Molecular characterization of DNAH6 and ATPase6 (Mitochondrial DNA) genes in asthenozoospermia patients in the northern region of India.

BACKGROUND: Male infertility due to spermatogenesis defects affects millions of men worldwide. However, the genetic etiology of the vast majority remains unclear. The present study was undertaken to assess the association of DNAH6 and ATPase6 genes in asthenozoospermia patients in the northern region of India. METHODS: A total of 60 semen samples were collected for the study, of which 30 were from the case group and 30 were from the control group. The semen samples for the case group (asthenozoospermia) and control groups were collected from IVF and Reproductive Biology Centre, Maulana Azad Medical College, New Delhi. Sperm count and motility were classified as per World Health Organization (WHO 2021) protocol. A total genomic DNA was extracted as per the stranded TRIZOL method with little modification. RESULTS: In-vitro molecular characterizations of DNAH6 and ATPase6 genes in both groups were checked by Polymerase Chain Reaction (PCR). The 675 bp and 375 bp amplicons were amplified using PCR for ATPase6 and DNAH6 genes. Our study results showed a significant (P ≤ 0.05) null deletion of DNAH6 and ATPase6 genes in asthenozoospermia patients as compared to the control. We found the significant null deletion of DNAH6 in case 45.0%, and the control group was 11.7%. However, in the case of APTase6, it was 26.7% and 10.0%, respectively. CONCLUSIONS: Our study concluded that the presence of DHAH6 and ATPase6 genes had a significant impact on male infertility.

Human Genetics of Defects of Situs.

Defects of situs are associated with complex sets of congenital heart defects in which the normal concordance of asymmetric thoracic and abdominal organs is disturbed. The cellular and molecular mechanisms underlying the formation of the embryonic left-right axis have been investigated extensively in the past decade. This has led to the identification of mutations in at least 33 different genes in humans with heterotaxy and situs defects. Those mutations affect a broad range of molecular components, from transcription factors, signaling molecules, and chromatin modifiers to ciliary proteins. A substantial overlap of these genes is observed with genes associated with other congenital heart diseases such as tetralogy of Fallot and double-outlet right ventricle, d-transposition of the great arteries, and atrioventricular septal defects. In this chapter, we present the broad genetic heterogeneity of situs defects including recent human genomics efforts.

A novel frameshift mutation in DNAH6 associated with male infertility and asthenoteratozoospermia.

Introduction: Asthenoteratozoospermia is one of the most common causes of male infertility. Several genes have been identified as genetic causative factors, but there is a considerable genetic heterogeneity underlying asthenoteratozoospermia. In this study, we performed a genetic analysis of two brothers from a consanguineous Uighur family in China to identify gene mutations causative for asthenoteratozoospermia-related male infertility. Methods: Two related patients with asthenoteratozoospermia from a large consanguineous family were sequenced by whole-exome sequencing and Sanger sequencing to identify disease-causing genes. Scanning and transmission electron microscopy analysis revealed ultrastructural abnormalities of spermatozoa. Quantitative real-time PCR (qRT-PCR) analysis and immunofluorescence (IF) analysis were used to assess the expression of the mutant messenger RNA (mRNA) and protein. Results: A novel homozygous frameshift mutation (c.2823dupT, p.Val942Cysfs*21) in DNAH6 was identified in both affected individuals and was predicted to be pathogenic. Papanicolaou staining and electron microscopy revealed multiple morphological and ultrastructural abnormalities of affected spermatozoa. qRT-PCR and IF analysis showed abnormal expression of DNAH6 in affected sperm, probably due to premature termination code and decay of abnormal 3' untranslated region (UTR) region of mRNA. Furthermore, intracytoplasmic sperm injection could achieve successful fertilization in infertile men with DNAH6 mutations. Discussion: The novel frameshift mutation identified in DNAH6 may contribute to asthenoteratozoospermia. These findings expand the spectrum of genetic mutations and phenotypes associated with asthenoteratozoospermia and may be useful for genetic and reproductive counseling in male infertility.

Identification of Novel Genetic Risk Factors for Focal Segmental Glomerulosclerosis in Children: Results From the Chronic Kidney Disease in Children (CKiD) Cohort.

RATIONALE & OBJECTIVE: Focal segmental glomerulosclerosis (FSGS) is a major cause of pediatric nephrotic syndrome, and African Americans exhibit an increased risk for developing FSGS compared with other populations. Predisposing genetic factors have previously been described in adults. Here we performed genomic screening of primary FSGS in a pediatric African American population. STUDY DESIGN: Prospective cohort with case-control genetic association study design. SETTING & PARTICIPANTS: 140 African American children with chronic kidney disease from the Chronic Kidney Disease in Children (CKiD) cohort, including 32 cases with FSGS. PREDICTORS: Over 680,000 common single-nucleotide polymorphisms (SNPs) were tested for association. We also ran a pathway enrichment analysis and a human leucocyte antigen (HLA)-focused association study. OUTCOME: Primary biopsy-proven pediatric FSGS. ANALYTICAL APPROACH: Multivariate logistic regression models. RESULTS: The genome-wide association study revealed 169 SNPs from 14 independent loci significantly associated with FSGS (false discovery rate [FDR]<5%). We observed notable signals for genetic variants within the APOL1 (P=8.6×10-7; OR, 25.8 [95% CI, 7.1-94.0]), ALMS1 (P=1.3×10-7; 13.0% in FSGS cases vs 0% in controls), and FGFR4 (P=4.3×10-6; OR, 24.8 [95% CI, 6.3-97.7]) genes, all of which had previously been associated with adult FSGS, kidney function, or chronic kidney disease. We also highlighted novel, functionally relevant genes, including GRB2 (which encodes a slit diaphragm protein promoting podocyte structure through actin polymerization) and ITGB1 (which is linked to renal injuries). Our results suggest a major role for immune responses and antigen presentation in pediatric FSGS through (1) associations with SNPs in PTPRJ (or CD148, P=3.5×10-7), which plays a role in T-cell receptor signaling, (2) HLA-DRB1∗11:01 association (P=6.1×10-3; OR, 4.5 [95% CI, 1.5-13.0]), and (3) signaling pathway enrichment (P=1.3×10-6). LIMITATIONS: Sample size and no independent replication cohort with genomic data readily available. CONCLUSIONS: Our genetic study has identified functionally relevant risk factors and the importance of immune regulation for pediatric primary FSGS, which contributes to a better description of its molecular pathophysiological mechanisms. PLAIN-LANGUAGE SUMMARY: We assessed the genetic risk factors for primary focal segmental glomerulosclerosis (FSGS) by simultaneously testing over 680,000 genetic markers spread across the genome in 140 children, including 32 with FSGS lesions. Fourteen independent genetic regions were significantly associated with pediatric FSGS, including APOL1 and ALMS1-NAT8, which were previously found to be associated with FSGS and chronic kidney diseases in adults. Novel genes with relevant biological functions were also highlighted, such as GRB2 and FGFR4, which play a role in the kidney filtration barrier and in kidney cell differentiation, respectively. Finally, we revealed the importance of immune regulation in pediatric FSGS through associations involving cell surface proteins presenting antigens to the immune system and interacting with T-cell receptors.

DNAH6 is a novel candidate gene associated with sperm head anomaly.

Globozoospermia and acephalic spermatozoa are two rare sperm head anomalies associated with male infertility. Combination of the two phenotypes in the same patient is extremely rare, so the underlying pathogenesis of this disorder remains unclear. Here, we report a 35-year-old infertile male, who presented with 30% of sperm-lacked heads and 69% of sperm round-headed or small-headed with neck thickening in his ejaculate. Subsequent whole-exome sequencing (WES) analysis identified compound heterozygous variants within the DNAH6 gene. DNAH6 is a testis-specific-expressed protein that was localised to the neck region in the spermatozoa of normal control; however, immunofluorescent staining failed to detect DNAH6 protein in the patient's spermatozoa. Quantitative real-time PCR analysis also showed the complete absence of DNAH6 mRNA in the patient's spermatozoa. Moreover, two cycles of in vitro fertilisation (IVF)-assisted reproduction were carried out, but pregnancy was not achieved after embryo transfer. Therefore, rare sequence variants in DNAH6 might be susceptibility risks for human sperm head anomaly.

Identification of a duplication within the GDF9 gene and novel candidate genes for primary ovarian insufficiency (POI) by a customized high-resolution array comparative genomic hybridization platform.

STUDY QUESTION: Can high-resolution array comparative genomic hybridization (CGH) analysis of DNA samples from women with primary ovarian insufficiency (POI) improve the diagnosis of the condition and identify novel candidate genes for POI? SUMMARY ANSWER: A mutation affecting the regulatory region of growth differentiation factor 9 (GDF9) was identified for the first time together with several novel candidate genes for POI. WHAT IS KNOWN ALREADY: Most patients with POI do not receive a molecular diagnosis despite a significant genetic component in the pathogenesis. STUDY DESIGN, SIZE, DURATION: We performed a case-control study. Twenty-six patients were analyzed by array CGH for identification of copy number variants. Novel changes were investigated in 95 controls and in a separate population of 28 additional patients with POI. The experimental procedures were performed during a 1-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA samples from 26 patients with POI were analyzed by a customized 1M array-CGH platform with whole genome coverage and probe enrichment targeting 78 genes in sex development. By PCR amplification and sequencing, the breakpoint of an identified partial GDF9 gene duplication was characterized. A multiplex ligation-dependent probe amplification (MLPA) probe set for specific identification of deletions/duplications affecting GDF9 was developed. An MLPA probe set for the identification of additional cases or controls carrying novel candidate regions identified by array-CGH was developed. Sequencing of three candidate genes was performed. MAIN RESULTS AND THE ROLE OF CHANCE: Eleven unique copy number changes were identified in a total of 11 patients, including a tandem duplication of 475 bp, containing part of the GDF9 gene promoter region. The duplicated region contains three NOBOX-binding elements and an E-box, important for GDF9 gene regulation. This aberration is likely causative of POI. Fifty-four patients were investigated for copy number changes within GDF9, but no additional cases were found. Ten aberrations constituting novel candidate regions were detected, including a second DNAH6 deletion in a patient with POI. Other identified candidate genes were TSPYL6, SMARCC1, CSPG5 and ZFR2. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study and no functional experiments were performed. WIDER IMPLICATIONS OF THE FINDINGS: The study illustrates the importance of analyzing small copy number changes in addition to sequence alterations in the genetic investigation of patients with POI. Also, promoter regions should be included in the investigation. STUDY FUNDING/COMPETING INTERESTS: The study was supported by grants from the Swedish Research council (project no 12198 to A.W. and project no 20324 to A.L.H.), Stockholm County Council (E.I., A.W. and K.R.W.), Foundation Frimurare Barnhuset (A.N., A.W. and M.B.), Karolinska Institutet (A.N., A.L.H., E.I., A.W. and M.B.), Novo Nordic Foundation (A.W.) and Svenska Läkaresällskapet (M.B.). The funding sources had no involvement in the design or analysis of the study. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.