Background Fetal fraction (FF) quantification is critical for prenatal cell-free DNA screening. Current methods based on Y chromosome (chrY), differential methylation, or single-nucleotide polymorphisms (SNPs) face limitations including sex dependency, complex workflows, or the need for parental genotyping. An FF quantification method that integrates directly into the digital polymerase chain reaction (dPCR) workflow is needed to support the clinical implementation of dPCR-based prenatal screening. Methods We developed a multiplex dPCR assay quantifying 9 autosomal SNPs and 3 chrY loci across 2 reaction wells. Key innovations include: concurrent SNP and chrY detection for sex-independent estimation; a single-probe, dual-allele discrimination design with a calculation algorithm that eliminates parental genotyping; and a triplex chrY assay to enhance accuracy. The assay was analytically validated using simulated DNA mixtures and clinically evaluated using 143 maternal plasma samples against a next-generation sequencing (NGS) reference. Results Analytical validation showed strong linearity between expected and observed FF for both chrY (R-2 = 0.971) and SNP (R-2 = 0.924) methods. The chrY assay demonstrated superior accuracy and sensitivity at low FF levels (mean absolute error: 0.94% chrY, 1.61% SNPs). Clinical evaluation showed a 92.3% informative SNP rate, a low 2.8% no-call rate, and strong concordance with NGS-based FF estimates (r = 0.799, P < 0.0001). Conclusions Our dPCR assay provides a practical QC tool for dPCR-based prenatal screening by enabling sex-independent FF quantification without parental genotyping or complex preanalytical steps. Future efforts will include multicenter validation and panel expansion. This methodology also shows potential for applications in transplantation medicine and obstetric monitoring.

Advances in bioinformatics have revealed the potential of whole exome sequencing (WES) for copy number variations (CNVs) detection. This study aimed to evaluate whether WES can replace low pass copy number variation sequencing (CNV-seq) for the detection of CNVs in clinical prenatal diagnosis. Based on a cohort of 1055 fetuses tested concurrently with trio-WES and CNV-Seq, we performed a systematic comparison of both methods for the detection of aneuploidies, mosaicism, and pathogenic/likely pathogenic (P/LP) CNVs. Both WES and CNV-Seq concordantly identified 11 aneuploidies, 4 mosaicism, and 29 P/LP CNVs. The aneuploidies comprised eight cases of trisomy 21, two cases of 45,X, and one case of 47,XXX. Four mosaicisms consisted of two whole-chromosome mosaicism (WCM) and two segmental aneuploid mosaicism. The two platforms demonstrated comparable accuracy in copy number (CN) detection for both aneuploidies and whole-chromosome mosaicism. However, WES showed comparatively lower precision than CNV-Seq in detecting CN alterations within segmental aneuploid mosaicism. For P/LP CNVs, both platforms demonstrated full concordance in the quantity and pathogenicity classification, although minor discrepancies were observed in their genomic locations and sizes determination. The Jaccard similarity coefficient values ranged from 57.9

Background To validate the clinical efficacy of non-invasive prenatal diagnosis (NIPD) for spinal muscular atrophy (SMA) in the first trimester and extend its applicability to families without probands.Method From December 2020 to October 2024, 288 high-risk pregnancies were recruited prospectively, with 81 qualifying for NIPD after genetic counseling. Among the eligible cases, parent-based haplotypes were successfully constructed in 75 families (92.6%), while grandparent-based haplotype reconstruction was performed for the remaining 6 cases (7.4%) where proband samples were unavailable. Through targeted sequencing of the SMN1/SMN2 gene and flanking informative SNPs in maternal plasma, fetal haplotypes were inferred by analyzing dosage changes in cell-free DNA (cfDNA) using Bayes factor. All NIPD results were subsequently validated through invasive diagnostic procedures (chorionic villus sampling or amniocentesis).Results The haplotypes were successfully constructed in 81 families through parents or grandparents of the identified variant carriers. 76 families (93.8%) successfully obtained NIPD results, among which the earliest gestational week for successful NIPD was 7+3 weeks, with a minimum fetal fraction of 1.9%. 5 cases were classified ‘no call’ results due to pathogenic variant-adjacent recombination events (2/5), insufficient or unevenly distributed informative SNPs (2/5), and subthreshold fetal fraction (1/5). The average gestational age of NIPD blood drawing is 9 weeks. Validation test showed the NIPD results accuracy was 100%.Conclusion This study demonstrates the clinical feasibility of grandparent-assisted haplotype construction for SMA families without probands and enables accurate early prenatal diagnosis of SMA in first-trimester pregnancies.

Noninvasive prenatal testing for single-gene disorders faces significant challenges due to low fetal DNA abundance in maternal blood, particularly in early gestation. Although next-generation sequencing (NGS) is widely used, it frequently encounters false-negative outcomes and high costs when detecting low-abundance fetal variants. To address these limitations, we developed a highly sensitive and cost-effective analytical method utilizing high-resolution tandem mass spectrometry, capable of accurately detecting single nucleotide variants (SNVs) as early as 5-8 weeks gestation. It employs gas-phase enrichment combined with fragment mass spectral analysis for precise isolation and identification of fetal DNA amidst predominant maternal DNA. Analytical validation with synthetic DNA and cultured cells confirmed exceptional sensitivity, achieving single copy level detection despite significant interference. Clinically, our method accurately detected paternally inherited SNVs in 134 cell-free fetal DNA samples collected at 5-8 weeks gestation, achieving 100% accuracy. Additionally, it reliably identified pathogenic SNVs linked to phenylketonuria from 12 samples collected at 11-13 weeks gestation. This approach significantly reduces operational costs, enhances sensitivity, and eliminates false-negative results compared to traditional NGS-based methods.

Evaluate the application efficacy of trio exome sequencing (Trio-ES) in prenatal families with a history of developmental delay/intellectual disability (DD/ID). This cohort study enrolled prenatal families with a family history of DD/ID from a single center between January 2020 and May 2025. We included 104 prenatal families that had excluded copy number variation abnormalities but had not undergone single nucleotide variant testing. Based on family history, these families were divided into two groups: those with a history of DD/ID births but phenotypically normal parents (lacking proband samples), and those without a history of DD/ID births but with one or both parents having ID. The overall positive rate of prenatal Trio-ES among 104 families with a history of DD/ID was 21.15% (22/104). Among 81 families with a history of DD/ID births but phenotypically normal parents, the positive rate was 13.58% (11/81), including 7.41% (6/81) affected fetuses and 6.17% (5/81) only parents as carriers, both primarily exhibiting a recessive inheritance pattern. Additionally, the positive rate in non-syndromic DD/ID families was 11.54% (6/52), while it reached 17.24% (5/29) in syndromic DD/ID families (associated with epilepsy, dystonia, hearing impairment, abnormal head circumference, etc.). Among 23 families without a history of DD/ID births but with one or both parents having ID, the positive rate significantly increased to 47.83% (11/23), primarily driven by dominant variants. This study represents the first focused evaluation of the clinical utility of Trio-ES in prenatal families with a history of DD/ID when proband samples are unavailable. We recommend actively employing Trio-ES for prenatal genetic evaluation in such families, particularly those presenting with a syndromic phenotype.

Introduction: Patients with hemophilia A (HA) can reach normal life expectancy and suffer from coronary artery disease (CAD). The study evaluates the likelihood of subclinical CAD evaluated by advanced electrocardiography (A-ECG), global hemostasis and extracellular vesicles (EVs) in Chinese patients with HA treated on-demand. Materials and Methods: Patients with HA (n = 42) and age-matched male controls (n = 37) were included. The likelihood of having CAD was evaluated by A-ECG. Fibrin formation and fibrinolysis were assessed by the overall hemostatic potential (OHP) assay. EVs derived from platelets (PEVs), endothelial cells (EEVs) and leukocytes (LEVs), and expressing phosphatidylserine (PS + EVs), tissue factor (TF + EVs) or P-selectin (CD62P + EVs) were measured by flow cytometry. Results: The likelihood of CAD evaluated by A-ECG did not differ between patients and controls. Fibrin formation and clot stability were significantly impaired in patients. Patients had higher PEVs and CD62P + EVs. CD62P + EVs counts were inversely correlated with OHP, velocity and clot lysis time. Subclinical CAD did not correlate with OHP or EVs. Conclusion: Controls and patients with HA treated on-demand exhibited no differences in A-ECG, but varied in fibrin formation and clot stability. Larger studies are required to explore the significance of this finding in the context of CAD risk in this population.

BackgroundNext-generation sequencing (NGS) facilitates simultaneous carrier screening for multiple single-gene disorders. However, conventional NGS methods struggle to detect complex variants, such as F8 inversions, CYP21A2 variations, and single-exon copy number variations (CNVs), resulting in residual risk.MethodsWe developed and validated MLDP-AS (Multiplex Long-Distance PCR followed by Amplicon Sequencing), a novel NGS assay, to screen for pathogenic variants in ten prevalent single-gene disorders in the Chinese population, including alpha- and beta-thalassemia, non-syndromic hearing loss, spinal muscular atrophy, Duchenne muscular dystrophy, phenylketonuria, 21-hydroxylase deficiency, Wilson disease, methylmalonic acidemia, and Hemophilia A. MLDP-AS detects routine variants and technically challenging types, such as F8 intron 22 inversions, CYP21A2 variations, and single-exon CNVs, in a single test. The assay was optimized using positive clinical samples, with sensitivity validated against gold-standard methods. Clinical applicability was evaluated through a prospective study of couples planning or undergoing pregnancy, with positive results confirmed by gold-standard techniques.ResultsMLDP-AS achieved 100% sensitivity, identifying all 255 pathogenic variants in known positive samples, including 36 technically challenging variants. In a prospective trial involving 5,209 individuals, carriers for all targeted disorders were detected, with an overall carrier rate of 22.15%. Thirty-four couples (1.09%) were identified as at-risk, spanning seven of the ten diseases. A total of 289 pathogenic variants were detected 1,290 times, including 169 technically challenging variants. The assay demonstrated a positive predictive value of 99.7% compared to gold-standard methods. MLDP-AS is rapid and cost-effective, completing testing within three days at a cost under $25.ConclusionsMLDP-AS integrates detection of multiple complex variants into a single, comprehensive assay, overcoming limitations of conventional NGS. It significantly enhances variant detection and provides an efficient, cost-effective tool for carrier screening in the Chinese population.

Renal ciliopathies encompass a spectrum of genetic disorders arising from structural or functional impairments of primary cilia, specialized organelles critical for mechanosensation and signal transduction within renal epithelial cells. These disorders are characterized by cystogenesis, driven by dysregulated ciliary signaling, leading to uncontrolled epithelial proliferation, aberrant growth, and loss of cellular polarity. The clinical trajectory evolves from initial cyst formation to advanced tubulointerstitial fibrosis and progressive renal failure. This progression is governed by pathogenic variants in genes encoding ciliary proteins. While advancements in genetic testing have established prenatal diagnosis as a pivotal tool for early identification, definitive diagnosis and therapeutic intervention remain challenging. These difficulties stem from several factors: incomplete understanding of the molecular mechanisms underlying cyst formation and fibrosis; limitations in prenatal diagnostic accuracy owing to phenotypic overlap and incomplete penetrance; and the marked genetic heterogeneity and diverse clinical trajectories of renal ciliopathies. Existing studies have primarily focused on unidirectional modulation of individual pathways, whereas the systematic integration of signaling network cascades remains largely unaddressed. This review systematically elucidates the molecular mechanisms and aberrant signaling pathways in renal ciliopathies, links genetic heterogeneity to clinical phenotypes, and lays a theoretical basis for prenatal diagnosis and novel therapies.

Abstract Objective Spinal muscular atrophy (SMA) is a motor neuron disorder encompassing 5q and non-5q forms, causing muscle weakness and atrophy due to spinal cord cell degeneration. Understanding its genetic basis is crucial for genetic counseling and personalized treatment options. Methods This study retrospectively analyzed families of patients suspected of SMA at our institution from February 2006 to March 2024. Various molecular techniques, including multiplex ligation-dependent probe amplification analysis, long-range polymerase chain reaction (PCR) combined with nested PCR, Sanger sequencing, and whole-exome sequencing were employed to establish a thorough genetic variant profile in 680 Chinese pedigrees with clinically suspected SMA. Results Out of 680 families suspected of having SMA, 675 exhibited mutations in the SMN1 gene, while three families were linked to mutations in the IGHMBP2 gene. One family exhibited a genetic variation in the NEB gene, and another family exhibited a variation in the SCO2 gene. Among the families with mutations in the SMN1 gene, 645 families exhibited either E7‒E8 or E7 homozygous deletion. Some families displayed E7‒8 heterozygous deletions along with other mutations, such as E1 or E1‒6 heterozygote deletion and point mutations. Furthermore, one family demonstrated a compound-heterozygous double mutation, while another carried a type “2 + 0” mutation alongside a point mutation. Conclusions This study comprehensively analyzed the genetics of suspected familial SMA cases in the Chinese population, providing insights into the molecular genetic mechanisms of SMA and the utility of various detection techniques. The findings revealed important implications for genetic counseling, prenatal diagnosis, and targeted therapies in clinical practice.

BackgroundCongenital adrenal hyperplasia (CAH) is a common metabolic genetic disease. Early diagnosis and intervention are crucial to improve the prognosis. Noninvasive prenatal diagnosis (NIPD) is an early, safe, and accurate method. This study aimed to evaluate the NIPD of CAH while guiding individualized intrauterine treatment.MethodsTwenty families with a 25% risk of having a baby with 21-hydroxylase deficiency (21-OHD) were included. Haplotypes were constructed based on targeted sequencing and family linkage analysis. Relative haplotype dosage (RHDO) combined with Bayes factor was used to infer fetal genotypes. Invasive prenatal diagnosis was performed to verify the reliability of NIPD. For affected-female fetuses, intrauterine treatment was applied until delivery.ResultsIn 20 families, NIPD successfully identified one female-affected fetus, four male-affected fetuses, nine heterozygotes, and five normal fetuses. The first-pass success rate of NIPD was 90% (18/20), the reporting rate was 95% (19/20), and the accuracy was 100% (19/19). Individualized intrauterine treatment avoided 88.9% (8/9) of unnecessary treatment of unaffected female fetuses. Moreover, no significant virilization was observed in the newborn of CAH16, which underwent intrauterine treatment.ConclusionNIPD has far-reaching implications for the early treatment and clinical management of pregnancy in families with 21-OHD.

BACKGROUND:Whole-genome sequencing (WGS) has been studied increasingly as a genetic testing technology in clinical applications, and its clinical validity has been preliminarily verified. In recent years, WGS has been employed in prenatal diagnosis. METHODS:This review synthesizes the current research and existing guidelines on the use of WGS for prenatal diagnosis. The methods, diagnostic scope, diagnostic rate, clinical usefulness, feasibility, limitations, and ethical issues of WGS in prenatal diagnosis are also presented. RESULTS:After reviewing the relevant studies, evidence indicated that WGS can improve the diagnostic rate for fetuses with abnormal development. At the same time, WGS also has significant challenges, such as a higher detection rate of variants of uncertain significance. CONCLUSION:WGS has great potential in prenatal diagnosis, but more research is needed to advance its clinical application.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare early-onset neurodegenerative disorder characterized by progressive cerebellar ataxia, spasticity, and sensorimotor peripheral neuropathy. This disorder is caused by homozygous or compound heterozygous variants in the sacsin (SACS) gene on chromosome 13q12.12. Three patients with ARSACS from two unrelated Chinese families were recruited for this study. Patient #1 was an 18-year-old male who had been walking unstably for 12 years. Patient #2, the younger sister of Patient #1, was a 5-year-old girl who had been walking unstably for 2 years. Patient #3 was a 19-year-old female who had been walking unstably and a tendency to fall for 17 years. For Patient #1, whole-exome sequencing (WES) identified a hemizygous variant c.8310_8313delAGAT (p.Asp2771fs4*) in SACS (NM_014363.6), with the father being heterozygous, the mother wild-type, and Patient #2 hemizygous, as verified by Sanger sequencing. Additional copy number variant analysis of the WES data indicated that Patient #1 had a heterozygous gross deletion of chr13q12.12 (chr13:23,808,732 − 24,890,322). Low-coverage whole-genome sequencing results revealed that Patient #2 carried a chr13q12.12 deletion (chr13:23,520,000–24,940,000). Together with Sanger sequencing results, this gross deletion was speculated to have been inherited from the mother, further explaining the hemizygous state of c.8310_8313delAGAT (p.Asp2771fs4*) in Patients #1 and #2. Through WES, Patient #3 was identified as having suspected compound heterozygous variants of c.2881 C > T (p.Arg961*) and c.6409 C > T (p.Gln2137*), inherited from the father and mother, respectively, as confirmed by Sanger sequencing. This study identified three variants in SACS. The c.8310_8313delAGAT (p.Asp2771fs4*) is novel, whereas c.2881 C > T (p.Arg961*) and c.6409 C > T (p.Gln2137*) have been reported previously. Moreover, this study highlights the growing trend that ARSACS has become increasingly prevalent worldwide rather than being localized to a specific region or race. As an increasing number of patients with ARSACS are diagnosed, the genetic spectrum of ARSACS will gradually broaden, providing an accurate genetic basis for prenatal diagnosis of mothers in the years ahead, if possible.

Abstract Background Noninvasive prenatal diagnosis (NIPD) has been proven feasible for non-syndromic hearing loss (NSHL) in singleton pregnancies. However, previous research is limited to the second trimester and the application in twin pregnancies is blank. Here we provide a novel algorithmic approach to assess singleton and twin pregnancies in the first trimester. Methods A 324.614 kb capture panel was designed to selectively enrich target regions. Parental haplotypes were constructed by target sequencing of blood samples from the parents and the proband. Then single nucleotide polymorphisms (SNP) within target regions were classified into four and six categories in singleton and twin pregnancy, respectively. Combining relative haplotype dosage change (RHDO) and the Bayes factor (BF), fetal fraction (FF) and fetal genotype were deduced in singleton and twin pregnancies. The pregnant women’s NIPD results were validated by invasive prenatal diagnosis and Sanger sequencing. Results Sixteen women with singleton pregnancies and one woman with a twin pregnancy were recruited. Among the 16 singleton pregnancies, NIPD was successfully applied in 15 families and the coincidence rate with invasive prenatal diagnosis was 100% (15/15). Only one family NIPD result is “no call” because the imbalance distribution of SNP sites makes it difficult to estimate recombination events. Most (13/15) of pregnant women were diagnosed in the first trimester and the earliest gestation week was the 7th week. The twin pregnancy was a dichorionic diamniotic twin (DCDA). NIPD confirmed one fetus is affected, and another is a carrier with c.299_300delAT of GJB2 gene. Conclusion This study represents the pioneering evidence in the field, demonstrating the feasibility of NIPD for NSHL in twin pregnancies. Moreover, it provides a novel and advanced diagnostic approach for families at high risk of NSHL during pregnancy, offering earlier detection, enhanced safety, and improved accuracy.

BACKGROUND:The clinical validity of whole-genome sequencing in postnatal settings is well documented, but studies of its use in prenatal settings are limited. OBJECTIVE:The objective of this study was to prospectively evaluate the performance of whole-genome sequencing for prenatal diagnosis of fetal structural anomalies compared with the commonly used testing strategy of copy-number variant sequencing plus exome sequencing. STUDY DESIGN:Whole-genome sequencing was performed in parallel with copy-number variant sequencing and exome sequencing for 96 parent-fetus trios with fetal structural anomalies. Single-nucleotide variants, small insertions/deletions, copy-number variations, structural variants, and absence of heterozygosity were classified according to the American College of Medical Genetics and Genomics, Association for Molecular Pathology, and ClinGen guidelines. RESULTS:Diagnostic variants were found by copy-number variant sequencing for 5/96 (5.2%) fetuses and by trio-exome sequencing for 26/96 (27.1%) fetuses. The combined diagnostic rate for copy-number variant sequencing plus trio-exome sequencing was 30/96 (31.2%). Whole-genome sequencing identified all diagnostic variants detected by copy-number variant sequencing and trio-exome sequencing plus 3 additional fetuses (one with maternal uniparental disomy 15, one with a complex chromosomal rearrangement, and one with compound heterozygous single-nucleotide variants in NADSYN1), increasing the diagnostic rate to 33/96 (34.4%). The highest diagnostic rate was observed in fetuses with craniofacial abnormalities (2/3, 66.7%), followed by those with hydrops (3/6, 50.0%). Ten families (10.5%, 10/96) were detected with incidental findings, of which, structural variants in 7 fetuses were detected only by whole-genome sequencing, including 6 inversions and one uniparental disomy 16. CONCLUSION:Trio-based whole-genome sequencing offers a valid alternative to copy-number variant sequencing plus exome sequencing, demonstrating the capacity for more comprehensive genomic analysis for fetuses with structural anomalies while permitting consolidation of laboratory workflows into a single test.

BackgroundUnexpected pregnancy loss can be a traumatic experience for fertile couples. The aim of the study was to assess the nature and type of chromosomal variants involved in early and late pregnancy loss and provide couples an explanation on the cause of their pregnancy loss.MethodsInvestigations were conducted on 2928 pregnancy loss cases where products of conception (POC) samples could be retrieved for genetic analysis. Chromosomal variants were detected by low pass copy number variation sequencing (CNV-seq).ResultsIn first-trimester miscarriages, 1272 of POC (60.4%) samples had a chromosome abnormality. Autosomal aneuploidy and monosomy X were the predominate variants (73.2%), followed by autosomal and sex chromosome mosaicism (10.7%), triploidy (9.6%), pathogenic CNVs (6.2%) and haploidy (0.3%). The chromosomal variants were similar in type and frequency regardless of whether the fetus had normal or abnormal ultrasound findings. In second trimester pregnancy loss where there was either a structural or non-structural ultrasound anomaly, only 15.3% of POC samples had a chromosome abnormality, involving mainly the smaller autosomes and monosomy X (55.7%), autosomal and sex chromosomal mosaicism (11.5%), triploidy (4.1%) and pathogenic CNVs (28.7%).ConclusionChromosomal variants contribute to fetal demise in almost two thirds of pregnancy losses.

Biallelic pathogenic variants in the lamin B receptor (LBR) with impaired sterol reductase function are associated with the development of perinatal lethal Greenberg dysplasia (GRBGD) and mild nonfatal skeletal dysplasia with or without Pelger-Huet anomaly (PHASK), as well as other related hereditary skeletal dysplasia. However, the underlying molecular mechanism remains unclear. In this study, we found two novel pathogenic variants of LBR, namely missense mutation (c.1011 T > G, NM_002296.4; p.Cys337Trp, NP_002287.2) and LBR gene deletion (Chr1q42.12 (225,515,082-225,633,464), NC_000001.10). LBR is a novel substrate of FBW7, which is degraded by GSK3β/FBW7-mediated proteasome pathway and whose C337W mutation promotes its degradation through enhanced interaction with FBW7. Wild-type but not C337W mutant LBR is upregulated by WNT3A-mediated inactivation of GSK3β/FBW7 axis and then participated in WNT3A-activated Wnt pathway through its mediated cholesterol synthesis. MC3T3-E1 cells with Lbr knockdown or cholesterol removal exhibited reduced mineralized nodules in the presence of WNT3A, but addition of cholesterol in the culture medium reversed this phenotype. Collectively, we detected two novel variants in LBR and our study revealed for the first time that disruption of cholesterol synthesis by LBR impairs Wnt pathway and thus disrupts the cell osteogenic differentiation, providing new insights into the pathogenesis of skeletal dysplasia caused by LBR variation.

OBJECTIVE:To analyze the clinical data and results of prenatal diagnosis for fetuses with high-risk for trisomy/monosomy 13 by non-invasive prenatal testing (NIPT). METHODS:Clinical data of pregnant women with fetus at a high risk for trisomy/monosomy 13 by NIPT at the First Affiliated Hospital of Zhengzhou University from May 2016 to May 2024 were reviewed, and relevant data such as Z-score, positive predictive value (PPV) and fetal fraction (FF) were analyzed to assess the correlation between them. This study was approved by the Ethics Committee of the Hospital (No. 2018-YB-08). RESULTS:71 fetuses were found to have a high risk by NIPT, including 58 cases for trisomy 13 (T13) and 13 cases for monosomy 13 (M13). 52 women had opted invasive prenatal diagnosis and 13 cases were confirmed, which yielded a positive prediction value (PPV) of 25%. 12 fetuses were confirmed as T13 (PPV = 29.3%; 12/41), 1 was confirmed as M13 (PPV = 9.1%; 1/11). The PPV had increased along with the Z-score. Fetal faction (FF) was not correlated with the age of woman but gestational age, and was negatively correlated with the body mass index. No statistical difference was found in FF and Z-score between true- and false-positive fetuses, and there was a weak correlation between the Z-score and FF. The PPV of the NIPT could be improved by combining the results of ultrasonography. CONCLUSION:The high false positive rate for T13 may be related to confined placental mosaicism, PPV is related to the Z-score, which in turn is related to FF. High-risk women are strongly recommended to undergo genetic counseling and prenatal diagnosis. Clinicians should consider relevant information such as the age of women, gestational age, indication for prenatal screening, Z-score, PPV, and FF in order to accurately interpret the result of NIPT, reduce anxiety, and avoid direct termination of the pregnancy.

Hearing loss is a prevalent sensory disability with strong genetic heterogeneity, affecting approximately 60% of patients due to genetic factors. To investigate the possible genetic causes of hearing loss in 768 unrelated Chinese patients and analyze the genetic diagnosis rates among patients with different clinical phenotypic characteristics, 768 patients were enrolled, and whole-exome sequencing (WES) was performed for genetic testing. Sanger sequencing, MLPA, or qPCR were used to verify the parental origin of identified variants. We identified possible genetic etiologies in 501 of the 768 patients (65.2%), including 456 with non-syndromic and 45 with syndromic hearing loss. A total of 214 variants from 30 genes were identified: 174 previously reported and 40 novel pathogenic/likely pathogenic variants, the 18 hotspot variants accounted for 71.9% of all identified variants. Notably, 15 patients carried de novo variants. The genetic diagnosis rate was significantly higher in patients with severe-profound hearing loss (95.8%, 474/495) compared to those with mild-moderate hearing loss (9.9%, 27/273), P < 0.001. Our findings expand the spectrum of genetic variants associated with hearing loss and demonstrate high genetic diagnosis rates in patients with severe-profound hearing loss and congenital onset. WES combined with parental origin verification is an effective and economical method for identifying the genetic etiology of hearing loss and can be considered a priority in clinical practice for guiding early intervention and preventing further hearing loss.