Browsing by Author "Seabra, Catarina"
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- An Alu-mediated 1Mb deletion removes Wilms’ tumor 1 (WT1) but not PAX6 in a patient with isolated cryptorchidismPublication . Seabra, Catarina; Quental, Sofia; Neto, Ana; Carvalho, Filipa; Gonçalves, João; Fernandes, Susana; Sousa, Mário; Barros, Alberto; Amorim, António; Lopes, Alexandra MObjective: We have recently performed an array-based genome-wide analysis of structural variants in a cohort of patients with non-obstructive azoospermia (NOA) and found a cryptic deletion of approximately 1Mb in 11p13, spanning the WT1 gene but not PAX6, in a Portuguese patient with clinical history of cryptorchidism during childhood?. Here we performed the molecular characterization of this novel deletion, to precisely map the breakpoints of this deletion, and evaluated the prevalence of focal WT1 genetic alterations in infertile Portuguese patients with cryptorchidism. Design: Fine molecular characterization of a heterozygous large deletion in 11p13 in one azoospermic patient (with clinical history of cryptorchidism) and screening for WT1 exonic microdeletions and mutations in a group of 31 Portuguese patients with uni- or bi-lateral cryptorchidism. Materials and Methods: Multiplex ligation-dependent probe amplification (MLPA), Long Range PCR; PCR amplification of the WT1 exons and proximal flanking sequences followed by Sanger sequencing. Results: We confirmed by MLPA the ~1Mb deletions at 11p13 spanning six genes - WT1, PRRG4, QSER1, TCP11L1, CSTF3 and HIPK3. Examination of the deletion breakpoint showed that it lies within highly homologous Alu Y sequences. Therefore the likely mechanism for this deletion was Alu-mediated non-allelic homologous recombination (NAHR). No mutations were found in the single allele present in this patient suggesting that the phenotype probably results from WT1 haploinsufficiency. We found no additional WT1 alterations in our group of patients with cryptorchidism. Conclusions: To our knowledge this is the smallest as yet described deletion encompassing the WT1 gene, which results in a non-syndromic clinical presentation of infertility. Repeat-mediated non-allelic recombination is an alternative mechanism for 11p13 deletions spanning WT1. Based on our results WT1 genetic defects are not frequently involved in isolated cryptorchidism, even though more patients should be analyzed. Support: This work was partially funded by the Portuguese Foundation for Science and Technology FCT/MCTES (PIDDAC) and co-financed by European funds (FEDER) through the COMPETE program, research grant PTDC/SAU-GMG/101229/2008 to AML. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology, and Higher Education and is partially supported by FCT. AML is the recipient of a postdoctoral fellowship from FCT (SFRH/BPD/73366/2010).
- 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.
- 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.