Browsing by Author "Lopes, Alexandra"
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- Assessing the impact of Copy Number Variation on severe spermatogenic impairment with exome dataPublication . Lopes, Alexandra; Nagirnaja, Liina; Filipa, Carvalho; Gonçalves, João; Fernades, Susana; Pereira-Caetano, Iris; Almstrup, Kristian; Rajpert-De Meyts, Ewa; Seixas, Susana; Houston, Brendan; Barros, Alberto; O’Bryan, Moira; Aston, Kenneth; Conrad, Donald; on Behalf of the GEMINI ConsortiumBackground: Azoospermia, the most severe form of male infertility, affects approximately 1% of men worldwide and in the great majority of the cases the etiology of the disease remains unidentified. Given the large number of genes involved in spermatogenesis it is likely that a proportion of cases of idiopathic azoospermia have a genetic basis. We have previously described, using SNP arrays, an excess of low frequency copy number variants (CNVs) in both the autosomes and the sex chromosomes in non-obstructive azoospermia (NOA) suggesting an heterogeneous genetic ethiology for this condition.
- Evaluation of the potential association of SOHLH2 polymorphisms with non-obstructive azoospermia susceptibility in a large European populationPublication . Sánchez, Maria I.S.; Martín, Miriam C.; Egea, Rocío R.; Puchalt, Nicolás G.; Marco, Saturnino L.; Magraner, Gema R.; Ribeiro, Samuel; Castilla Alcalá, José A.; Gonzalvo López, Maria C.; Gilabert, Ana C.; Vicente Prados, F. Javier; Ezequiel, Vicente M.; Poyatos, Miguel B.; Rodrigo, Olga L.; Peraza Godoy, María F.; Caetano, Iris; Marques, Patricia; Arnau, Lluis B.; Seixas, Susana; Gonçalves, João; Bartolomé, Sara L.; Lopes, Alexandra; Carmona López, F. David; Palomino Morales, Rogelio J.Non-obstructive azoospermia (NOA) or spermatogenic failure is a complex disease with an important genetic component that causes infertility in men. Known genetic factors associated with NOA include AZF microdeletions of the Y chromosome or karyotype abnormalities; however, most causes of NOA are idiopathic. During the last decade, a large list of associations between single-nucleotide polymorphisms (SNP) and NOA have been reported. However, most of the genetic studies have been performed only in Asian populations. We aimed to evaluate whether the previously described association in Han Chinese between NOA and two SNPs of the SOHLH2 gene (involved in the spermatogenesis process) may also confer risk for NOA in a population of European ancestry. We genotyped a total of 551 NOA patients (218 from Portugal and 333 from Spain) and 1,050 fertile controls (226 from Portugal and 824 from Spain) for the genetic variants rs1328626 and rs6563386 using TaqMan assays. To test for association, we compared the allele and genotype frequencies between cases and controls using an additive model. A haplotype analysis and a meta-analysis using the inverse variance method with our data and those of the original Asian study were also performed. No statistically significant differences were observed in any of the analyses described above. Therefore, considering the high statistical power of our study, it is not likely that the two analysed SOHLH2 genetic variants are related with an increase susceptibility to NOA in the European population.
- Human spermatogenic failure purges deleterious mutation load from the autosomes and both sex chromosomes, including the gene DMRT1Publication . Lopes, Alexandra; Aston, Kenneth I.; Thompson, Emma E; Carvalho, Filipa; Gonçalves, João; Huang, N.; Matthiesen, Rune; Noordam, Michiel J.; Quintela, Ines; Ramu, Avinash; Seabra, Catarina; Wilfert, Amy B.; Dai, Juncheng; Downie, Jonathan; Fernandes, Susana; Guo, Xuejiang; Shah, Jiahao; Amorim, Antonio; Barros, Alberto; Carracedo, A.; Hu, Z.; Hurles, M.E.; Moskovtsev, S.; Ober, C.; Paduch, D.A.; Schiffman, J.D.; Schlegel, P.N.; Sousa, M.; Carrell, D.T.; Conrad, D.F.Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man’s risk of disease by 10% (OR 1.10 [1.04–1.16], p,261023), rare X-linked CNVs by 29%, (OR 1.29 [1.11–1.50], p,161023), and rare Y-linked duplications by 88% (OR 1.88 [1.13–3.13], p,0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.261025). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.
- New insight into the genetic contribution of common variants to the development of extreme phenotypes of unexplained male infertility: a multicenter genome-wide association studyPublication . Cerván-Martín, Miriam; Tüttelmann, Frank; Lopes, Alexandra; Castillo, Lara B.; Garrido, Nicolás; Luján, Saturnino; Castilla, José A.; Azoonomic, S.G.; Gromoll, J.; Seixas, Susana; Gonçalves, João; Larriba, Sara; Kliesch, Sabine; Palomino-Morales, Rogelio; Carmona, Francisco D.Study question: What is the contribution of the common genetic variation to the development of unexplained male infertility due to severe spermatogenic failure (SPGF)? Summary answer: Genetic polymorphisms of key immune and spermatogenesis loci are involved in the etiology of the most severe SPGF cases, defined by Sertoli cell-only (SCO) phenotype. What is known already: Male infertility is a rising worldwide concern that affects millions of couples. Non-obstructive azoospermia (NOA) and severe oligospermia (SO) are two extreme manifestations characterized by SPGF. A genetic cause can be established in only around 20% of affected men, with the remaining cases being classified as otherwise unexplained. To date, the genomewide association study (GWAS) strategy, although already successfully applied in several other complex traits and diseases, was less fruitful in studies that attempted to decipher the genetic component of unexplained SPGF, mainly due to both a lack of well-powered samples in different ancestries and limitations in study design. Study design, size, duration: We designed a GWAS for unexplained male infertility due to SPGF including a total of 1,274 affected cases and 1,951 fertile controls from the Iberian Peninsula (Spain and Portugal) and Germany. Different biostatistics and bioinformatics approaches were used to evaluate the possible effect of single-nucleotide polymorphisms (SNPs) across the whole genome in the susceptibility to specific subtypes of unexplained SPGF. Participants/materials, setting, methods: The case cohort comprised 502 SO and 772 NOA patients, who were subdivided according to histological phenotypes (SCO, maturation arrest, and hypospermatogenesis) and the outcome of testicular sperm extraction techniques (TESE) from testis biopsies. Genotyping was performed with the GSA platform (Illumina). After quality-control and genotype imputation, 6,539,982 SNPs remained for the analysis, which was performed by logistic regression models. The datasets went through a meta-analysis by the inverse variance weighted method under fixed effects. Main results and the role of chance: Genetic associations with SCO at the genome-wide-level of significance were identified in the major histocompatibility (MHC) class II region (rs1136759, OR=1.80, P=1.32E-08) and in a regulatory region of chromosome 14 nearby the vaccinia-related kinase 1 (VRK1) gene (rs115054029, OR=3.14, P=4.37-08). VRK1 is a relevant proliferative factor for spermatogenesis that causes progressive loss of spermatogonia when disrupted in mouse models. The role of the MHC system in SCO susceptibility was comprehensively evaluated through a validated imputation method that infers classical MHC alleles and polymorphic amino acid positions. A serine at position 13 of the HLA-DRβ1 protein (defined by the risk allele of the lead variant rs1136759) explained most of the SCO association signals within the MHC class II region. This residue is located in the binding pocket of the HLA-DR molecule and interacts directly with the presented antigen. Interestingly, position 13 of HLADRβ1 is the most relevant risk amino acid position for a wide spectrum of immune-mediated disorders. The HLA-DRB1*13 haplotype (which includes the serine at position 13 and represents the strongest NOA-associated marker in Asians to date) was the strongest signal amongst the classical MHC alleles in our study cohort (OR=1.93, P=9.90E-07). Limitations, reasons for caution: Although the statistical power for the overall analysis was appropriate, the subphenotype analyses performed had considerably lower counts, which may influence the identification of genetic variants conferring low to moderate risk effects. Independent studies in larger SCO study cohorts should be performed to confirm our findings. Wider implications of the findings: The molecular mechanisms underlying unexplained SPGF are largely unknown. Our data suggest a relevant role of common genetic variation in the development of SCO, the most extreme histological phenotype of NOA. SCO is characterized by the loss of germ cells and, therefore, implies a considerably higher probability of unsuccessful TESE.
- Validation of Rare Structural Variants in Portuguese Azoospermic PatientsPublication . Seabra, Catarina; Carvalho, Filipa; Gonçalves, João; Matthiesen, Rune; Fernandes, Susana; Ana, Neto; de Sousa, Mário; Barros, Alberto; Amorim, António; Donald, Conrad; Lopes, AlexandraAzoospermia affects approximately 15% of infertile males. Despite considerable research efforts in the last decades, in the majority of cases the cause remains unidentified. Chromosomal abnormalities and Yq microdeletions have been thoroughly studied, yet only account for 17% of azoospermic men. In fact, little is known about the contribution of the hemizygous X-linked and autosomal genes to male infertility. This study focuses on the validation of rare deletions encompassing candidate genes on the X chromosome and on the autosomes, previously identified by Affymetrix 6.0 SNP Array, in a cohort of 166 Portuguese individuals with severe spermatogenic impairment (non-obstructive azoospermia and severe oligozoospermia). As expected, the protein-coding genes CXORF48, and MAGEA8, as well as a miRNA (hsa-mir-4330) could not be amplified by PCR from the single X chromosome of the patients suspected of carrying deletions. These rearrangements will be further validated by aCGH (array Comparative Genomic Hybridization). Additionally, by MLPA analysis on 11p13 we confirmed a large deletion (~1Mb) spanning the WT1 gene - a conserved transcription factor known to play a crucial role in gonadal differentiation. A retrospective clinical evaluation of this patient revealed partial gonadal dysgenesis, consistent with a causal role for the newly discovered deletion. These results reveal new candidate genes for a role in spermatogenic pathways and suggest that haploinsufficiency of proteins important for the development of the male reproductive system can lead to spermatogenic dysfunction.