Browsing by Author "Quental, Sofia"
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- Alu-Alu Recombination Underlying the First Large Genomic Deletion in GlcNAc-Phosphotransferase Alpha/Beta (GNPTAB) Gene in a MLII Alpha/Beta PatientPublication . Coutinho, Maria Francisca; da Silva Santos, Liliana; Lacerda, Lúcia; Quental, Sofia; Wibrand, F.; Lund, A.M.; Johansen, K.B.; Prata, Maria João; Alves, SandraMucolipidosis type II α/β is a severe, autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the α/β subunits of the GlcNAc-phosphotransferase. To date, over 100 different mutations have been identified in MLII α/β patients, but no large deletions have been reported. Here we present the first case of a large homozygous intragenic GNPTAB gene deletion (c.3435-386_3602 + 343del897) encompassing exon 19, identified in a ML II α/β patient. Long-range PCR and sequencing methodologies were used to refine the characterization of this rearrangement, leading to the identification of a 21 bp repetitive motif in introns 18 and 19. Further analysis revealed that both the 5' and 3' breakpoints were located within highly homologous Alu elements (Alu-Sz in intron 18 and Alu-Sq2, in intron 19), suggesting that this deletion has probably resulted from Alu-Alu unequal homologous recombination. RT-PCR methods were used to further evaluate the consequences of the alteration for the processing of the mutant pre mRNA GNPTAB, revealing the production of three abnormal transcripts: one without exon 19 (p.Lys1146_Trp1201del); another with an additional loss of exon 20 (p.Arg1145Serfs*2), and a third in which exon 19 was substituted by a pseudoexon inclusion consisting of a 62 bp fragment from intron 18 (p.Arg1145Serfs*16). Interestingly, this 62 bp fragment corresponds to the Alu-Sz element integrated in intron 18.This represents the first description of a large deletion identified in the GNPTAB gene and contributes to enrich the knowledge on the molecular mechanisms underlying causative mutations in ML II.
- Alu-Alu recombination underlying the first large genomic deletion in GlcNAc-phosphotransferase α/β (GNPTAB) gene in a MLII α/β patientPublication . Coutinho, Maria Francisca; da Silva Santos, Liliana; Lacerda, Lúcia; Quental, Sofia; Flemming, W; Lund, AM; Johansen, KB; Prata, Maria João; Alves, SandraMucolipidosis type II alpha/beta is a severe, autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the alpha/beta subunits of the GlcNAc-phosphotransferase. To date, over 100 different mutations have been identified in MLII alpha/beta patients but no large deletions have been reported. Here we present the first case of a large homozygous intragenic GNPTAB gene deletion (c.3435-386_3602+343del897) encompassing exon 19, identified in a ML II alpha/beta patient. Long range PCR and sequencing methodologies were used to refine the characterization of this rearrangement, leading to the identification of a 21bp repetitive motif in introns 18 and 19. Further analysis revealed that both the 5’ and 3’ breakpoints were located within highly homologous Alu elements (Alu-Sz in intron 18 and Alu-Sq2, in intron 19), suggesting that this deletion has probably resulted from Alu-Alu unequal homologous recombination. RT-PCR methods were used to further evaluate the consequences of the alteration for the processing of the mutant pre mRNA GNPTAB, revealing the production of three abnormal transcripts: one without exon 19 (p.Lys1146_Trp1201del); another with an additional loss of exon 20 (p.Arg1145Serfs*2), and a third in which exon 19 was substituted by a pseudoexon inclusion consisting of a 62 bp fragment from intron 18 (p.Arg1145Serfs*16). Interestingly, this 62 bp fragment corresponds to the Alu-Sz element integrated in intron 18. This represents the first description of a large deletion identified in the GNPTAB gene and contributes to enrich the knowledge on the molecular mechanisms underlying causative mutations in ML II.
- 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).
- Incidence of maple syrup urine disease in PortugalPublication . Quental, Sofia; Vilarinho, Laura; Martins, Esmeralda; Teles, Elisa Leão; Rodrigues, Esmeralda; Diogo, Luísa; Garcia, Paula; Eusébio, Filomena; Gaspar, Ana; Sequeira, Sílvia; Amorim, António; Prata, Maria JoãoMaple syrup urine disease is an autosomal recessive disorder of branched-chain amino acids metabolism with a worldwide frequency of 1/185,000 live newborns. In Portugal, the incidence of the disease has not been assessed. Based on the review of the cases diagnosed by tandem mass spectrometry an incidence of 1/86,800 live newborns was estimated in Portugal, indicating that the disease is more frequent in this country than reported in most populations.
- A novel Alu-mediated microdeletion at 11p13 removes WT1 in a patient with cryptorchidism and azoospermiaPublication . Seabra, Catarina M.; Quental, Sofia; Paula Neto, Ana; Carvalho, Filipa; Gonçalves, João; Paulo Oliveira, João; Fernandes, Susana; Sousa, Mário; Barros, Alberto; Amorim, António; Lopes, Alexandra M.This article describes a patient with cryptorchidism and nonobstructive azoospermia presenting a novel microdeletion of approximately 1 Mb at 11p13. It was confirmed by multiplex ligation-dependent probe amplification that this heterozygous deletion spanned nine genes (WT1, EIF3M, CCDC73, PRRG4, QSER1, DEPDC7, TCP11L1, CSTF3 and HIPK3) and positioned the breakpoints within highly homologous repetitive elements. As far as is known, this is the smallest deletion as-yet described encompassing the WT1 gene and was detected only once in a total of 32 Portuguese patients with isolated uni- or bilateral cryptorchidism. These findings suggest that molecular analysis in patients with genitourinary features suggestive of WT1 impairment, namely cryptorchidism and renal abnormalities, may reveal cryptic genetic defects.
- The mutational spectrum of WT1 in male infertilityPublication . Seabra, Catarina M.; Quental, Sofia; Lima, Ana C; Carvalho, Filipa; Gonçalves, João; Fernandes, Susana; Pereira, Iris; Silva, Júlia; Marques, Patrícia I.; Sousa, Mário; Barros, Alberto; Seixas, Susana; Amorim, António; Lopes, Alexandra M.PURPOSE: We evaluated the impact of WT1 mutations in isolated severe spermatogenic impairment in a population of European ancestry. WT1 was first identified as the gene responsible for Wilms tumor. It was later associated with a plethora of clinical phenotypes often accompanied by urogenital defects and male infertility. The recent finding of WT1 missense mutations in Chinese azoospermic males without major gonadal malformations broadened the phenotypic spectrum of WT1 defects and motivated this study. MATERIALS AND METHODS: We analyzed the WT1 coding region in a cohort of 194 Portuguese patients with nonobstructive azoospermia and in 188 with severe oligozoospermia with increased depth for the exons encoding the regulatory region of the protein. We also analyzed a group of 31 infertile males with a clinical history of unilateral or bilateral cryptorchidism and 1 patient with anorchia. RESULTS: We found 2 WT1 missense substitutions at higher frequency in patients than in controls. 1) A novel variant in exon 1 (p.Pro130Leu) that disrupted a mammalian specific polyproline stretch in the self-association domain was more frequent in azoospermia cases (0.27% vs 0.13%, p = 0.549). 2) A rare variant in a conserved residue in close proximity to the first zinc finger (pCys350Arg) was more frequent in severe oligozoospermia cases (0.80% vs 0.13%, p = 0.113). CONCLUSIONS: Results suggest a role for rare WT1 damaging variants in severe spermatogenic failure in populations of European ancestry. Large multicenter studies are needed to fully assess the contribution of WT1 genetic alterations to male infertility in the absence of other disease phenotypes.