A novel homozygous TSGA10 missense variant causes acephalic spermatozoa syndrome in a Pakistani family.

BACKGROUND: Acephalic spermatozoa syndrome is a rare type of teratozoospermia causing male infertility due to detachment of the sperm head and flagellum, which precludes fertilization potential. Although loss-of-function variations in several genes, including TSGA10, have been associated with acephalic spermatozoa syndrome, the genetic cause of many cases remains unclear. RESULTS: We recruited a Pakistani family with two infertile brothers who suffered from acephalic spermatozoa syndrome. Through whole-exome sequencing (WES) followed by Sanger sequencing, we identified a novel missense variant in TSGA10 (c.1112T > C, p. Leu371Pro), which recessively co-segregated with the acephalic spermatozoa syndrome within this family. Ultrastructural analyses of spermatozoa from the patient revealed that 98% of flagellar cross-sections displayed abnormal axonemal ultrastructure, in addition to the head-flagellum detachment. Real-time quantitative PCR analysis revealed almost no detectable TSAG10 mRNA and western blot analysis also failed to detect TSAG10 protein in patient's sperm samples while TSGA10 expression was clearly detected in control samples. Consistently, immunofluorescence analysis demonstrated the presence of TSGA10 signal in the midpiece of sperm from the control but a complete absence of TSGA10 signal in sperm from the patient. CONCLUSION: Altogether, our study identifies a novel TSGA10 pathogenic variant as a cause of acephalic spermatozoa syndrome in this family and provides information regarding the clinical manifestations associated with TSGA10 variants in human.

RéSUMé: CONTEXTE: Le syndrome des spermatozoïdes acéphaliques est un type rare de tératozoospermie provoquant une infertilité masculine en raison du détachement de la tête et du flagelle des spermatozoïdes, ce qui exclut une potentielle fécondation. Bien que des variations de perte de fonction dans plusieurs gènes, y compris TSGA10, aient été associées au syndrome des spermatozoïdes acéphaliques, la cause génétique de nombreux cas reste incertaine. RéSULTATS: Nous avons recruté une famille pakistanaise avec deux frères infertiles qui souffraient du syndrome des spermatozoïdes acéphaliques. Grâce au séquençage de l'exome entier (WES) suivi du séquençage Sanger, nous avons identifié un nouveau variant faux-sens dans TSGA10 (c.1112T > C, p. Leu371Pro), qui co-ségréguait de manière récessive avec le syndrome des spermatozoïdes acéphaliques au sein de cette famille. Les analyses ultrastructurales des spermatozoïdes des patients ont révélé que 98% des coupes transversales flagellaires présentaient une ultrastructure axonémiques anormales, en plus du décollement tête-flagelle. L'analyse quantitative par PCR en temps réel n'a révélé presque aucun ARNm TSAG10 détectable; l'analyse par transfert Western n'a pas non plus réussi à détecter la protéine TSAG10 dans les échantillons de sperme des patients, tandis que l'expression de TSGA10 a été clairement détectée dans les échantillons du témoin. De manière cohérente, l'analyse par immunofluorescence a démontré la présence du signal TSGA10 dans la partie médiane des spermatozoïdes du témoin, mais une absence totale de signal TSGA10 chez ceux des patients. CONCLUSION: Dans l'ensemble, notre étude identifie un nouveau variant pathogène de TSGA10 comme cause du syndrome des spermatozoïdes acéphaliques dans cette famille et fournit des informations concernant les manifestations cliniques associées aux variants de TSGA10 chez l'homme. MOTS-CLéS: Infertilité, TSGA10, Spermatozoïdes acéphaliques, Variations faux-sens.

Screening of high-risk deleterious missense variations in the CYP1B1 gene implicated in the pathogenesis of primary congenital glaucoma: A comprehensive in silico approach.

Background: Primary congenital glaucoma (PCG) is the most common subtype of glaucoma caused by defects in the cytochrome P450 1B1 (CYP1B1) gene. It is developing among infants in more than 80% of cases who exhibit impairments in the anterior chamber angle and the trabecular meshwork. Thus, a comprehensive in silico approach was performed to evaluate the effect of high-risk deleterious missense variations in the CYP1B1 gene. Material and methods: All the information for CYP1B1 missense variants was retrieved from the dbSNP database. Seven different tools, namely: SIFT, PolyPhen-2, PROVEAN, SNAP2, PANTHER, PhD-SNP, and Predict-SNP, were used for functional annotation, and two packages, which were I-Mutant 2.0 and MUpro, were used to predict the effect of the variants on protein stability. A phylogenetic conservation analysis using deleterious variants was performed by the ConSurf server. The 3D structures of the wild-type and mutants were generated using the I-TASSER tool, and a 50 ns molecular dynamic simulation (MDS) was executed using the GROMACS webserver to determine the stability of mutants compared to the native protein. Co-expression, protein-protein interaction (PPI), gene ontology (GO), and pathway analyses were additionally performed for the CYP1B1 in-depth study. Results: All the retrieved data from the dbSNP database was subjected to functional, structural, and phylogenetic analysis. From the conducted analyses, a total of 19 high-risk variants (P52L, G61E, G90R, P118L, E173K, D291G, Y349D, G365W, G365R, R368H, R368C, D374N, N423Y, D430E, P442A, R444Q, F445L, R469W, and C470Y) were screened out that were considered to be deleterious to the CYP1B1 gene. The phylogenetic analysis revealed that the majority of the variants occurred in highly conserved regions. The MD simulation analysis exhibited that all mutants' average root mean square deviation (RMSD) values were higher compared to the wild-type protein, which could potentially cause CYP1B1 protein dysfunction, leading to the severity of the disease. Moreover, it has been discovered that CYP1A1, VCAN, HSD17B1, HSD17B2, and AKR1C3 are highly co-expressed and interact with CYP1B1. Besides, the CYP1B1 protein is primarily involved in the metabolism of xenobiotics, chemical carcinogenesis, the retinal metabolic process, and steroid hormone biosynthesis pathways, demonstrating its multifaceted and important roles. Discussion: This is the first comprehensive study that adds essential information to the ongoing efforts to understand the crucial role of genetic signatures in the development of PCG and will be useful for more targeted gene-disease association studies.

Investigating the Effects of Amino Acid Variations in Human Menin.

Human menin is a nuclear protein that participates in many cellular processes, as transcriptional regulation, DNA damage repair, cell signaling, cell division, proliferation, and migration, by interacting with many other proteins. Mutations of the gene encoding menin cause multiple endocrine neoplasia type 1 (MEN1), a rare autosomal dominant disorder associated with tumors of the endocrine glands. In order to characterize the structural and functional effects at protein level of the hundreds of missense variations, we investigated by computational methods the wild-type menin and more than 200 variants, predicting the amino acid variations that change secondary structure, solvent accessibility, salt-bridge and H-bond interactions, protein thermostability, and altering the capability to bind known protein interactors. The structural analyses are freely accessible online by means of a web interface that integrates also a 3D visualization of the structure of the wild-type and variant proteins. The results of the study offer insight into the effects of the amino acid variations in view of a more complete understanding of their pathological role.

Splicing analysis of SLC40A1 missense variations and contribution to hemochromatosis type 4 phenotypes.

Hemochromatosis type 4, or ferroportin disease, is considered as the second leading cause of primary iron overload after HFE-related hemochromatosis. The disease, which is predominantly associated with missense variations in the SLC40A1 gene, is characterized by wide clinical heterogeneity. We tested the possibility that some of the reported missense mutations, despite their positions within exons, cause splicing defects. Fifty-eight genetic variants were selected from the literature based on two criteria: a precise description of the nucleotide change and individual evidence of iron overload. The selected variants were investigated by different in silico prediction tools and prioritized for midigene splicing assays. Of the 15 variations tested in vitro, only two were associated with splicing changes. We confirm that the c.1402G>A transition (p.Gly468Ser) disrupts the exon 7 donor site, leading to the use of an exonic cryptic splicing site and the generation of a truncated reading frame. We observed, for the first time, that the p.Gly468Ser substitution has no effect on the ferroportin iron export function. We demonstrate alternative splicing of exon 5 in different cell lines and show that the c.430A>G (p.Asn144Asp) variant promotes exon 5 inclusion. This could be part of a gain-of-function mechanism. We conclude that splicing mutations rarely contribute to hemochromatosis type 4 phenotypes. An in-depth investigation of exon 5 auxiliary splicing sequences may help to elucidate the mechanism by which splicing regulatory proteins regulate the production of the full length SLC40A1 transcript and to clarify its physiological importance.

Targeted pharmacotherapies for defective ABC transporters.

Human ABC (ATP Binding Cassette) transporters form a superfamily of forty-eight transmembrane proteins, which transport their substrates across biological membranes against important concentration gradients, in an energy-dependent manner. Gene variations in approximately half of these transporters have been identified in subjects with rare and often severe genetic diseases, highlighting the importance of their biological function. For missense variations leading to defects in ABC transporters, the current challenge is to identify new molecules with therapeutic potential able to rescue the induced molecular deficiency. In this review, we first address the progress provided by emerging pharmacotherapies in cystic fibrosis, the most frequent monogenic disease caused by variations of an ABC transporter, i.e. ABCC7/CFTR. Then, we enlarge the topic to the other ABC transporters, more notably to canalicular ABC transporters, the variations of which cause rare hepatobiliary diseases, and we discuss the first promising attempts aiming to correct molecular defects of these proteins.