OBJECTIVE: This study aims to evaluate the clinical management of incidental findings of copy number variations (CNVs) in the DMD gene detected through prenatal single nucleotide polymorphism array (SNP-array). METHODS: Prenatal SNP-array testing was performed on samples exhibiting CNVs in the DMD locus, followed by parental analysis using the same technique. Additionally, multiplex ligation-dependent probe amplification (MLPA) testing was conducted on prenatal cases and their parents. Pregnancy outcomes were documented, and postnatal follow-up was conducted. RESULTS: SNP-array analysis identified copy number variations at Xp21 affecting either the entire DMD gene or only a portion of it in 14 fetuses. In 11 cases, MLPA testing confirmed the presence of deletions or duplications detected by the SNP-array. CONCLUSION: High-density SNP-array platforms with low reporting thresholds may incidentally detect a subset of exon-level copy number variations involving the DMD gene during routine prenatal testing and thereby contribute to early recognition of potential dystrophinopathy-related variants. Suspected DMD-related CNVs, especially exon-level alterations, require confirmation by targeted assays such as MLPA or next-generation sequencing, together with cautious clinical interpretation. Assessing the pathogenicity of prenatally detected DMD copy number variations remains challenging, particularly for duplications, which require careful evaluation. Furthermore, the sequential, time-intensive nature of confirmatory and familial studies often limits definitive risk assessment within the prenatal decision-making window, and introduces broader familial implications that must be navigated through careful, multidisciplinary counseling.

PURPOSE:To assess the feasibility of using the cell-free DNA barcode-enabled single-molecule test (cfBEST) for non-invasive prenatal testing (NIPT) of α-thalassemia and β-thalassemia. METHODS:Seventy two thalassemia carrier families participated in the study. The cfBEST method was employed to identify thalassemia genotypes in fetal cell-free DNA extracted from maternal plasma, targeting four α-thalassemia gene mutations and 13 common β-thalassemia gene mutations. Validation was conducted using gap-PCR and PCR-RDB through invasive prenatal diagnosis. All prenatal diagnosis results were later confirmed for accuracy. RESULTS:cfBEST successfully identified 94.6% (88/93) of fetal alleles, achieving a sensitivity of 94% (95% CI, 83.45%-98.75%) and a specificity of 95.35% (95% CI, 84.19%-99.43%). All prenatal diagnosis results were followed up, and the follow-up findings were consistent with the prenatal diagnosis results. CONCLUSION:The findings indicate that cfBEST is a reliable, precise, simple, and cost-efficient method suitable for non-invasive prenatal testing of α-thalassemia and β-thalassemia.

This study aimed to summarize the ultrasound and genetic features in a case series of fetuses presenting vertebral defects, to provide useful information for prenatal counseling and prognostic evaluation. Fetuses with vertebral anomalies by a second or third trimester ultrasound screening between January 2020 and April 2024 at a single center were included in the study. Chromosome microarray analysis (CMA) as a first-line diagnostic test was performed. Whole exome sequencing (WES) was further applied to the cases with negative CMA results. A total of 12 fetuses presenting vertebral defects were included. Isolated and non-isolated vertebral malformations were reported for eight (66.7

Most of fetal cardiac rhabdomyomas (CRs) are associated with tuberous sclerosis (TSC), an autosomal dominant inherited disorder caused by mutations in the TSC1 or TSC2 genes. In this study, 12 fetuses with sonographically identified CR were included. A comprehensive analysis integrating prenatal echocardiographic findings, parental phenotypic characteristics, and genetic profiles of fetuses with CR were conducted. Karyotype and SNP-array/CNV-seq were performed simultaneously with Trio-WES. All CRs were observed in the ventricle, interventricular septum, and the atrium, while the left ventricle were the most common areas. All subjects did not detect arrhythmia during pregnancy. Beside CRs, irregular low echo in the brain was detected in Case 12. TSC1 and TSC2 variants were identified in all 12 fetuses (100

X-linked recessive chondrodysplasia punctata 1 (CDPX1) is a rare congenital skeletal dysplasia characterized by stippled epiphyses, nasal hypoplasia, and brachytelephalangy. ARSL (formerly known as ARSE), a member of the sulfatase gene family located on Xp22.3, has been identified as the causative gene for CDPX1. The high clinical and genetic heterogeneity of CDPX1 presents a challenge to prenatal diagnosis. A G1P0 woman in her 30s with an unremarkable prenatal course presented in the second trimester. Maternal diseases, tobacco, alcohol, and drug history during pregnancy were denied. Obstetrical ultrasound examination revealed a flattened nose and a flattened midface with echogenic alterations of lumbar spinous process in the fetus. Amniocentesis was performed for genetic testing. A normal karyotype and a negative result of CNV-seq were obtained. However, Whole exome sequencing (WES) in trios revealed a hemizygous ARSL variant [NM_000047.3:c.1108del p.(Trp370Glyfs*35)] in the fetus, which was maternally inherited as confirmed by Sanger sequencing. This variant was absent from the genomAD and HGMD databases. According to the ACMG guidelines, this variant was interpreted as likely pathogenic (PVS1 + PM2_Supporting). The couple decided to terminate the pregnancy. After induction of labour, a severe nasal hypoplasia was noted; and brachytelephalangy was not remarkable. Postmortem digital X-ray imaging revealed symmetrical stippled epiphyses of the vertebrae in all spine regions and enlargement of spinous process of L1-L4 vertebrae. A novel frameshift deletion variant of ARSL and the associated fetal phenotype have been identified. This study provides useful information for prenatal diagnosis and genetic counseling of CDPX1.

Background Recurrent pregnancy loss (RPL) is a common pregnancy complication that brings great pain to pregnant women and their families. Genetic factors are an important cause reason of RPL. However, clinical research on monogenic diseases with recurrent miscarriage is insufficient. Case presentation Here we reported a Chinese family with RPL and genetic analysis of the abortion and parents. A paternally inherited heterozygous missense variant c.1415T > G (p.V472G) and a maternally inherited heterozygous nonsense variant c.2314del (p.M772*) in TMEM67 gene were identified by trio-exome sequencing. c.2314del (p.M772*) generated a premature stop codon and truncated protein, was classified as "pathogenic". c.1415T > G (p.V472G) located in extra-cellular region, was classified as "likely pathogenic". Biallelic variants in TMEM67 gene cause lethal Meckel syndrome 3, consistent with the proband's prenatal phenotype. Conclusion The current study of the Chinese family expands the pathogenic variant spectrum of TMEM67 and emphasizes the necessity of exome sequencing in RPL condition.

Objective:This study was aimed at investigating the carrier rate of, and molecular variation in, α- and β-globin gene mutations in Hunan Province.Methods:We recruited 25,946 individuals attending premarital screening from 42 districts and counties in all 14 cities of Hunan Province. Hematological screening was performed, and molecular parameters were assessed.Results:The overall carrier rate of thalassemia was 7.1%, including 4.83% for α-thalassemia, 2.15% for β-thalassemia, and 0.12% for both α- and β-thalassemia. The highest carrier rate of thalassemia was in Yongzhou (14.57%). The most abundant genotype of α-thalassemia and β-thalassemia was -α 3.7/αα (50.23%) and β IVS-II-654/β N (28.23%), respectively. Four α-globin mutations [CD108 (ACC>AAC), CAP +29 (G>C), Hb Agrinio and Hb Cervantes] and six β-globin mutations [CAP +8 (C>T), IVS-II-848 (C>T), -56 (G>C), beta nt-77 (G>C), codon 20/21 (-TGGA) and Hb Knossos] had not previously been identified in China. Furthermore, this study provides the first report of the carrier rates of abnormal hemoglobin variants and α-globin triplication in Hunan Province, which were 0.49% and 1.99%, respectively.Conclusion:Our study demonstrates the high complexity and diversity of thalassemia gene mutations in the Hunan population. The results should facilitate genetic counselling and the prevention of severe thalassemia in this region.

Chromosomal mosaicism remains a perpetual diagnostic and clinical dilemma. In the present study, we detected two prenatal trisomy 9 mosaic syndrome cases by using multiple genetic testing methods. The non-invasive prenatal testing (NIPT) results suggested trisomy 9 in two fetuses. Karyotype analysis of amniocytes showed a high level (42%-50%) of mosaicism, and chromosomal microarray analysis (CMA) of uncultured amniocytes showed no copy number variation (CNV) except for large fragment loss of heterozygosity. Ultrasound findings were unmarkable except for small for gestational age. In Case 1, further umbilical blood puncture confirmed 22.4% and 34% trisomy 9 mosaicism by CMA and fluorescent in situ hybridization (FISH) respectively. After comprehensive consideration of the genetic and ultrasound results, the two gravidas decided to receive elective termination and molecular investigations of multiple tissue samples from the aborted fetus and the placenta. The results confirmed the presence of true fetoplacental mosaicism with levels of trisomy 9 mosaicism from 76% to normal in various tissues. These two cases highlight the necessity of genetic counseling for gravidas whose NIPT results highly suggest the risk of chromosome 9 to ascertain the occurrence of mosaicism. In addition, the comprehensive use of multiple genetic techniques and biological samples is recommended for prenatal diagnosis to avoid false-negative results. It should also be noted that ultrasound results of organs with true trisomy 9 mosaicism can be free of structural abnormalities during pregnancy.

Background Short-rib polydactyly syndrome (SRPS) refers to a group of lethal skeletal dysplasias that can be difficult to differentiate between subtypes or from other non-lethal skeletal dysplasias such as Ellis-van Creveld syndrome and Jeune syndrome in a prenatal setting. We report the ultrasound and genetic findings of four unrelated fetuses with skeletal dysplasias. Methods Systemic prenatal ultrasound examination was performed in the second or third trimester. Genetic tests including GTG-banding, single nucleotide polymorphism (SNP) array and exome sequencing were performed with amniocytes or aborted fetal tissues. Results The major and common ultrasound anomalies for the four unrelated fetuses included short long bones of the limbs and narrow thorax. No chromosomal abnormalities and pathogenic copy number variations were detected. Exome sequencing revealed three novel variants in the DYNC2H1 gene, namely NM_001080463.2:c.6809G > A p.(Arg2270Gln), NM_001080463.2:3133C > T p.(Gln1045Ter), and NM_001080463.2:c.337C > T p.(Arg113Trp); one novel variant in the IFT172 gene, NM_015662.3:4540-5 T > A; and one novel variant in the WDR19 gene, NM_025132.4:c.2596G > C p.(Gly866Arg). The genotypes of DYNC2H1, IFT172 and WDR19 and the phenotypes of the fetuses give hints for the diagnosis of short-rib thoracic dysplasia (SRTD) with or without polydactyly 3, 10, and 5, respectively. Conclusion Our findings expand the mutation spectrum of DYNC2H1, IFT172 and WDR19 associated with skeletal ciliopathies, and provide useful information for prenatal diagnosis and genetic counseling on rare skeletal disorders.

Objective:To analyze the costs and effectiveness of five common screening modes and genetic screening for thalassemia in China in order to find the optimal way and provide evidence for the implementation of thalassemia prevention and control projects in Hunan Province.Methods:From June 2020 to April 2021, 12 971 couples from 14 cities and autonomous prefectures in Hunan Province were selected as the study population. The diagnosis of thalassemia was based on the results of genetic testing. Results of routine blood test and hemoglobin electrophoresis were collected and analyzed. The efficacy of five screening modes, at the cut-off value of <80 fl or 82 fl for the mean corpuscular volume (MCV), was analyzed by positive predictive value, negative predictive value, Jorden index and cost-effectiveness ratio. Sensitivity analysis was used to assess the feasibility of genetic screening at different costs after fixing the costs of routine blood and hemoglobin electrophoresis. The five thalassemia screening models are as follows: Mode 1: The woman had a blood routine test first. If the result was positive, the spouse required a blood routine test. If both results were positive, a thalassemia gene test should be offered to the couple. Mode 2: Both husband and wife were screened by blood routine and hemoglobin electrophoresis. If one or both of them were positive, both would be tested for thalassemia gene. Mode 3: The couple received blood routine tests initially. If either was positive, both should receive hemoglobin electrophoresis testing. If either was positive, both parties will conduct thalassemia gene testing. Mode 4: The woman was screened by blood routine and hemoglobin electrophoresis. If any one of them was positive, the woman would be tested for thalassemia gene. If the gene test result was positive, the spouse should receive thalassemia gene. Mode 5: Both spouses conducted a blood routine test. If either was positive, both would conduct hemoglobin electrophoresis test. If both were positive, both spouses should receive thalassemia gene testing. Gene testing mode: The woman would be tested for thalassemia, and her spouse would have thalassemia test too if her result was positive.Results:When using MCV<80 fl as the cut-off for diagnosing thalassemia, the Youden indices of the five prenatal screening modes in Hunan Province were 0.551, 0.639, 0.898, 0.555 and 0.356, while when using MCV<82 fl as the cut-off, the Youden indices were 0.549, 0.629, 0.851, 0.548 and 0.356. When the MCV cut-off value was <80 fl, the missed diagnosis rates of the five screening modes were 44.44%, 0.00, 0.00, 18.52% and 62.96%, and the cost-effectiveness ratios were 21 709, 250 939, 76 870, 138 463 and 92 860 yuan (RMB)/couple, respectively. When the price of genetic testing was lower than 55 yuan (RMB), the cost-effectiveness ratio of genetic screening was lower than that of Mode 3.Conclusions:MCV<80 fl can be considered as the positive criteria in blood routine screening for thalassemia in Hunan Province, and the cost-effectiveness ratio of Mode 3 (the couple received blood routine tests initially. If either was positive, both should receive hemoglobin electrophoresis testing. If either was positive, both parties will conduct thalassemia gene testing) is the best. Genetic screening has certain advantages with the decreasing price.

Background: Citrullinemia type I (CTLN1) is a rare autosomal recessive inborn error of the urea cycle caused by mutations in the gene encoding the arginosuccinate synthetase (ASS1) enzyme. Classic CTLN1 often manifests with acute hyperammonemia and neurological symptoms. Molecular genetic testing is critical for patient diagnosis. Methods: Three unrelated families with clinically suspected CTLN1 were included in this study. Potential pathogenic variants were identified using whole exome sequencing (WES) and validated using Sanger sequencing. Western blotting, quantitative PCR, immunofluorescent staining, and ELISA were used to assess functional changes in candidate ASS1 variants. Results: Five variants were identified, two of which were novel, and one has been reported, but its pathogenicity was not validated. The novel variant c.649-651del (p.P217del) and the 5'UTR variant (c.-4C>T) resulted in a decrease in ASS1 expression at both the protein and transcription levels. The other novel variant, c.1048C>T (p.Q350*), showed a marked decrease in expression at the protein level, with the formation of truncated proteins but an increased transcription. Both c.649_651del (p.P217del) and c.1048C>T (p.Q350*) showed a highly significant reduction in enzyme activity, while c.-4C>T had no effect. Conclusion: We identified two novel variants and a hypomorphic non-coding variant in ASS1 and validated the pathogenicity using functional studies. Our findings contribute to expanding the spectrum of ASS1 variants and understanding the genotype-phenotype relationships of CTLN1.

Objective:To investigate the power and prenatal diagnosis strategies of cell-free fetal DNA (cffDNA) testing for chromosomal aneuploidy screening apart from trisomy-13/18/21.Methods:This study collected the clinical data of three cases at high risk of trisomy-16 indicated by cffDNA testing in Hunan Provincial Maternal and Child Health Care Hospital from March 2019 to March 2020. Results of the conventional G-banding karyotype analysis of amniotic fluid, single nucleotide polymorphism array (SNP-array) and low-coverage massively parallel copy number variation sequencing (CNV-seq) of placenta/fetal skin samples were analyzed.Results:(1) cffDNA testing results suggested that case 1-3 were at high risk of trisomy-16 and the Z values of chromosome 16 were 20.57, 24.88 and 17.87, respectively. (2) Karyotype analysis of amniotic fluid samples did not identify any abnormalities in Case 1 and 2, while SNP-array revealed a 19.2 Mb and 23.0 Mb heterozygous deletion at 16p13.3p12.3 and 16q22.1q24.3 in Case 1, and a 16.0 Mb loss of heterozygosity at 16q22.3q24.3 in Case 2. Case 3 had a mosaicism karyotype of 47,XY,+16[3]/46,XY[97] and SNP-array analysis showed no heterozygous deletion greater than 5 Mb or copy number variation. (3) Ultrasonography indicated fetal growth restriction in Case 1 and 2 and fetal death in Case 3. All three pregnancies were terminated. CNV-seq analysis of placental tissue in the center of both fetal and maternal side revealed mosaic trisomy 16, with the copy numbers of chromosome 16 of 2.56/2.70, 2.73/2.82, 2.80/2.81, respectively. However, no copy number variation was detected in Case 1 or 2 by CNV-seq analysis of fetal skin tissues. Conclusions:cffDNA testing has a certain power in detecting trisomy-16 apart from trisomy-13/18/21. For high-risk cases of trisomy-16 indicated by cffDNA testing, SNP-array analysis combined with karyotype analysis is suggested to rule out low-level mosaicism and loss of heterozygosity.

Background: Fragile X syndrome (FXS) is the most common inherited form of intellectual disability. Prenatal screening of FXS allows for early identification and intervention. The present study explored the feasibility of FXS carrier screening during prenatal diagnosis for those who were not offered screening early in pregnancy or prior to conception. Methods: Pregnant women to be offered amniotic fluid testing were recruited for the free voluntary carrier screening at a single center between August, 2017 and September, 2019. The number of CGG repeats in the 5' un-translated region of the fragile X mental retardation gene 1 (FMR1) was determined. Results: 4286 of 7000 (61.2%) pregnant women volunteered for the screening. Forty (0.93%), five (0.11%), and three (0.07%) carriers for intermediate mutation (45-54 repeats), premutation (55-200 repeats) and full mutation (>200 repeats) of the FMR1 gene were identified respectively. None of the detected premutation alleles were inherited by the fetuses. Of the three full mutation carrier mothers, all had a family history and one transmitted a full mutation allele to her male fetus. Conclusion: Implementation of FXS carrier screening during prenatal diagnosis may be considered for the need to increase screening for FXS.

Objective:To study whether there were significant difference among different choriontity for guiding the clinical twin's first-trimester prenatal screening.Method:Collecting 170 cases of natural twin pregnancy samples from July 2014 to December 2014.All cases were dichorionic diamniotic (DCDA).Using different chodontity such as dichorionic diamniotic,monochorionic diamniotic (MCDA),monochorionic monoamniotic (MCMA) calculated the risk of Down's syndrome at the first-trimester pregnancy.The difference were analyzed combined with the pregnancy results were followed up.Results:According to different choriontity (DCDA,MCDA,MCMA),the multiple of median PAPP-A were 2.19,1.20,1.37.The multiple of media free HCG β were 2.02,1.09,1.36.The high risk cases number of Down's syndrome were 13,10,7.The false positive rate of Down's syndrome were 7.65％,5.88％,4.12％.The pregnancy results found 6 abnormal cases.Chromosome abnormality occurred in two cases.Single intrauterine fetal death (sIUFD) happened in two cases.One case miscarry at 13 weeks.Two babies were diagnosed as Congenital hypothyroidism in one case.Conclusion:Using different choriontity calculate the first-trimester Down's syndrome risk,the multiple media of serum marker were significant difference.The risk values were also significant different.Hence,we should describe the choriontity of twin pregnancy,calculate the Down's syndrome risk correctly to avoid the mistakes.

目的探讨孕早期、中期和整合筛查对唐氏综合征筛查对检出胎儿染色体异常和妊娠不良结局的实用价值。方法应用时间分辨荧光免疫法对7802例孕9周～13+6周和11 911例孕15周～20+6周妇女进行血清标记物妊娠相关蛋白（PAPP-A）和游离β绒毛膜促性腺激素（freeβ-HCG）或甲胎蛋白（AFP）、游离β绒毛膜促性腺激素（freeβ-HCG）和游离雌三醇（uE3）进行检测,并对其中1014例孕妇进行整合筛查。筛查结果应用Lifecycle3.0软件计算唐氏综合征风险。唐氏综合征风险切割值为1∶270,当其值≥1∶270时为唐氏高危孕妇,于孕16周以后抽羊水或脐血进行胎儿核型分析,追踪胎儿和孕妇的情况。结果产前筛查19 713例孕妇,唐氏综合征筛查阳性孕妇1059例,假阳性率为5.37（1059/19713）。有607例接受了羊水或脐血穿刺产前诊断,占总筛查高危孕妇的57.32%（607/1059）。发现胎儿染色体异常24例,异常检出率为3.95%（24/607）,其中4例唐氏综合征,3例18-三体,17例其他染色体异常,5例NTD畸形在孕中期筛查中发现。1例唐氏综合征在出生后被证实。三种筛查方法的假阳性率分别为:5.79%,5.10%,4.04%。结论三种筛查方法间存在显著性差异（P＜0.05）,整合筛查的假阳性率低于孕早期和孕中期筛查,如果有条件,我们推荐选择整合筛查。