Clinical Research On Newborn Hearing Concurrent Genetic Screening In106,513Neonates And New Technology Development

Congenital hearing impairment is a distressing disorder which occurs inabout1-3in1000live births. According to the correlated data reported by theWHO,278million people worldwide have moderate to profound hearing loss inbilateral ears. Most people who have hearing impairment live in developingcountries. In China, there are approximately21million people with hearingimpairment out of the60million people classified as disabled，and of those21million with hearing loss, approximately0.8million are younger than7years old.This number has continued to increase by more than30,000newborns withcongenital deafness annually.To date,64nonsyndromic deafness genes and more than1000discretedeafness causing mutations have been described (http://hereditaryhearingloss.org).Despite the genetic heterogeneity, there are still a limited number of mutationalhot spots of hereditary deafness. Among Chinese children, epidemiologicalstudies indicated that at least36%cases of congenital hearing impairment were resulted from GJB2, SLC26A4or mitochondrial12S rRNA (MTRNR1) mutations(GJB2,18.31%; SLC26A4,13.73%; MTRNR1,3.96%).Therefore, for early disclosure of genetic causes leading to onset inchildhood or aminoglycoside-sensitive hearing loss and identification of carrierswith those pathogenic mutations, integration of genetic tests into the currenthearing screening programs is essential for maintaining hearing health of childrenwho are most vulnerable to both genetic and environmental risk exposures. In thisreport, we first thoroughly analyzed the universal newborn hearing and the newlydeveloped genetic screening conducted106,513neonates in13provinces ofChina. Then, we develop a new Tetra-primer Amplification Refractory MutationSystem-PCR (ARMS-PCR) Kit to detect the four most commonly or specificpoint mutations of the three genes (GJB2c.235delC; SLC26A4c.919-2A>G;MTRNR1mt.1555A>G and mt.1494C>T). A simple and economical genotypingmethod involving a single PCR reaction followed by gel electrophoresis. Inlarge-scale newborn screening this rapid, method for point mutation genotypingwill provide both cost and time benefits compared to current methods. At last, weproposed several strategies for improving the concurrent audiological and genetictesting based on the practical evidence from the largest population in the world.These studies were eomposed of three parts below: PART1: A Report of Newborn Hearing Concurrent GeneticScreening in106,513Neonates in ChinaThis study was undertaken in13provinces of China.106,513newbornbabies received hearing concurrent genetic screening. The hearing screening wasperformed in two step protocol with OAE or AABR. Blood sample wascollected with a universal newborn genetic screening card. And three commongene, MTRNR1, GJB2and SLC26A4were screened with standard protocol.Among all the106,513neonates,91.48%(97,433/106,513）individuals passed thefirst-step hearing screening,3.41%(3638/106,513) babies passed only one side,and the other5.11%(5,442/106,513) were bilaterally referred. Gene screeningfound3,055individuals had one or two mutant alleles, the carrier rate is2.87%(3,055/106,513) among the entire newborn population. The risk for hearing losswas100%(33/33) for those newborns carrying causative GJB2or SLC26A4mutations (homozygotes or compound heterozygotes). Total169newborns withMTRNR1mt.1555A>G or mt.1494C>T pathogenic mutation, who would sufferfrom sudden hearing loss once applying aminoglycoside drugs. PART2: Operate a Newborn Genetic Screening Procedure forHigh Risk Deafness-associated Mutations with a NewTetra-primer ARMS PCR KitThe new ARMS-PCR screening kit was designed to detect four high riskdeafness-associated mutations (GJB2c.235delC, SLC26A4c.919-2A>G, mtDNA12S rRNA mt.1555A>G and mt.1494C>T). The kit was able to amplify bothwild-type and mutant alleles with a control fragment. The proposed method wasconducted to genotype the above four deafness gene mutations in four PCRreactions. Each mutation was genotyped by a set of four primers, twoallele-specific inner primers, and two common outer primers. A mismatch at thepenultimate or antepenult nucleotide of the3’ terminus was introduced in order tomaximize specificity. The16primers were used for the amplification of genomicDNA as a template. Amplified fragments were separated by electrophoresis. Wedesigned and validated the kit with wild and mutant type DNA samples that hadbeen previously been confirmed by Sanger sequencing. Then12,224newbornswere enrolled, and those samples with mutations were further validated withsequencing too. Among12,224newborns,258individuals had one or two mutantalleles, with the carrier rate being2.11%(258/12,224). For GJB2c.235delCmutation,3cases was homozygote and129cases were heterozygote carriers;For SLC26A4c.919-2A>G mutation,104cases were heterozygotes carriers, andno homozygotes were found; for MTRNR1mt.1555A>G mutation,16cases wasidentified; six cases of MTRNR1mt.1494C>T mutation were detected. All mutations were detected with high specificity. Mutation samples were confirmedvia Sanger sequencing. No false positive was found. We developed a user-friendlyscreening kit for deafness-associated mutations. It provided rapid, reproducible,and cost-effective detection of deafness gene mutation without special equipment.The kit allowed the detection of the four high risk deafness-associated mutationswith only4single tube PCR reactions. In the future, the kit could be applied tolarge population-based epidemiological studies for newborn hearing defectsscreening.PART3: Analyze Different Results of a Newborn HearingConcurrent Genetic Screening Procedure and toDevelop a Spread-ModelThis study indicated that the conventional newborn hearing screeningprogram for newborns could be improved by adding the genetic component. Thegenetic tests could immediately confirm hearing impaired babies referred byconventional hearing screening test and identified the cause of deafness withinone month. And genetic tests have the advantage of immediately determining themost important cause for preventable deafness, in particular resulting fromMTRNR1m.1555A> G or m.1494C>T, which can be efficiently eliminated byaverting the use of risky aminoglycoside antibiotics. The genetic tests aid in theidentification of newborn carriers of causative alleles, who tended to have a higher incidence of hearing loss and should be given more attention in future. To avoidthese causative alleles to be transmitted to next generation and maintain thecarriers’ normal hearing. So, genetic tests complements the current newbornhearing screening program and provides additional insights beyond whatconventional audiological tests reveal, thus significantly advancing the currentpractice to improve the newborn hearing screening as a means of early discoveryof genetic risk factors.