Identification of a Homozygous Mutation of CCDC40 in a Chinese Infertile Man with MMAF and PCD-like Phenotypes.

Aims: Asthenozoospermia is the most common factor of male infertility, mainly caused by multiple morphological abnormalities of the sperm flagella (MMAF) and primary ciliary dyskinesia (PCD). Previous studies have shown that genetic factors may contribute to MMAF and PCD. The study aimed to identify novel potentially pathogenic gene mutations in a Chinese infertile man with MMAF and PCD-like phenotypes. Methods: A Chinese infertile man with MMAF and PCD was enrolled in this study. Whole exome sequencing and Sanger sequencing were performed to identify potential causative genes and mutations. Results: A novel homozygous missense mutation (c.1450G>A; p.E484K) of CCDC40 was finally identified and Sanger sequencing confirmed that the patient carried the homozygous mutation, which was inherited from his parents. We reported the first homozygous missense CCDC40 mutation in infertile men with MMAF but had other milder PCD symptoms. Conclusion: Our findings not only broaden the disease-causing mutation spectrum of CCDC40 but also provide new insight into the correlation between CCDC40 mutations and MMAF.

HBV integrations reshaping genomic structures promote hepatocellular carcinoma.

OBJECTIVE: Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), mostly characterised by HBV integrations, is prevalent worldwide. Previous HBV studies mainly focused on a few hotspot integrations. However, the oncogenic role of the other HBV integrations remains unclear. This study aimed to elucidate HBV integration-induced tumourigenesis further. DESIGN: Here, we illuminated the genomic structures encompassing HBV integrations in 124 HCCs across ages using whole genome sequencing and Nanopore long reads. We classified a repertoire of integration patterns featured by complex genomic rearrangement. We also conducted a clustered regularly interspaced short palindromic repeat (CRISPR)-based gain-of-function genetic screen in mouse hepatocytes. We individually activated each candidate gene in the mouse model to uncover HBV integration-mediated oncogenic aberration that elicits tumourigenesis in mice. RESULTS: These HBV-mediated rearrangements are significantly enriched in a bridge-fusion-bridge pattern and interchromosomal translocations, and frequently led to a wide range of aberrations including driver copy number variations in chr 4q, 5p (TERT), 6q, 8p, 16q, 9p (CDKN2A/B), 17p (TP53) and 13q (RB1), and particularly, ultra-early amplifications in chr8q. Integrated HBV frequently contains complex structures correlated with the translocation distance. Paired breakpoints within each integration event usually exhibit different microhomology, likely mediated by different DNA repair mechanisms. HBV-mediated rearrangements significantly correlated with young age, higher HBV DNA level and TP53 mutations but were less prevalent in the patients subjected to prior antiviral therapies. Finally, we recapitulated the TONSL and TMEM65 amplification in chr8q led by HBV integration using CRISPR/Cas9 editing and demonstrated their tumourigenic potentials. CONCLUSION: HBV integrations extensively reshape genomic structures and promote hepatocarcinogenesis (graphical abstract), which may occur early in a patient's life.

A novel missense variant in LAMC1 identified in a POI family by whole exome sequencing.

OBJECTIVE: This study aimed to identify novel pathogenic genes and variants in a Chinese family with premature ovarian insufficiency (POI). METHODS: A Chinese POI family was enrolled in this study. Whole exome sequencing was performed on the proband and her mother to identify the potential causative genes and variants and Sanger sequencing was used to confirm the finally identified potential pathogenic variant in the family. RESULTS: An assessment of the family pedigree suggested that POI was inherited in an autosomal dominant manner in this family. A novel missense variant of the laminin subunit gamma-1 gene (LAMC1; NM_002293.4: c.3281A > T, p.D1094V) was finally identified in the proband and her affected mother. This variant was not found in any public databases. In silico analysis indicated the amino acid encoded at the variant site was highly conserved among mammals and associated with decreased protein stability and disrupted protein function. Its presence in the POI family was confirmed by Sanger sequencing. CONCLUSIONS: This study firstly reported a novel missense variant of LAMC1 in a Chinese POI family, which was inherited in an autosomal dominant manner. This variant may result in the development of POI. Our results provide supporting evidence for a causative role for LAMC1 variants in POI.

Molecular diagnosis of pancreatobiliary tract cancer by detecting mutations and methylation changes in bile samples.

Background: Patients with pancreatobiliary tract cancer usually have a poor clinical outcome, with a 5-year overall survival rate below 20%. This is mainly associated with the late diagnosis. In addition, the standard-of-care for patients with malignant biliary stenosis involves a major surgery, the Whipple procedure. An accurate preoperative diagnosis, including differentiation from benign diseases, is critical to avoid unnecessary treatment. Here we developed BileScreen, a sensitive detection modality for the diagnosis of pancreatobiliary tract cancer based on massively parallel sequencing mutation and methylation changes in bile samples. Methods: A total of 338 patients, from five hospitals in China, with pancreatobiliary system disorders were enrolled in this study between November 2018 and October 2020, and 259 were included for the analysis of BileScreen. We profiled 23 gene mutations and 44 genes with methylation modifications in parallel from bile samples, and set up a model for the detection of malignancy based on multi-level biomarkers. Findings: We applied the BileScreen assay in a training cohort (n = 104) to set up the model and algorithm. The model was further evaluated in a validation cohort (n = 105), resulting in 92% sensitivity and 98% specificity. The performance of BileScreen was further assessed in a prospective test cohort (n = 50) of patients diagnosed with suspicious or negative pathology by endoscopic retrograde cholangiopancreatography and were confirmed in follow-up. BileScreen yielded 90% sensitivity and 80% specificity, and outcompeted serum carbohydrate antigen 19-9 in detecting pancreatobiliary tract cancer in all three cohorts, especially in terms of specificity. Interpretation: Taken together, BileScreen has the ability to interrogate mutations and methylation changes in bile samples in parallel, thus rendering it a potentially sensitive detection method to help in the diagnosis of pancreatobiliary tract cancer in a safe, convenient and less-invasive manner. Funding: This study was supported by the Capital's Funds for Health Improvement and Research (2020-2-4025 to S.H.), the National Natural Science Foundation of China (81972311 to H.Z.), CAMS Innovation Fund for Medical Sciences (CIFMS) (2017-12M-4-002 to H.Z.), the CAMS Innovation Fund for Medical Sciences(CIFMS) (2021-I2M-1-066 to CJQ), the Non-profit Central Research Institution Fund of Chinese Academy of Medical Sciences (2019PT310026 to H.Z.) and Sanming Project of Medicine in Shenzhen (SZSM202011010 to H.Z.).

A draft genome of the medicinal plant Cremastra appendiculata (D. Don) provides insights into the colchicine biosynthetic pathway.

Cremastra appendiculata (D. Don) Makino is a rare terrestrial orchid with a high market value as an ornamental and Chinese traditional medicinal herb with a wide range of pharmacological properties. The pseudobulbs of C. appendiculata are one of the primary sources of the famous traditional Chinese medicine "Shancigu", which has been clinically used for treating many diseases, especially, as the main component to treat gout. The lack of genetic research and genome data restricts the modern development and clinical use of C. appendiculata. Here, we report a 2.3 Gb chromosome-level genome of C. appendiculata. We identify a series of candidates of 35 candidate genes responsible for colchicine biosynthesis, among which O-methyltransferase (OMT) gene exhibits an important role in colchicine biosynthesis. Co-expression analysis reveal purple and green-yellow module have close relationships with pseudobulb parts and comprise most of the colchicine pathway genes. Overall, our genome data and the candidate genes reported here set the foundation to decipher the colchicine biosynthesis pathways in medicinal plants.

Novel compound heterozygous variants of DNAH17 in a Chinese infertile man with multiple morphological abnormalities of sperm flagella.

Multiple morphological abnormalities of the sperm flagellum (MMAF) have been reported to be an important cause of male infertility and reflect a heterogeneous genetic disorder. Previous studies have identified dozens of candidate pathogenic genes for MMAF, but the aetiology in approximately 50% of cases remains unexplained. The present study aimed to identify novel potentially pathogenic gene variants of MMAF. A Chinese family with a 32-year-old infertile proband presenting with MMAF was recruited, and sperm morphology of the patient was examined by Papanicolaou staining. Whole exome sequencing was performed on the proband and Sanger sequencing was used to identify genetic variants in the family. The frequencies of variants were assessed using public databases and the effects on protein structure and function were predicted by online bioinformatics tools. The patient exhibited asthenozoospermia and a MMAF phenotype. Novel compound heterozygous variants (c.5368C > T, p.R1790C and c.13183C > T, p.R4395W) of the DNAH17 gene were identified in the patient, and showed autosomal recessive inheritance in this family. These variants were very rare in the GnomAD database. The two mutated amino acids were located in a highly conserved region of the DNAH17 protein. In silico analysis revealed that the compound heterozygous variants may compromise the function of DNAH17. Our findings expand upon the spectrum of pathogenic DNAH17 variants that are responsible for MMAF, and provide new knowledge for genetic counselling of male infertility due to MMAF.

Case Report: Tetralogy of Fallot in a Chinese Family Caused by a Novel Missense Variant of MYOM2.

Background: Rare genetic variants have been identified to be important contributors to the risk of Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease (CHD). But relatively limited familial studies with small numbers of TOF cases have been reported to date. In this study, we aimed to identify novel pathogenic genes and variants that caused TOF in a Chinese family using whole exome sequencing (WES). Methods: A Chinese family whose twins were affected by TOF were recruited for this study. A WES was performed for the affected twins, their healthy brother, and parents to identify the potential pathogenic mutated gene(s). Heterozygous variants carried by the twins, but not the unaffected brother, were retained. Public databases were used to assess the frequencies of the selected variants, and online prediction tools were accessed to predict the influences of these variants on protein function. The final candidate variant was further confirmed by Sanger sequencing in other members of the family. Results: After several filtering processes, a heterozygous missense variant in the MYOM2 gene (NM_003970.4:c.3097C>T:p.R1033C) was identified and confirmed by Sanger sequencing in the affected twins and their unaffected father, suggesting an inheritance pattern with incomplete penetrance. The variant was found to be extremely rare in the public databases. Furthermore, the mutated site was highly conserved among mammals, and as shown using multiple online prediction tools, this variant was predicted to be a detrimental variant. Conclusion: We assessed a family with TOF caused by a rare heterozygous missense variant of MYOM2. Our findings not only further confirm the significant role of genetics in the incidence of TOF but also expand the spectrum of the gene variants that lead to TOF.

Novel missense variant of CIITA contributing to endometriosis.

RESEARCH QUESTION: What are the novel pathogenic variants leading to endometriosis? DESIGN: A Chinese family, in which three female offspring were diagnosed with ovarian endometriosis by pathological biopsy and the mother had ovarian cysts, were recruited to this study, plus another 111 unrelated endometriosis patients. Whole-exome sequencing was performed on the affected offspring and the parents of the family. Sanger sequencing was used to screen all the coding regions of candidate genes. Scratch wound assays, transwell migration and invasion assays were used to determine whether the mutation could affect cell migration and invasion. RESULTS: A novel missense variant in the CIITA gene (NM_001286402;c.C1949G;p.Ala650Gly) was identified in three affected sisters by exome sequencing, which was inherited from their mother, who had a suspected diagnosis of ovarian endometriosis. This variant was absent from all public databases. Another two rare missense variants of CIITA (c.1031G>T;p.Arg344Leu and c.1535T>C;p.Leu512Pro) were identified in two unrelated endometriosis patients. The scratch wound and transwell assays showed that the mutation c.C1949G;p.Ala650Gly significantly (P = 0.0307; P = 0.0162; P = 0.0117, respectively) affected cell migration and invasion ability. CONCLUSIONS: The present study demonstrates that CIITA variants may contribute to the development of endometriosis.

Identification of KANSL1 as a novel pathogenic gene for developmental dysplasia of the hip.

Developmental dysplasia of the hip (DDH) is a common anomaly leading to adult osteoarthritis. Environmental and genetic factors contribute to DDH, but its exact genetic mechanism is unclear. In this study, we used whole exome sequencing to identify the causative gene of a DDH pedigree. A rare missense variant in KANSL1 (c.C767T; p.S256F) was identified as the pathogenic cause of DDH. Subsequent mutation screening showed another missense variant in 1 of 200 sporadic patients. Kansl1-mutated mice showed reduced chondrocytes in the acetabulum and a decrease in the cartilage matrix, which may be DDH phenotype-related abnormalities. Furthermore, functional studies showed that cell proliferation was delayed and Mmp13 expression was abnormally upregulated in chondrocytes differentiated from Kansl1 mutant mouse embryonic stem cells. In conclusion, our findings suggest that KANSL1 is a novel pathogenic gene for DDH. The identification of KANSL1 variants has great diagnostic value for identifying individuals with DDH. KEY MESSAGES: Developmental dysplasia of the hip (DDH) is a common anomaly causing adult osteoarthritis. Environmental and genetic factors contribute to DDH, but its exact genetic mechanism is unclear. Using high-throughput whole exome sequencing, we found a novel variant in KANSL1 that was co-inherited by all severely affected individuals diagnosed with DDH from a three-generation family. Further analysis revealed that a Kansl1 variant in mice reduced the number of chondrocytes and decreased cartilage matrix, and mouse embryonic stem differentiation assay showed cartilage defects. These findings indicate a direct association between KANSL1 and hip development, expanding the pathogenic gene spectrum in DDH and providing insight into potential new targets for diagnosing and treating hip dysplasia.

Identification of a novel heterozygous SOX9 variant in a Chinese family with congenital heart disease.

BACKGROUND: Previous studies of individuals with hereditary or sporadic congenital heart disease (CHD) have provided strong evidence for a genetic basis for CHD. The aim of this study was to identify novel pathogenic genes and variants in a Chinese CHD family. METHODS: Three generations of a family with CHD were recruited. We performed whole exome sequencing for the affected individuals and the proband's unaffected aunt to investigate the genetic causes of CHD in this family. Heterozygous variants carried by the proband and her maternal grandmother, but not the proband's aunt, were selected. The frequencies of the variants detected were assessed using public databases, and their influences on protein function were predicted using online prediction software. The candidate variant was further confirmed by Sanger sequencing of other members of the family. RESULTS: On the basis of the family's pedigree, the mode of inheritance was speculated to be autosomal dominant with incomplete penetrance. We identified a novel heterozygous missense variant in SOX9 in all affected individuals and one asymptomatic family member, suggesting an inheritance pattern with incomplete penetrance. The variant was not found in any public database. In addition, the variant was highly conserved among mammals, and was predicted to be deleterious by online software programs. CONCLUSIONS: We report for the first time a novel heterozygous missense variant in SOX9 (NM_000346:c.931G>T:p.Gly311Cys) in a Chinese CHD family. Our results provide further evidence supporting a causative role for SOX9 variants in CHD.

Association Between miR-143/145 rs4705343 Polymorphism and Risk of Congenital Heart Disease in a Chinese Tibetan Population.

Objective: Congenital heart disease (CHD) is the most common birth defect worldwide and is caused by both genetic and environmental factors. The microRNA (miR)-143/145 cluster is involved in various biological processes related to cardiovascular development. The functional single nucleotide polymorphism (SNP) rs4705343 of miR-143/145 may influence the expression of these miRNAs. In this study, we aimed to estimate the association between miR-143/145 rs4705343 and the risk of CHD in a Chinese Tibetan population. Methods: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry assays were performed to genotype the miRNA-143/145 rs4705343 SNP in 510 CHD Tibetan patients and 681 unrelated Tibetan healthy controls. The associations between the SNP frequencies and the CHD risk were analyzed by χ2 test/Fisher's test and assessed by odds ratios (ORs) and 95% confidence intervals (95% CIs). Results: We successfully genotyped 1165 subjects with a SNP call rate of 97.8%. Under the allelic model we found that rs4705343 was not associated with the risk of CHD (p = 0.082), but under the recessive model the CC genotype at this locus was associated with a significantly increased risk of CHD compared with the other genotypes (CC vs TT+TC: OR = 1.60, 95% CI = 1.08-2.37, p = 0.017). Conclusion: The present study suggests that the rs4705343 CC genotype of miR-143/145 is associated with CHD risk in a Chinese Tibetan population.

Exome-Sequencing Identifies Novel Genes Associated with Recurrent Pregnancy Loss in a Chinese Cohort.

Recurrent pregnancy loss (RPL) is a common reproductive problem affecting around 5% of couples worldwide. At present, about half of RPL cases remained unexplained. Previous studies have suggested an important role for genetic determinants in the etiology of RPL. Here, we performed whole-exome sequencing (WES) analysis on 100 unrelated Han Chinese women with a history of two or more spontaneous abortions. We identified 6736 rare deleterious nonsynonymous variants across all patients. To focus on possible candidate genes, we generated a list of 95 highly relevant genes that were functionally associated with miscarriage according to human and mouse model studies, and found 35 heterozygous variants of 28 RPL-associated genes in 32 patients. Four genes (FOXA2, FGA, F13A1, and KHDC3L) were identified as being strong candidates. The FOXA2 nonsense variant was for the first time reported here in women with RPL. FOXA2 knockdown in HEK-293T cells significantly diminished the mRNA and protein expression levels of LIF, a pivotal factor for maternal receptivity and blastocyst implantation. The other genes, with 29 variants, were involved in angiogenesis, the immune response and inflammation, cell growth and proliferation, which are functionally important processes for implantation and pregnancy. Our study identified several potential causal genetic variants in women with RPL by WES, highlighting the important role of genes controlling coagulation, confirming the pathogenic role of KHDC3L and identifying FOXA2 as a newly identified causal gene in women with RPL.

Analysis of copy number variations of WNT4 gene in a Chinese population with Müllerian anomalies.

BACKGROUND: To investigate the genetic contribution of copy number variations (CNVs) in Wingless-type MMTV integration site family, member 4 (WNT4), in a Chinese population with Müllerian anomalies (MA), copy number analysis of WNT4 by Multiplex ligation-dependent probe amplification (MLPA) was performed on 248 female patients. Some studies have shown that heterozygous missense mutation of WNT4 can lead to MA. However, few studies on the relationship between WNT4 CNVs and MA have been performed. RESULTS: Among the 248 Chinese women affected by MA in this study, heterozygous deletion of WNT4 was detected in a single patient. CONCLUSIONS: MLPA identified one heterozygous deletion in WNT4 in a single female patient among 248 Chinese women affected by MA. This study firstly reports CNVs of WNT4 in a large sample of MA patients from the Chinese population, which suggests that CNVs of WNT4 cannot be excluded in the occurrence of MA. This provides a genetic basis for precise treatment in the future.

Prognostic factors for overall survival in patients with primary duodenal adenocarcinoma.

BACKGROUND: The complete resection of primary duodenal adenocarcinoma (PDA) offers a chance for a cure, but the clinical and pathological characteristics of survivors have not been well studied. METHODS: Patients with stage I-III PDA who underwent surgical resection between 2013 and 2018 were identified retrospectively and followed until December 2019. All patients are from the Cancer Hospital Chinese Academy of Medical Sciences. The clinical and pathological information of the patients, such as age, gender, tumor location, operative procedure, pathologic features, TNM stage, common presenting symptoms, lymph node dissection status, serum tumor markers, etc., was collected in detail. The KaplanMeier method and a Cox proportional hazards model were used for the survival analysis. RESULTS: In total, 85 patients with PDA were eligible for this study. Among these patients, 48 were male (56.5%), 37 were female (43.5%), the median age was 59 (range, 22-79) years, 44 (51.8%) patients were aged <60 years, and 41 (48.2%) patients were aged ≥60 years. The 1-, 3-, and 5-year survival rates were 93.7%, 79.4%, and 64.9%, respectively. The median overall survival (OS) was 27 months (range, 2-82 months), and the median follow-up was 27 months (range, 3-82 months). The patients with stage III disease had the worst prognosis (P=0.001). The univariate analysis showed that lymph node positivity (P=0.000), the N stage (P=0.000), the TNM stage (P=0.001) and carbohydrate antigen 19-9 (CA19-9) positivity (P=0.038) were related to OS. However, the total number of lymph nodes (LN) retrieved (P=0.723), tumor differentiation (P=0.136), carcinoembryonic antigen (CEA) (P=0.812), gender (P=0.477), operation type (P=0.860), tumor size (P=0.869), tumor site (P=0.120), age (P=0.733), intraoperative blood loss (P=0.660), and intraoperative blood transfusion (P=0.748) were not correlated with OS. The multivariate analysis suggested that the lymph node status was an independent prognostic risk factor for OS. CONCLUSIONS: In our study the median OS was 27 months (range, 2-82 months), and the 5-year survival rates was 64.9%. The lymph node status was the only prognostic factor for OS in PDA.

Analysis of CNVs of CFTR gene in Chinese Han population with CBAVD.

BACKGROUND: Congenital bilateral absence of vas deferens (CBAVD) is an important disease of male infertility, which affects 1%-2% of infertile population. In addition to common mutations of CFTR, copy number variants (CNVs) have also been implicated as one of the pathogenesis of CBAVD. The present study aimed to investigate the genetic contribution of CFTR CNVs in Chinese Han population with CBAVD. METHODS: Two hundred and sixty-three CBAVD patients were recruited. Genomic DNA was extracted from peripheral blood samples. The Multiplex Ligation-dependent Probe Amplification assay was performed which targets the region of the CFTR gene. RESULTS: Among 263 Chinese men affected with CBAVD in this study, 5 (1.90%) patients were detected for copy number variants in the region of CFTR gene (4 of them carried partial deletions and 1 of them carried partial duplication of CFTR gene). CONCLUSIONS: The study showed that the rate of CFTR CNVs in Chinese population with CBAVD were basically consistent with the previous reports. And the study first revealed genetic risk of CNVs of CFTR on a large sample size of CBAVD patients in Chinese Han population, which prompted that it was necessary to detect CNVs of CFTR in Chinese Han people with CBAVD.

Serum Metabolic Profiling Analysis of Gout Patients Treated with Traditional Chinese Medicine Tongfengtai Granules Based on Gas Chromatography-Mass Spectrometry.

Gout has become a public health problem that seriously threatens human health. Traditional Chinese medicines (TCMs) have a long history of treating gout and have some advantages compared with the conventional medicines. Compound TCM Tongfengtai granules are gradually being used for clinical treatment of gout, but its mechanism is still unclear. The purpose of this study was to explore the metabolic profiling of serum from gout patients before and after treatment with Tongfengtai granules and identify the differential metabolites and related metabolic pathways. A total of 40 gout patients hospitalized in Shenzhen Traditional Chinese Medicine Hospital from 2018 to March 2019 were recruited in the current study, and serum samples from these patients before and after treatment with Tongfengtai granules were collected. Gas chromatography-mass spectrometry (GC-MS) assay was used to identify serum metabolites. The OPLS-DA VIP method was used to screen for potential metabolic biomarkers, and MetaboAnalyst 4.0 was used to identify related metabolic pathways. The result showed that there was a significant difference in the concentrations of six metabolites in the serum after treatment: D-galactose, lactic acid, 3-hydroxybutyric acid, D-pyran (type) glucose, alanine, and L-isoleucine. Except D-pyran (type) glucose, the serum concentrations of the other five metabolites were all significantly reduced. Besides, pathway enrichment analysis found that these potential metabolic biomarkers were mainly involved in lactose degradation and the glucose-alanine cycle. Thus, the serum metabolic profiling of gout patients treated with Tongfengtai granules changed, and the differential metabolites and related metabolic pathways might provide clues for understanding the mechanism of Tongfengtai granules.

New Gene Variants Associated with the Risk of Chronic HBV Infection.

Some patients with chronic hepatitis B virus (HBV) infection failed to clear HBV, even persistently continue to produce antibodies to HBV. Here we performed a two stage genome wide association study in a cohort of Chinese patients designed to discover single nucleotide variants that associate with HBV infection and clearance of HBV. The first stage involved genome wide exome sequencing of 101 cases (HBsAg plus anti-HBs positive) compared with 102 control patients (anti-HBs positive, HBsAg negative). Over 80% of individual sequences displayed 20 × sequence coverage. Adapters, uncertain bases > 10% or low-quality base calls (> 50%) were filtered and compared to the human reference genome hg19. In the second stage, 579 chronic HBV infected cases and 439 HBV clearance controls were sequenced with selected genes from the first stage. Although there were no significant associated gene variants in the first stage, two significant gene associations were discovered when the two stages were assessed in a combined analysis. One association showed rs506121-"T" allele [within the dedicator of cytokinesis 8 (DOCK8) gene] was higher in chronic HBV infection group than that in clearance group (P = 0.002, OR = 0.77, 95% CI [0.65, 0.91]). The second association involved rs2071676-A allele within the Carbonic anhydrase (CA9) gene that was significantly elevated in chronic HBV infection group compared to the clearance group (P = 0.0003, OR = 1.35, 95% CI [1.15, 1.58]). Upon replication these gene associations would suggest the influence of DOCK8 and CA9 as potential risk genetic factors in the persistence of HBV infection.

NOTCH2 variant D1853H is mutated in two non-syndromic premature ovarian insufficiency patients from a Chinese pedigree.

BACKGROUND: Premature ovarian insufficiency (POI) is a severe disorder of female infertility, characterized by 4-6 months of amenorrhea before the age of 40 years, with elevated follicle stimulating hormone (FSH) levels (> 25 IU/L). Although several genes have been reported to contribute to the genetic basis of POI, the molecular mechanism of POI remains unclear. METHODS: Whole-exome sequencing (WES) was performed. Sanger sequencing was carried out to validate the variant in the proband and her mother. In silico algorithms were used to analyze the mutational effect of the variant. Protein 3D structural modeling was used for predicting mutated protein structures. Vector construction and plasmids transfection were performed, and subsequently RNA-sequencing (RNA-seq) was carried out in each group to dissect the differentially expressed genes in wild-type (WT) and D1853H NOTCH2 mutant expressing groups. Gene Ontology analysis was also used to analyze the enriched biological processes or pathways among the differentially expressed genes. RESULTS: We report two non-syndromic POI patients from a Chinese pedigree. The FSH level of the proband (the daughter) was 46 IU/L at the age of 22. Her menarche was at the age of 12, but she was amenorrhea at the age of 20. By WES, a rare heterozygous variant (c.5557G > C;p.D1853H) in the NOTCH2 gene was identified. In silico analysis suggested that p.D1853H was a pathogenic allele. Protein 3D structural modeling suggested that D1853H may enhance or weaken the electrostatic surface potential. By molecular analysis, we found that cells expressing the D1853H NOTCH2 mutant had similar effect in activating the NOTCH signaling pathway downstream target genes. However, 106 protein-coding genes were differentially expressed between D1853H expressing cells and WT NOTCH2 expressing cells, and these genes were enriched for collagen degradation, NCAM1 interactions and HDACs deacetylate histones, revealing a unknown underlying mechanism of the pathology that leads to POI. CONCLUSIONS: We conclude that the rare heterozygous variant in NOTCH2 may be associated with POI. This finding provides researchers and clinicians with a better understanding of the etiology, molecular mechanism and genetic consulting of POI.