Preliminary Study On Clinical Application Of Gene Screening For Neonatal Wilson Disease

Part I: Genetic Analysis of ATP7 B GeneObjective(s): To study genetic spectrum differences in Wilson disease(WD)caused by mutations in the adenosine triphosphate copper transporting beta gene(ATP7B).Methods: Firstly,this study included(1)single nucleotide polymorphism variants,small fragment structure insertion or deletion,and copy number variations in the ATP7 B gene with an allele frequency(AF)of<5% from the Chinese Children’s Rare Disease Genetic Testing Clinical Collaboration System(CCGT)between December2015 and December 2021;(2)From April 1996 to December 2021,variants in the ATP7 B gene in the Human Gene Mutation Database(HGMD),Clin Var,and Wilson Gen were curated as pathogenic(P)or likely pathogenic(LP);(3)From December 2020 to December 2021,Pub Med literature searched for variants in the ATP7 B gene that were reported to be curated P/LP.After inclusion of sites from three pathways,variants curated as variant of unknown significance/likely benign/benign,and variants whose names do not meet Human Genome Variation Society(HGVS)specifications were excluded,and identical variants from different sources were de duplicated.Secondly,referring to the genetic variation classification standards and guidelines of the American College of Medical Genetics and Genomics(ACMG),pathogenicity ratings were conducted for all variants included in the study based on information such as CCGT population’s AF,gnom AD public population’s AF,and genetic patterns.Two senior genetic analysts independently reviewed and confirmed the final evaluation results,and selected P/LP variants.We analyzed the critical neonatal cohort of the China Neonatal Genomes Project(CNGP),the healthy neonatal cohort of the Sixth Affiliated Hospital of Sun Yat-sen University(abbreviated as ZL),the metabolic disease analysis cohort of the China Metabolic Analytics Project(China MAP),and the above-mentioned gnom AD international population cohort,calculate the AF of each P/LP variant in four cohorts to obtain the genetic spectrum of the ATP7 B gene in each cohort.Compare the AF differences of each variant in different cohorts through Fisher’s precision probability test,and perform Bonferroni correction for the statistical values.The prevalence and 95% confidence interval of WD in each cohort were estimated using the Bayesian framework.Results: A total of 1826 variants in the ATP7 B gene were included in this study,and a total of 875 P/LP variants were selected.This study found that two variants(c.2605G>A,c.3207C>A)are the most common P/LP variants in the international population(gnom AD_TOTAL,abbreviated as TOTAL),while c.3316G>A,c.2333G>T,and c.2975C>T are the most common P/LP variants in the Chinese population.In the four cohorts of CNGP,ZL,China MAP,and TOTAL,a total of 15 variants showed statistical differences in pairwise comparisons,with 2 variants(c.3207C>A and c.1934T>G)having significantly higher AF values in TOTAL than in three Chinese cohorts,and 5 variants(c.2333G>T,c.2304 dup,c.2975C>T,c.3316G>A and c.3443T>C)having significantly higher AF values in three Chinese cohorts(all P values<0.05).The lower estimated prevalence of WD is in the gnom AD-Finnish in Finland population: 1/183348(95% confidence interval:1/464760-1/92439)and the gnom AD-African American population: 1/175290(95%confidence interval: 1/267302-1/121924).The prevalence is higher in three Chinese population cohorts,including the CNGP cohort: 1/3976(95% confidence interval:1/4707-1/3394),the China MAP cohort: 1/4181(95% confidence interval:1/5232-1/3402),and the ZL cohort: 1/5774(95% confidence interval: 1/6960-1/4851)Conclusion(s): There are differences in the genetic spectrum and estimated prevalence of ATP7 B gene in the Chinese and foreign population cohorts.The high prevalence in the Chinese population suggests that the Chinese population can build a genetic screening package suitable for their own genetic characteristics.Part II: Analysis of neonatal ATP7 B gene screening in different clinical settingsObjective(s): To explore the effects of neonatal ATP7 B gene screening in different clinical settings.Methods: The 875 P/LP variants of the manually screened ATP7 B gene were regarded as a preliminary single gene screening package(Panel),which was screened in the critical neonatal cohort of the CNGP and the healthy neonatal cohort of the ZL.Patients with the P/LP variant in the ATP7 B gene on both chromosomes were selected for outpatient or telephone follow-up at least one year and follow up every six months to clarify their clinical phenotype,ceruloplasmin levels,low copper diet,and drug treatment effects.Further,in order to refine the number of variants in a simple gene screening package,875 P/LP variants in two cohorts were sequentially descending by AF,and the Bayesian framework was used to simulate how many P/LP variants were required for 90%,95%,and 100% of the patients screened in each cohort.To assess whether it is necessary to design different panels for the severe neonatal cohort and the healthy neonatal cohort,this study compared the differences between the top 10 and top 30 high-frequency variants in the two cohorts through Fisher’s precision probability test and Bonferroni correction,and finally screened and compared the differences in AF values of the high-frequency variants(AF>1:10000)in the two cohorts and whether the rankings based on AF values tend to be consistent.Results: Out of a total of 17724 genetic testing samples in the healthy neonatal ZL cohort and screened for 7 suspicious patients.Out of a total of 17861 samples in the severe neonatal CNGP cohort and screened for 6 suspicious patients.During follow-up,none of the suspicious patients showed any symptoms related to WD,6patients showed a decrease in complete ceruloplasmin testing,and 3 patients had started low copper diet and zinc treatment without adverse reactions.In addition,90%of WD patients screened in the severe neonatal cohort theoretically need to include the top 66 P/LP variants,while in the healthy neonatal cohort,the top 49 variants are required,of which 33 variants overlap.Screening for 95% of WD patients theoretically requires inclusion of variants in the top 81,while in the healthy neonatal cohort,variants in the top 61 are required,with 39 variants overlapping.In the CNGP cohort,screening for 100% of WD patients requires 95 variants,while in the healthy neonatal cohort,72 variants are required,with 45 variants overlapping.Seven of the top 10 variants in the two cohorts overlap,namely,c.3316G>A,c.2333G>T,c.3443T>C,c.2975C>T,c.2755C>G,c.2605G>A,and c.3859G>A.Among the top 30 variants,22 overlap.For 32 high-frequency P/LP variants with AF>1:10000,the AF values and rankings in the two cohorts tend to be consistent,and imply that different cohorts can use the same Panel.Conclusion(s): Gene screening for neonatal Wilson disease in the Chinese population is a feasible early screening and diagnosis strategy,and a gene screening package consisting of high-frequency pathogenic variants can be selected for rapid screening of patients.