The Effect and Mechanism of Gene Variation on Neonatal Hyperbilirubinemia
Conditions: Neonatal Hyperbilirubinemia
Sponsor: Sixth Affiliated Hospital, Sun Yat-sen University

 
 
 
 
Clinical research protocol 
 
Project name: The effect and mechanism of gene variation on 
neonatal hyperbilirubinemia 
Leading unit: The Sixth Affiliated Hospital, Sun Yat-sen 
University 
Project leader: Hu Hao 
Department: Pediatrics  
Research period: September 1, 2023 to December 31, 2024 
Project number: EK-NHB-20230216 
NCT No.: none. 
Version No.: V1.0  
Version Date: February 16, 2023 
 

 
      
 
Abstract 
object name 
The effect and mechanism of gene variation on neonatal 
hyperbilirubinemia 
goal of study 
( 1 ) To analyze the carrying and pathogenicity of neonatal 
jaundice-related genes, and collect the sequencing data of 
jaundice-related genes ( 24 genes of 29 NHB-related genetic 
diseases ) of 2000 newborns in the cooperative hospital to 
complete the construction of the gene database.  
( 2 ) To analyze the incidence of NHB-related genes, pathogenic 
mutations, suspected pathogenic mutations, and the carrying rate 
of unknown mutations in 2000 cases, and to count the distribution 
of high-frequency mutation sites in the population, so as to 
provide a scientific basis for the selection of mutation sites for 
large-scale neonatal hyperbilirubinemia gene screening.  
( 3 ) The influence of jaundice-related genes on neonatal jaundice 
was analyzed. The clinical data of newborns and the daily 
percutaneous bilirubin level were collected through the hospital 
hospitalization system and the " percutaneous jaundice instrument 
home monitoring + software doctor-patient interconnection " 
method to complete the construction of intelligent neonatal 
jaundice clinical database.  
( 4 ) Analyze the integrated monitoring data, statistically analyze 
the gene and jaundice clinical data of 2000 newborns, understand 
whether there is a difference in the carrying rate of related 
pathogenic genes in different degrees of jaundice, and explore 
whether there is a difference in the degree of jaundice carrying 
multiple and single jaundice pathogenic genes. To understand the 

 
      
 
correlation between gene polymorphism and clinical 
manifestations ( phenotypic polymorphism ) of neonatal 
hyperbilirubinemia, so as to better evaluate the effect of genes on 
neonatal jaundice. 
research design 
prospective observational study 
Research period 
September 2022 to December 2024 
Participating 
Newborns 
Inclusion criteria 
( 1 ) Informed consent of parents or guardians ;  
( 2 ) Age : 1-28 days, gestational age ≥ 35 weeks ;  
( 3 ) Birth weight ≥ 2.5 kg and < 4 kg. 
Excluded criteria ( 1 ) Neonatal data with unclear clinical basic information ;  
( 2 ) Lack of traceability core information data ;  
( 3 ) data that the test results cannot be analyzed and interpreted ;  
( 4 ) Sample collection is not qualified and unwilling to cooperate 
with re-sampling.  
( 5 ) Newborns with severe deformity and severe lethal inherited 
metabolic diseases 
Grouping and 
sample size 
A total of 2000 neonates were included, including 500 cases of non-
significant hyperbilirubinemia ( TSB / TCB < 205umol / L ) ; ( 2 ) 
500 cases of significant hyperbilirubinemia ( 205umol / L ≤ TSB / 
TCB < 342umol / L ) ; ( 3 ) 500 cases of severe hyperbilirubinemia 
( TSB / TCB ≥ 342umol / L ) ; 4 Extremely severe hyperbilirubinemia 
500 cases ( TSB / TCB ≥ 428umol / L ). 
data collection 
( 1 ) Basic information : gender, gestational age, date of birth, birth 
weight, height, head circumference, mode of conception, Apgar score 
and parents ' information ;  
( 2 ) Perinatal information : parity, parity, mode of delivery, fetal 

 
      
 
membranes, amniotic fluid, placenta, umbilical cord, intrauterine 
distress, group B streptococcal infection, pregnancy complications 
such as gestational hypertension, diabetes, etc.  
( 3 ) Jaundice information : blood types of parents and newborns, 
feeding methods, monitoring jaundice values, jaundice treatment 
methods and related test results ;  
( 4 ) Test results : Collect genetic analysis results, notes, etc. 
Statistical 
processing 
IBM-SPSS 26.0 statistical software was used to analyze the test 
results. Logistic multivariate regression analysis was used to 
evaluate the correlation between genotype and clinical phenotype, 
and mediating variables were used to analyze the correlation 
between gene-gene interaction and genotype-phenotype. 
abbreviation 
neonatal hyperbilirubinemia, NHB; total serum bilirubin, TSB;
transcutaneous bilirubin, TCB; Principal Investigator, PI. 
 

                                           
 
1 
 
1. Research background 
Neonatal disease screening is one of the most effective public health initiatives, 
which has been carried out for more than 60 years. Since the single screening of 
phenylketonuria ( PKU ) in 1961, with the improvement of screening technology, it has 
developed into a screening of more than 50 diseases including endocrine system 
diseases ( congenital hypothyroidism, congenital adrenal hyperplasia ), hemoglobin 
disease, infectious diseases, cystic fibrosis, and congenital metabolic diseases. The 
incidence of birth defects is related to genetic and environmental factors. Among the 
patients with birth defects diagnosed by etiology, 94.4 % are caused by chromosomal 
abnormalities and genetic variations. Because most patients have a genetic basis, early 
screening and diagnosis have important scientific value and social significance for the 
prevention and control of these diseases.  
At present, neonatal disease screening methods are diverse, including : neonatal 
metabolic disease screening ( hypothyroidism, phenylketonuria, adrenocortical 
hyperplasia, G-6-PD deficiency and metabolic disease tandem mass spectrometry 
screening ), hearing screening, fundus screening, cardiac ultrasound, etc. However, 
although the above-mentioned routine screening techniques play an important role in 
the prevention and control of birth defects, there are still obvious deficiencies : 1 ) There 
are many interference factors, such as susceptibility to sample quality, blood collection 
time, gestational age, birth weight and other factors, and false positive or false negative 
are more likely to occur, which brings a greater psychological burden to the children 's 
families. At the same time, biochemical tests, tandem mass spectrometry, urinary 
organic acids and other methods are needed to assist diagnosis after recall ; 2 ) For 
children with positive screening, further gene sequencing is needed to confirm the 
diagnosis and typing, and further develop an accurate treatment plan. During this period, 
the diagnosis may be delayed for 1-2 months. For children with severe illness, the 
damage to the nervous system is often irreversible, and even the treatment is not timely 
and life-threatening during the detection period ; 3 ) Traditional methods can only make 
a preliminary diagnosis, and can not be clearly classified, and more birth defects of 

 
      
 
different types, treatment options vary. At present, the mainstream technology of 
neonatal screening has the problems of limited methods, long time for diagnosis, easy 
delay in disease treatment, and direct impact on the prognosis of children. Therefore, 
efficient and accurate screening techniques need to be developed and applied. 
The development of genomics technology has pushed the diagnosis and prevention 
of hereditary birth defects to a new height. With the rapid development of gene 
sequencing technology and the deepening of medical genetics research, more and more 
genetic screening and related clinical studies of genetic diseases caused by genetic 
variation have been widely carried out at home and abroad, highlighting the broad 
prospects of neonatal disease screening and diagnosis based on gene detection in early 
diagnosis and early prevention of birth defects.  
Neonatal hyperbilirubinemia, also known as neonatal jaundice, is the most 
common clinical manifestation of newborns. It has many causes and is difficult to 
diagnose. Genetic factors are one of the common causes of metabolic-related jaundice. 
According to statistics, about 60 % to 80 % of newborns can have varying degrees of 
jaundice, and severe cases can cause a series of body damage, including nerve, hearing, 
cardiovascular and kidney function, and even cause death. Within 7-27 days after birth, 
the incidence of death in children due to neonatal jaundice is about 107.1 / 100,000, 
ranking ninth in the global causes of late neonatal death. The common causes of 
neonatal jaundice include neonatal ABO hemolysis, RH hemolysis, neonatal sepsis, and 
congenital hypothyroidism. Genetic factors play an important role in the metabolism of 
bilirubin. Hyperbilirubinemia is mainly caused by bilirubin metabolism or circulatory 
disorders. Clinically, it can be manifested as high unconjugated hyperbilirubinemia and 
hyperconjugated hyperbilirubinemia. At present, the gold standard for the detection of 
neonatal jaundice is to use venous blood to detect bilirubin as the main laboratory 
examination method. In addition, it also includes micro-blood bilirubin meter detection, 
percutaneous bilirubin measurement and visual inspection. However, such detection 
does not have high diagnostic sensitivity and specificity and cannot determine whether 
it is jaundice caused by genetic factors. Genetic metabolism-related jaundice has great 

 
      
 
individual differences in age of onset and clinical manifestations. Even if the same 
pathogenic gene mutation is carried out, the clinical phenotype can be different. 
Therefore, the application of genetic testing in this type of disease can improve the early 
diagnosis of the disease. Genetic testing of children without jaundice or hospitalized 
jaundice can obtain the proportion of genetic factors and their genotypes in children 
with jaundice, so as to continuously improve the efficiency of disease diagnosis. 
However, previous studies have focused on the role of one or two specific genes 
in patients with hyperbilirubinemia. For example, a number of studies have shown that 
the UGT1A1 gene hotspot c.211G > A polymorphism is a risk factor for NHB, and is 
closely related to the severity of jaundice, and can aggravate the symptoms of breast 
milk 
jaundice, 
glucose-6-phosphate 
dehydrogenase 
deficiency, 
hereditary 
spherocytosis, and hemolytic jaundice caused by congenital erythropoietic anemia. 
Yang et al.showed that the c.211G > A locus was a risk factor for severe 
hyperbilirubinemia in patients with glucose-6-phosphate dehydrogenase deficiency. 
Watchko et al.showed that a single G6PD gene mutation or UGT1A1 gene mutation 
only caused mild jaundice, but the coexistence of the two mutations may cause severe 
NHB or even nuclear jaundice. Up to now, there is no literature report on the study of 
gene polymorphism and clinical manifestation polymorphism of jaundice in large data 
population. 
The transcutaneous jaundice instrument ( YSJ-20 ) has obtained the ' People 's 
Republic of China Class II Medical Device Registration Certificate '.It can accurately, 
quickly, safely and non-invasively detect the current bilirubin level of the newborn 
through the skin appearance. It is portable, easy to operate, and has a low threshold for 
use. It is very suitable for family members to monitor the level of neonatal jaundice at 
home.  
In summary, this study intends to collect heel peripheral blood samples from 2000 
newborns [ including 500 non-significant hyperbilirubinemia newborns, 500 significant 
hyperbilirubinemia newborns, 500 severe hyperbilirubinemia newborns and 500 
extremely severe hyperbilirubinemia newborns ]. The gene capture sequencing 

 
      
 
technology containing 24 genes of neonatal common hereditary jaundice genes was 
used to detect and collect the clinical data of these 2000 newborns. At the same time, 
the percutaneous jaundice meter ( YSJ-20 ) was used to monitor the daily jaundice at 
home. According to the analysis results of gene detection and clinical data of jaundice 
in 2000 children, the carrying rate of pathogenic genes of common hereditary jaundice 
in neonates was studied, and the carrying rate of pathogenic genes in different degrees 
of jaundice was understood. Whether there is a difference in the degree of jaundice with 
multiple and single jaundice pathogenic genes, and the correlation between gene 
polymorphism and clinical manifestations ( phenotypic polymorphism ) of neonatal 
hyperbilirubinemia were analyzed. 
2. Research objective 
( 1 ) To analyze the carrying and pathogenicity of neonatal jaundice-related genes, 
and to collect the sequencing data of jaundice-related genes ( 24 genes of 29 NHB-
related genetic diseases ) of 2000 newborns in the cooperative hospital, and to complete 
the construction of the gene database.  
( 2 ) To analyze the incidence of NHB-related genes, pathogenic mutations, 
suspected pathogenic mutations, and the carrying rate of unknown mutations in 2000 
cases, and to count the distribution of high-frequency variation sites in the population, 
so as to provide a scientific basis for the selection of mutation sites for large-scale 
neonatal hyperbilirubinemia gene screening.  
( 3 ) The influence of jaundice-related genes on neonatal jaundice was analyzed. 
The clinical data of newborns and the daily percutaneous bilirubin level were collected 
through the hospital hospitalization system and the " percutaneous jaundice instrument 
home monitoring + software doctor-patient interconnection " method, and the 
construction of intelligent neonatal jaundice clinical database was completed.  
( 4 ) Analyze the integrated monitoring data, statistically analyze the gene and 
jaundice data of 2000 newborns, understand whether there is a difference in the carrying 
rate of related pathogenic genes in different degrees of jaundice, and explore whether 
there is a difference in the degree of jaundice carrying multiple and single jaundice 
pathogenic genes. To understand the correlation between gene polymorphism and 
clinical manifestations (phenotypic polymorphism) of neonatal hyperbilirubinemia, 
thereby increasing the accuracy of genetic diagnosis. 

 
      
 
3. Research Design 
This study is a prospective observational study. Three drops of heel peripheral 
blood samples were collected from 2000 newborns, and 24 genes of 29 NHB-related 
genetic diseases were analyzed. All dry blood spot samples that met the inclusion 
criteria could be included in the genetic analysis. At the same time, the clinical data of 
these 2000 newborns were collected, and the daily jaundice monitoring at home was 
performed using the percutaneous jaundice meter (YSJ-20). The data were 
synchronized to the mobile phone jaundice follow-up doctor assistant small program 
for storage statistics. The family members can view the jaundice risk level and obtain 
free doctor consultation in the small program.  
The purpose of this study is to study the carrying rate of pathogenic genes of 
common hereditary jaundice diseases in newborns, to understand whether there is a 
difference in the carrying rate of pathogenic genes in different degrees of jaundice, to 
explore whether there is a difference in the degree of jaundice carrying multiple and 
single jaundice pathogenic genes, and to analyze the correlation between gene 
polymorphism and clinical manifestations ( phenotypic polymorphism ) of neonatal 
hyperbilirubinemia, so as to evaluate the feasibility of jaundice gene screening program 
in the detection of jaundice-related genetic metabolic diseases.  
Based on the ' Classification Criteria and Guidelines for Genetic Variation ( 2017 ) 
', the variation pathogenicity of genetic testing is divided into five categories : known 
pathogenic, possible pathogenic, unclear significance, possible benign and benign. In 
this study, known pathogenic and possible pathogenic are classified as positive results. 
The pathogenicity of the mutation site is mainly based on the pathogenic mutation 
database and the latest literature reports. 
The classification criteria of jaundice were based on the American Academy of 
Pediatrics ( AAP ) 2022 edition of neonatal hyperbilirubinemia management guidelines 
for gestational age ≥ 35 weeks and the latest version of 2023 guidelines to interpret the 
management of neonatal hyperbilirubinemia-the diagnosis and intervention criteria of 
neonatal hyperbilirubinemia in the American Academy of Pediatrics and multi-national 
clinical management guidelines. The 2000 neonates were divided into 500 cases of non-

 
      
 
significant hyperbilirubinemia ( TSB / TCB < 205umol / L), 500 cases of significant  
hyperbilirubinemia ( 205umol / L ≤ TSB / TCB < 342umol/L ), 500 cases of severe 
hyperbilirubinemia ( TSB/TCB ≥ 342umol/L) and 500 cases of extremely severe 
hyperbilirubinemia ( TSB/TCB ≥ 428umol /L).  
4. Inclusion criteria 
( 1 ) Informed consent of parents or guardians ;  
( 2 ) Age : 1-28 days, gestational age ≥ 35 weeks ;  
( 3 ) Birth weight ≥ 2.5 kg and < 4 kg. 
5. Excluded criteria 
( 1 ) Neonatal data with unclear clinical basic information ;  
( 2 ) Lack of traceability core information data ;  
( 3 ) data that the test results cannot be analyzed and interpreted ;  
( 4 ) Sample collection is not qualified and unwilling to cooperate with re-sampling.  
( 5 ) Newborns with severe deformity and severe lethal inherited metabolic diseases 
6. termination criteria 
( 1 ) The family members of the newborn included in the study requested termination 
at any time ;  
( 2 ) The relevant management departments require termination. 
7. outcome index 
Through the comprehensive analysis of genetic variation information and jaundice 
clinical data, the carrying rate of pathogenic genes of common hereditary jaundice 
diseases in newborns was counted, and the difference of carrying rate of pathogenic 
genes in different degrees of jaundice was understood. To explore whether there is a 
difference in the degree of jaundice carrying multiple and single jaundice pathogenic 
genes, and to analyze the correlation between gene polymorphism and clinical 
manifestations ( phenotypic polymorphism ) of neonatal hyperbilirubinemia. 
8. Grouping and sample size 
This study is a prospective observational study. According to the positive mutation rate 
of NHB-related genes in neonatal hyperbilirubinemia, P is about 30 %, the test level α 

 
      
 
= 0.05 and the allowable error δ = 2 %, n ( sample size ) = ( Uα / δ ) 2 * p * ( 1-P ) ≈ 
2000 cases. A total of 2000 neonates were included, including 500 cases of non-
significant hyperbilirubinemia ( TSB / TCB < 205umol / L ) ; ( 2 ) 500 cases of 
significant hyperbilirubinemia ( 205umol / L ≤ TSB / TCB < 342umol / L ) ; ( 3 ) 500 
cases of severe hyperbilirubinemia ( TSB / TCB ≥ 342umol / L ) ; (4) Extremely severe 
hyperbilirubinemia 500 cases ( TSB / TCB ≥ 428umol / L ). 
9. Recruiting 
This study will recruit subjects in the outpatient and inpatient departments of pediatrics, 
neonatology, and obstetrics in the South China Neonatal Genetic Screening Alliance 
( including cooperation units of 49 hospitals ). 
10. Clinical evaluation index 
( 1 ) The carrying rate, positive rate, negative rate and high-frequency mutation sites of 
common NHB-related gene mutation sites in neonates ;  
( 2 ) The difference in the carrying rate of NHB-related pathogenic genes in neonates 
with different degrees of jaundice ;  
( 3 ) The difference of jaundice degree between carrying multiple and single jaundice 
pathogenic genes ;  
( 4 ) Gene-gene interaction and genotype-phenotype correlation.  
11. Methods 
11.1 gene sequencing 
A total of 24 genes were included in 29 NHB-related genetic diseases, including 
bilirubin metabolic disorders, congenital bile acid synthesis or transport disorders, red 
blood cell metabolic defects, lipid metabolic abnormalities, and metal metabolic 
abnormalities. The detected genes included ABCB11, ABCB4, ABCC2, ABCD3, 
ACOX2, AKR1D1, AMACR, ATP7B, ATP8B1, CYP7B1, G6PD, GBA, HBB, 
HSD3B7, JAG1, NOTCH2, NPC1, NPC2, NR1H4, SLC10A1, SLC25A13, SMPD1, 
TJP2 and UGT1A1. Gene and genetic disease related information refer to OMIM and 
Orphanet database. 
Target gene sequence capture and library construction. Three drops of heel peripheral 

 
      
 
blood were collected by filter paper, and genomic DNA was extracted by magnetic bead 
method. The target gene sequence was captured by multiplex PCR amplification 
method, covering all exon regions and adjacent intron regions ( ± 50bp ) of the target 
gene. A total of 900 amplicons were designed for 24 genes and completed in two 
amplification systems. The target sequence library was obtained after amplification-
purification-re-amplification-re-purification. The library was quantified using a Qubit 
® 3.0 fluorescence quantitative instrument ( Thermo Fisher Scientific ), and the library 
length was determined using an Aglient 2100 biological analyzer ( Agilent ) ( 228-378 
bp ). 
High-throughput sequencing and bioinformatics analysis. The library was diluted, 
added sequencing primers, and sequenced on the Illumina NextSeq 550 platform 
( PE150 ). The effective sequencing data were aligned to the human reference genome 
( Human _ B37 ) by BWA ( Burrows-Wheeler Aligner ) software. GATK ( Genome 
Analysis ToolKit ) was used to analyze the variation information, and ANNOVAR 
software was used to annotate the variation, including the annotation information of 
dbSNP database, Thousand Human Genome Project and other existing databases. The 
annotation content covers population frequency, mutation type, function prediction 
( SIFT, Polyphen2, Mutation Taster, etc. ), HGMD, Clinvar and other pathogenic 
classification information. Based on the ' Classification Criteria and Guidelines for 
Genetic Variation ( 2017 ) ', the pathogenic interpretation of genetic variation was 
divided into five categories : ' pathogenic ', ' possible pathogenic ', ' unclear significance 
', ' possible benign ' and ' benign '. Among them, ' pathogenic ', ' possible pathogenic ' 
and ' unclear significance ' were included in the statistics of test results. 
Validation of positive loci. For children with autosomal recessive double heterozygosity 
and children with autosomal dominant single heterozygosity, Sanger sequencing was 
used to verify the loci of their parents. 
11.2 Information and data collection 
Researchers should truthfully fill in the test results of the subjects and other relevant 
information, and ensure that the information is true and reliable. The specific 

 
      
 
information and data to be collected are as follows :  
( 1 ) Sign informed consent ;  
( 2 ) Jaundice gene detection : Take out the special filter paper blood collection card, 
disinfect with alcohol and dry the heel blood collection site, collect heel peripheral 
blood, drop three drops on the filter paper.  
( 3 ) Detection of jaundice value by percutaneous jaundice meter : The family members 
of the subjects rented the percutaneous jaundice meter for free under the guidance of 
the doctor, and used the ' single measurement mode ' to measure the forehead, chest and 
inner thigh of the subjects every day for a total of 2 weeks.  
( 4 ) Collect demographic characteristics : screening number, name, gender, gestational 
age, date of birth, birth weight, body length, head circumference, mode of conception, 
Apgar score and parental information ;  
( 5 ) Perinatal information : parity, parity, mode of delivery, fetal membranes, amniotic 
fluid, placenta, umbilical cord, intrauterine distress, group B streptococcal infection, 
pregnancy complications such as gestational hypertension, diabetes, etc.  
( 6 ) Jaundice information : parents and newborns blood type, hemolysis three tests, 
feeding methods, percutaneous jaundice value, jaundice treatment and related test 
results ;  
( 7 ) Gene detection results : Collect gene analysis results, notes, etc. 
12. Statistical processing 
IBM-SPSS 26.0 statistical software and R language ( Version 4.1.0 ) were used for 
statistical analysis of the test results. The carrying rate of common NHB-related gene 
mutation sites in neonates was counted, and the differences in the carrying rate of NHB-
related pathogenic genes in neonates with different degrees of jaundice were compared. 
To explore the difference in the degree of jaundice carrying multiple and single 
jaundice-related genes. The measurement data were expressed as mean ± standard 
deviation ( x  ± s ), and the t test was used for comparison between groups. The count 
data were expressed as values or rates ( % ), and the chi-square test was used for 
comparison between groups. Unconditional logistic multivariate regression analysis 

 
      
 
was used to evaluate the correlation between genotype and clinical phenotype, and 
mediating variables were used to analyze the correlation between gene-gene interaction 
and genotype-phenotype. 
13. data monitoring 
( 1 ) Case Report FormThe case report form ( CRF ) was used in this study. The 
researcher ensures the authenticity, accuracy and integrity of the data in the original 
case of each enrolled ( signed informed consent ) newborn. The researcher or authorized 
researcher confirms and fills in the original medical record and submits it to the 
researcher / clinical coordinator. The data points in the original medical record are 
entered into the case report form system by the researcher / clinical coordinator.  
( 2 ) Data audit and locking After all data-related queries have been resolved, the main 
researchers, data managers, and statistical analysts will confirm the statistical analysis 
of the population, lock the database, and sign the audit report at the same time. The 
locked data must not be changed.  
( 3 ) Data privacy regulations All members of the research team, including lead 
researchers and their authorized researchers, must comply with all local and regional 
regulatory requirements and applicable privacy regulations ( such as the ' Health 
Insurance Carriage and Liability Act ' ).  
Each newborn participating in this study will be assigned a unique number and an 
institutional identifier.  
All research data will be stored in a confidential electronic system and stored for at least 
3 years after the completion of the study.  
The coding table for recording neonatal information and research will be kept by the 
researcher ( PI ) in a locked file cabinet or in a password-protected database. Only 
researchers and their authorized researchers have the right to access the coding table.  
The coding table that records the name of the hospital and the institution identification 
number will be saved by the research team in a locked file cabinet, which is only limited 
to access by limited research members.  
The identity of each subject of observation should be kept confidential in research 

 
      
 
reports and related publications. 
14. adverse reactions 
In this study, only 3 drops of neonatal heel peripheral blood were collected for genetic 
analysis and statistical analysis of percutaneous jaundice values. No human trials were 
involved. Family members were willing to know the genetic test results and the genetic 
test results were positive. Family members may have a certain psychological burden. 
We will provide free professional genetic counseling, as detailed as possible to inform 
the hazards of this genetic defect, treatment and other related information to help. 
Psychiatrists can be arranged for free psychological counseling if necessary. 
15. Ethical approval 
This clinical study follows the Helsinki Declaration ( 2013 edition ), the ethical 
principles of human medical research and the relevant clinical research norms and 
regulations in China.  
This clinical study was approved by the Research Ethics Committee / Institutional 
Review Boards ( REC / IRBs ).  
This clinical study is an observational clinical study initiated by researchers. The 
clinical research protocol ( including informed consent and case report forms, etc. ) and 
other information provided to the family members or guardians of the newborn were 
reviewed and approved by the ethics committee of the clinical research team leader unit 
and the participating units. 
16. informed consent 
Before the start of the clinical study, the researchers completely and comprehensively 
introduced the purpose, process, method, and the interests and risks of the newborn to 
the parents or legal representatives who met the inclusion criteria. A written informed 
consent form was given to each parent or guardian of the newborn before enrollment, 
so that he / she had sufficient time to consider whether to consent to the collection of 
neonatal information to participate in this clinical study. After obtaining the informed 
consent of each newborn 's parent or legal representative and signing the informed 
consent form, researchers can begin to collect relevant data. 

 
      
 
17. privacy protection 
All members of the research team, including the primary researchers and their 
authorized researchers, must comply with all local and regional regulatory requirements 
and applicable privacy regulations.  
Each newborn participating in this study will be assigned a unique number and an 
institutional identifier.  
All research data will be stored in a confidential electronic system and stored for at least 
3 years after the completion of the study.  
The coding table for recording neonatal information and research will be kept by the 
researcher ( PI ) in a locked file cabinet or in a password-protected database. Only 
researchers and their authorized researchers have the right to access the coding table.  
The coding table that records the name of the hospital and the institution identification 
number will be saved by the research team in a locked file cabinet, which is only limited 
to access by limited research members. The identity of each subject of observation 
should be kept confidential in research reports and related publications. 
18. Research progress 
September 01, 2023 to March 31, 2024: The samples were grouped and tested : ( 1 ) 
contact parents for informed consent ; ( 2 ) Collect 3 drops of heel peripheral blood 
samples, extract genomic DNA by magnetic bead genomic DNA extraction kit, and 
detect the samples by high-throughput sequencing technology ; ( 3 ) Collect neonatal 
clinical data and daily percutaneous bilirubin levels through the hospital inpatient 
system and the ' percutaneous jaundice instrument home monitoring + software doctor-
patient interconnection ' method. 
April 01, 2024 to June 30, 2024: Data analysis : ( 1 ) Statistics of neonatal common 
jaundice-related gene screening results : the positive rate of common jaundice gene 
screening ( common positive disease cases, high-frequency pathogenic sites ), the 
carrying rate of pathogenic mutations, the carrying rate of pathogenic genes with 
different degrees of jaundice, and the degree of jaundice carrying multiple and single 
jaundice pathogenic genes. ( 2 ) Combined with clinical data and data uploaded by 

 
      
 
transcutaneous jaundice meter : Correlation analysis and comparison of jaundice-
related gene detection results and jaundice clinical data to evaluate the feasibility of 
jaundice gene detection for neonatal hyperbilirubinemia screening. 
July 01, 2024 - December 31, 2024: Write the conclusion report, complete the paper, 
submission and publication work. 
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