Novel PATL2 variants cause female infertility with oocyte maturation defect.

PURPOSE: Oocyte maturation defect (OOMD) is a rare cause of in vitro fertilization failure characterized by the production of immature oocytes. Compound heterozygous or homozygous PATL2 mutations have been associated with oocyte arrest at the germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) stages, as well as morphological changes. METHODS: In this study, we recruited three OOMD cases and conducted a comprehensive multiplatform laboratory investigation. RESULTS: Whole exome sequence (WES) revealed four diagnostic variants in PATL2, nonsense mutation c.709C > T (p.R237*) and frameshift mutation c.1486_1487delinsT (p.A496Sfs*4) were novel mutations that have not been reported previously. Furthermore, the pathogenicity of these variants was predicted using in silico analysis, which indicated detrimental effects. Molecular dynamic analysis suggested that the A496S variant disrupted the hydrophobic segment, leading to structural changes that affected the overall protein folding and stability. Additionally, biochemical and molecular experiments were conducted on cells transfected with wild-type (WT) or mutant PATL2 (p.R237* and p.A496Sfs*4) plasmid vectors. CONCLUSIONS: The results demonstrated that PATL2A496Sfs*4 and PATL2R237* had impacts on protein size and expression level. Interestingly, expression levels of specific genes involved in oocyte maturation and early embryonic development were found to be simultaneously deregulated. The findings in our study expand the variation spectrum of the PATL2 gene, provide solid evidence for counseling on future pregnancies in affected families, strongly support the application of in the diagnosis of OOMD, and contribute to the understanding of PATL2 function.

Comprehensive strategy improves the genetic diagnosis of different polycystic kidney diseases.

Polycystic kidney disease (PKD) is known to occur in three main forms, namely autosomal dominant PKD (ADPKD), autosomal recessive PKD (ARPKD) and syndromic PKD (SPKD), based on the clinical manifestations and genetic causes, which are diagnosable from the embryo stage to the later stages of life. Selection of the genetic test for the individuals with diagnostic imaging reports of cystic kidneys without a family history of the disease continues to be a challenge in clinical practice. With the objective of maintaining a limit on the time and medical cost of the procedure, a practical strategy for genotyping and targeted validation to resolve cystogene variations was developed in our clinical laboratory, which combined the techniques of whole-exome sequencing (WES), Long-range PCR (LR-PCR), Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to work in a stepwise approach. In this context, twenty-six families with renal polycystic disorders were enrolled in the present study. Thirty-two variants involving four ciliary genes (PKD1, PKHD1, TMEM67 and TMEM107) were identified and verified in 23 families (88.5%, 23/26), which expanded the variant spectrum by 16 novel variants. Pathogenic variations in five foetuses of six families diagnosed with PKD were identified using prenatal ultrasound imaging. Constitutional biallelic and digenic variations constituted the pathogenic patterns in these foetuses. The preliminary clinical data highlighted that the WES + LR PCR-based workflow followed in the present study is efficient in detecting divergent variations in PKD. The biallelic and digenic mutations were revealed as the main pathogenic patterns in the foetuses with PKD.

Identification of novel variations in the NTRK1 gene causing congenital insensitivity to pain with anhidrosis.

BACKGROUND: Congenital insensitivity to pain (CIP) conditions are a group of Mendelian disorders with clinical and genetic heterogeneity. CIP with anhidrosis (CIPA) is a distinct subtype caused by biallelic variants in the NTRK1 gene. METHODS: In this study, six families with CIPA were recruited and submitted to a series of clinical and genetic examinations. Whole-exome sequencing and whole-genome sequencing were applied to perform a comprehensive genetic analysis. Sanger sequencing was used as a validation method. RESULTS: These patients exhibited phenotypic variability. All probands in the six families were positive for biallelic pathogenic variants in NTRK1. Five individual variants, namely NTRK1: (NM_002529.3) c.851-33T>A, c.717+2T>C, c.1806-2A>G, c.1251+1G>A, and c.851-794C>G, including three novel ones, were identified, which were carried by the six patients in a homozygous or compound heterozygous way. The validation results indicated that all the parents of the six probands, except for one father and one mother, were monoallelic carriers of a single variant. CONCLUSIONS: The findings in our study extended the variation spectrum of the NTRK1 gene and highlighted the advantage of the integrated application of multiplatform genetic technologies.

Investigation of a Novel NTRK1 Variation Causing Congenital Insensitivity to Pain With Anhidrosis.

Background: Congenital insensitivity to pain with anhidrosis (CIPA), a rare autosomal recessive sensory neuropathy, was caused mainly by biallelic mutations in the NTRK1 gene. The pathogenesis of CIPA still needs further elucidation. Methods: Here, we recruited a CIPA case and introduced whole-exome sequencing (WES) to identify the causative variation. Subsequently, an in silico molecular dynamic (MD) analysis was performed to explore the intramolecular impact of the novel missense variant. Meanwhile, in vitro functional study on the novel variant from a metabolomic perspective was conducted via the liquid chromatography-mass spectrometry (LC-MS) approach, of which the result was verified by quantitative real-time PCR (qRT-PCR). Results: A novel compound heterozygous variation in NTRK1 gene was detected, consisting of the c.851-33T > A and c.2242C > T (p.Arg748Trp) variants. MD result suggested that p.Arg748Trp could affect the intramolecular structure stability. The results of the LC-MS and metabolic pathway clustering indicated that the NTRK1Arg748Trp variant would significantly affect the purine metabolism in vitro. Further analysis showed that it induced the elevation of NT5C2 mRNA level. Conclusion: The findings in this study extended the variation spectrum of NTRK1, provided evidence for counseling to the affected family, and offered potential clues and biomarkers to the pathogenesis of CIPA.

Novel Biallelic DNAH1 Variations Cause Multiple Morphological Abnormalities of the Sperm Flagella.

Sperm motility is vital to human reproduction, and malformed sperm flagella can cause male infertility. Individuals with multiple morphological abnormalities of the flagella mostly have absent, short, coiled, bent, and/or irregular-caliber flagella. In this study, a patient with male infertility underwent a physical examination along with his wife. Genetic testing was performed by whole-exome sequencing of the couple, and Sanger sequencing was performed for validation. Novel biallelic variations in the DNAH1: (NM_015512.4) gene consisting of c.1336G>C (p.E446Q) and c.2912G>A (p.R971H) were identified. In silico structural analysis revealed that the amino acid residues affected by the variation were evolutionarily conserved, and the variant p.R971H influenced the stability of the DNAH1 protein. Morphological studies of the patient's sperm showed defects in its flagella. Results of Papanicolaou staining and scanning electron microscopy demonstrated coiled and short flagella with multiple anomalies. Transmission electron microscopy of the sperm flagella showed that the inner dynein arm and radial spoke were absent, and the dense fiber and microtubule doublets were displaced. Quantitative PCR of the mRNA of the patient's sperm showed that the expression of DNALI1 was dramatically reduced. Collectively, these findings elucidated the genetic cause of the family's infertility and provided insight into the functioning of the DNAH1 gene.