Application of whole exome sequencing in carrier screening for high-risk families without probands.

Purpose: This study aimed to screen the genetic etiology for the high-risk families including those with an adverse pregnancy history, a history of consanguineous marriages, or a history of genetic diseases, but lack of proband via whole exome sequencing (WES). Methods: 128 individuals from high-risk family were tested by WES. The candidate variants were analyzed according to the ACMG criteria to screen the potential carriers. At-risk couples (ARCs) who harbored the same causative gene were provided with precise fertility guidance to avoid the birth of children with birth defects. Results: The total detection rate was 36.72%, with pathogenic/likely pathogenic (P/LP) variants found in 47 individuals, and variants of uncertain significance (VUS) were found in 34. Among couples with adverse pregnancy history: P/LP variants were found in 38 individuals, and VUS were found in 26, for a detection rate of 34.55%; among members of family history of genetic disease or consanguineous marriages: P/LP variants were found in nine individuals, and VUS were found in 8, for a detection rate of 50.00%. Otherwise, we detected 19 ARCs who both carried P/LP variants in the same gene, with a theoretical offspring prevalence of up to 7.42%. Conclusion: In the absence of probands, carrier screening using WES can provide an efficient tool for screening the molecular etiology of high-risk families.

Genetic etiology study in a large cohort with congenital insensitivity to pain with anhidrosis.

ABSTRACT: Pathogenic variations in the NTRK1 can cause congenital insensitivity to pain with anhidrosis (CIPA), a rare autosomal recessive inherited neuropathy. The precise diagnosis of CIPA relies on the identification of pathogenic genotypes. Therefore, it is essential to expand the NTRK1 variation spectrum and improve molecular diagnosis methods. In this study, 74 probands with typical manifestations of CIPA but unknown genotypes were recruited. A comprehensive molecular genetic analysis was performed to identify variations in the NTRK1, using techniques including Sanger and next-generation sequencing, bioinformatic analysis, quantitative polymerase chain reaction (qPCR), gap-PCR, short tandem repeat (STR) genotyping, and reverse-transcription PCR. In addition, functional assays were conducted to determine the pathogenicity of variants of uncertain significance (VUS) and further characterized changes in glycosylation and phosphorylation of 14 overexpressed mutant vectors with variants at different domains in the TrkA protein, which is encoded by NTRK1. A total of 48 variations in the NTRK1 were identified, including 22 novel ones. When combined with data from another 53 CIPA patients examined in our previous work, this study establishes the largest genotypic and phenotypic spectra of CIPA worldwide, including 127 CIPA families. Moreover, functional studies indicated that the pathogenicity of VUS mainly affected insufficient glycosylation in the extracellular domain and abnormal phosphorylation in the intracellular domain. This study not only provides important evidence for precise diagnosis of CIPA but also further enriches our understanding of the pathogenesis of this disease.

[Expert consensus on the genetic counseling for Dystrophinopathies].

Dystrophinopathies caused by variants of DMD gene are a group of muscular diseases including Duchenne muscular dystrophy, Becker muscular dystrophy, and DMD-associated dilated cardiomyopathy. With the advancement of genetic testing techniques and wider implementation of genetic screening, especially the expanded carrier screening, more and more individuals carrying DMD gene variants have been identified, whereas the genetic counseling capacity is relatively insufficient. Currently there is still a lack of professional norms for genetic counseling on dystrophinopathies. In this consensus, the main points to be covered in the pre- and post-test consultation have been discussed, with an aim to provide genetic counseling guidance for the disease diagnosis, treatment, and family reproduction.

Sensitive and visual detection of SARS-CoV-2 using RPA-Cas12a one-step assay with ssDNA-modified crRNA.

BACKGROUND: 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. RESULTS: 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 %. SIGNIFICANCE: 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.

iPSC-derived NK cells with site-specific integration of CAR19 and IL24 at the multi-copy rDNA locus enhanced antitumor activity and proliferation.

The generation of chimeric antigen receptor-modified natural killer (CAR-NK) cells using induced pluripotent stem cells (iPSCs) has emerged as one of the paradigms for manufacturing off-the-shelf universal immunotherapy. However, there are still some challenges in enhancing the potency, safety, and multiple actions of CAR-NK cells. Here, iPSCs were site-specifically integrated at the ribosomal DNA (rDNA) locus with interleukin 24 (IL24) and CD19-specific chimeric antigen receptor (CAR19), and successfully differentiated into iPSC-derived NK (iNK) cells, followed by expansion using magnetic beads in vitro. Compared with the CAR19-iNK cells, IL24 armored CAR19-iNK (CAR19-IL24-iNK) cells showed higher cytotoxic capacity and amplification ability in vitro and inhibited tumor progression more effectively with better survival in a B-cell acute lymphoblastic leukaemia (B-ALL) (Nalm-6 (Luc1))-bearing mouse model. Interestingly, RNA-sequencing analysis showed that IL24 may enhance iNK cell function through nuclear factor kappa B (NFκB) pathway-related genes while exerting a direct effect on tumor cells. This study proved the feasibility and potential of combining IL24 with CAR-iNK cell therapy, suggesting a novel and promising off-the-shelf immunotherapy strategy.

Identification of the Efficient Enhancer Elements in FVIII-Padua for Gene Therapy Study of Hemophilia A.

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.

Motor patterns of patients with spinal muscular atrophy suggestive of sensory and corticospinal contributions to the development of locomotor muscle synergies.

Complex locomotor patterns are generated by combination of muscle synergies. How genetic processes, early sensorimotor experiences, and the developmental dynamics of neuronal circuits contribute to the expression of muscle synergies remains elusive. We shed light on the factors that influence development of muscle synergies by studying subjects with spinal muscular atrophy (SMA, types II/IIIa), a disorder associated with degeneration and deafferentation of motoneurons and possibly motor cortical and cerebellar abnormalities, from which the afflicted would have atypical sensorimotor histories around typical walking onset. Muscle synergies of children with SMA were identified from electromyographic signals recorded during active-assisted leg motions or walking, and compared with those of age-matched controls. We found that the earlier the SMA onset age, the more different the SMA synergies were from the normative. These alterations could not just be explained by the different degrees of uneven motoneuronal losses across muscles. The SMA-specific synergies had activations in muscles from multiple limb compartments, a finding reminiscent of the neonatal synergies of typically developing infants. Overall, while the synergies shared between SMA and control subjects may reflect components of a core modular infrastructure determined early in life, the SMA-specific synergies may be developmentally immature synergies that arise from inadequate activity-dependent interneuronal sculpting due to abnormal sensorimotor experience and other factors. Other mechanisms including SMA-induced intraspinal changes and altered cortical-spinal interactions may also contribute to synergy changes. Our interpretation highlights the roles of the sensory and descending systems to the typical and abnormal development of locomotor modules.NEW & NOTEWORTHY This is likely the first report of locomotor muscle synergies of children with spinal muscular atrophy (SMA), a subject group with atypical developmental sensorimotor experience. We found that the earlier the SMA onset age, the more the subjects' synergies deviated from those of age-matched controls. This result suggests contributions of the sensory/corticospinal activities to the typical expression of locomotor modules, and how their disruptions during a critical period of development may lead to abnormal motor modules.

Identification of six novel mutations in EDA from 20 hypohidrotic ectodermal dysplasia families.

OBJECTIVE: 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. SUBJECTS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

A homozygous variant in INTS11 links mitosis and neurogenesis defects to a severe neurodevelopmental disorder.

The INTS11 endonuclease is crucial in modulating gene expression and has only recently been linked to human neurodevelopmental disorders (NDDs). However, how INTS11 participates in human development and disease remains unclear. Here, we identify a homozygous INTS11 variant in two siblings with a severe NDD. The variant impairs INTS11 catalytic activity, supported by its substrate's accumulation, and causes G2/M arrest in patient cells with length-dependent dysregulation of genes involved in mitosis and neural development, including the NDD gene CDKL5. The mutant knockin (KI) in induced pluripotent stem cells (iPSCs) disturbs their mitotic spindle organization and thus leads to slow proliferation and increased apoptosis, possibly through the decreased neurally functional CDKL5-induced extracellular signal-regulated kinase (ERK) pathway inhibition. The generation of neural progenitor cells (NPCs) from the mutant iPSCs is also delayed, with long transcript loss concerning neurogenesis. Our work reveals a mechanism underlying INTS11 dysfunction-caused human NDD and provides an iPSC model for this disease.

Chinese experts' consensus guideline on preimplantation genetic testing of monogenic disorders.

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.

A de novo heterozygous POU3F3 genotype for the p.(Q214*) variant in a fetus with transient isolated bilateral mild ventriculomegaly: a case report and review of the literature.

The prenatal prevalence of isolated ventriculomegaly is 0.039%-0.087%. Most isolated mild ventriculomegaly (MV) fetuses (>90%) have a favorable prognosis. However, 5.6% to 7.9% of fetuses with isolated MV have adverse neurodevelopmental outcomes. In this study, we reported the first case of prenatal Snijders Blok-Fisher syndrome (OMIM: #618604) caused by a truncating variant of POU3F3 (OMIM: *602480) in a fetus with transient isolated bilateral MV. The results of karyotype analysis, chromosomal microarray analysis, and TORCH infection evaluation for the fetus were all negative. However, a de novo likely pathogenic nonsense variant of NM_006236.3 (POU3F3): c.640C > T [rs1254251078] p.(Q214*) was identified by whole-exome sequencing (WES). Despite sufficient genetic counseling, the mother refused to undertake further brain magnetic resonance imaging (MRI) and decided to keep the fetus. She gave birth to a male infant through a full-term vaginal delivery. With a long-term follow-up, the infant unfortunately gradually presented with delayed motor development. The postnatal brain MRI of the proband showed dysplasia of the corpus callosum and ventriculomegaly. Considering the high probability of misdiagnosis for such cases, we further summarized the prenatal phenotypes from 19 reported patients with variants in POU3F3. The results revealed that 14 patients displayed a normal prenatal ultrasonographic manifestation, while only approximately 26.32% of fetuses showed MV or cysts without structural deformity. Thus our findings expand the variant spectrum of POU3F3 and suggest the importance of undertaking WES and brain MRI when the fetus has isolated bilateral MV.

CCNK Gene Deficiency Influences Neural Progenitor Cells Via Wnt5a Signaling in CCNK-Related Syndrome.

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.

Rapid and sensitive Cas12a-based one-step nucleic acid detection with ssDNA-modified crRNA.

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.

Functional identification of two novel variants and a hypomorphic variant in ASS1 from patients with Citrullinemia type I.

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

A protospacer adjacent motif-free, multiplexed, and quantitative nucleic acid detection platform with barcode-based Cas12a activity.

Clustered regularly interspaced short palindromic repeat (CRISPR)-based biosensors have been developed to facilitate the rapid and sensitive detection of nucleic acids. However, most approaches using CRISPR-based detection have disadvantages associated with the limitations of CRISPR RNA (crRNA), protospacer adjacent motif (PAM) or protospacer flanking sequence restriction, single channel detection, and difficulty in quantitative detection resulting in only some target sites being detected qualitatively. Here, we aimed to develop a barcode-based Cas12a-mediated DNA detection (BCDetection) strategy, which overcomes the aforementioned drawbacks and enables (1) detection with a universal PAM and crRNA without PAM or crRNA restriction, (2) simultaneous detection of multiple targets in a single reaction, and (3) quantitative detection, which can significantly distinguish copy number differences up to as low as a two-fold limit. We could efficiently and simultaneously detect three ß-thalassemia mutations in a single reaction using BCDetection. Notably, samples from normal individuals, spinal muscular atrophy (SMA) carriers, and SMA patients were significantly and accurately distinguished using the quantitative detection ability of BCDetection, indicating its potential application in ß-thalassemia and SMA carrier screening. Therefore, our findings demonstrate that BCDetection provides a new platform for accurate and efficient quantitative detection using CRISPR/Cas12a, highlighting its bioanalytical applications.

Association analysis between chromosomal abnormalities and fetal ultrasonographic soft markers based on 15,263 fetuses.

BACKGROUND: Soft markers are common prenatal ultrasonographic findings that indicate an increased risk for fetal aneuploidy. However, the association between soft markers and pathogenic or likely pathogenic copy number variations is still unclear, and clinicians lack clarity on which soft markers warrant a recommendation for invasive prenatal genetic testing of the fetus. OBJECTIVE: This study aimed to provide guidance on ordering prenatal genetic testing for fetuses with different soft markers and to elucidate the association between specific types of chromosomal abnormalities and specific ultrasonographic soft markers. STUDY DESIGN: Low-pass genome sequencing was performed for 15,263 fetuses, including 9123 with ultrasonographic soft markers and 6140 with normal ultrasonographic findings. The detection rate of pathogenic or likely pathogenic copy number variants among fetuses with various ultrasonographic soft markers were compared with that of fetuses with normal ultrasonography. The association of soft markers with aneuploidy and pathogenic or likely pathogenic copy number variants were investigated using Fisher exact tests with Bonferroni correction. RESULTS: The detection rate of aneuploidy and pathogenic or likely pathogenic copy number variants was 3.04% (277/9123) and 3.40% (310/9123), respectively, in fetuses with ultrasonographic soft markers. An absent or a hypoplastic nasal bone was the soft marker in the second trimester with the highest diagnostic rate for aneuploidy of 5.22% (83/1591) among all isolated groups. Four types of isolated ultrasonographic soft markers, namely a thickened nuchal fold, single umbilical artery, mild ventriculomegaly, and absent or hypoplastic nasal bone, had higher diagnostic rates for pathogenic or likely pathogenic copy number variants (P<.05; odds ratio, 1.69-3.31). Furthermore, this study found that the 22q11.2 deletion was associated with an aberrant right subclavian artery, whereas the 16p13.11 deletion, 10q26.13-q26.3 deletion, and 8p23.3-p23.1 deletion were associated with a thickened nuchal fold, and the 16p11.2 deletion and 17p11.2 deletion were associated with mild ventriculomegaly (P<.05). CONCLUSION: Ultrasonographic phenotype-based genetic testing should be considered in clinical consultations. Copy number variant analysis is recommended for fetuses with an isolated thickened nuchal fold, a single umbilical artery, mild ventriculomegaly, and an absent or a hypoplastic nasal bone. A comprehensive definition of genotype-phenotype correlations in aneuploidy and pathogenic or likely pathogenic copy number variants could provide better information for genetic counseling.

Comprehensive Analysis of Hemophilia A (CAHEA): Towards Full Characterization of the F8 Gene Variants by Long-Read Sequencing.

BACKGROUND: Hemophilia A (HA) is the most frequently occurring X-linked bleeding disorder caused by heterogeneous variants in the F8 gene, one of the largest genes known. Conventional molecular analysis of F8 requires a combination of assays, usually including long-range polymerase chain reaction (LR-PCR) or inverse-PCR for inversions, Sanger sequencing or next-generation sequencing for single-nucleotide variants (SNVs) and indels, and multiplex ligation-dependent probe amplification for large deletions or duplications. MATERIALS AND METHODS: This study aimed to develop a LR-PCR and long-read sequencing-based assay termed comprehensive analysis of hemophilia A (CAHEA) for full characterization of F8 variants. The performance of CAHEA was evaluated in 272 samples from 131 HA pedigrees with a wide spectrum of F8 variants by comparing to conventional molecular assays. RESULTS: CAHEA identified F8 variants in all the 131 pedigrees, including 35 intron 22-related gene rearrangements, 3 intron 1 inversion (Inv1), 85 SNVs and indels, 1 large insertion, and 7 large deletions. The accuracy of CAHEA was also confirmed in another set of 14 HA pedigrees. Compared with the conventional methods combined altogether, CAHEA assay demonstrated 100% sensitivity and specificity for identifying various types of F8 variants and had the advantages of directly determining the break regions/points of large inversions, insertions, and deletions, which enabled analyzing the mechanisms of recombination at the junction sites and pathogenicity of the variants. CONCLUSION: CAHEA represents a comprehensive assay toward full characterization of F8 variants including intron 22 and intron 1 inversions, SNVs/indels, and large insertions and deletions, greatly improving the genetic screening and diagnosis for HA.

[Consensus on laboratory diagnosis of congenital adrenal hyperplasia due to 21 hydroxylase deficiency].

21 hydroxylase deficiency (21-OHD), the most common form of congenital adrenal hyperplasia, is caused by defects in CYP21A2 gene, which encodes the cytochrome P450 oxidase (P450C21) involved in glucocorticoid and mineralocorticoid synthesis. The diagnosis of 21-OHD is based on the comprehensive evaluation of clinical manifestation, biochemical alteration and molecular genetics results. Due to the complex structure of CYP21A2, special techniques are required to perform delicate analysis to avoid the interference of its pseudogene. Recently, the state-of-the-art diagnostic methods were applied to the clinic gradually, including the steroid hormone profiling and third generation sequencing. To standardize the laboratory diagnosis of 21-OHD, this consensus was drafted on the basis of the extensive knowledge, the updated progress and the published consensuses and guidelines worldwide by expert discussion organized by Rare Diseases Group of Pediatric Branch of Chinese Medical Association, Medical Genetics Branch of Chinese Medical Doctor Association, Birth Defect Prevention and Molecular Genetics Branch of China Maternal and Child Health Association. and Molecular Diagnosis Branch of Shanghai Medical Association.

CRISPR-Mediated In Situ Introduction or Integration of F9-Padua in Human iPSCs for Gene Therapy of Hemophilia B.

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.

Noninvasive prenatal testing, ultrasonographic findings and poor prenatal diagnosis rates for twin pregnancies: a retrospective study.

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.