Viruses can escape from host recognition by degradation of RIG-I or interference with the RIG-I signalling to establish persistent infections. However, the mechanisms by which host cells stabilize RIG-I protein for avoiding its degradation are largely unknown. We report here that, upon virus infection, the E3 ubiquitin ligase FBXW7 translocates from the nucleus into the cytoplasm and stabilizes RIG-I. FBXW7 interacts with SHP2 and mediates the degradation and ubiquitination of SHP2, thus disrupting the SHP2/c-Cbl complex, which mediates RIG-I degradation. When infected with VSV or influenza A virus, FBXW7 conditional knockout mice (Lysm+FBXW7f/f) show impaired antiviral immunity. FBXW7-deficient macrophages have decreased RIG-I protein levels and type-I interferon signalling. Furthermore, PBMCs from RSV-infected children have reduced FBXW7 mRNA levels. Our results identify FBXW7 as an important interacting partner for RIG-I. These findings provide insights into the function of FBXW7 in antiviral immunity and its related clinical significance.
High-throughput ligation-dependent probe amplification is a rapid and accurate method for aneuploidy detection. It can be used as a cost-effective screening procedure in clinical spontaneous abortions. © 2016 John Wiley & Sons, Ltd.
Primary ovarian insufficiency (POI) leads to infertility and premature menopause in young women. The genetic etiology of this disorder remains unknown in most patients. Using whole exome sequencing of a large Chinese POI pedigree, we identified a heterozygous 5 bp deletion inducing a frameshift in BNC1, which is predicted to result in a non-sense-mediated decay or a truncated BNC1 protein. Sanger sequencing identified another BNC1 missense mutation in 4 of 82 idiopathic patients with POI, and the mutation was absent in 332 healthy controls. Transfection of recombinant plasmids with the frameshift mutant and separately with the missense mutant in HEK293T cells led to abnormal nuclear localization. Knockdown of BNC1 was found to reduce BMP15 and p-AKT levels and to inhibit meiosis in oocytes. A female mouse model of the human Bnc1 frameshift mutation exhibited infertility, significantly increased serum follicle-stimulating hormone, decreased ovary size and reduced follicle numbers, consistent with POI. We report haploinsufficiency of BNC1 as an etiology of human autosomal dominant POI.
Objective To evaluate the usefulness and incremental diagnostic yield of chromosomal microarray analysis (CMA) compared with standard karyotyping in fetuses with congenital anomalies of the kidney and urinary tract (CAKUT). Methods A prospective cohort study and systematic review of the literature were conducted. In the prospective cohort study, 123 fetuses with CAKUT, as detected by prenatal ultrasound at our center, were enrolled and evaluated using karyotyping and CMA. In the meta‐analysis, articles in PubMed and ISI Web of Knowledge databases describing copy number variations (CNVs) in prenatal cases of CAKUT were included. Results Among the 123 fetuses in our prospective cohort study, 13 fetuses were detected with chromosomal abnormalities or submicroscopic chromosomal abnormalities by both karyotyping and CMA. In the remaining 110 fetuses, four pathogenic CNVs in four fetuses were only detected by CMA, indicating an excess diagnostic yield of 3.6%. Six publications and our own study met the inclusion criteria for the meta‐analysis. In total, 615 fetuses with CAKUT were included. The pooled data from all of the reviewed studies indicate that the incremental yield of CMA over karyotyping was 3.8%. Conclusion The use of CMA provides a 3.8% incremental yield of detecting pathogenic CNVs in fetuses with CAKUT and normal karyotype.
This study investigated the association between premature ovarian failure (POF), MTHFR C677T/A1298C and MTRR A66G genotypes and serum homocysteine (Hcy) concentration. A prospective study was conducted in Chinese women, which included POF patients (n = 180) and controls (n = 195). Peripheral blood samples were used to determine MTHFR C677T/A1298C and MTRR A66G genotypes, and serum Hcy and sex hormone concentrations. Results showed that serum Hcy concentrations of POF patients were significantly higher than those of controls (P < 0.0001). In POF patients, serum Hcy concentrations were significantly correlated with oestradiol and FSH concentrations (r = -0.174, P = 0.037 and r = +0.238, P = 0.006, respectively). There were no significant differences in the distributions of MTHFR C677T/A1298C or MTRR A66G genotypes between the two groups. However, these genetic variants influenced serum Hcy concentrations in POF patients, especially for MTRR 66 AA/AG/GG genotypes, which were significantly correlated with the patients' Hcy concentrations (τ = 0.166, P = 0.033). These results suggest that serum Hcy concentrations in Chinese POF patients are increased and correlated with serum oestradiol/FSH concentrations. In conclusion, MTHFR C667T/A1298C and MTRR A66G genotypes are not associated with POF development, but they affect the patients' serum Hcy concentrations.
Targeted, capture-based next-generation sequencing for identification of informative markers together with Sanger sequencing is an easy and efficient method for the PGD of monogenic diseases such as MEN2.
Non-invasive prenatal testing (NIPT) for common fetal trisomies is effective. However, the usefulness of cell-free DNA testing to detect other chromosomal abnormalities is poorly understood. We analyzed the positive rate at different read depths in next-generation sequencing (NGS) and identified a strategy for fetal copy number variant (CNV) detection in NIPT. Pregnant women who underwent NIPT by NGS at read depths of 4–6 M and fetuses with suspected CNVs were analyzed by amniocentesis and chromosomal microarray analysis (CMA). These fetus samples were re-sequenced at a read depth of 25 M and the positive detection rate was determined. With the increase in read depth, the positive CNV detection rate increased. The positive CNV detection rates at 25 M with small fragments were higher by NGS than by karyotype analysis. Increasing read depth in NGS improves the positive CNV detection rate while lowering the false positive detection rate. NIPT by NGS may be an accurate method of fetal chromosome analysis and reduce the rate of birth defects.
STUDY QUESTION Can whole genome sequencing (WGS) offer a relatively cost-effective approach for embryonic genome-wide haplotyping and preimplantation genetic testing (PGT) for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR)? SUMMARY ANSWER Reliable genome-wide haplotyping, PGT-M, PGT-A and PGT-SR could be performed by WGS with 10× depth of parental and 4× depth of embryonic sequencing data. WHAT IS KNOWN ALREADY Reduced representation genome sequencing with a genome-wide next-generation sequencing haplarithmisis-based solution has been verified as a generic approach for automated haplotyping and comprehensive PGT. Several low-depth massively parallel sequencing (MPS)-based methods for haplotyping and comprehensive PGT have been developed. However, an additional family member, such as a sibling, or a proband, is required for PGT-M haplotyping using low-depth MPS methods. STUDY DESIGN, SIZE, DURATION In this study, 10 families that had undergone traditional IVF-PGT and 53 embryos, including 13 embryos from two PGT-SR families and 40 embryos from eight PGT-M families, were included to evaluate a WGS-based method. There were 24 blastomeres and 29 blastocysts in total. All embryos were used for PGT-A. Karyomapping validated the WGS results. Clinical outcomes of the 10 families were evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS A blastomere or a few trophectoderm cells from the blastocyst were biopsied, and multiple displacement amplification (MDA) was performed. MDA DNA and bulk DNA of family members were used for library construction. Libraries were sequenced, and data analysis, including haplotype inheritance deduction for PGT-M and PGT-SR and read-count analysis for PGT-A, was performed using an in-house pipeline. Haplotyping with a proband and parent-only haplotyping without additional family members were performed to assess the WGS methodology. Concordance analysis between the WGS results and traditional PGT methods was performed. MAIN RESULTS AND THE ROLE OF CHANCE For the 40 PGT-M and 53 PGT-A embryos, 100% concordance between the WGS and single-nucleotide polymorphism (SNP)-array results was observed, regardless of whether additional family members or a proband was included for PGT-M haplotyping. For the 13 embryos from the two PGT-SR families, the embryonic balanced translocation was detected and 100% concordance between WGS and MicroSeq with PCR-seq was demonstrated. LIMITATIONS, REASONS FOR CAUTION The number of samples in this study was limited. In some cases, the reference embryo for PGT-M or PGT-SR parent-only haplotyping was not available owing to failed direct genotyping. WIDER IMPLICATIONS OF THE FINDINGS WGS-based PGT-A, PGT-M and PGT-SR offered a comprehensive PGT approach for haplotyping without the requirement for additional family members. It provided an improved complementary method to PGT methodologies, such as low-depth MPS- and SNP array-based methods. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the research grant from the National Key R&D Program of China (2018YFC0910201 and 2018YFC1004900), the Guangdong province science and technology project of China (2019B020226001), the Shenzhen Birth Defect Screening Project Lab (JZF No. [2016] 750) and the Shenzhen Municipal Government of China (JCYJ20170412152854656). This work was also supported by the National Natural Science Foundation of China (81771638, 81901495 and 81971344), the National Key R&D Program of China (2018YFC1004901 and 2016YFC0905103), the Shanghai Sailing Program (18YF1424800), the Shanghai Municipal Commission of Science and Technology Program (15411964000) and the Shanghai ‘Rising Stars of Medical Talent’ Youth Development Program Clinical Laboratory Practitioners Program (201972). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.
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