Sequence analysis of 37 candidate genes for male infertility: challenges in variant assessment and validating genes.

BACKGROUND: The routine genetic analysis for diagnosing male infertility has not changed over the last twenty years, and currently available tests can only determine the etiology of 4% of unselected infertile patients. Thus, to create new diagnostic assays, we must better understand the molecular and genetic mechanisms of male infertility. Although next-generation sequencing allows for simultaneous analysis of hundreds of genes and the discovery of novel candidates related to male infertility, so far only a few gene candidates have enough sound evidence to support the gene-disease relationship. OBJECTIVE: Since complementary studies are required to validate genes, we aimed to analyze the presence of potentially pathogenic rare variants in a set of candidate genes related to azoospermia in a hitherto understudied South American population. SUBJECTS AND METHODS: We performed whole exome sequencing in a group of 16 patients with non-obstructive azoospermia from Ribeirão Preto, Brazil. Based on a recent systematic review of monogenic causes of male infertility, we selected a set of 37 genes related to azoospermia, Sertoli-Cell-Only histology, and spermatogenic arrest to analyze. The identified variants were confirmed by Sanger sequencing, and their functional consequence was predicted by in silico programs. RESULTS: We identified potential pathogenic variants in seven genes in six patients. Two variants, c.671A>G (p.(Asn224Ser)) in DMRT1 and c.91C>T (p.(Arg31Cys)) in REC8, have already been described in association with azoospermia. We also found new variants in genes that already have moderate evidence of being linked to spermatogenic failure (TEX15, KLHL10), in genes with limited evidence (DNMT3B, TEX14) and in one novel promising candidate gene that has no evidence so far (SYCE1L). DISCUSSION: Although this study included a small number of patients, the process of rationally selecting genes allowed us to detect rare potentially pathogenic variants, providing supporting evidence for validating candidate genes associated with azoospermia.

The polymorphism in MUC1 gene in Nelore cattle.

MUC1 is a transmembrane glycoprotein expressed on the apical surfaces of the uterine epithelial tissue with predicted functions in protection and cell-cell adhesion. These properties are closely related with the repetitive region [variable number of tandem repeats (VNTR)] of the extracellullar domain and with the O-glycosylation in their serine and threonine residues. This study describes a polymerase chain reaction (PCR) protocol to analyse MUC1 bovine genetic polymorphism and demonstrates the existence of a VNTR within the sites for O-glycosylation. Oligonucleotide primers based on the Bos taurus mucin (MUC1) gene sequence GenBank AF399757 were used to amplify five VNTR MUC1 alleles from a study group of 56 pure Nelore bovines. The number of repeats varied between 10 and 24, being more prevalent than the alleles with less number of repeats. The DNA sequence analysis revealed two repeats and one of them presented 100% homology with the bovine consensus sequence already reported. The second repeat showed codons that translate to serine and proline amino acids, which are conserved in the MUC1 of several species. This study is the first description of allelic variation and the VNTR structure in the Nelore breed MUC1 gene, and we suggest that this genetic variability can be tested for association with variation in reproductive traits in Nelore cattle.

Recurrent 22q11.2 deletion in a sibship suggestive of parental germline mosaicism in velocardiofacial syndrome.

Deletions of chromosome 22q11.2 are recognized as the main cause of a number of clinical phenotypes, including velocardiofacial syndrome (VCFS) and DiGeorge syndrome (DGS). Velocardiofacial syndrome is a relatively common developmental disorder that is characterized by craniofacial anomalies and conotruncal heart defects. Most 22q11.2 deletions occur sporadically, although the deletion may be transmitted in some cases. The present performed a molecular analysis in one family including a patient with clinical diagnosis of VCFS and his sister with a suggestive phenotype. Six polymorphic 22q11.2 markers (i.e. D22S420, D22S264, D22S941, D22S306, D22S425 and D22S257) were used for genotype analysis of the DNA from the patients and unaffected relatives. The results revealed a 22q11.2 deletion in the patient and his sister from one of six markers (i.e. D22S941). Genotype analysis demonstrated that the deletion in this sib was of maternal origin. The results suggest that the mother probably has gonadal mosaicism. The other relatives present normal DNA profiles for all markers. These results have implications for genetic counseling because of a risk of transmission by germ cells carrying the deletion, even when parents present with a normal DNA profile in their blood cells.