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Hemophilia A (HA) is a common X-linked recessive hereditary bleeding disorder. Coagulation factor VIII (FVIII) is insufficient in patients with HA due to the mutations in the F8 gene. The restoration of plasma levels of FVIII via both recombinant B-domain-deleted FVIII (BDD-FVIII) and B-domain-deleted F8 (BDDF8) transgenes was proven to be helpful. FVIII-Padua is a 23.4 kb tandem repeat mutation in the F8 associated with a high F8 gene expression and thrombogenesis. Here we screened a core enhancer element in FVIII-Padua for improving the F8 expression. In detail, we identified a 400 bp efficient enhancer element, C400, in FVIII-Padua for the first time. The core enhancer C400 extensively improved the transcription of BDDF8 driven by human elongation factor-1 alpha in HepG2, HeLa, HEK-293T and induced pluripotent stem cells (iPSCs) with different genetic backgrounds, as well as iPSCs-derived endothelial progenitor cells (iEPCs) and iPSCs-derived mesenchymal stem cells (iMSCs). The expression of FVIII protein was increased by C400, especially in iEPCs. Our research provides a novel molecular target to enhance expression of FVIII protein, which has scientific value and application prospects in both viral and nonviral HA gene therapy strategies. [ABSTRACT FROM AUTHOR]

Objective Subjects and Methods Results Conclusions To investigate the genetic causes of 22 patients with clinically high suspicion of X‐linked hypohidrotic ectodermal dysplasia from 20 unrelated Chinese families, expand the spectrum of ectodysplasin‐A mutations, and provide more evidence for variants of uncertain significance.Whole‐exome sequencing was performed and potentially pathogenic variants were verified by Sanger sequencing. Western blotting, real‐time PCR and immunofluorescence analyses were performed to investigate the preliminary functions of the candidate variants.Nineteen ectodysplasin‐A variants were identified, six of which were not previously reported. Among these variants, we identified a patient who carried two mutations in ectodysplasin‐A and exhibited more severe phenotypes. Additionally, mutant protein expression levels decreased, whereas mRNA transcription levels increased. Cellular sublocalisation of the variants located in the tumour necrosis factor homologous domain showed that the proteins accumulated in the nucleus, whereas wild‐type proteins remained in the cell membrane. A rare indel variant and two classical splicing variants that lead to exon 7 skipping were detected.This study provides definitive diagnoses for 20 families with suspected X‐linked hypohidrotic ectodermal dysplasia and additional information on clinical heterogeneity and genotype–phenotype relationships. [ABSTRACT FROM AUTHOR]

Objective: Rare variants of CCNK (cyclin K) give rise to a syndrome with intellectual disability. The purpose of this study was to describe the genotype–phenotype spectrum of CCNK‐related syndrome and the underlying molecular mechanisms of pathogenesis. Methods: We identified a number of de novo CCNK variants in unrelated patients. We generated patient‐induced pluripotent stem cells (iPSCs) and neural progenitor cells (NPCs) as disease models. In addition, we constructed NPC‐specific Ccnk knockout (KO) mice and performed molecular and morphological analyses. Results: We identified 2 new patients harboring CCNK missense variants and followed‐up 3 previous reported patients, which constitute the largest patient population analysis of the disease. We demonstrate that both the patient‐derived NPC models and the Ccnk KO mouse displayed deficient NPC proliferation and enhanced apoptotic cell death. RNA sequencing analyses of these NPC models uncovered transcriptomic signatures unique to CCNK‐related syndrome, revealing significant changes in genes, including WNT5A, critical for progenitor proliferation and cell death. Further, to confirm WNT5A's role, we conducted rescue experiments using NPC and mouse models. We found that a Wnt5a inhibitor significantly increased proliferation and reduced apoptosis in NPCs derived from patients with CCNK‐related syndrome and NPCs in the developing cortex of Ccnk KO mice. Interpretation: We discussed the genotype–phenotype relationship of CCNK‐related syndrome. Importantly, we demonstrated that CCNK plays critical roles in NPC proliferation and NPC apoptosis in vivo and in vitro. Together, our study highlights that Wnt5a may serve as a promising therapeutic target for the disease intervention. ANN NEUROL 2023;94:1136–1154 [ABSTRACT FROM AUTHOR]

Recent developments in molecular biological technologies and genetic diagnostic methods, accompanying with updates of relevant terminologies, have enabled the improvements of new strategies of preimplantation genetic testing for monogenic (single gene) disorders (PGT-M) to prevent the transmission of inherited diseases. However, there has been much in the way of published consensus on PGT-M. To properly regulate the application of PGT-M, Chinese experts in reproductive medicine and genetics have jointly developed this consensus statement. The consensus includes indications for patient selection, genetic and reproductive counseling, informed consent, diagnostic strategies, report generation, interpretation of results and patient follow-ups. This consensus statement serves to assist in establishment of evidence-based clinical and laboratory practices for PGT-M. [ABSTRACT FROM AUTHOR]

Hemophilia B (HB) is an X-linked recessive disease caused by F9 gene mutation and functional coagulation factor IX (FIX) deficiency. Patients suffer from chronic arthritis and death threats owing to excessive bleeding. Compared with traditional treatments, gene therapy for HB has obvious advantages, especially when the hyperactive FIX mutant (FIX-Padua) is used. However, the mechanism by which FIX-Padua works remains ambiguous due to a lack of research models. Here, in situ introduction of F9-Padua mutation was performed in human induced pluripotent stem cells (hiPSCs) via CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs). The hyperactivity of FIX-Padua was confirmed to be 364% of the normal level in edited hiPSCs-derived hepatocytes, providing a reliable model for exploring the mechanism of the hyperactivity of FIX-Padua. Moreover, the F9 cDNA containing F9-Padua was integrated before the F9 initiation codon by CRISPR/Cas9 in iPSCs from an HB patient (HB-hiPSCs). Integrated HB-hiPSCs after off-target screening were differentiated into hepatocytes. The FIX activity in the supernatant of integrated hepatocytes showed a 4.2-fold increase and reached 63.64% of the normal level, suggesting a universal treatment for HB patients with various mutations in F9 exons. Overall, our study provides new approaches for the exploration and development of cell-based gene therapy for HB. [ABSTRACT FROM AUTHOR]

Background: Noninvasive prenatal testing (NIPT) is increasingly used in the clinical prenatal screening of twin pregnancies, and its screening performance for chromosomal abnormalities requires further evaluation. For twin pregnancies with indications for prenatal diagnosis, there is a lack of clinical data to assess the prenatal diagnosis rate (PDR). The aim of this study was to evaluate the screening performance of NIPT for foetal chromosomal abnormalities in twin pregnancies and the PDR in the second and third trimesters. Methods: Ultrasound scans were carried out for all twin pregnancies between 11 and 13+ 6 gestational weeks. For twin pregnancies with nuchal translucency thickness˂3.0 mm and no foetal structural malformations, NIPT was performed after blood sampling, followed by routine ultrasound monitoring. Women with twin pregnancies who underwent NIPT at the prenatal diagnostic centre of Xiangya Hospital from January 2018 to May 2022 were included in the study. Genetic counselling was offered to each pregnant woman when the NIPT result indicated a high risk of abnormalities or abnormal ultrasonographic (USG) findings were detected. We followed up twin pregnancies for NIPT results, USG findings, prenatal diagnosis results and pregnancy outcomes. Results: In 1754 twin pregnancies, the sensitivity, specificity and positive predictive value of NIPT for trisomy 21 were 100%, 99.9% and 75%, and the corresponding values for sex chromosome aneuploidy (SCA) were 100%, 99.9% and 50%, respectively. For the 14 twin pregnancies for which the NIPT results indicated a high risk of abnormalities, the PDR was 78.6% (11/14). For the 492 twin pregnancies for which the NIPT results indicated a low risk of abnormalities, the rate of USG findings in the second and third trimesters was 39.4% (194/492); of these pregnancies, prenatal diagnosis was recommended for 16.7% (82/492), but it was actually performed in only 8.3% (41/492), and the PDR was 50% (41/82). There was no significant difference in the PDR between the NIPT high-risk and low-risk groups. Conclusions: The screening performance of NIPT for SCA in twin pregnancies needs to be further evaluated. When abnormal NIPT results or USG findings are used as the main prenatal diagnostic indicator in the second and third trimesters, the PDR is poor. [ABSTRACT FROM AUTHOR]

Gene therapy is a new technology that provides potential for curing monogenic diseases caused by mutations in a single gene. Hemophilia and Duchenne muscular dystrophy (DMD) are ideal target diseases of gene therapy. Important advances have been made in clinical trials, including studies of adeno-associated virus vectors in hemophilia and antisense in DMD. However, issues regarding the high doses of viral vectors required and limited delivery efficiency of antisense oligonucleotides have not yet been fully addressed. As an alternative strategy to classic gene addition, genome editing based on programmable nucleases has also shown promise to correct mutations in situ. This review describes the recent progress made by Chinese researchers in gene therapy for hemophilia and DMD. [ABSTRACT FROM AUTHOR]

Contributed to the development of next-generation sequencing (NGS) technology, more and more chromosomally mosaic and aneuploid embryos are discovered during the preimplantation genetic testing for aneuploidy (PGT-A) cycles. Because mosaicism and aneuploidy are routine phenomena throughout human pre- and post-implantation development. The benefit of implanting such mosaicism or aneuploidies detected by precise NGS remains controversial. This study aimed to investigate chromosomal concordance between babies produced by PGT-A and trophectoderm (TE) biopsies, and whether precise NGS resolution would reduce the development of an abnormal embryo in PGT cycles. Peripheral blood samples from 17 PGT-A babies were collected to compare with TE biopsy results at different NGS resolutions. 16 euploid embryos diagnosed by 10 Mb resolution developed into 16 healthy babies with normal copy number variations (CNVs). One mosaic embryo diagnosed by both 10 Mb and 4 Mb resolution also produced a euploid baby finally. Among them, four euploid embryos diagnosed by 10 Mb NGS, showed segmental aneuploidy at 4 Mb NGS resolution. Four of them developed into euploid babies with normal CNVs finally. NGS at 10 Mb resolution is accurate enough to diagnose viable embryos. A more precise NGS resolution (e.g., 4 Mb resolution) results in discard of some potentially viable embryos. It is suggested to analyze the TE biopsy at both 10 Mb and 4 Mb resolutions to identify embryos with adverse chromosomal aberrations, but using 10 Mb resolution for guide transfer to increase a development chance of an embryo. Trial registration: www.ClinicalTrials.gov , identifier ChiCTR2100042522. [ABSTRACT FROM AUTHOR]

Primary congenital hypothyroidism (CH) is a common neonatal endocrine disorder characterized by elevated concentrations of thyroid stimulating hormone (TSH) and low concentrations of free thyroxine (FT4). PAX8 and NKX2-1 are important transcription factors involved in thyroid development. In this study, we detected three novel variants in PAX8 (c.149A > C and c.329G > A) and NKX2-1 (c.706A > G) by whole exome sequencing (WES) in three unrelated CH patients with variable phenotypes. The results of Western blot and immunofluorescence analysis showed that the three variants had no effect on protein expression and subcellular localization. However, the results of the electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay suggested that the three variants in PAX8 and NKX2-1 both affected their DNA-binding ability and reduced their transactivation capacity. Moreover, a dominant-negative effect in K236E−NKX2-1 was identified by dual-luciferase reporter assay. To sum up, our findings extend our knowledge of the current mutation spectrum of PAX8 and NKX2-1 and provide important information for diagnosing, treating, and preventing CH in these families. [ABSTRACT FROM AUTHOR]

• Explores the aberrant genetic profiles of a Chinese ID cohort composed of 321 patients. • Provides a new reference for genotype-phenotype analysis and broadens the mutation spectrum of ID-related genes. • Provides evidence for the advantage of using WES as an early diagnostic strategy. Intellectual disability is a heterogeneous neurodevelopmental disorder with complex genetic architectures. Different sequential methodologies are usually applied to identify the genetic aetiologies of ID patients. We collected 321 consecutive ID patients. All patients underwent karyotyping, while 293 and 164 cases further received copy number variation sequencing (CNV-seq) and whole-exome sequencing (WES). The updated WES technology can detect CNVs simultaneously. The diagnostic data from 137 patients who received WES and CNV-seq were used to define the approach that could be recommended as the first-tier test. WES obtains the highest diagnostic yield of 50% (82/164), compared with karyotyping (7.79%, 25/321) and CNV-seq (19.80%, 58/293). Among the variants detected by WES, 66.67% (44/66) de novo and 57.58% (38/66) novel pathogenic/likely pathogenic (P/LP) variants were identified in patients with ID. Besides, 24 out of 25P/LP CNVs discovered by CNV-seq can also be accurately identified using WES in 137 patients who received WES and CNV-seq. Thus, genetic abnormalities found through karyotyping, CNV-seq, and WES can be completely detected by combined karyotyping and WES. This study illustrates the genetic aberrations of a Chinese ID cohort and expands the mutation spectrum of ID-related genes. Compared with the conventional diagnostic strategy, a combination of karyotype analysis and WES could be recommended as the first-tier diagnostic strategy for ID patients. [ABSTRACT FROM AUTHOR]

Simple Summary: Galloway–Mowat syndrome is a rare genetic disease, classically characterized by a combination of various neurological symptoms and nephrotic syndrome. WDR73 is the pathogenic gene responsible for Galloway–Mowat syndrome. However, the pathological and molecular mechanisms of Galloway–Mowat syndrome, especially nephrotic syndrome caused by WDR73 deficiency, remains unknown. In this study, we knocked out the WDR73 in human embryonic kidney 293 cells to observe the morphological characteristics of the cells and elucidate the functions of WDR73. Additionally, we used a combination of proteomics, transcriptomics, and biochemical assays to identify the regulated targets of WDR73. We aimed to discover directly interacting molecules and the regulatory pathway of WDR73 and to illustrate the molecular mechanism between the WDR73 pathway and nephrotic disease in Galloway–Mowat syndrome. From the molecular mechanism we found in vitro, we draw a hypothesis that the damage to focal adhesion of podocytes caused by WDR73 defect is the key issue of kidney dysfunction. Finally, we verified the hypothesis in a podocyte-specific conditional knockout Wdr73 mouse model. (1) Background: Galloway–Mowat syndrome (GAMOS) is a rare genetic disease, classically characterized by a combination of various neurological symptoms and nephrotic syndrome. WDR73 is the pathogenic gene responsible for GAMOS1. However, the pathological and molecular mechanisms of GAMOS1, especially nephrotic syndrome caused by WDR73 deficiency, remain unknown. (2) Methods and Results: In this study, we first observed remarkable cellular morphological changes including impaired cell adhesion, decreased pseudopodia, and G2/M phase arrest in WDR73 knockout (KO) HEK 293 cells. The differentially expressed genes in WDR73 KO cells were enriched in the focal adhesion (FA) pathway. Additionally, PIP4K2C, a phospholipid kinase also involved in the FA pathway, was subsequently validated to interact with WDR73 via protein microarray and GST pulldown. WDR73 regulates PIP4K2C protein stability through the autophagy–lysosomal pathway. The stability of PIP4K2C was significantly disrupted by WDR73 KO, leading to a remarkable reduction in PIP2 and thus weakening the FA formation. In addition, we found that podocyte-specific conditional knockout (Wdr73 CKO) mice showed high levels of albuminuria and podocyte foot process injury in the ADR-induced model. FA formation was impaired in primary podocytes derived from Wdr73 CKO mice. (3) Conclusions: Since FA has been well known for its critical roles in maintaining podocyte structures and function, our study indicated that nephrotic syndrome in GAMOS1 is associated with disruption of FA caused by WDR73 deficiency. [ABSTRACT FROM AUTHOR]

Background: It has been proved that mutations in the PADI6 gene can cause early embryo arrest. This study describes a newly discovered mutation in PADI6 that expands the genetic spectrum of early embryo arrest. Methods: Peripheral blood of a patient diagnosed with early embryo arrest was collected for whole-exome sequencing. Sanger sequencing was performed to confirm this mutation. The effects of the variant were investigated in human embryonic kidney 293T (HEK293T) cells using western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence. Results: A novel homozygous mutation in PADI6 was identified in the proband. The patient carried a frameshift insertion mutation c.558dupA (p.Thr187Asnfs*48), which was located in the protein arginine deiminase middle domain. The variant destroyed PADI6 protein expression and reduced PADI6 mRNA expression in HEK293T cells. Conclusions: The newly identified mutation in PADI6 accounts for early embryo arrest. It expands the spectrum of genetic causes and phenotypes of infertility in humans. These findings also provide an additional possible diagnostic marker for patients with recurrent in vitro fertilization/intracytoplasmic sperm injection failure. Plain Language Summary: Some infertile patients experience multiple in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) failure owing to recurrent early embryo arrest. However, the underlying mechanisms remain largely unknown. Due to the development of whole-exome sequencing, early embryo arrest has been confirmed as a type of Mendelian disease. This study aimed to identify the genetic cause of early embryo arrest in patients and to expand the genetic spectrum. Furthermore, it can help doctors offer better suggestions to such patients and prevent patients from suffering from multiple IVF/ICSI failures. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is the most common fatal muscle disease, with an estimated incidence of 1/3500–1/5000 male births, and it is associated with mutations in the X-linked DMD gene encoding dystrophin, the largest known human gene. There is currently no cure for DMD. The large size of the DMD gene hampers exogenous gene addition and delivery. The genetic correction of DMD patient-derived induced pluripotent stem cells (DMD-iPSCs) and differentiation into suitable cells for transplantation is a promising autologous therapeutic strategy for DMD. In this study, using CRISPR/Cas9, the full-length dystrophin coding sequence was reconstructed in an exon-50-deleted DMD-iPSCs by the targeted addition of exon 50 at the junction of exon 49 and intron 49 via homologous-directed recombination (HDR), with a high targeting efficiency of 5/15, and the genetically corrected iPSCs were differentiated into cardiomyocytes (iCMs). Importantly, the full-length dystrophin expression and membrane localization were restored in genetically corrected iPSCs and iCMs. Thus, this is the first study demonstrating that full-length dystrophin can be restored in iPSCs and iCMs via targeted exon addition, indicating potential clinical prospects for DMD gene therapy. [ABSTRACT FROM AUTHOR]

Due to uneven space–time distribution of vehicles, Internet of Vehicles (IoV) has problems with load imbalance and low resource utilization of Base Stations (BSs) in the Coordinated Multi-Point (CoMP) communication scenario. This paper proposes a dynamic load balancing algorithm based on vehicle prediction. It is assumed that the number of vehicles arriving at the BSs obeys the segmented Poisson distribution to determine the current and predicted load statuses of BSs. First, analyze the load status of each BS and the location of users (vehicles). Then, screen out BSs whose load below the full load threshold as a switchable low-load cooperative cluster, which can convert interference signals into useful signals and reduce the interference between adjacent BSs. Finally, complete load balancing by redistributing the communication service of edge users through sharing channel information and user date among coordinated BSs. Because IoV is a dynamic network, the proposed algorithm runs dynamically in cycles. Simulation results show that the algorithm can perform balance the load of BSs well, the overload rates of BSs during the traffic off-peak period and peak period are reduced significantly, and the average information rate of users is greatly improved. [ABSTRACT FROM AUTHOR]

CRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step assay is reduced because the Cas12a protein competes with the isothermal amplification enzymes for the target DNA and cleaves it. Therefore, the key to improving the sensitivity of the one-step assay is to address the imbalance between isothermal amplification and CRISPR detection. In previous study, we developed a Cas12a one-step assay using single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA) and applied this method for the detection of pathogenic DNA. Here, we utilized mD-crRNA to establish a sensitive one-step assay that enables the visual detection of SARS-CoV-2 under ultraviolet light, achieving a detection limit of 5 aM without cross-reactivity. The sensitivity of mD-crRNA in the one-step assay was 100-fold higher than that of wild-type crRNA. Mechanistic studies revealed that the addition of ssDNA at the 3′ end of mD-crRNA attenuates the binding affinity between the Cas12a-mD-crRNA complex and the target DNA. Consequently, this reduction in binding affinity decreases the cis -cleavage activity of Cas12a, mitigating its cleavage of the target DNA in the one-step assay. As a result, there is an augmentation in the amplification and accumulation of target DNA, thereby enhancing detection sensitivity. In the clinical testing of 40 SARS-CoV-2 RNA samples, the concordance between the results of the one-step assay and known qPCR results was 97.5 %. The one-step assay using mD-crRNA proves to be highly sensitive and specificity and visually effective for the detection of SARS-CoV-2. Our study delves into the application of the mD-crRNA-mediated one-step assay in nucleic acid detection and its associated reaction mechanism. This holds great significance in addressing the inherent incompatibility issues between isothermal amplification and CRISPR detection. [Display omitted] • The one-step assay using mD-crRNA for detecting the SARS-CoV-2 was established. • The 3′ end ssDNA in mD-crRNA was crucial for enhancing one-step assay sensitivity. • Reduced affinity of Cas12a-mD-crRNA and target DNA lowered Cas12a cleavage activity. • This assay demonstrated a visual detection limit of 5 aM for SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a devastating autosomal recessive motor neuron disease associated with mutations in the survival motor neuron 1 (SMN1) gene, the leading genetic cause of infant mortality. A nearly identical copy gene (SMN2) is retained in almost all patients with SMA. However, SMN2 fails to prevent disease development because of its alternative splicing, leading to a lack of exon 7 in the majority of SMN2 transcripts and yielding an unstable truncated protein. Several splicing regulatory elements, including intronic splicing silencer-N1 (ISS-N1) of SMN2 have been described. In this study, targeted-deletion of ISS-N1 was achieved using prime editing (PE) in SMA patient-specific induced pluripotent stem cells (SMA-iPSCs) with a high efficiency of 7/24. FL-SMN expression was restored in the targeted-deletion iPS clones and their derived motor neurons (iMNs). Notably, the apoptosis of the iMNs, caused by the loss of SMN protein that leads to the hyperactivity of endoplasmic reticulum (ER) stress, was alleviated in targeted-deletion iPSCs derived-iMNs. Thus, this is the first study to demonstrate that the targeted-deletion of ISS-N1 via PE for restoring FL-SMN expression holds therapeutic promise for SMA. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the efficacy and safety of short-term insemination and early-rescue intracytoplasmic sperm injection (ICSI), an approach that rescued oocytes with unclear second polar body 6 h after initial insemination by ICSI (early R-ICSI) to avoid total or near-total fertilization failure in conventional in vitro fertilization (IVF). Methods: We performed a retrospective study in 16,769 patients (short-term IVF, n = 12,094; ICSI, n = 3452; early R-ICSI, n = 1223) who received IVF/ICSI treatment in our hospital from January 2009 to October 2018. Fertilization and clinical outcomes were compared among those three groups. Results: When considering the R-ICSI embryos in the early R-ICSI group independently, the rates of fertilization and day-3 cleaved embryos in 2PN oocytes were comparable, the rates of fertilization (2PN) and high-quality embryos were lower, whereas the multi-PN fertilization rate (3.27%) was significantly higher than the ICSI group (1.26%). The difference of clinical pregnancy rate between the part of transferred R-ICSI embryos (40.81%) and the ICSI group (44.73%) remained nonsignificant. Furthermore, the rate of congenital birth defects in the early R-ICSI group (0.99%) was not significantly different from those in the short-term IVF (0.76%) and ICSI groups (1.07%). Conclusion: Despite the multi-PN fertilization rate, our study highlights early R-ICSI as a safe and effective alternative in assisted reproduction to decrease complete IVF fertilization failure and reduce ICSI utilization. Additional large amount and long-term follow-up studies are needed to further validate the use of early R-ICSI. [ABSTRACT FROM AUTHOR]

• Genetic etiology was clarified in 54 families, with a positive rate of 48%. • 19 novel P/LP variants and 22 VUS variants were identified. All of these findings broadened the mutation spectrum of hearing loss genes. Hearing loss is a group of diseases with high genetic heterogeneity. About 160 genes have been reported to be associated with hereditary hearing loss. 113 families with hearing loss were collected, and WES was used to detect SNV, InDel, CNV and mitochondrial gene variants. For some probands with negative WES test results, the copy number of STRC and OTOA were determined by using real-time fluorescence quantitative PCR. Pathogenic or likely pathogenic variants were found in 54 probands, of which 98% (53/54) were SNVs or InDels and 2% (1/54) were CNVs, a positive rate of 48%. 16 families (14%) were detected with candidate variants of uncertain significance. 19 novel pathogenic or likely pathogenic variants and 22 candidate variants of uncertain significance were identified in this study. The most common hearing loss gene in the families was GJB2 , accounting for 28% (15/53), followed by SLC26A4 and MYO15A , accounting for 21% (11/53) and 11% (6/53), respectively. Heterozygous gene deletion was detected in 3 probands, including 2 with STRC and 1 with OTOA in 43 families with WES negative test. Genetic etiology was clarified in 54 families. All of these findings broadened the mutation spectrum of hearing loss genes, thus providing new variant information for the future diagnosis of patients with hearing loss. [ABSTRACT FROM AUTHOR]

• This study makes definitive diagnosis for 6 families with suspected XLA and further expands the spectrum of BTK mutations, providing new information for the diagnosis of the disease. • Six BTK mutations were identified, four of which were novel. • The classification of the novel missense variant c.1900 T > G can be upgraded to "likely pathogenic". • A novel deletion covering splice-site resulted in 35 bp skipping of exon and exact break point of a novel gross deletion was located. The defect of Bruton's tyrosine kinase (BTK) gene resulted in X-linked agammaglobulinemia (XLA), which is characterized by recurrent bacterial infections, immunodeficiency with low B-cell numbers and immunoglobulin. Diagnosis of XLA depends on clinical phenotype and genetic testing. Six unrelated Chinese families with high suspicion of XLA were enrolled in this study. Potential pathogenic variants were detected and validated by Whole Exome Sequencing (WES) and Sanger Sequencing. Western blot, Quantitative PCR (qPCR) analysis and immunofluorescence analysis were used to evaluate the preliminary function of candidate BTK variants. A total of six variants were identified, four of which were not reported before. The novel missense mutation(c.1900 T > G) and deletion(c.897delG) were found that the mutant protein and mRNA expression levels have fallen by Western Blot and qPCR identification. We also constructed minigene expression vector to determine the deletion (c.1751-6_1755delttctagGGGTT) resulting a 35 bp skipping in exon 18. Meanwhile, the break point of gross deletion (Exon2-5) discovered based on WES was confirmed to be located at site ChX:101367539_101376531 through qPCR and Gap-PCR. This study makes definitive diagnosis for 6 families with suspected XLA and further expands the spectrum of BTK mutations, providing new information for the diagnosis of the disease. [ABSTRACT FROM AUTHOR]

Background: Oocyte maturation arrest at metaphase I leads to fertilization failure in humans. In early embryos, the tubulin beta 8 class VIII (TUBB8) encodes a β-tubulin isotype and aids in the assembling of the human oocyte spindle. Mutations in the TUBB8 potentially interfere with human oocyte maturation—a crucial prerequisite for fertilization and subsequent embryonic development. This study aims to investigate the novel mutations in TUBB8 and their prevalence. Results: Hundred fertile women (controls) and eleven infertile women with oocyte maturation arrest were chosen for the study. A total of five TUBB8 heterozygous/homozygous mutations were found in eleven infertile females (p.A313V, p.C239W, p.R251Q, p.P358L, and p.G96R). The Exome Aggregation Consortium (ExAC), SIFT, and PolyPhen-2 analyses revealed that p. A313V has unknown pathogenicity and p.C239W, p.R251Q, p.P358L, and p.G96R have possible pathogenicity. The wild-type (WT) and four mutant gene constructs were transfected to Hela cells. The Western blot analysis indicates that the TUBB8 expression of the p.C239W, p.R251Q, and p.G96R mutations was significantly decreased than that of WT. The immunofluorescence assay showed that the Hela cells transfected with either p.C239W, p.R251Q, or p.G96R mutations exhibited the disrupted microtubule structure, revealing a significant difference in the organization of the microtubule network compared to the WT. Conclusions: We identified three novel variants and two reported variants out of 11 infertile women with oocyte metaphase I arrest. According to the present data, TUBB8 gene variants account for 31.96% of all participants (109/341) with oocyte maturation arrest. [ABSTRACT FROM AUTHOR]

[Display omitted] • Identification of two novel variants in OSGEP gene. • Reanalysis WES data together with the follow-up provided accurate diagnosis. • The variants reduced OSGEP expression and altered its subcellular localization. • The activated DNA damage response signaling was found in patient's cell. Galloway-Mowat syndrome (GAMOS) is an extremely rare clinically heterogeneous autosomal or X-linked inherited recessive disease characterized by early-onset steroid-resistant nephrotic syndrome (SRNS), microcephaly and neurological impairment. In this study, two siblings mainly presenting with decreased head circumference, hypotonia, gross motor delay, and dysmorphic features were initially detected without pathogenic variants by karyotyping, SNP-array and WES. After a 3 year's follow-up, the proband manifested additional proteinuria, hematuria and "deeper sulci" with a sign of brain atrophy. By reanalysis on the proband's previous WES data, two novel compound heterozygous variants of OSGEP (c.133dupA; c.608C > T) were identified. Furthermore, functional studies showed that the variants reduced the expression of OSGEP protein and activated the DNA damage response (DDR) signaling in the lymphoblastoid cell lines (LCLs) obtained from the patient. The analysis of protein localization with confocal microscopy revealed that the EGFP-tagged/HA-tagged mutant OSGEP proteins were abnormal aggregation or retained inside the cytosol, respectively. Our study not only expanded the pathogenic variant spectrum of OSGEP but also carried on regular follow-up for kidney involvement and established a strategy for evaluation on the function of mutant OSGFP by subcellular localization assay. [ABSTRACT FROM AUTHOR]

• Missed diagnoses have been presented in whole-exome sequencing (WES) analysis. • WES reanalysis improves the diagnostic yield. • Diagnosed by upgraded bioinformatics pipeline and variant interpretation strategy. Some missed diagnoses have been presented in whole-exome sequencing (WES) analysis for cases with possible Mendelian diseases. To assess how much contributions of WES reanalysis might improve diagnostic yield, we reviewed the WES data of 174 undiagnosed cases. We performed reanalysis with an updated bioinformatics pipeline involving better algorithms and updated databases so that CNVs and SNVs in intron regions and InDels within 10–50 bp can be detected. Upgraded variant interpretation processes, including updated software packages, databases and literature, expanded knowledge of genes and diseases, extended filtering conditions and phenotype reevaluation, were also implemented for reanalysis. Candidate variants were classified by ACMG guidelines and certified by Sanger sequencing, qPCR or MLPA. Fourteen additional cases received new diagnosis in the reanalysis. The results which became positive were sorted according to the following aspects: detection of CNVs; diagnosis by SNVs in intron regions or InDels within 10–50 bp; reclassification due to new reports of variants or gene-disease relationships; digenic inheritance leading to disease; disease caused by frequent variations in the general population; and accurate phenotype assessment enabling the establishment of the molecular diagnosis. Our study improved diagnosis yield through an optimized bioinformatics pipeline and variant interpretation strategy of WES and provided analysis experience learned from the WES reanalysis to reduce missed diagnoses. [ABSTRACT FROM AUTHOR]

Skeletal dysplasias (SDs) are common birth defects, but they are difficult to diagnose accurately according to only the limited phenotypic information available from ultrasound during the pregnancy. To evaluate the application of whole‐exome sequencing (WES) and expand the data in the prenatal molecular diagnosis of fetuses with SDs, we collected 55 fetuses with SDs based on ultrasonographic features. WES of the fetuses or parent‐fetus trio were subjected to sequential tests and produced a diagnostic yield of 64% (35/55). 65% (11/17) of families with a history of adverse pregnancies were diagnosed, 16 genes were involved and 37 different pathogenic or likely pathogenic variants were identified, including 14 novel variants, which were first reported in this study. De novo variants were identified in 21 cases (60%, 21/35) among the fetuses with a genetic diagnosis. The pathogenicity of two novel splice‐site variants was confirmed by constructing minigene in vitro. Our results revealed that WES can provide new evidence for the relationship between the genotype and phenotype of fetuses with SDs, as well as broaden the mutation spectrum of detected genes, which is significant for prenatal diagnosis and genetic counseling. [ABSTRACT FROM AUTHOR]

PIGK gene, encoding a key component of glycosylphosphatidylinositol (GPI) transamidase, was recently reported to be associated with inherited GPI deficiency disorders (IGDs). However, little is known about the specific downstream effects of PIGK on neurodevelopment due to the rarity of the disease and the lack of in vivo study. Here, we described 2 patients in a Chinese family presented with profound global developmental delay, severe hypotonia, seizures, and postnatal progressive global brain atrophy including hemisphere, cerebellar and corpus callosum atrophy. Two novel compound heterozygous variants in PIGK were identified via genetic analysis, which was proved to cause significant decrease of PIGK protein and reduced cell surface presence of GPI-APs in the patients. To explore the role of Pigk on embryonic and neuronal development, we constructed Pigk knock-down zebrafish and knock-in mouse models. Zebrafish injected with a small dose of morpholino oligonucleotides displayed severe developmental defects including small eyes, deformed head, curly spinal cord, and unconsumed yolk sac. Primary motor neuronal dysplasia and extensive neural cell apoptosis were further observed. Meanwhile, the mouse models, carrying the two variants respectively homologous with the patients, both resulted in complete embryonic lethality of the homozygotes, which suggested the intolerable effect caused by amino acid substitution of Asp204 as well as the truncated mutation. Our findings provide the in vivo evidence for the essential role of PIGK during the embryonic and neuronal development. Based on these data, we propose a basis for further study of pathological and molecular mechanisms of PIGK-related neurodevelopmental defects. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD), the most common lethal muscular disorder, affects 1 in 5000 male births. It is caused by mutations in the X-linked dystrophin gene (DMD), and there is no effective treatment currently. Gene addition is a promising strategy owing to its universality for patients with all gene mutations types. In this study, we describe a site-specific gene addition strategy in induced pluripotent stem cells (iPSCs) derived from a DMD patient with exon 50 deletion. By using transcription activator-like effector nickases (TALENickases), the mini-dystrophin cassette was precisely targeted at the ribosomal RNA gene (rDNA) locus via homologous recombination with high targeting efficiency. The targeted clone retained the main pluripotent properties and was differentiated into cardiomyocytes. Significantly, the dystrophin expression and membrane localization were restored in the genetic corrected iPSCs and their derived cardiomyocytes. More importantly, the enhanced spontaneous contraction was observed in modified cardiomyocytes. These results provide a proof of principle for an efficient targeted gene addition for DMD gene therapy and represents a significant step toward precisely therapeutic for DMD. • Site-specific integrated mini-dystrophin into rDNA locus in DMD-iPSCs. • Dystrophin expression and localization were restored in targeted iPSCs and iCMs. • The enhanced spontaneous contraction was observed in the targeted iCMs. [ABSTRACT FROM AUTHOR]

GZF1 was recently reported as a genetic factor associated with Larsen syndrome. Two patients presenting hip dislocation, scoliosis and severe myopia, as well as hearing loss and other abnormal features, were found to carry two novel compounds heterozygous variants in GZF1 (c.397400del, p. Leu133fs; and c.1474del, p. Met492fs) through whole‐exome sequencing. The mRNA expression level of L133fs‐GZF1 did not significantly differ from that of WT‐GZF1. However, no HA‐conjugated mutant protein was detected by western blotting, which was also confirmed by immunofluorescence staining. In addition, both mRNA transcription and protein expression levels of M492fs‐GZF1 were significantly lower than those of wild type, and HA‐tagged M492fs‐GZF1 was mainly distributed in the cytoplasm of HEK 293 T cells. These results suggested that the two variants could lead to loss of function of GZF1. Our study was the second to report the association between GZF1 variants and Larsen syndrome. We also provided functional evidence for the pathogenicity of GZF1 variants, which expands the mutation spectrum and offers a basis for functional research on the role of GZF1 in the development of Larsen syndrome. [ABSTRACT FROM AUTHOR]

Background: Current copy number variation (CNV) identification methods have rapidly become mature. However, the postdetection processes such as variant interpretation or reporting are inefficient. To overcome this situation, we developed REDBot as an automated software package for accurate and direct generation of clinical diagnostic reports for prenatal and products of conception (POC) samples. Methods: We applied natural language process (NLP) methods for analyzing 30,235 in‐house historical clinical reports through active learning, and then, developed clinical knowledge bases, evidence‐based interpretation methods and reporting criteria to support the whole postdetection pipeline. Results: Of the 30,235 reports, we obtained 37,175 CNV‐paragraph pairs. For these pairs, the active learning approaches achieved a 0.9466 average F1‐score in sentence classification. The overall accuracy for variant classification was 95.7%, 95.2%, and 100.0% in retrospective, prospective, and clinical utility experiments, respectively. Conclusion: By integrating NLP methods in CNVs postdetection pipeline, REDBot is a robust and rapid tool with clinical utility for prenatal and POC diagnosis. [ABSTRACT FROM AUTHOR]

Background: Treacher Collins syndrome (TCS) is the most common mandibulofacial dysostosis with an autosomal dominant or rarely recessive manner of inheritance. It is still challenging to make a definite diagnosis for affected fetuses with TCS only depending on the ultrasound screening. Genetic tests can contribute to the accurate diagnosis for those prenatal cases. Methods: Targeted exome sequencing was performed in a fetus of a Chinese family, who presenting an abnormal facial appearance by prenatal 2D and 3D ultrasound screening, including micrognathia, nasal bridge pit, and abnormal auricle. The result was validated with multiplex ligation‐dependent probe amplification (MLPA) and real‐time quantitative PCR (qPCR). Results: A novel 2–6 exons deletion of TCOF1 gene was identified and confirmed by the MLPA and qPCR in the fetus, which was inherited from the affected father with similar facial anomalies. Conclusion: The heterozygous deletion of 2–6 exons in TCOF1 results in the TCS of this Chinese family. Our findings not only enlarge the spectrum of mutations in TCOF1 gene, but also highlight the values of combination of ultrasound and genetics tests in diagnosis of craniofacial malformation‐related diseases during perinatal period. [ABSTRACT FROM AUTHOR]

• The causes of androgen insensitivity in 17 Chinese patients were identified. • Six novel mutations in AR were detected. • The stop codon mutation of AR was first discovered. Androgen insensitivity syndrome (AIS) is the most common type of 46, XY disorders of sex development (DSD), with a wide range of clinical heterogeneity, from male infertility, hypospadias to completely normal female external genitalia. Mutation of the androgen receptor (AR) gene on the X chromosome (Xq11.2q12) is the main cause of AIS. By phenotype evaluation, hormone test, ultrasound scan and G-banding karyotype, 17 unrelated Chinese patients were clinical diagnosed with AIS. Sanger sequencing of the AR was performed in these 17 patients. Functional studies were carried out for the novel mutations. We identified 16 mutations in all patients, including six novel mutations (Q59*, F171Sfs*4, E204*, G209E, I870T, *921R). It is the first time that a stop codon mutation (*921R) in AR has been identified. Expression and nuclear localization analysis showed the *921R mutation caused an elongated abnormal polypeptide chain of the AR protein, and the abnormal protein could not be transported to the nucleus to stimulate the expression of downstream genes after androgenic treatment. Expression analysis showed the protein level of G209E mutation was obviously decreased. Our study expands the spectrum of AR mutations and could provide evidence for the genetic and reproductive counseling of families with AIS. All of these findings broadened the mutation spectrum of AR , which were significantly valuable for patient gender assignment, genetic counseling and the clinical and psychological management. [ABSTRACT FROM AUTHOR]

Background: Charcot–Marie–Tooth (CMT) disease is a group of hereditary neuropathies with high phenotypic and genetic heterogeneity. In this study, we report a large family with X‐linked CMT (CMTX) caused by a novel GJB1 mutation. Methods: A family with the clinical diagnosis of CMTX was investigated. For mutation analysis, the coding region of GJB1 was sequenced using DNA from 15 family members. The identified GJB1 mutation was investigated by DHPLC in 120 normal controls. Mutation reanalysis was performed based on whole‐exome sequencing (WES). Cell transfection studies were performed to characterize the function of the novel mutation. Results: A missense mutation (c.605T>A) in GJB1 was detected in five patients and eight female carriers but not in two unaffected members of the family. The mutation was not found in 120 healthy controls and has not been previously reported. WES excluded other pathogenic mutations in the family. The pathogenicity of the mutation was confirmed by disrupting the membrane localization of the encoded proteins. Conclusion: Our findings demonstrate that a novel mutation (c.605T>A) in GJB1 is associated with CMTX and adds to the repertoire of GJB1 mutations related to CMTX. [ABSTRACT FROM AUTHOR]

• Compound heterozygous c.27delC and de novo complex rearrangement sequences in GAN were found. • The first report of the chimerism of multiple de novo complex rearrangement sequences in GAN. • QPCR is an effective way to detect LOH in patients with homozygous mutations. Giant axonal neuropathy (GAN) is a rare and grievous autosomal recessive neurodegenerative disease due to loss-of-function mutation in GAN. However, the chimerism of complex rearrangement sequences of GAN has not been reported so far, and the mechanism for its complex rearrangements remains to be determined. We identified a family with clinical symptoms of GAN and aimed to reveal a genetic cause underlying this disease. By whole-exome sequencing in the patient we identified a novel homozygous frameshift mutation with 1 bp deletion (c.27delC) in GAN. However, when analyzed the patient's genomic DNA (gDNA) by quantitative real-time PCR and breakpoint DNA sequencing, we found the chimerism of multiple complex rearrangement sequences encompassing exon 1 of GAN in the patient's genome. The microhomology and localization of the breakpoint indicated that they may be caused by Alu repeat elements. We also found that the mRNA expression level of GAN in patient's lymphocyte was decreased, confirming the pathogenicity of these mutations. Our study is the first reported on many complex rearrangement sequences mosaic in GAN mediated by Alu element. The patient here is not a simple homozygous frameshift mutation, but a compound heterozygous paternal c.27delC mutation and the chimerism of multiple de novo complex rearrangement sequences in GAN. Our results may also provide new insights into the formation and pathogenicity of complex rearrangement in GAN , and may be helpful to genetic counseling and genetic testing. It also enriches the Alu -mediated disease-associated database which are important for correct clinical interpretation. [ABSTRACT FROM AUTHOR]

• Gene panel can be an economical and effective way to detected cilia-related kidney disorders. • 70% (28/40) of the mutations were first found in patients. • A hypomorphic nontruncating variant was identified in an atypia PKD family. • Fetus with DYNC2H1 compound heterozygous missense mutations can show mainly kidney dysplasia without skeletal abnormalities. • Novel long-range PCR primers for PKD1 were efficient and accurate. Cilia play an important role in cellular signaling pathways. Defective ciliary function causes a variety of disorders involve retina, skeleton, liver, kidney or others. Cilia-related kidney disorders are characterized by cystic renal disease, nephronophthisis and renal failure in general. In this study, we collected 33 families clinically suspected of cilia-related kidney disorders. Capture-based next-generation sequencing (NGS) of 88 related genes, Sanger sequencing, pedigree analysis and functional study were performed to analyze their genetic cause. 40 mutations in PKD1 , PKD2 , PKHD1 , DYNC2H1 and TMEM67 genes were identified from 27 of 33 affected families. 70% (28/40) of the mutations were first found in patients. We reported a very early-onset autosomal dominant polycystic kidney disease (ADPKD) family caused by a novel heterozygous PKD1 mutation; another fetus with DYNC2H1 compound heterozygous missense mutations showed mainly kidney dysplasia instead of skeletal abnormalities; and a novel PKD1 mutation, c.12445-3C > G, was confirmed to cause two wrong splicing modes. As for previously reported mutations, such as PKD1 , c.6395 T > G (p.F2132C) and c.6868G > T (p.D2290Y), we had new and different findings. The findings provided new references for genotype-phenotype analyses and broadened the mutation spectrum of detected genes, which were significantly valuable for prenatal diagnosis and genetic counseling. [ABSTRACT FROM AUTHOR]

Background: Interleukin-24 (IL-24) is a therapeutic gene for melanoma, which can induce melanoma cell apoptosis. Mesenchymal stem cells (MSCs) show promise as a carrier to delivery anti-cancer factors to tumor tissues. Induced pluripotent stem cells (iPSCs) are an alternative source of mesenchymal stem cells (MSCs). We previously developed a novel non-viral gene targeting vector to target IL-24 to human iPSCs. This study aims to investigate whether MSCs derived from the iPSCs with the site-specific integration of IL-24 can inhibit the growth of melanoma in a tumor-bearing mouse model via retro-orbital injection. Methods: IL-24-iPSCs were differentiated into IL-24-iMSCs in vitro, of which cellular properties and potential of differentiation were characterized. The expression of IL-24 in the IL-24-iMSCs was measured by qRT-PCR, Western Blotting, and ELISA analysis. IL-24-iMSCs were transplanted into the melanoma-bearing mice by retro-orbital intravenous injection. The inhibitory effect of IL-24-iMSCs on the melanoma cells was investigated in a co-culture system and tumor-bearing mice. The molecular mechanisms underlying IL-24-iMSCs in exerting anti-tumor effect were also explored. Results: iPSCs-derived iMSCs have the typical profile of cell surface markers of MSCs and have the ability to differentiate into osteoblasts, adipocytes, and chondroblasts. The expression level of IL-24 in IL-24-iMSCs reached 95.39 ng/106 cells/24 h, which is significantly higher than that in iMSCs, inducing melanoma cells apoptosis more effectively in vitro compared with iMSCs. IL-24-iMSCs exerted a significant inhibitory effect on the growth of melanoma in subcutaneous mouse models, in which the migration of IL-24-iMSCs to tumor tissue was confirmed. Additionally, increased expression of Bax and Cleaved caspase-3 and down-regulation of Bcl-2 were observed in the mice treated with IL-24-iMSCs. Conclusion: MSCs derived from iPSCs with the integration of IL-24 at rDNA locus can inhibit the growth of melanoma in tumor-bearing mouse models when administrated via retro-orbital injection. [ABSTRACT FROM AUTHOR]

Chromosome classification is critical for karyotyping in abnormality diagnosis. To expedite the diagnosis, we present a novel method named Varifocal-Net for simultaneous classification of chromosome’s type and polarity using deep convolutional networks. The approach consists of one global-scale network (G-Net) and one local-scale network (L-Net). It follows three stages. The first stage is to learn both global and local features. We extract global features and detect finer local regions via the G-Net. By proposing a varifocal mechanism, we zoom into local parts and extract local features via the L-Net. Residual learning and multi-task learning strategies are utilized to promote high-level feature extraction. The detection of discriminative local parts is fulfilled by a localization subnet of the G-Net, whose training process involves both supervised and weakly supervised learning. The second stage is to build two multi-layer perceptron classifiers that exploit features of both two scales to boost classification performance. The third stage is to introduce a dispatch strategy of assigning each chromosome to a type within each patient case, by utilizing the domain knowledge of karyotyping. The evaluation results from 1909 karyotyping cases showed that the proposed Varifocal-Net achieved the highest accuracy per patient case (%) of 99.2 for both type and polarity tasks. It outperformed state-of-the-art methods, demonstrating the effectiveness of our varifocal mechanism, multi-scale feature ensemble, and dispatch strategy. The proposed method has been applied to assist practical karyotype diagnosis. [ABSTRACT FROM AUTHOR]

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. • We collected five infants who were diagnosed were rare E3 deficiency MSUD, and corrected the clinical diagnosis finally. • We found 6 novel mutations that cause rare MSUD, and verified the pathogenicity of related mutations. • We reviewed previously reported mutations in Chinese patients and summarized their genotypic and phenotypic correlation. [ABSTRACT FROM AUTHOR]

Hemophilia A is an X-linked recessive bleeding disorder caused by FVIII gene deficiency, which may result in spontaneous joint hemorrhages or life-threatening bleeding. Currently, cell-based gene therapy via ex vivo transduction of transplantable cells with integrating gene-expressing vectors offers an attractive treatment for HA. In present study, we targeted an expression cassette of B-domain-deleted FVIII into the ribosomal DNA (rDNA) locus of human embryonic stem cells (hESCs) by transfection with a nonviral targeting plasmid pHrn. The targeted hESCs clone could be expanded and retained the main pluripotent properties of differentiation into three germ layers both in vitro and in vivo. Importantly, under defined induction conditions, the targeted hESCs could differentiated into functional mesenchymal stem cells (MSCs) and hepatocytes, as validated by relevant specific cell markers and functional examination. Tumorgenesis assay demonstrated that these cells are relatively safe for future applications. Analysis on gene expression revealed that exogenous FVIII mRNA and FVIII proteins were both present in differentiated MSCs and hepatocytes. These results indicated that through gene targeting at hESCs rDNA locus a persistent cell source of transplantable genetic-modified cells can be accomplished for HA therapy. • Targeting FVIII into hESCs rDNA locus using nonviral vector for HA therapy. • Differentiation of genetic-modified hESCs into MSCs and hepatocytes. • Derived MSCs and hepatocytes stably secret exogenous FVIII protein. • The strategy provided transplantable cells for HA therapy. [ABSTRACT FROM AUTHOR]

CRISPR-Cas12a RNA-guided complexes have been developed to facilitate the rapid and sensitive detection of nucleic acids. However, they are limited by the complexity of the operation, risk of carry-over contamination, and degradation of CRISPR RNA (crRNA). In this study, a Cas12a-based single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA)-mediated one-step diagnostic method (CasDOS) was established to overcome these drawbacks. mD-crRNA consisted of wild-type crRNA (Wt-crRNA) with ssDNA extensions at the 3′ and 5′ ends. Compared to Wt-crRNA, mD-crRNA exhibited a 100–1000-fold increase in sensitivity in the one-step assay, reducing the cis- cleavage activity of Cas12a to avoid excessive cleavage of the target DNA in the early stages of the reaction, leading to increased amplification and accumulation of the target amplicons, and improved the speed and intensity of the generated fluorescence signal. The detectability of CasDOS was 16.6 aM for the constructed plasmids of Streptococcus agalactiae (GBS), human papillomavirus type 16 (HPV16), and type 18 (HPV18). In clinical trials, CasDOS achieved 100% accuracy in identifying the known genotypes of the five HPV DNA samples. Moreover, CasDOS showed complete concordance with the qPCR results for GBS detection in ten vaginal or cervical swab samples, with a turnaround time from sampling to results within 30 min. In addition, mD-crRNA remained stable after Ribonuclease R treatment, suggesting that it might be more suitable as a raw material for the CRISPR detection kit. In conclusion, we have developed a universal, rapid, and highly sensitive one-step CRISPR detection assay. [Display omitted] • The first time both ends ssDNA modified-crRNA utilize in one-step assay. • The modified crRNA showed 100–1000-fold increased sensitivity in one-step assay. • The modified crRNA was more resistant to RNase R treatment than wild-type crRNA. • The turnaround time with CasDOS from sampling to results was within 30 min. [ABSTRACT FROM AUTHOR]

Autosomal recessive cornea plana is a very rare hereditary ocular disease, characterized by a flattened corneal curvature, marked hyperopia due to low refractive power and frequently consequent accommodative esotropia. Other features include various cornea anterior segment abnormalities, without systemic problems. The purpose of the present study was to investigate the clinical and molecular alterations in a Chinese family with cornea plana. Full ophthalmic examinations of the patients were performed, including slit-lamp examination, fundus examination and ocular ultrasound. Whole-exome sequencing data were screened for pathological variants in the proband, which were confirmed by Sanger sequencing. One novel missense mutation, c.242A>G (p.N81S) and another novel 7 base-pair deletion mutation, c.772-779del (p.G258Cfs*30), were detected in the keratocan (KERA) gene; two affected siblings inherited these variations in a compound heterozygous state, which were derived from the clinically unaffected heterozygous father (c.772_779del) and mother (c.242A>G), respectively. Neither mutation was observed in unrelated healthy controls (n=200). Multiple computer software predictions supported the pathogenicity of the two variants. Furthermore, protein modeling prediction was performed to better understand the molecular basis of cornea plana, particularly the importance of the leucine-rich repeat domain. This study presents the 14th pathogenic KERA mutations identified worldwide and the first in East Asia so far, to the best of our knowledge. These findings guided prenatal diagnosis for the family in question and expand on the variant spectrum of KERA, therefore facilitating genetic counseling. [ABSTRACT FROM AUTHOR]

Background: The detection and interpretation of CNVs are of clinical importance in genetic testing. Several databases and web services are already being used by clinical geneticists to interpret the medical relevance of identified CNVs in patients. However, geneticists or physicians would like to obtain the original literature context for more detailed information, especially for rare CNVs that were not included in databases. Results: The resulting CNVdigest database includes 440,485 sentences for CNV-disease relationship. A total number of 1582 CNVs and 2425 diseases are involved. Sentences describing CNV-disease correlations are indexed in CNVdigest, with CNV mentions and disease mentions annotated. Conclusions: In this paper, we use a systematic text mining method to construct a database for the relationship between CNVs and diseases. Based on that, we also developed a concise front-end to facilitate the analysis of CNV/disease association, providing a user-friendly web interface for convenient queries. The resulting system is publically available at http://cnv.gtxlab.com/. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a kind of neuromuscular disease characterized by progressive motor neuron loss in the spinal cord. It is caused by mutations in the survival motor neuron 1 (SMN1) gene. SMN1 has a paralogous gene, survival motor neuron 2 (SMN2), in humans that is present in almost all SMA patients. The generation and genetic correction of SMA patient-specific induced pluripotent stem cells (iPSCs) is a viable, autologous therapeutic strategy for the disease. Here, c-Myc-free and non-integrating iPSCs were generated from the urine cells of an SMA patient using an episomal iPSC reprogramming vector, and a unique crRNA was designed that does not have similar sequences (≤3 mismatches) anywhere in the human reference genome. In situ gene conversion of the SMN2 gene to an SMN1-like gene in SMA-iPSCs was achieved using CRISPR/Cpf1 and single-stranded oligodeoxynucleotide with a high efficiency of 4/36. Seamlessly gene-converted iPSC lines contained no exogenous sequences and retained a normal karyotype. Significantly, the SMN expression and gems localization were rescued in the gene-converted iPSCs and their derived motor neurons. This is the first report of an efficient gene conversion mediated by Cpf1 homology-directed repair in human cells and may provide a universal gene therapeutic approach for most SMA patients. [ABSTRACT FROM AUTHOR]

Neurodevelopment is a transcriptionally orchestrated process. Cyclin K, a regulator of transcription encoded by CCNK , is thought to play a critical role in the RNA polymerase II-mediated activities. However, dysfunction of CCNK has not been linked to genetic disorders. In this study, we identified three unrelated individuals harboring de novo heterozygous copy number loss of CCNK in an overlapping 14q32.3 region and one individual harboring a de novo nonsynonymous variant c.331A>G (p.Lys111Glu) in CCNK. These four individuals, though from different ethnic backgrounds, shared a common phenotype of developmental delay and intellectual disability (DD/ID), language defects, and distinctive facial dysmorphism including high hairline, hypertelorism, thin eyebrows, dysmorphic ears, broad nasal bridge and tip, and narrow jaw. Functional assay in zebrafish larvae showed that Ccnk knockdown resulted in defective brain development, small eyes, and curly spinal cord. These defects were partially rescued by wild-type mRNA coding CCNK but not the mRNA with the identified likely pathogenic variant c.331A>G, supporting a causal role of CCNK variants in neurodevelopmental disorders. Taken together, we reported a syndromic neurodevelopmental disorder with DD/ID and facial characteristics caused by CCNK variations, possibly through a mechanism of haploinsufficiency. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Reporter embryonic stem cell (ESC) lines with tissue-specific reporter genes may contribute to optimizing the differentiation conditions in vitro as well as trafficking transplanted cells in vivo. To optimize and monitor endothelial cell (EC) differentiation specifically, here we targeted the enhanced green fluorescent protein (EGFP) reporter gene at the junction of 5'UTR and exon2 of the endothelial specific marker gene CD144 using TALENs in human ESCs (H9) to generate a EGFP-CD144-reporter ESC line. The reporter cells expressed EGFP and CD144 increasingly and specifically without unexpected effects during the EC differentiation. The EC differentiation protocol was optimized and applied to EC differentiation from hiPSCs, resulting in an efficient and simplified endothelial differentiation approach. Here we created our own optimized and robust protocol for EC differentiation of hESCs and hiPSCs by generating the lineage- specific site- specific integration reporter cell lines, showing great potential to be applied in the fields such as trafficking gene and cell fate in vivo in preclinical animal models. [ABSTRACT FROM AUTHOR]

Galloway-Mowat syndrome (GMS) is a very rare autosomal-recessive disorder characterized by nephrotic syndrome associated with microcephaly, and various central nervous system abnormalities, mostly cerebral hypoplasia or cerebellar atrophy, intellectual disability and neural-migration defects. WDR73 is the only gene known to cause GMS, and has never been implicated in other disease. Here we present a Chinese consanguineous family with infantile onset intellectual disability and cerebellar hypoplasia but no microcephaly. Whole exome sequencing identified a WDR73 p.W371G missense mutation. The mutation is confirmed to be segregated in this family by Sanger sequencing according to a recessive inheritance pattern. It is predicted to be deleterious by multiple algorithms and affect highly conserved site. Structural modeling revealed conformational differences between the wild type protein and the p.W371G protein. Real-time PCR and Western blotting revealed altered mRNA and protein levels in mutated samples. Our study indicates the novel WDR73 p.W371G missense mutation causes infantile onset intellectual disability and cerebellar hypoplasia in recessive mode of inheritance. Our findings imply that microcephaly is a variable phenotype in WDR73 -related disease, suggest WDR73 to be a candidate gene of severe intellectual disability and cerebellar hypoplasia, and expand the molecular spectrum of WDR73 -related disease. [ABSTRACT FROM AUTHOR]

Osteogenesis imperfecta (OI) is a highly clinically and genetically heterogeneous group of disorders. It is difficult to identify severe OI in the perinatal period. Here, a Chinese woman with a suspected history of fetal OI was referred to our institution at 19 weeks of gestation, due to ultrasound inspection during antenatal screening, which revealed bulbous metaphyses, short humeri, and short thick bent femora in the fetus. Using targeted exome sequencing of 248 genes known to be involved in skeletal system diseases, we identified novel compound heterozygous mutation in the P3H1 gene in the fetus with OI type VIII: c.105_120del (p.D36Rfs*16) and c.2164C > T (p.Q722*). These two mutations were inherited from the father and mother, respectively. The mRNA level of P3H1 wasn't changed suggested that mRNA with this mutation escaped from nonsense-mediated RNA decay. Besides, the level of P3H1 was absence while the CRTAP was mildly decreased. In conclusion, our findings imply this novel compound heterozygous mutation as the molecular pathogenetic in a Chinese fetus with OI type VIII, and demonstrate that targeted next-generation sequencing (NGS) is an accurate, rapid, and cost-effective method in the genetic diagnosis of fetal skeletal dysplasia with genetic and clinical heterogeneity, especially for autosomal recessive skeletal disorders. [ABSTRACT FROM AUTHOR]

Background Tooth agenesis is a common developmental dental anomaly. The aim of the study was to identify the causal genetic mutation in a four-generation Chinese family affected with non-syndromic autosomal dominant tooth agenesis. Methods Genome-wide scanning was performed using the Illumina Linkage-12 array. Genotyping of short tandem repeat markers was used to finely map the causative locus. Haplotype analysis and Sanger sequencing was performed to precisely locate the position and nature of the gene defect. Results Clinical examination of the available 23 family members showed variable tooth agenesis in 10 subjects, ranging from oligodontia to mild hypodontia. Genome-wide scanning and haplotype analyses identified the 4p16.1–p16.3 region with a maximum multi-point LOD score of 3.50, which overlapped with the MSX1 gene. A single heterozygous point mutation IVS1–5 G > A in the MSX1 gene was exclusively detected in the 10 family members affected with tooth agenesis. Sequencing of MSX1 cDNA revealed that the intronic mutation did not affect the normal splicing pattern of the pre-mRNA. However, real-time qPCR analysis of lymphocyte RNA showed that the level of MSX1 mRNA was significantly decreased in individuals heterozygous for the mutation. Conclusions We identified and characterized a novel intronic mutation in the MSX1 gene in a large Chinese pedigree, adding to the small repertoire of MSX1 mutations associated with autosomal dominant tooth agenesis. We hypothesize that the variable degree of tooth agenesis observed in each affected individual may be due to sub-optimal levels of MSX1 expression during critical stages tooth development. [ABSTRACT FROM AUTHOR]

Objective Familial adenomatous polyposis (FAP) is mainly caused by germline mutations in the adenomatous polyposis coli ( APC ) gene. This study aimed to detect pathogenic variants in five Chinese FAP families and review all previously reported pathogenic variants of APC gene in Chinese population. Methods Five non-consanguineous FAP families and 100 unrelated ethnicity-matched controls were included in the study. Sanger sequencing was performed to screen for APC coding and splicing variants. Chinese and English literature on APC germline mutations were reviewed to compile the mutation spectrum of APC gene in Chinese FAP patients. Results One pathogenic variant was detected in each family for the five pedigrees we tested. Three variants (c.3183_3187delACAAA, c.2626C > T and c.1312 + 1G > A) were previously reported as pathogenic. The other two variants were novel: c.794_795insG/p.Val266SerfsTer11 and c.2142_2143insG/p.His715AlafsTer19. They are absent from public databases (1000 Genomes, dbSNP, ESP and ExAC) and 100 normal controls, and are classified as pathogenic based on the new ACMG/AMP variant classification guidelines. Literature review and current study revealed a total of 82 different pathogenic variants from 127 Chinese FAP families. Among these families, 83 families had frameshift variants (65.35%), 26 with nonsense variants (20.47%), six with splice site variants (4.72%), three with missense variants (2.36%) and nine with large deletion or duplication variants (7.09%). Apart from the two previously reported mutation hotspots c.3927_3931delAAAGA (20.47%) and c.3183_3187delACAAA (7.09%), c.847C > T/p.Arg283Ter variant occurred with a frequency of 3.15% (4 out of 127) in Chinese FAP patients. Conclusions We reported two novel pathogenic variants. The comprehensive compilation of variants and comparison revealed largely similar mutation spectrum between Chinese and Western patient populations. Some unique features noticed in Chinese patient population may help to better understand the pathogenesis of FAP. [ABSTRACT FROM AUTHOR]

Glutaric aciduria type I (GA-I) is a rare autosomal recessive metabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase (GCDH), leading to an abnormal metabolism of lysine, hydroxylysine and tryptophan. It results in accumulations of glutaric acid, 3-hydroxyglutaric acid and glutaconic acid. Clinical features include the sudden onset of encephalopathy, hypotonia and macrocephaly usually before age 18 months. Here we report five cases of GA-I confirmed with mutation analysis. GCDH gene mutations were identified in all five probands with GA-I. Three of them had compound heterozygous mutations and two had homozygous mutations. Mutations of two alleles (c.334G > T and IVS11-11A > G) were novel and both of them were confirmed to be splice site mutations by reverse transcription PCR. [ABSTRACT FROM AUTHOR]

Increased risk of monozygotic twinning (MZT) has been shown to be associated with assisted reproduction techniques, particularly blastocyst culture. Interestingly, inner cell mass (ICM) splitting in human ‘8’-shaped hatching blastocysts that resulted in MZT was reported. However, the underlying cause of MZT is not known. In this study, we investigated in a mouse model whether in vitro culture leads to ICM splitting and its association with hatching types. Blastocyst hatching was observed in: (i) in vivo developed blastocysts and (ii–iii) in vitro cultured blastocysts following in vivo or in vitro fertilization. We found that ‘8’-shaped hatching occurred with significantly higher frequency in the two groups of in vitro cultured blastocysts than in the group of in vivo developed blastocysts (24.4% and 20.4% versus 0.8%, respectively; n = 805, P < 0.01). Moreover, Oct4 immunofluorescence staining was performed to identify the ICM in the hatching and hatched blastocysts. Scattered and split distribution of ICM cells was observed around the small zona opening of ‘8’-shaped hatching blastocysts. This occurred at a high frequency in the in vitro cultured groups. Furthermore, we found more double OCT4-positive masses, suggestive of increased ICM splitting in ‘8’-shaped hatching and hatched blastocysts than in ‘U’-shaped hatching and hatched blastocysts (12.5% versus 1.9%, respectively; n = 838, P < 0.01). Therefore, our results demonstrate that extended in vitro culture can cause high frequencies of ‘8’-shaped hatching, and ‘8’-shaped hatching that may disturb ICM herniation leading to increased risk of ICM splitting in mouse blastocysts. These results may provide insights into the increased risk of human MZT after in vitro fertilization and blastocyst transfer. [ABSTRACT FROM AUTHOR]