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Objective To evaluate the clinical feasibility of noninvasive prenatal diagnosis (NIPD) for β-thalassaemia using circulating single molecule amplification and re-sequencing technology (cSMART). Design Through carrier screening, 102 pregnant Chinese couples carrying pathogenic HBB gene variants were recruited to the study. Pregnancies were managed using traditional invasive prenatal diagnosis (IPD). Retrospectively, we evaluated the archived pregnancy plasma DNA by NIPD to evaluate the performance of our cSMART assay for fetal genotyping. Setting Chinese prenatal diagnostic centres specialising in thalassaemia testing. Population Chinese carrier couples at high genetic risk for β-thalassaemia. Methods Fetal cell sampling was performed by amniocentesis and HBB genotypes were determined by reverse dot blot. NIPD was performed by a newly designed HBB cSMART assay and fetal genotypes were called by measuring the allelic ratios in the maternal cell-free DNA. Main outcome measures Concordance of HBB fetal genotyping between IPD and NIPD and the sensitivity and specificity of NIPD. Results Invasive prenatal diagnosis identified 29 affected homozygotes or compound heterozygotes, 54 heterozygotes and 19 normal homozygotes. Compared with IPD results, 99 of 102 fetuses (97%) were correctly genotyped by our NIPD assay. Two of three discordant samples were false positives and the other sample involved an incorrect call of a heterozygote carrier as a homozygote normal. Overall, the sensitivity and specificity of our NIPD assay was 100% (95% CI 88.06-100.00%) and 97.26% (95% CI 90.45-99.67%), respectively. Conclusions This study demonstrates that our cSMART-based NIPD assay for β-thalassaemia has potential clinical utility as an alternative to IPD for pregnant HBB carrier couples. Tweetable abstract A new noninvasive test for pregnancies at risk for β-thalassaemia.

Background: This study explores the diagnostic efficiency of different prenatal diagnostic approaches for women with positive non-invasive prenatal testing (NIPT) results by analyzing their clinical information and pregnancy outcome. Methods: We collected data on 626 NIPT-positive pregnant women from January 2017 to June 2021 and arranged subsequent prenatal diagnostic operations for them after genetic counseling, along with long-term intensive follow-up. A total of 567 women accepted invasive prenatal diagnosis (IPD) (90.57%), and 262 cases were confirmed as true positives of NIPT. Results: The positive predictive value for trisomy 21 (T21), trisomy 18 (T18), trisomy 13 (T13), sex chromosome aneuploidies (SCAs), rare autosomal trisomies (RATs), and microdeletion and microduplication syndromes (MMS) were 81.13%, 37.93%, 18.42%, 48.83%, 18.37%, and 41.67%, respectively. Discordant results between NIPT and IPD were seen in 53 cases, with the discordance rate being 9.35%. Additionally, there were 43 cases with discordant results between karyotyping and chromosomal microarray analysis (CMA)/copy number variation sequencing. Conclusions: Additional reporting of RATs and MMS with routine NIPT that only detects T21/T18/T13 and SCAs can yield diagnosis more accurately. However, NIPT cannot be used as a substitute for IPD owing to its high false positive rate and discordances with other diagnostic methods. We recommend CMA combined with karyotyping as the preferred method for accurate diagnosis of NIPT-positive women.

Background: Tetrasomy 18p syndrome is a very rare chromosomal disorder that is caused by the presence of isochromosome 18p.Most tetrasomy 18p cases are de novo cases,maternal origin trisomy 18p is a very rare condition.At present, only 4 cases of maternal origin trisomy 18p have been reported.This was the the fifth from maternal trisomy 18p,the mother has no apparent disease phenotype.Case presentation: We hereby report a case of a fetus with normal ultrasound features, the Karyotyping and Single Nucleotide Polymophism array (SNP array) confirmed tetrasomy 18p.The mother and grandfather are phenotypically normal and healthy,but with trisomy 18p was confirmed by conventional karyotyping and SNP array.Conclusions: We report a family with an 18p trisomic mother and grandfather,18p tetrasomic fetus,the mother and grandfather are phenotypically normal. The findings could provide a reference for the genetic counseling of trisomy

To evaluate the efficacy of non-invasive prenatal screening (NIPT) for detecting fetal sex chromosome abnormalities, a total of 639 women carrying sex chromosome abnormalities were selected from 222,107 pregnant women who participated in free NIPT from April 2018 to December 2020. The clinical data, prenatal diagnosis results, and follow-up pregnancy outcomes of participants were collected. The positive predictive value (PPV) was used to analyze the performance of NIPT. Around 235 cases were confirmed with sex chromosome abnormalities, including 229 cases with sex chromosome aneuploidy (45, X (n = 37), 47, XXX (n = 37), 47, XXY (n = 110), 47, XYY (n = 42)) and 6 cases with structural abnormalities. The total incidence rate was 0.11% (235/222,107). The PPV of NIPT was 45.37% (235/518). NIPT accuracy for detecting sex chromosome polysomes was higher than that for sex chromosome monomers. The termination of pregnancy rate for fetal diagnosis of 45, X, and 47, XXY was higher than that of 47, XXX, and 47, XYY. The detection rate of fetal sex chromosome abnormalities was higher in 2018–2020 than in 2010–2012 (χ2 = 69.708, P < 2.2 × 10−16), indicating that NIPT is greatly efficient to detect fetal sex chromosome abnormalities.

BACKGROUND Lysosomal storage diseases (LSDs), a group of rare inherited metabolic disorders, result from specific lysosomal proteins deficiencies in the degradation of biomacromolecule, including over 70 different diseases, most of which are autosomal recessive. LSDs are multisystem disorders, and the clinical manifestations are usually broad and severe, involving the skeletal system, central nervous system (CNS), cardiovascular system, etc. Besides, patients with some subtypes of LSD have distinctive facial features. MATERIAL AND METHODS We performed next generation sequencing on 4 suspected mucopolysaccharidosis (MPS) cases to determine the genetic causes of the disease. By in vitro molecular cell assay, such as real-time polymerase chain reaction (RT-PCR) and western blot, we tested the pathogenicity of candidate variants. RESULTS We detected 5 novel mutations in 4 patients. The mutations were: c.211_214del and c.1270C>T in GUSB; c.1284+1C>A and c.2404C>T in GNPTAB; and c.717C>A in FUCA1). We identified a rare mucopolysaccharidosis VII patient, a rare fucosidosis patient, and 2 rare mucolipidosis II patients, one of which was an atypical patient. We also present a new pathogenic conjecture about a small deletion in GUSB. CONCLUSIONS Our study described rare diseases in Chinese patients and our results enrich the phenotype spectrum of related diseases, as well as mutation spectrum of related genes, which might be significant for clinical disease diagnosis and prenatal diagnosis.

To assess the clinical performance of an expanded noninvasive prenatal screening (NIPS) test (“NIPS-Plus”) for detection of both aneuploidy and genome-wide microdeletion/microduplication syndromes (MMS). A total of 94,085 women with a singleton pregnancy were prospectively enrolled in the study. The cell-free plasma DNA was directly sequenced without intermediate amplification and fetal abnormalities identified using an improved copy-number variation (CNV) calling algorithm. A total of 1128 pregnancies (1.2%) were scored positive for clinically significant fetal chromosome abnormalities. This comprised 965 aneuploidies (1.026%) and 163 (0.174%) MMS. From follow-up tests, the positive predictive values (PPVs) for T21, T18, T13, rare trisomies, and sex chromosome aneuploidies were calculated as 95%, 82%, 46%, 29%, and 47%, respectively. For known MMS (n = 32), PPVs were 93% (DiGeorge), 68% (22q11.22 microduplication), 75% (Prader–Willi/Angleman), and 50% (Cri du Chat). For the remaining genome-wide MMS (n = 88), combined PPVs were 32% (CNVs ≥10 Mb) and 19% (CNVs

Maple syrup urine disease (MSUD) is a rare autosomal recessive genetic metabolic disease, with a high incidence rate in infants. We analyzed the data of molecular genetic analysis of five infants whose metabolism screening suspected MSUD and described their clinical symptoms. Further, we performed next-generation sequencing and Sanger sequencing to determine the genetic causes of the disease. Bioinformatics tools were used to predict the pathogenicity of novel mutations by performing structural modeling. All the five infants showed symptoms before one year of age and had elevated plasma leucine and valine levels. Among them, four infants presented an obvious increase in the urine lactic acid level. We identified the genetic cause of the disease in four infants and analyzed the pathogenicity of six novel mutations, viz., two mutations in BCKDHA (p.Gly180Asp and p.Arg265Gln), three in BCKDHB (p.Tyr169Cys, p.Ala331Thr, and p.Gly336Ser), and one in DBT (p.Leu69Arg), using in silico analysis. We also reviewed previously reported mutations in Chinese patients and summarized their genotypic and phenotypic characteristics. Our study has confirmed or corrected the clinical diagnosis and enriched the mutation spectrum of BCKDHA, BCKDHB, and DBT. We suggest blood and urine metabolism screening combined with next generation sequencing to diagnose MSUD, especially in infants, to achieve early diagnosis and early treatment.

Joubert syndrome (JBTS) is a clinically and genetically heterogeneous group of ciliary diseases. To date, 34 subtypes of JBTS have been classified due to different causative genes or extra clinical features. Most of them are autosomal recessive, while only the subtype 10 (JBTS10) is a quite rare X-linked recessive disorder caused by OFD1 mutations with few reports. In this study, by using whole exome sequencing (WES), a novel OFD1 splicing mutation (c.2488+2T>C) was identified in a male fetus with suspected Dandy-Walker variant (DWV) and syndactyly, for whom abnormal karyotype and pathogenic CNV have been excluded. This mutation was inherited from the mother who has experienced two similar pregnancies before. An abnormal skipping of exon 18 in OFD1 mRNA was confirmed by RT-PCR and sequencing. Result from quantitative RT-PCR also showed that total OFD1 mRNA in the index fetus was significantly lower than the control. After a combined analysis of genetic testing results and genotype-phenotype correlations, the novel mutation c.2488+2T>C in OFD1 was considered to be the genetic cause for the affected fetus. Thus the diagnosis should be JBTS10 rather than the primary clinical diagnosis of DWV. We report the first prenatal case of JBTS10 in Chinese population, which not only helps the family to predict recurrence risks for future pregnancies but also provides more information for understanding such a rare disease. The results also present evidence that WES is an effective method in prenatal diagnosis for those fetuses with Joubert syndrome.

There are many inherited disorders associated with thoracic aortic aneurysms and dissections (TAADs), like Marfan syndrome and Loeys-Dietz syndrome (LDS). The 4 patients in this study all had TAADs and were initially diagnosed with suspected Marfan syndrome. We collected peripheral blood samples from the patients and their family members and then attempted to identify the causal mutation using different methods including PCR, Sanger sequencing, and next generation sequencing. We identified 3 novel heterozygous mutations including 2 splicing mutations of FBN1 and 1 missense mutation of TGFBR2 in our patients. Although these mutation sites have been reported in the Human Gene Mutation Database, the nucleotide changes are different. All novel mutations found in this study were confirmed to be absent in 50 unrelated normal individuals of the same ethnic background. The RT-PCR results of 2 splicing mutations verified that the mutations can lead to the skipping of exons. The RT-qPCR results indicated that FBN1 mRNA levels were nearly 50 percent lower in the patients than in normal controls, indicating that there is almost no expression of truncated fibrillin-1 because of the nonsense-mediated mRNA decay (NMD) mechanism. To the best of our knowledge, we are the first to report these 3 novel mutations. However, the pathogenicity of these mutations still needs further confirmation. Our study has confirmed or corrected the clinical diagnosis, and enlarged the mutation spectrum of FBN1 and TGFBR2. The results should be helpful for prenatal diagnosis and genetic counseling.

Idiopathic hypogonadotropic hypogonadism (IHH) is characterized by low levels of gonadotropins and delayed or absent sexual development. Most of the patients are diagnosed in late adolescence or early adulthood. Determining the diagnosis of IHH in prepubertal patients can be challenging. Making a timely, correct diagnosis has important clinical implications. Here we aimed to identify the genetic cause of IHH in three prepubertal siblings from a Chinese Han family and give appropriate treatment advice. Using whole exome sequencing (WES), we identified a novel homozygous GNRHR mutation (NM_000406; c.364C>T, p.L122F) in two prepubertal boys with cryptorchidism and micropenis. Sanger sequencing showed that their younger asymptomatic sister also had the homozygous GNRHR mutation. This mutation was inherited from the father and the mother. Immunofluorescence analysis showed that in permeabilized cells, expression of the mutant receptor on the cell membrane was significantly lower than that of wild-type. Calcium mobilization assays demonstrated that c.364C>T in the GNRHR gene is a complete loss-of-function mutation that caused IHH. These results may contribute to the genetic diagnosis of the three prepubertal siblings with IHH. According to this diagnosis, timely hormonal treatment can be given for the three prepubertal patients to induce pubertal development, especially for the asymptomatic female.

3-hydroxyisobutryl-CoA hydrolase (HIBCH) deficiency is a rare inborn error of valine metabolism characterized by neurodegenerative symptoms and caused by recessive mutations in the HIBCH gene. In this study, utilizing whole exome sequencing, we identified two novel splicing mutations of HIBCH (c.304+3A>G; c.1010_1011+3delTGGTA) in a Chinese patient with characterized neurodegenerative features of HIBCH deficiency and bilateral syndactyly which was not reported in previous studies. Functional tests showed that both of these two mutations destroyed the normal splicing and reduced the expression of HIBCH protein. Through a literature review, a potential phenotype-genotype correlation was found that patients carrying truncating mutations tended to have more severe phenotypes compared with those with missense mutations. Our findings would widen the mutation spectrum of HIBCH causing HIBCH deficiency and the phenotypic spectrum of the disease. The potential genotype-phenotype correlation would be profitable for the treatment and management of patients with HIBCH deficiency.

Purpose To detect the underlying pathogenesis of congenital cataract in a four-generation Chinese family. Methods Whole-exome sequencing (WES) of family members (III:4, IV:4, and IV:6) was performed. Sanger sequencing and bioinformatics analysis were subsequently conducted. Full-length WT-MIP or K228fs-MIP fused to HA markers at the N-terminal was transfected into HeLa cells. Next, quantitative real-time PCR, western blotting and immunofluorescence confocal laser scanning were performed. Results The age of onset for nonsyndromic cataracts in male patients was by 1-year old, earlier than for female patients, who exhibited onset at adulthood. A novel c.682_683delAA (p.K228fs230X) mutation in main intrinsic protein (MIP) cosegregated with the cataract phenotype. The instability index and unfolded states for truncated MIP were predicted to increase by bioinformatics analysis. The mRNA transcription level of K228fs-MIP was reduced compared with that of WT-MIP, and K228fs-MIP protein expression was also lower than that of WT-MIP. Immunofluorescence images showed that WT-MIP principally localized to the plasma membrane, whereas the mutant protein was trapped in the cytoplasm. Conclusions Our study generated genetic and primary functional evidence for a novel c.682_683delAA mutation in MIP that expands the variant spectrum of MIP and help us better understand the molecular basis of cataract.

The cover image is based on the Original Article A novel GJB1 mutation associated with X‐linked Charcot–Marie–Tooth disease in a large Chinese family pedigree by Yingdi Liu et al., https://doi.org/10.1002/mgg3.1127. [Image: see text]

PurposeTo reveal the underlying genetic defect in two four-generation Chinese families with aniridia and explore the pathologic mechanism.MethodsFull ophthalmic examinations were performed in two families with aniridia. The PAX6 gene was directly sequenced in patients of two families, and the detected variants were screened in unaffected family members and two hundred unrelated healthy controls. Real-time quantitative PCR was used to explore pathologic mechanisms of the two variants.ResultsAniridia, cataract, and oscillatory nystagmus were observed in patients of the two families. In addition, we observed corneal opacity and microphthalmus in family 1, and strabismus, left ectopia lentis, microphthalmus, and microcornea in family 2. Sanger sequencing detected a novel 1-bp duplication (c.50dupA) in family 1 and a novel 2-bp splice site deletion (c.765+1_765+2delGT) in family 2. Sequencing of cDNA indicated skipping of exon 9 caused by the splice site deletion, being predicted to cause a premature stop codon, as well as the duplication. The PAX6 mRNA significantly lower in patients with aniridia than in unaffected family members in both families, suggesting that the duplication and splice site deletion caused nonsense-mediated mRNA decay.ConclusionsOur study identified two novel PAX6 variants in two families with aniridia and revealed the pathogenicity of the variants; this would expand the variant spectrum of PAX6 and help us better understand the molecular basis of aniridia, thus facilitating genetic counseling.

Background Cornelia de Lange syndrome (CdLS) and Rubinstein-Taybi syndrome (RSTS) are both rare congenital multiple malformation disorders caused by genes associated with transcription. They share a number of similar features clinically. In addition, it is difficult to make a molecular diagnosis rapidly and detect the mosaic mutation when only sanger sequencing is taken. This study aims to report three novel mutations in three Chinese children identified by next generation sequencing. Results We describe patient 1 and patient 2 presenting with characteristics of CdLS with mutations in NIPBL and patient 3 with a frame shift mutation in CREBBP who can be diagnosed as RSTS clinically and also have similar symptoms with CdLS to some extent. The splicing site c.4321-1G > A transversion in NIPBL is a mosaic mutation and produces an abnormal transcript bearing the loss of exon 20. The nonsense mutation c.218C > A in NIPBL and the frame shift c.1715delC mutation in CREBBP generate stop codon and yield the premature termination of proteins. Conclusions In general, we detect three novel heterozygous mutations including a splicing mutation and a nonsense mutation in NIPBL and a frame shift in CREBBP. And several similar features observed in patients indicate the clinical complexity and clinically overlapping of CdLS and RSTS termed “transcriptomopathies”, suggest the underlying molecular mechanism and emphasize the utilization of next generation sequencing technologies.

Joubert syndrome (JBTS) is a clinically and genetically heterogeneous group of ciliary diseases. To date, 34 subtypes of JBTS have been classified due to different causative genes or extra clinical features. Most of them are autosomal recessive, while only the subtype 10 (JBTS10) is a quite rare X-linked recessive disorder caused by OFD1 mutations with few reports. In this study, by using whole exome sequencing (WES), a novel OFD1 splicing mutation (c.2488+2T>C) was identified in a male fetus with suspected Dandy-Walker variant (DWV) and syndactyly, for whom abnormal karyotype and pathogenic CNV have been excluded. This mutation was inherited from the mother who has experienced two similar pregnancies before. An abnormal skipping of exon 18 in OFD1 mRNA was confirmed by RT-PCR and sequencing. Result from quantitative RT-PCR also showed that total OFD1 mRNA in the index fetus was significantly lower than the control. After a combined analysis of genetic testing results and genotype-phenotype correlations, the novel mutation c.2488+2T>C in OFD1 was considered to be the genetic cause for the affected fetus. Thus the diagnosis should be JBTS10 rather than the primary clinical diagnosis of DWV. We report the first prenatal case of JBTS10 in Chinese population, which not only helps the family to predict recurrence risks for future pregnancies but also provides more information for understanding such a rare disease. The results also present evidence that WES is an effective method in prenatal diagnosis for those fetuses with Joubert syndrome.

There are many inherited disorders associated with thoracic aortic aneurysms and dissections (TAADs), like Marfan syndrome and Loeys-Dietz syndrome (LDS). The 4 patients in this study all had TAADs and were initially diagnosed with suspected Marfan syndrome. We collected peripheral blood samples from the patients and their family members and then attempted to identify the causal mutation using different methods including PCR, Sanger sequencing, and next generation sequencing. We identified 3 novel heterozygous mutations including 2 splicing mutations of FBN1 and 1 missense mutation of TGFBR2 in our patients. Although these mutation sites have been reported in the Human Gene Mutation Database, the nucleotide changes are different. All novel mutations found in this study were confirmed to be absent in 50 unrelated normal individuals of the same ethnic background. The RT-PCR results of 2 splicing mutations verified that the mutations can lead to the skipping of exons. The RT-qPCR results indicated that FBN1 mRNA levels were nearly 50 percent lower in the patients than in normal controls, indicating that there is almost no expression of truncated fibrillin-1 because of the nonsense-mediated mRNA decay (NMD) mechanism. To the best of our knowledge, we are the first to report these 3 novel mutations. However, the pathogenicity of these mutations still needs further confirmation. Our study has confirmed or corrected the clinical diagnosis, and enlarged the mutation spectrum of FBN1 and TGFBR2. The results should be helpful for prenatal diagnosis and genetic counseling.

Oculocutaneous albinism is an autosomal recessive disorder characterized by either a complete lack of or reduction in melanin biosynthesis in the skin, hair, and eyes. The aim of the present study was to identify the molecular basis for 59 Chinese OCA families. In this study, compound heterozygous or homozygous pathogenic variants were found in 53 families, 4 families possessed only one heterozygous variant, and the pathogenic variants of 2 families remain undiscovered by using Sanger sequencing, whole exome sequencing and multiplex ligation-dependent probe amplification. We have identified a total of 55 variants including 21 novel variants in TYR, OCA2, SLC45A2, SLC24A5, and HPS1. The 21 novel variants include 11 missense changes, 4 nonsense changes, 2 splice site changes, 1 frameshift and 3 gross deletions. Forty-six variants including 14 novel variants were segregated with the phenotype in 37 families. We conducted RT-PCR of the novel splicing site variant (c.399-14G > A) of HPS1 and verified that the variant would result in the inclusion of 12 bp of intronic material in exon 6 of HPS1. The results of platelet whole mount electron microscopy further confirmed the diagnosis of HPS1. These novel variants identified in our study expand the mutational spectrum of the disease, which contributes to prenatal diagnosis and genetic counselling.

Supplemental Digital Content is available in the text, Rationale: Hereditary multiple exostoses (HMEs) is an autosomal dominant skeletal disorder. Patient concerns: Six probands of the 6 unrelated Han Chinese families were identified as having HME. These patients had exostoses at multiple sites and significantly affected joints malformation and movement. Diagnoses: Hereditary multiple exostoses. Interventions: To detect the genetic mechanism of HME in 6 unrelated Chinese families, whole-exome sequencing (WES) and multiplex ligation-dependent probe amplification (MLPA) were used after genomic DNA was isolated from peripheral blood leucocytes. Point mutations identified by these methods were verified by Sanger sequencing after PCR amplification. Outcomes: Six mutations in the EXT1 and EXT2 genes were identified, including a heterozygous deletion mutation from exon 2 to exon 8 (Family 1), a c.448C>T, p.(Gln150X) heterozygous nonsense mutation (Family 4), a c.1057-2A>T heterozygous splicing substitution (Family 5), and a c.1468dupC, p.(Leu490fs519X) (Family 6) heterozygous duplication mutation in the EXT1 gene in addition to a heterozygous deletion mutation from exon 2 to exon 3 (Family 2) and a c.1197C>G, p.(Tyr399X) heterozygous nonsense mutation (Family 3) in the EXT2 gene. Lessons: Overall, we identified 5 novel mutations and 1 recurrent mutation in the EXT1 and EXT2 genes in 6 Chinese families with HME. Our findings expand the mutational spectrum of the EXT1 and EXT2 genes and are useful for genetic counseling and prenatal diagnosis.

Nearly half of severe Hemophilia A (HA) cases are caused by F8 intron 22 inversion (Inv22). This 0.6-Mb inversion splits the 186-kb F8 into two parts with opposite transcription directions. The inverted 5′ part (141 kb) preserves the first 22 exons that are driven by the intrinsic F8 promoter, leading to a truncated F8 transcript due to the lack of the last 627 bp coding sequence of exons 23–26. Here we describe an in situ genetic correction of Inv22 in patient-specific induced pluripotent stem cells (iPSCs). By using TALENs, the 627 bp sequence plus a polyA signal was precisely targeted at the junction of exon 22 and intron 22 via homologous recombination (HR) with high targeting efficiencies of 62.5% and 52.9%. The gene-corrected iPSCs retained a normal karyotype following removal of drug selection cassette using a Cre-LoxP system. Importantly, both F8 transcription and FVIII secretion were rescued in the candidate cell types for HA gene therapy including endothelial cells (ECs) and mesenchymal stem cells (MSCs) derived from the gene-corrected iPSCs. This is the first report of an efficient in situ genetic correction of the large inversion mutation using a strategy of targeted gene addition.