BackgroundPerrault syndrome (PS) is a rare autosomal recessive disorder characterized by sensorineural hearing loss (SNHL) and primary ovarian insufficiency in females. LARS2, encoding mitochondrial leucyl-tRNA synthetase, is the most common causative gene for PS. However, the genetic spectrum and clinical variability of PS remain underexplored. Expanding the catalog of LARS2 variants and correlating them with phenotypic data are critical for delineating genotype-phenotype relationships.MethodsTwo unrelated Chinese probands with hearing loss were enrolled, and comprehensive clinical evaluations were performed. Whole-exome sequencing (WES) was used to identify genetic variants, followed by Sanger sequencing for family co-segregation verification. Minigene assays and RT-PCR were conducted to assess the splicing effect of the novel canonical splice-site variant LARS2 c.235-2A>G. For the novel missense variant LARS2 c.1661T>C, 3-D structural modeling and evolutionary conservation analysis were performed to evaluate its pathogenicity. Moreover, we comprehensively summarized all LARS2 variants associated with PS via an extensive literature review.ResultsProband 1 (12-year-old female) harbors compound heterozygous variants LARS2 c.235-2A>G (novel) and LARS2 c.880G>A, presenting with profound SNHL, primary ovarian insufficiency, and developmental delay. Proband 2 (7-year-old male) carries compound heterozygous variants LARS2 c.1661T>C (novel) and LARS2 c.1886C>T, manifesting severe SNHL with an unusual upsloping audiogram pattern and comprehension difficulties. Functional assays confirmed that LARS2 c.235-2A>G disrupts canonical splicing, leading to exon 4 skipping and in-frame deletions. 3-D structural modeling and conservation analysis revealed that LARS2 c.1661T>C likely impairs protein stability by altering residue interactions, with Val554 being highly conserved across species. According to the ACMG/AMP guideline, both novel LARS2 variants were classified as likely pathogenic.ConclusionWe identified two novel LARS2 variants associated with PS in Chinese patients, thereby expanding the LARS2 genetic spectrum and providing precise molecular evidence for clinical management and genetic counseling. This study enhances understanding of genotype-phenotype correlations in PS, thereby revealing the phenotypic heterogeneity of LARS2 variants.

ABSTRACTBACKGROUNDThe genetic complexity of hemoglobin genes, characterized by high GC content and homologous sequences, poses significant challenges for detecting hemoglobin variants in clinical settings.METHODSA long-read indexed PCR method utilizing the novel CycloneSEQ nanopore sequencing platform was developed to detect all variant types, including single nucleotide variants (SNVs), deletions, structural variants (SVs) inHBA, HBB, HBD, andHBGgenes. The method was validated using 507 clinical samples to assess its performance.RESULTSThe long-read indexed PCR system employed 13 primers targeting the hemoglobin gene clusters. This design enabled the detection of 37 types ofHBAdeletions, 5 SV (3 multicopies (αααα, αααanti3.7, αααanti4.2) and 2 fusion allele (HKαα andanti-HKαα)), 37HBBdeletions, and all SNVs in the targeted regions. Validation across 507 samples (84 withHBAvariants, 60 withHBBvariants, 256 with bothHBAandHBBvariants, and 107 with no known variants) demonstrated 100.0% sensitivity and specificity. Additionally, the long-read sequencing enabled phasing of variants within hemoglobin genes, providing insights critical for clinical interpretation.CONCLUSIONSThe long-read indexed PCR method, combined with the CycloneSEQ nanopore sequencing platform, proved to be a robust and efficient solution for detecting hemoglobinopathy variants. The integration of indexed primers and barcoding enhances scalability, making this method ideal for large-scale population screening programs in the future.

Genomic sequencing can identify nucleotide changes for underlying monogenic disorders, making it a promising newborn screening method for enabling early intervention and reducing false positives. Here, in this prospective study, we enrolled 9,992 newborns from the West Coast New District of Qingdao, China, within 3 days after birth; positive cases were followed until 31 March 2025 to assess the effectiveness of whole-genome sequencing (WGS) in neonatal screening. Among 9,992 newborns screened by WGS, 268 (2.7%) were positive. By the date of follow-up, 19 were clinically confirmed (11 hearing loss, 3 glucose-6-phosphate dehydrogenase deficiency, 2 Wilson disease, 2 phenylketonuria and 1 methylmalonic aciduria), of which 8 were missed by traditional screening. Among 19 symptomatic infants who underwent reanalysis, 8 (42.1%) were diagnosed with potentially pathogenic or pathogenic variants associated with the phenotype. Our findings indicate that integrating WGS into routine newborn screening could substantially improve early detection of monogenic diseases and enhance clinical outcomes in China. In a prospective cohort study of 9,992 newborns in Qingdao, China, whole-genome sequencing (WGS) identified 268 screen-positive cases and clinically confirmed 19 disorders, 8 of which were missed by traditional newborn screening. These findings highlight that WGS can detect monogenic conditions more effectively than standard methods and could improve early diagnosis in routine neonatal screening.

Abstract Background Thalassemia is one of the most common monogenic disorders worldwide. Current screening strategies combining hematological testing with molecular assays still carry a risk of missed diagnoses and suboptimal efficiency, particularly for complex structural variants and rare mutations. Methods In this prospective, double-blind, multicenter cohort study of 3,842 participants (3,362 pregnant women and 480 male partners), we conducted a head-to-head comparison to systematically evaluate the incremental clinical value and detection performance of single-molecule nanopore sequencing in thalassemia (SMITH) against conventional hematological testing and next-generation sequencing (NGS). Findings The overall concordance rate between NGS and SMITH was 98.6% (3789/3842). The discrepant cases (n=53) were directly attributed to the superior detection capabilities of SMITH, which successfully identified complex structural rearrangements—including 45 α-globin gene triplications and four HKαα alleles—that were missed by NGS. Furthermore, SMITH accurately detected four rare variants (αα c.134_135insT /αα, αα c.-22(C>T) /αα, β N /β c.316-290delinsAGGGCAATAATTT , and β 3.5 kb deletion /β N ) and resolved ten trans and three cis configurations within the globin gene alleles. Clinically, these technical advantages translated to a 9.3% (5/54) increase in the detection rate of high-risk prenatal couples, effectively preventing one birth affected by moderate-to-severe thalassemia. Additionally, SMITH corrected a diagnostic discrepancy in one case (HKαα vs. -α 3.7 ), sparing the couple from an unnecessary invasive procedure. Interpretation Our findings demonstrate that SMITH provides a powerful platform for resolving globin gene rearrangements, detecting rare variants, and enabling direct haplotype phasing. By effectively eliminating diagnostic blind spots, SMITH is expected to become an optimal method for thalassemia prevention programs. Funding This study was supported by the Chinese National Natural Science Foundation Projects 81760037 and 82271894. Research in context Evidence before this study Next-generation sequencing (NGS) has substantially advanced carrier screening for thalassemia. However, its analytical blind spots—particularly in highly homologous regions, complex structural variants (SVs), and haplotype phasing—directly compromise the accuracy of genetic counseling and prenatal diagnosis. Long-read sequencing holds promise to address these gaps by generating reads that span entire gene clusters. Prior to this study, research on long-read sequencing for thalassemia screening had been extensive. To systematically evaluate its clinical value, we searched the literature published over the past decade. However, most previous studies compared long-read sequencing with conventional PCR-based methods, which do not allow assessment of its incremental value relative to current NGS-based screening pathways. To date, no large-scale, prospective, head-to-head study has compared NGS with single-molecule nanopore sequencing in a real-world prenatal screening setting. Therefore, the incremental clinical value of long-read sequencing in this context remains to be systematically quantified. Added value of this study In this prospective, double-blind, multicenter cohort study of 3,842 participants, we performed a head-to-head comparison between standard NGS and our novel Single-Molecule nanopore sequencing In THalassemia (SMITH) in a real-world prenatal screening setting. To our knowledge, this is the first large-scale, prospective study to directly compare long-read sequencing with standard NGS and to link technical performance with clinical outcomes. SMITH increased the detection rate of high-risk couples by 9.3%, prevented one moderate-to-severe thalassemia birth, and corrected a diagnostic misclassification that spared a couple from unnecessary invasive amniocentesis. These findings provide detailed, clinically actionable insights not previously reported. Implications of all the available evidence These findings establish SMITH as a major advance in thalassemia prevention. By eliminating key diagnostic blind spots—complex structural variants, rare mutations, and unresolved allelic phases—SMITH improves the accuracy of genetic counseling and prenatal diagnosis. The reduction in missed high-risk couples and preventable severe thalassemia births has direct implications for public health policy, particularly in high-prevalence regions. Future work should prioritise cost-effectiveness analyses and large-scale implementation studies to guide the integration of SMITH into routine population-based screening programmes.

BackgroundPreimplantation genetic testing for structural rearrangements (PGT-SR) facilitates the selection of embryos with balanced karyotype prior to implantation. Currently, there is a critical need for reference materials (RMs) to ensure PGT-SR development.ResultsThis study designed a novel and comprehensive strategy to produce renewable RMs using quartet families of structural rearrangements. A panel of quartet RMs for PGT-SR was successfully developed from 13 families, including 8 reciprocal translocations, 2 Robertsonian translocations, 2 inversions, and 1 normal family. Each quartet RMs comprised 3 tubes of DNA and 1 tube of sorted cells mimicking embryonic cells, all passed stringent quality assessments. The panel of quartet RMs was characterized by three volunteer laboratories with 100% accuracy and 100% specificity, using different sequencing platforms to assess the biomimetics. Additionally, while both short-read and long-read genome sequencing could detect exact breakpoints of the reciprocal translocations and standard inversion, long-read genome sequencing outperformed short-read sequencing for complex structural rearrangements.ConclusionsThis panel of quartet RMs for PGT-SR is well-characterized, renewable, and publicly accessible, playing a crucial role in proficiency test and quality assurance for structural rearrangement detection. The methods developed in this study are adaptable for other preimplantation genetic tests, thereby enhancing the advancement of assisted reproduction techniques.

Background Physiologic newborn hearing screening (NBHS) has several weaknesses requiring improvement. Studies have explored variant-based genetic NBHS as a complement to physiologic NBHS. However, this genetic NBHS strategy has fundamental limitations that challenge logistics. Methods Given findings that a small set of genes accounts for the majority of genetic etiologies of hearing loss (HL), we designed a gene-based genetic NBHS strategy that screens for genes instead of limited variants, targeting the coding regions of twelve HL-related genes and the aminoglycoside-induced HL risk gene MT-RNR1. Our cohort study was conducted at Nanjing Women and Children's Healthcare Hospital in China, between March 2022 and July 2023. All participants were offered concurrent physiologic and gene-based genetic NBHS. Infants with positive physiologic or genetic NBHS results were scheduled for audiological assessments. Results Of 20,997 participants with eligible physiologic and genetic NBHS results, 164 (0.8%) had positive physiologic or genetic NBHS results. Of 150 who underwent audiological assessments, 77 had confirmed HL. Among them, 32 were identified by genetic NBHS only, 24 by both physiologic and genetic NBHS, and 21 by physiologic NBHS only. Compared with conventional physiologic NBHS protocol (identified 45 HL cases), incorporating gene-based genetic NBHS into physiologic NBHS (additionally identified 32 HL cases) only required an additional 0.4% (87/20,997) of infants to undergo audiological assessments, yet achieved a 71% (32/45) increase in HL identification and provided genetic etiological information for 73% (56/77) of all HL cases. For 21 HL cases identified by physiologic NBHS only, genome sequencing identified genetic findings in three additional cases, showing that up to 77% (59/77) of infants with HL were caused by genetic factors in this general newborn population. Conclusions Our new comprehensive NBHS protocol, concurrent physiologic and gene-based genetic NBHS, significantly improved the identification of infants with HL. The results demonstrated that the gene-based genetic NBHS addressed fundamental limitations of the variant-based strategy and was a powerful complement to physiologic NBHS.

Purpose: Exon–level duplications in the DMD gene present interpretive challenges due to limitations in resolving their genomic context. We aimed to assess the utility of long–read genome sequencing (lrGS) in characterizing DMD duplications and guiding clinical interpretation. Methods: We applied low coverage lrGS (3–10× depth; ~8.2 kb mean read length) to 18 individuals with DMD duplications identified via short–read sequencing. Structural variant calling and breakpoint localization were validated by Sanger sequencing. In addition, the genomic characteristics of the duplication breakpoints were systematically analyzed. Results: lrGS resolved duplication architecture in all cases. Two duplications (11%, 2/18) were extragenic and reclassified as benign; 16 (89%, 16/18) were tandem events within DMD. Among tandem duplications, 50% (8/16) were classified as pathogenic/likely pathogenic and 50% (8/16) as variants of uncertain significance. Breakpoints were consistently located in intronic regions, often flanked by repetitive elements. Conclusion: Low–coverage lrGS enables high–resolution mapping of DMD duplications and improves variant classification. This approach addresses a key gap in carrier screening and molecular diagnosis of dystrophinopathies, and provides lrGS as a prototype for decoding duplication architecture of monogenic disorders, which is a critical advance in genetic diagnosis. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study did not receive any funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the institutional review board of BGI (IRB25049). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data generated by this study is available from the corresponding authors upon request

Non-invasive prenatal testing (NIPT) has been endorsed by the American College of Medical Genetics and Genomics as the preferred method for screening fetal 22q11.2 deletion syndrome (22q11.2 DS). Maternal genomic aberrations represent a significant source of false positives in NIPT, and there are currently no solutions that effectively address this challenge. We have devised an innovative NIPT bioinformatics pipeline designed to discern the origins of copy number variations (CNVs). Then, we recruited a cohort of 39cases of 22q11.2 DS to validate the effectiveness of our methodology. Follow-up tests including amniocentesis and genome sequencing of maternal leukocytes were conducted. Leveraging a dataset of over 900 CNVs, we developed a new pipeline that classifies CNVs into those of fetal, maternal, and maternal-fetal origin based on NIPT data. The use of our pipeline led to a notable increase in the positive predictive value of NIPT for detecting 22q11.2 DS from 87% (34/39) to 94% (34/36). Furthermore, our approach has the potential to reduce the number of invasive tests by 8% (3/39). Our innovative and reliable bioinformatics pipeline has enabled the accurate differentiation of CNV origin into fetal, maternal, and maternal-fetal categories. Incorporating this pipeline into the analytical workflow could reduce false positives in NIPT results and minimize the need for invasive prenatal diagnoses.

The application of next-generation sequencing (NGS) technology is increasingly used in newborn screening (NBS) to detect monogenic disorders. However, its capability to identify chromosomal aneuploidies and its potential clinical value have not been fully explored. This study investigates the feasibility of using an NGS panel for aneuploidy screening and examines the incidence of aneuploidies in newborns. We designed an NBS panel targeting 142 genes associated with 128 disorders and conducted chromosomal copy number analysis on 29,601 newborns across eight hospitals in China. The presence of chromosomal aneuploidies was confirmed through karyotyping or genome sequencing, and follow-up visits were conducted to assess prenatal screening outcomes and postnatal phenotypes. Among the 29,601 newborns, 47 were identified with various aneuploidies. Further investigation confirmed 30 of these cases, yielding a positive predictive value (PPV) of 100%. The estimated incidence of aneuploidies among live births was 0.16%, with significant regional discrepancies ranging from 0.04–0.23%. Sex chromosome aneuploidy (SCA) was the most prevalent at 0.15%, while trisomy 21 occurred at a lower rate of 0.01%. The NBS panel demonstrated potential effectiveness and accuracy in detecting chromosomal aneuploidies, suggesting it could play a valuable role in future genetic NBS clinical practice.

Sensorineural hearing loss is a prevalent disorder with significant genetic involvement, which is often challenging to diagnose due to genetic heterogeneity. Exome sequencing (ES) has been a standard diagnostic tool for sensorineural hearing loss, but its limitations in detecting copy number variants (CNVs) and intronic variants have prompted the exploration of genome sequencing (GS) for improved diagnostic yield. We conducted GS on 46 hearing loss families with previously negative ES results and an additional cohort of 36 patients with a monoallelic pathogenic variant in GJB2 (the most common deafness gene). Additionally, the impact of a previously unrecognized novel 125‐kb deletion in the DFNB1 locus on GJB2 expression was assessed using quantitative polymerase chain reaction (qPCR), and haplotype analysis was performed to characterize the deletion. GS diagnosed eight cases (17%, 8/46) in the ES‐negative cohort, primarily attributed to CNVs (6/8). Notably, a previously unrecognized 125 kb deletion in the DFNB1 region was identified, affecting GJB2 expression and characterizing it as a founder effect in East Asian. In 47 patients with a monoallelic GJB2 variant, 15% (95% CI, 7.4%–28%) were diagnosed with DFNB1 deletions. Analysis of the gnomAD database revealed the prevalence and ethnic diversity of DFNB1 deletions, with the novel 125 kb deletion emerging as a prominent pathogenic variant in East Asian, non‐Finnish European, and admixed American populations. Our study highlights the utility of GS in diagnosing sensorineural hearing loss. The identification of DFNB1 deletions underscores their significant contribution to hearing loss etiology, advocating for their inclusion in routine diagnostic testing. We propose GS as a primary genetic testing approach for patients with hearing loss, offering comprehensive genomic analysis and the potential for improved diagnostic accuracy.

BACKGROUND:Deafness, autosomal recessive 16 (DFNB16) is caused by compound heterozygous or homozygous variants in STRC and is the second most common form of genetic hearing loss. Due to the nearly identical sequences of STRC and the pseudogene STRCP1, analysis of this region is challenging in clinical testing.METHODS:We developed a method that accurately identifies the copy number of STRC and STRCP1 using standard short-read genome sequencing. Then, we used whole genome sequencing (WGS) data to investigate the population distribution of STRC copy number in 6813 neonates and the correlation between STRC and STRCP1 copy number.RESULTS:The comparison of WGS results with multiplex ligation-dependent probe amplification demonstrated high sensitivity (100%; 95% CI, 97.5%-100%) and specificity (98.8%; 95% CI, 97.7%-99.5%) in detecting heterozygous deletion of STRC from short-read genome sequencing data. The population analysis revealed that 5.22% of the general population has STRC copy number changes, almost half of which (2.33%; 95% CI, 1.99%-2.72%) were clinically significant, including heterozygous and homozygous STRC deletions. There was a strong inverse correlation between STRC and STRCP1 copy number.CONCLUSIONS:We developed a novel and reliable method to determine STRC copy number based on standard short-read based WGS data. Incorporating this method into analytic pipelines would improve the clinical utility of WGS in the screening and diagnosis of hearing loss. Finally, we provide population-based evidence of pseudogene-mediated gene conversions between STRC and STRCP1.

IMPORTANCE Newborn screening via biochemical tests is in use worldwide. The availability of genetic sequencing has allowed rapid screening for a substantial number of monogenic disorders. However, the outcomes of this strategy have not been evaluated in a general newborn population. OBJECTIVE To evaluate the outcomes of applying gene panel sequencing as a first-tier newborn screening test.DESIGN, SETTING, AND PARTICIPANTS This cohort study included newborns who were prospectively recruited from 8 screening centers in China between February 21 and December 31, 2021. Neonates with positive results were followed up before July 5, 2022. EXPOSURES All participants were concurrently screened using dried blood spots. The screen consisted of biochemical screening tests and a targeted gene panel sequencing test for 128 conditions. The biochemical and genomic tests could both detect 43 of the conditions, whereas the other 85 conditions were screened solely by the gene panel.MAIN OUTCOMES AND MEASURES The primary outcomes were the number of patients detected by gene panel sequencing but undetected by the biochemical test.RESULTS This study prospectively recruited 29 601 newborns (15 357 [51.2%] male). The mean (SD) gestational age was 39.0 (1.5) weeks, and the mean (SD) birth weight was 3273 (457) g. The gene panel sequencing screened 813 infants (2.7%; 95% CI, 2.6%-2.9%) as positive. By the date of follow-up, 402 infants (1.4%; 95% CI, 1.2%-1.5%) had been diagnosed, indicating the positive predictive value was 50.4% (95% CI, 50.0%-53.9%). The gene panel sequencing identified 59 patients undetected by biochemical tests, including 20 patients affected by biochemically and genetically screened disorders and 39 patients affected by solely genetically screened disorders, which translates into 1 out of every 500 newborns (95% CI, v385-v625) benefiting from the implementation of gene panels as a first-tier screening test.CONCLUSIONS AND RELEVANCE In this cohort study, the use of gene panel sequencing in a general newborn population as a first-tier screening test improved the detection capability of traditional screening, providing an evidence-based suggestion that it could be considered as a crucial method for first-tier screening.

BACKGROUNDGenome-wide noninvasive prenatal testing identifies several rare autosomal trisomies in the general obstetrical population, but its use is questioned by its low positive predictive value. Furthermore, the origin of rare autosomal trisomies and the clinical effect of reporting them has not been sufficiently investigated. In addition, professional societies express their need for data assessing the clinical use of genome-wide noninvasive prenatal testing for rare autosomal trisomies for years.OBJECTIVEThis study aimed to investigate the origin of rare autosomal trisomies and the clinical effect of disclosing rare autosomal trisomies in clinical settings.STUDY DESIGNWomen who received noninvasive prenatal testing between March 2021 and March 2022 were prospectively enrolled. Clinical follow-up and cytogenetic and molecular investigations were performed. Posthoc analysis was performed to investigate the association between placental mosaicism and clinical outcomes.RESULTSOverall, 154 rare autosomal trisomies were identified in 89,242 pregnancies (0.17%) through noninvasive prenatal testing. In the 120 cases in which cytogenetic and molecular investigations were carried out, the rare autosomal trisomies were found to originate from true fetal mosaicism (n=5), uniparental disomy (n=5), maternal mosaic trisomy (n=3), maternal malignancy (n=1), and confined placental mosaicism (n=106). Clinical follow-up showed that 40% of all rare autosomal trisomy cases had adverse perinatal outcomes. In women with false-positive noninvasive prenatal testing results originating from confined placental mosaicism, the frequency of adverse perinatal outcomes was 26%. More importantly, the placental mosaicism ratio revealed by noninvasive prenatal testing was significantly higher in women who experienced adverse perinatal outcomes than those who did not (0.688 vs 0.332; P<.001).CONCLUSIONWomen with noninvasive prenatal testing results indicative of rare autosomal trisomies are at risk of adverse perinatal outcomes, and that risk can be stratified using chromosomes and the mosaicism ratio revealed by noninvasive prenatal testing. Our data are valuable for obstetrical caregivers advising a patient with a noninvasive prenatal testing result indicative of a rare autosomal trisomy and a false-positive diagnosis and for managing risks during pregnancy.

Abstract Background Noninvasive prenatal testing (NIPT) is the testing of blood samples from pregnant women to screen for fetal risk of chromosomal disorders. Even though in vitro hemolysis of blood specimens is common in clinical laboratories, its influence on NIPT has not been well investigated. Methods Peripheral blood samples were collected from 205 pregnant women and categorized according to the concentration of free hemoglobin in the plasma. After performing NIPT using massively parallel sequencing, the quality control metrics were analyzed and compared with samples that did not undergo hemolysis or samples redrawn from the same women. Results The specimens were divided into four groups based on the concentration of free hemoglobin: Group I (0–1 g/L, n = 53), Group II (1–2 g/L, n = 97), Group III (2–4 g/L, n = 30), and Group IV (> 4 g/L, n = 25). There was no significant difference in the quality control metrics of clinical samples with slight or moderate hemolysis (Group II and III). However, samples with severe hemolysis (Group IV) showed a significantly increased rate of duplicated reads (duplication rate) and fetal fraction, as well as decreased library concentration compared with samples without hemolysis. Moreover, the increase in fetal fraction caused by hemolysis was confirmed by redrawing blood samples in Group IV. Conclusion For NIPT using massively parallel sequencing, samples with slight or moderate hemolysis (≤ 4 g/L) are acceptable. However, careful consideration should be taken regarding the use of severely hemolyzed samples (> 4 g/L), since they might increase the risk of test failure.

To improve the etiological diagnosis of congenital hearing loss by combining whole-exome sequencing (WES) with cytomegalovirus (CMV) testing and to explore the potential benefits of adding CMV screening to newborn hearing screening, 80 children under 2 years of age with bilateral sensorineural hearing loss were recruited. Peripheral venous blood was extracted from the children for WES analysis. Saliva after mouthwash and the first urine in the morning were collected and used as samples to quantify CMV DNA copy number in urine and saliva by qPCR; among the 80 children with congenital deafness, 59 (74%) were found to have genetic variants that may cause congenital deafness, including 44 with GJB2 or SLC26A4 gene variant, 1 with STRC gene variant, and 14 with other genetic variants. A total of 12 children carried deafness gene variants associated with a syndrome; CMV test results showed that in two children, the CMV DNA copy number in saliva was >1000/mL, which indicates that they were CMV-positive, and their genetic test results were negative. A neonatal CMV test combined with genetic screening can improve the etiological diagnosis rate of congenital deafness, and the direct evidence of neonatal CMV infection deserves further verification.

Purpose Genetic testing is widely used in diagnosing genetic hearing loss in patients. Other than providing genetic etiology, the benefits of genetic testing in pediatric patients with hearing loss are less investigated. Methods From 2018-2020, pediatric patients who initially presented isolated hearing loss were enrolled. Comprehensive genetic testing, including GJB2/SLC26A4 multiplex amplicon sequencing, STRC/OTOA copy number variation analysis, and exome sequencing, were hierarchically offered. Clinical follow-up and examinations were performed. Results A total of 80 pediatric patients who initially presented isolated hearing loss were considered as nonsyndromic hearing loss and enrolled in this study. The definitive diagnosis yield was 66% (53/80) and the likely diagnosis yield was 8% (6/80) through comprehensive genetic testing. With the aid of genetic testing and further clinical follow-up and examinations, the clinical diagnoses and medical management were altered in eleven patients (19%, 11/59); five were syndromic hearing loss; six were nonsyndromic hearing loss mimics. Conclusion Syndromic hearing loss and nonsyndromic hearing loss mimics are common in pediatric patients who initially present with isolated hearing loss. The comprehensive genetic testing provides not only a high diagnostic yield but also valuable information for clinicians to uncover subclinical or pre-symptomatic phenotypes, which allows early diagnosis of SHL, and leads to precise genetic counseling and changes the medical management.

Background: Hearing loss affects approximately two out of every 1,000 newborns. Genetic factors and congenital cytomegalovirus (CMV) infections account for around 90% of the etiology. The purpose of this study was to develop and test a whole genome sequencing (WGS) approach to detect deafness-related genetic variants and CMV infections simultaneously in newborns. Method: Deafness-related genes causing congenital or childhood hearing loss were curated and selected for newborn screening. Nine dried blood spots from newborns with known genetic variants (n = 6) or CMV infections (n = 3) were employed to develop and validate the WGS testing and analytic pipeline. We then pilot tested the WGS analysis on 51 de-identified clinical samples. Results: 92 gene-disease pairs were selected for screening hearing loss in newborns. In the validation test, WGS accurately detected all types of genetic variants, including single nucleotide variations, insertions/deletions, and copy number variations in the nuclear or mitochondrial genome. Sequence reads mapping to the CMV reference genome were discovered in CMV infected samples. In the pilot test, WGS identified nine out of 51 (18%) newborns carrying pathogenic variants associated with deafness. Conclusion: WGS can simultaneously detect genetic variants and CMV infections in dried blood spot specimens from newborns. Our study provides proof of principle that genome sequencing can be a promising alternative for newborn screening of hearing loss.

Genetic variants in GJB2 are the most frequent cause of congenital and childhood hearing loss worldwide. The purpose of this study was to delineate the genetic and phenotypic landscape of GJB2 SNV variants. All possible single-nucleotide substitution variants of the coding region of GJB2 (N = 2043) were manually curated following the ACMG/AMP hearing loss guidelines. As a result, 60 (2.9%), 177 (8.7%), 1499 (73.4%), 301 (14.7%) and 6 (0.3%) of the variants were classified as pathogenic, likely pathogenic, variant of uncertain significance, likely benign, and benign, respectively. 53% (84/158) of the pathogenic/likely pathogenic missense variants were not present in ClinVar. The second transmembrane domain and the 3(10) helix were highly enriched for pathogenic missense variants, while the intracellular loops were tolerant to variation. The N-terminal tail and the extracellular loop showed high clustering of variants that are associated with syndromic or dominant non-syndromic hearing loss. In conclusion, our study interpreted all possible single-nucleotide substitution coding variants, characterized novel clinically significant variants in GJB2, and revealed significant genotype-phenotype correlations at this common hearing loss locus. Our work provides a prototype for other genes with similarly high genetic and phenotypic heterogeneity.

Background The American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) presented technical standards for interpretation and reporting of constitutional copy-number variants in 2019 (the standards). Although ClinGen developed a web-based CNV classification calculator based on scoring metrics, it can only track and tally points that have been assigned based on observed evidence. Here, we developed AutoCNV (a semiautomatic automated CNV interpretation system) based on the standards, which can automatically generate predictions on 18 and 16 criteria for copy number loss and gain, respectively. Results We assessed the performance of AutoCNV using 72 CNVs evaluated by external independent reviewers and 20 illustrative case examples. Using AutoCNV, it showed that 100 % (72/72) and 95 % (19/20) of CNVs were consistent with the reviewers’ and ClinGen-verified classifications, respectively. AutoCNV only required an average of less than 5 milliseconds to obtain the result for one CNV with automated scoring. We also applied AutoCNV for the interpretation of CNVs from the ClinVar database and the dbVar database. We also developed a web-based version of AutoCNV (wAutoCNV). Conclusions AutoCNV may serve to assist users in conducting in-depth CNV interpretation, to accelerate and facilitate the interpretation process of CNVs and to improve the consistency and reliability of CNV interpretation.