) and is alerted to any unusual activity. If warranted, the institution will 
immediately as well as automatically ban offending IP addresses at the perimeter 
before they reach the server. The application itself also rejects and bans IP 
addresses of anything it considers abnormal access. REDCap Server(s) are 
regularly backed up. All transactions are securely delivered to the application 
using SSL (SHA‐1 with RSA Encryption; 2048‐bits). It is then transmitted 
internally (behind the firewall) to the database server. All transactions are logged 
at the server layer (http logging), application layer (REDCap logs activity to a 
database table), and the database layer. 
 
 

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10.1.4 
FUTURE USE OF STORED SPECIMENS AND DATA  
  
Blood samples/DNA  
Blood samples/DNA will be kept by the laboratories for the duration of the study period 
(up to 5 years) for the purpose of further testing of the infant. The specimens will then 
be destroyed at the conclusion of the study.   
 
Raw Sequencing Data  
The raw sequencing data generated by this research will be kept by Athena (Quest) 
Diagnostics and RCIGM for at least the length of the 5-year study period. The 
laboratories may destroy raw sequencing data after the conclusion of the study but 
should not do so without permission from the study wide PIs. Prior to destruction of any 
raw sequencing data, genomic sequencing data may be: 
1. Used to re-analyze VUS; in the event an infant’s diagnosis changes, we will 
attempt to contact the parents 
2. De-identified and transferred to a third party for use in a future study to further 
understand neonatal and childhood diseases  
 
Sharing of Genomic Research Data 
This prospective, multi-center clinical trial will generate genomic sequencing data that is 
classified as ‘Level 4’Data by the Genomic Data Sharing (GDS) Policy of the National 
Institutes of Health. Sequencing data that relates genomic data to phenotype or other 
biological states will be generated. As such, all human genomic data will be released in 
accordance to the NIH GDS Policy which establishes the timelines for submission and 
subsequent release of data for access by secondary investigators. Specifically, data 
(results of testing) will be provided to infants and their caregivers when complete and 
validated (with the exception of provisional results which are given prior to confirmation). 
In addition, data, including genome sequences (fastq files), variants (vcf files), and 
associated HIPAA compliant clinical metadata will be deposited in the Longitudinal 
Pediatric Data Resource (LPDR; https://www.nbstrn.org/research-tools/longitudinal-
pediatric-data-resource). The LPDR is being developed by the Newborn Screening 
Translational Research Network (NBSTRN). The LPDR is a secure informatics system 
designed to enable enhanced data collection, sharing, management and analysis for 
conditions identified as part of newborn screening or for conditions that may benefit 
from newborn screening, including NICU genomic sequencing. The NBSTRN is funded 
by the NICHD (contract #HHSN275201300011C). The LPDR is currently being used to 
deposit data from infants in the NICU and PICU who have already had rWGS performed 
related to the NICHD/NHGRINSIGHT program (PI Dr. Kingsmore). Much of the 
infrastructure for those datasets will serve as a template for the dissemination of data 
from this proposal. The LPDR, in turn, will deposit data in the Controlled Access section 
of the NCBI dbGAP. Variants with ACMG recommended pathogenicity assessments will 
be deposited in ClinVar. Novel disorder gene assertions will be deposited in ClinGen 
(https://clinicalgenome.org/). We anticipate annual data submissions supplemented by 
specific dataset deposits as manuscripts arising from this work are submitted for 
publication. 
 

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10.1.5 
KEY ROLES AND STUDY GOVERNANCE   
  
Co-Principal Investigators: 
 
Jonathan M. Davis, MD 
Tufts Medical Center 
800 Washington Street, Box 44, Boston, MA 02111 
Tel: 617-636-5322 
jdavis@tuftsmedicalcenter.org 
 
Jill Maron, MD, MPH 
Tufts Medical Center 
800 Washington Street, Box 44, Boston, MA 02111 
Pager: 617-647-2971 
jmaron@tuftsmedicalcenter.org 
 
10.1.6 
SAFETY OVERSIGHT 
 
DSMB 
Safety oversight will be under the direction of a Data and Safety Monitoring Board 
(DSMB). The DSMB is composed of individuals with the appropriate expertise, including 
an ethicist, geneticist, neonatologist and statistician:  
 
Tamsin Knox, MD, MPH (CHAIR) 
Associate Director, Regulatory Group, Tufts Clinical and Translational Science Institute 
Tufts University School of Medicine 
150 Harrison Avenue, Jaharis 2, Boston, MA 02111 
Tel: 617.636.3558 
tamsin.knox@tufts.edu 
 
Jonathan M. Fanaroff MD, JD  
Professor of Pediatrics 
Case Western Reserve University School of Medicine 
Director, Rainbow Center for Pediatric Ethics 
Co-Medical Director, Neonatal Intensive Care Unit 
Rainbow Babies & Children's Hospital 
Cleveland, Oh 44106 
 
Gina M. Geis, MD, MS 
Attending Neonatologist 
Associate Director, Neonatal-Perinatal Medicine Fellowship Program 
Associate Professor of Pediatrics 
Associate Professor in the Center for Bioethics, Education and Research 
Albany Medical Center 
Department of Pediatrics, Division of Neonatology 
Albany, NY 12208     

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Stephanie Sacharow, MD, FACMG 
Assistant Professor, Harvard Medical School 
Medical Genetics, Medical Biochemical Genetics 
Boston Children’s Hospital 
Division of Genetics and Genomics 
 
Hong Chang, PhD 
Statistician and Senior Project Manager, Biostatistics, Epidemiology, and Research 
Design (BERD) Center 
Assistant Professor 
Tufts University School of Medicine 
Boston, MA 
 
Members of the DSMB are independent from the study conduct and free of conflict of 
interest. The DSMB will meet at least annually to assess safety data or more often if 
needed. The DMSB will operate under the rules of an approved charter that will be 
written and reviewed at the organizational meeting of the DSMB. At this time, each data 
element the DSMB needs to assess will be clearly defined. The DSMB will provide its 
input to NCATS. 
 
Steering Committee 
The Steering Committee (Drs. Davis, Diacovo, Gelb, Ginns, Kingsmore, Maron, 
Poindexter, Powell and Vockley) with significant expertise in neonatal clinical trials and 
genomic medicine/testing will oversee the conduct of the trial. The Steering Committee 
will have conference calls every three months (or more often if needed) to review any 
problems, issues, and operations for the study.  
 
10.1.7 
CLINICAL MONITORING 
Clinical site monitoring will be conducted remotely by Tufts Medical Center research 
staff to ensure that 1) the rights and well-being of trial participants are protected; 2) the 
reported trial data are accurate, complete, and verifiable; and 3) the conduct of the trial 
is in compliance with the currently approved protocol/ amendment(s) and with 
applicable requirement(s) as outlined in the DSMP.  
 Data monitoring will be conducted remotely (enrolling sites will upload de-identified 
medical records and source documents to the REDCap system for reference by 
monitoring personnel) 
 Data monitoring will consist of examination of data and processes related to 
informed consent, subject eligibility, the accuracy of study primary endpoints, and 
safety/adverse events as outlined in the DSMP 
 Although monitoring will occur throughout the study, the frequency of monitoring will 
vary for each specific process or data point as outlined in the DSMP   

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 The study-wide Co-PIs will be provided copies of monitoring reports quarterly 
 Monitoring will include, but are not limited to, review of essential documents, 
accountability records, CRFs, ICFs, medical and laboratory reports, and protocol 
compliance 
 Each site principal investigator will provide direct access to study-related documents 
for monitoring and auditing by the data coordinating center or its representative, and 
inspection by local and regulatory authorities 
 If a significant number of issues at a site are identified, additional training, increased 
remote monitoring or in-person monitoring may occur 
10.1.8 
QUALITY ASSURANCE AND QUALITY CONTROL 
 
The site PI will provide direct access to all trial-related sites, source data/documents, 
case report forms, and reports for the purpose of monitoring and auditing by Tufts 
Medical Center, and inspection by local and regulatory authorities. The site PI will also 
ensure that all study personnel are appropriately trained and the applicable 
documentation is maintained on site. 
 
Tufts Medical Center research staff will verify that the clinical trial is conducted, and the 
data generated, documented (recorded), and reported in compliance with the protocol 
and 45 CFR Part 46 as outlined in the DSMP. 
 
The DCC will implement quality control procedures beginning with the data entry system 
and generate data quality control checks, such as automatic range checks, to identify 
data that appear inconsistent, incomplete, or inaccurate. Any missing data or data 
anomalies will be communicated to the site(s) for prompt clarification and resolution.   
 
10.1.9 
DATA HANDLING AND RECORD KEEPING 
 
10.1.9.1 
DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  
 
Data collection is the responsibility of site staff under the supervision of the site PI. The 
investigator is responsible for ensuring the accuracy, completeness, legibility, and 
timeliness of the data reported. Adverse events must be graded, assessed for severity 
and causality, and reviewed by the site principal investigator or designee.  
The DCC for this study will be responsible for data management, quality review, 
analysis, and reporting of the study data. 
 
Data for each participant will be captured on eCRFs. Study personnel at each site will 
enter clinical data from the medical record or source document directly into REDCap. 
The data system includes password protection and internal quality checks, such as 
automatic range checks, tracking of missing data, and the ability to create and track 
manual queries. The medical record will serve as the source documents in most cases. 
However, some data may need to be captured separately on a source document (for 

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example, date and time of sample collection). All source documents should be 
completed to ensure accurate interpretation of data. When making a change or 
correction, the original entry should be crossed out with a single line, and the change 
should be initialed and dated. Avoid the use of erasers, overwriting, or correction 
fluid/tape on the original. Data reported in the eCRF should be consistent with the 
source documents or the discrepancies should be documented in a note-to-file.   
 
10.1.9.2 
STUDY RECORDS RETENTION  
 
Records and documents pertaining to the conduct of this study, including source 
documents, consent forms, and laboratory results must be retained by the investigator 
for at least 7 years after the end of the study or longer as applicable to 
local/state/federal regulations. No records will be destroyed without the written consent 
of NIH. It is the responsibility of the overall study PIs to inform the site investigators 
when these documents no longer need to be retained. 
 
10.1.10 
PROTOCOL DEVIATIONS 
 
A protocol deviation is any non-compliance with the clinical trial protocol or manual of 
procedures requirements. The non-compliance may be on the part of the participant, the 
investigator, or the study site staff. It is the responsibility of the site to identify protocol 
deviations and to promptly develop and implement corrective actions. All deviations 
from the protocol must be reported in REDCap under the protocol deviation eCRF within 
5 working days of identification. A completed and investigator-signed copy of the 
protocol deviation form must be maintained in the file of essential documents. Protocol 
deviations must be submitted to the IRB per their guidelines. The site PI/study staff is 
responsible for knowing and adhering to the central IRB requirements.   
 
10.1.11 
PUBLICATION AND DATA SHARING POLICY 
 
This study will be conducted in accordance with the following publication and data 
sharing policies and regulations: 
 The National Institutes of Health (NIH) Public Access Policy, which ensures that 
the public has access to the published results of NIH funded research, requires 
scientists to submit final peer-reviewed journal manuscripts that arise from NIH 
funds to the digital archive PubMed Central upon acceptance for publication. The 
policy stipulates that these papers must be accessible to the public on PubMed 
Central no later than 12 months after publication.   
 This study will comply with the NIH Data Sharing Policy and Policy on the 
Dissemination of NIH-Funded Clinical Trial Information and the Clinical Trials 
Registration and Results Information Submission rule. As such, this trial will be 
registered at ClinicalTrials.gov, and results information from this trial will be 
submitted to ClinicalTrials.gov. In addition, every attempt will be made to publish 
results in peer-reviewed journals.  Data from this study may be requested from 
other researchers after the completion of the primary endpoint by contacting the 
Co-PIs Jonathan Davis or Jill Maron.  

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 This study will also comply with the NIH Genomic Data Sharing Policy, which 
applies to all NIH-funded research that generates large-scale human or non-
human genomic data, as well as the use of these data for subsequent research. 
Large-scale data include genome-wide association studies (GWAS), single 
nucleotide polymorphisms (SNP) arrays, and genome sequence, transcriptomic, 
epigenomic, and gene expression data. Refer to section 10.1.4 of this protocol 
for further information. 
 
In addition, the following groups may share the data to improve new treatments or other 
clinical trials (this language will be included in the ICF): 
 Health authorities throughout the world (e.g., Food and Drug Administration, 
European Medicines Agency, NIH, etc.) 
 Institutional Review Boards 
 Other groups such as academic, government, or industry researchers; public-
private partnerships; and/or external research collaborations. These entities will 
have oversight committees that will supervise the ethical use of the data  
 
The Steering Committee will be responsible for developing publication procedures and 
resolving authorship issues.     
 
If the parent/guardian would like to enroll the infant or him or herself in another research 
study that will utilize sequencing data, the enrolling site/labs may give the raw 
sequencing data from the GEMINI study directly to the other study upon receipt of a 
HIPAA authorization form signed by the infant’s parent/guardian. 
 
10.1.12 
CONFLICT OF INTEREST POLICY 
 
The independence of this study from any actual or perceived influence is critical.  
Therefore, any conflict of interest of persons who have a role in the design, conduct, 
analysis, publication, or any aspect of this trial will be disclosed. Furthermore, persons 
who have a perceived conflict of interest will be required to have such conflicts 
managed in a way that is appropriate to their participation in the design and conduct of 
this trial. Site investigators/study team members must adhere to their local requirements 
for disclosure of conflicts of interest and the local site reviews and manages any 
reported conflicts of interests. The local site must provide a copy of each research team 
members’ reported conflict of interests to 1) the JHU IRB and 2) the study-wide co-PIs. 
The JHU IRB will receive any management plans from relying sites and review them as 
part of oversight/review responsibilities but does not perform the initial review instead of 
the local site nor manage reported interests. 
 
 
 

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11 ABBREVIATIONS 
 
ACMG 
American College of Medical Genetics and Genomics 
AE 
Adverse Event 
ASHG 
American Society of Human Genetics 
BW 
Birth Weight 
BWA 
Burrows-Wheeler Aligner 
CFR 
Code of Federal Regulations 
CGA  
Corrected Gestational Age 
CLIA 
Clinical Laboratory Improvement Amendments 
CNVs 
Copy Number Variants 
COC 
Certificate of Confidentiality 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
DBS 
Dried Blood Spot 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DRE 
Disease-Related Event 
DRG  
Diagnosis-Related Group 
DSMB 
Data Safety Monitoring Board 
DSMP 
Data Safety and Monitoring Plan 
EC 
Ethics Committee 
eCRF 
Electronic Case Report Forms 
EMA 
European Medicines Agency 
EMR 
Electronic Medical Record 
FDA 
Food and Drug Administration 
GA 
Gestational Age 
GATK  
Genome Analysis Toolkit 
GCP 
Good Clinical Practice 
GDS   
Genomic Data Sharing 
GINA 
Genetic Information Nondiscrimination Act 
GLP 
Good Laboratory Practices 

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GWAS 
Genome-Wide Association Studies 
HIPAA 
Health Insurance Portability and Accountability Act  
HPO 
Human Phenotype Ontology 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation  
ICMJE 
International Committee of Medical Journal Editors 
IGV 
 
Integrated Genome Viewer 
INC 
 
International Neonatal Consortium 
IRB 
Institutional Review Board 
LAR 
Legally Authorized Representative 
MOP 
Manual of Procedures 
N 
 
Number (typically refers to participants) 
NBS  
Newborn Screening 
NCATS 
 National Center for Advancing Translational Sciences 
NICU 
Neonatal Intensive Care Unit 
NIH  
National Institutes of Health 
NPV 
 Negative Predictive Value 
OHRP 
Office for Human Research Protections 
OMIM  
Online Mendelian Inheritance in Man 
PCR  
Polymerase Chain Reaction 
PHI 
 
Protected Health Information 
PI 
Principal Investigator 
PMA 
Postmenstrual age (gestational age plus postnatal age) 
PNA 
Postnatal age 
PPV  
Positive Predictive Value 
QA 
Quality Assurance 
QALY  
Quality-Adjusted-Life Year 
QC 
Quality Control 
RCIGM 
 Rady Children’s Institute for Genomic Medicine 
rWES 
Rapid Whole Exome Sequencing 
rWGS 
Rapid Whole Genome Sequencing 

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SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SNP 
Single nucleotide polymorphism 
SOA 
Schedule of Activities 
SOC 
Standard of Care 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
STR  
Short Tandem Repeat 
Sub-I  
Sub Investigator 
SVs 
 
Structural Variants 
NewbornDx  NewbornDx Sequencing Evaluation 
UP 
Unanticipated Problem 
US 
United States 
WES 
Whole Exome Sequencing 
WGS 
Whole Genome Sequencing 
 
 
 

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12 PROTOCOL AMENDMENT HISTORY 
 
The table below is intended to capture changes of IRB-approved versions of the 
protocol, including a description of the change and rationale.  
 
Version & Date 
Description of Change  
Brief Rationale 
2.0, 05DEC2019 
The term incidental has been 
added to sections that refer to 
secondary findings. 
The RCIGM laboratory report 
will use the term incidental 
findings, instead of secondary 
findings, to reflect that the lab 
does not conduct a targeted 
search of variants not related to 
the infant’s phenotype.  
2.0, 05DEC2019 
When samples for the infant 
and parent(s) are received at 
the same time, RCIGM will 
routinely generate infant and 
parent sequences 
simultaneously and the data all 
analyzed together as a duo/trio 
as applicable. 
To increase diagnostic yield 
and/or decrease the time to 
result for the subject; 
NewbornDx is run as a trio 
when parent samples are 
available.  
2.0, 05DEC2019 
A retrospective analysis of 
infant-only data among infants 
who were initially analyzed as a 
duo or trio will be conducted. 
To provide data about how 
often a trio is needed to obtain 
the infant’s result. 
2.0, 05DEC2019 
The laboratory testing 
processes for subjects from 
Mount Sinai has been revised 
to reflect the new status of 
NewbornDx as conditionally 
approved by New York State 
and the ramifications of this 
approval on existing processes. 
To match the already IRB 
approved processes included in 
the Mount Sinai Local Context 
Questionnaire as a result of the 
conditional approval of 
NewbornDx by New York State.  
2.0, 05DEC2019 
The adjudication of discordant 
results procedures has been 
revised and includes the view 
by the 1) clinician directly caring 
for the subject and 2) a subset 
of the steering committee of the 
result that aligns best with the 
infant when discordance is due 
to variant classification. 
To better structure the process 
and to obtain the view of the 
clinician caring for the infant 
and an impartial sub-committee.  

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2.0, 05DEC2019 
The exclusion criteria “Infants 
who are not expected to receive 
medical care in the US 
healthcare system from time of 
discharge home until 1 year 
CGA” has been added. 
To ensure comparable follow-up 
economic data can be used in 
the health economic analysis.  
2.0, 05DEC2019 
Text to clarify that enrollment in 
this study is not intended to 
replace targeted available 
genetic testing for obvious 
suspected disorders have been 
added. 
To clarify that enrollment should 
not be prohibited nor delayed 
for return of clinical genetic test 
results unless the suspected 
disorder is of an obvious nature 
and therefore rWGS is not 
needed.  
2.0, 05DEC2019 
Terminology within the primary 
endpoints definitions has been 
updated. 
To use consistent terminology 
across endpoints and 
definitions. 
2.0, 05DEC2019 
The option to obtain 1ml of 
blood in an EDTA tube in lieu of 
the blood spot card when the 3-
hour drying time for the blood 
spot card pushes the shipment 
of samples to the next day and 
the case is urgent.  
To allow flexibility in sample 
collection vehicle in urgent 
cases when waiting for the 
drying time of a dried blood spot 
card would adversely affect time 
to potential life-saving 
treatment. 
2.0, 05DEC2019 
Details about the following have 
been deleted and moved to the 
MOP and/or eCRF instructions: 
Identification number 
definitions; description of eCRF; 
blood sample collection 
procedures; Athena specimen 
ID creation example; RCIGM 
portal instructions; shipping 
instructions and shipping 
addresses; contact information; 
source document types. 
To streamline repetitive 
information that is more 
appropriate for the MOP. 
2.0, 05DEC2019 
A licensed genetic counselor 
may return results without an 
attending physician present if 
clinical practice is for the 
licensed genetic counselors to 
do so outside of this study. 
To align with the existing clinical 
workflow at a site in a state for 
whom genetic counseling 
licensing allows for return of 
results. 
2.0, 05DEC2019 
Procedures for when, how and 
by whom results are returned 
To ensure consistent 
procedures across sites.  

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after an infant is discharged 
home have been added. 
2.0, 05DEC2019 
The site should wait until results 
from both NewbornDx and 
rWGS are returned before 
speaking with the 
parent/guardian about the 
results if waiting would not harm 
the infant (i.e. the result is not 
provisional or the infant does 
not require urgent testing). 
To clarify the intended timing of 
communication with the infant’s 
parent/guardian(s). 
 
2.0, 05DEC2019 
The timeframe in which to 
collect the Clinical Utility Survey 
has been expanded to include 
at discharge home or for infants 
who discharge home prior to 
the return of results, within one 
month after the return of results. 
To accommodate varying 
scenarios of when results are 
returned/acted upon. 
2.0, 05DEC2019 
In cases in which the infant is 
discharged home prior to the 
return of results, the Clinical 
Utility Survey should be 
completed by the clinical 
geneticist, most relevant 
subspecialist or by the site PI 
after speaking with the 
pediatrician. 
To account for different 
physicians who will follow the 
subject and act upon results as 
an outpatient.   
2.0, 05DEC2019 
Physician CPT codes have 
been added to the types of data 
captured. 
To better describe the types of 
billing data collected. 
2.0, 05DEC2019 
A parent may only opt-in to 
receive secondary findings if 
the parent/guardian opted-in for 
infant secondary findings. 
Inclusion of a table detailing the 
results returned for the infant 
and parent based on opt-in 
status, disorder onset, if the 
result is included on the 
ACMG59 or medically 
actionable. 
To clarify under what 
circumstances a parent may 
opt-in for additional results and 
the possible results returned for 
the infant and parent.  
2.0, 05DEC2019 
Removed clinical geneticist as a 
required input to eligibility 
discussion. The study team will 
discuss eligibility of the infant 
Depending on the site and 
condition, genetics is not always 
consulted per SOC. 

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with the attending physician of 
record and if in agreement, the 
family will be approached for 
the study.  
2.0, 05DEC2019 
The screening data captured in 
REDCap has been changed to 
replace maternal education and 
income level data with 5-digit 
zip code and primary symptoms 
with primary system 
involvement related to patient 
nomination.  
The 5-digit zip code will be used 
in the health economic analysis. 
The change from clinical 
symptoms to primary system(s) 
involvement related to patient 
nomination of infants who have 
not provided consent is to 
reduce the risk of identification 
given the addition of zip code. 
2.0, 05DEC2019 
Revised consent via telephone 
procedures. If the infant’s 
clinical status is life threatening 
and in-person consent or 
written documentation of 
consent via telephone is not 
feasible, verbal consent by 
telephone as documented by 
the research staff is sufficient to 
obtain, ship and process blood 
samples; however 
documentation of written 
consent must be obtained prior 
to return of any results. 
To enable the timely conduct of 
research procedures for infants 
whose clinical status is life 
threatening, whose 
parent/guardian verbally 
consents to participate in the 
study but does not have 
immediate access to a 
fax/scanner to return the signed 
ICF, and the potential delay in 
written consent may prevent 
life-saving treatment.  
 
 
 

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3. McCandless SE, Brunger JW, Cassidy SB. The Burden of Genetic Disease on 
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4. Yang Y, Muzny DM, Xia F, Niu Z, Person R, Ding Y et al. Molecular findings 
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5. Weiner J, Sharma J, Lantos J, Kilbride H. How infants die in the neonatal 
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6. Wilkinson DJ, Fitzsimons JJ, Dargaville PA, Campbell NT, Loughnan PM, 
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14 APPENDICES 
 
 Informed Consent Form  
 Secondary Findings Parent Handout 
 Follow-up Contact Information Form 
 Resource Utilization Follow-up Email and Questionnaire 
 SF-12 Your Health and Well Being 
 Child Visual Analog Scale instrument 
 Authorization to Release Protected Health Information 
 Clinician Assessment of Clinical Utility