Browsing by Author "da Silva Santos, Liliana"
Now showing 1 - 6 of 6
Results Per Page
Sort Options
- 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.
- Alu-Alu recombination underlying the first large genomic deletion in GlcNAc-phosphotransferase α/β (GNPTAB) gene in a MLII α/β patient [Poster]Publication . Coutinho, Maria Francisca; da Silva Santos, Liliana; Lacerda, Lúcia; Flemming, Wibrand; Lund, Allan M; Johansen, Klaus 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 including missense, nonsense, small deletions, small insertions and splice site mutations (Human Gene Mutation Database website [http://www.hgmd.org] and references therein). Large genomic rearrangements were rarely reported (1,6%) with only two large insertions having been described up to now (Tappino et al., 2008; Otomo et al., 2009) but no known large deletions. Results: In this work, through long range PCR and sequencing methodologies we identified a large homozygous intragenic GNPTAB gene deletion, encompassing exon 19, in a ML II α/β patient and refined the characterization of this rearrangement. As a result, it was possible to identify the deletion breakpoints and determine the deletion extension which was 897 bp and included the last 386 nucleotides of intron 18, exon 19, and the first 343 bp of intron 19. A 21bp repetitive motif in introns 18 and 19 was observed at both deletion breakpoints. 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; another with an additional loss of exon 20, and a third in which exon 19 was substituted by a pseudoexon inclusion consisting of a 62 bp fragment from intron 18. Interestingly, this 62 bp fragment corresponds to the Alu-Sz element integrated in intron 18. Conclusion: To the best of our knowledge, this represents the first description of a large deletion identified in the GNPTAB gene. Furthermore, the work adds on the knowledge of the molecular mechanisms underlying causative mutations in ML II and highlights the importance of cDNA analysis on the prediction of the impact of large deletions at protein levels, since a simple gDNA analysis might be misleading.
- Biochemical analysis of GNPTAB missense mutations associated with ML IIPublication . da Silva Santos, Liliana; Coutinho, Maria Francisca; Girisha, Katta Mohan; Lacerda, Lúcia; Tappino, Barbara; Stefano, Regis; Filocammo, Mirella; Prata, Maria João; Alves, SandraMucolipidosis type II and type III (ML II and III) are rare autosomal recessive disorders of lysosomal hydrolase trafficking respectively caused by completely absent or reduced activity of the enzyme GlcNAc-phosphotransferase, which catalyzes the initial step in the synthesis of mannose-6-phosphate recognition marke. This heterohexameric enzyme composed of three subunits (alpha2 beta2 gamma2), is a product of two distinct genes GNPTAB and GNPTG. Mutations in GNPTAB result ML II and ML III, while mutations in GNPTG are only associated with ML III. To date more than 100 different GNPTAB mutations have been described, causing either ML II alpha\beta or ML III alpha\beta. Although splicing and frameshift mutations are usually associated with more severe phenotypes and missense mutations with milder ones, this typical pattern is not observed for all ML II patients. Here we report the impact of two GNPTAB missense mutations upon the protein: while W81L occurred in the portion of the gene that encodes the apha-subunit, R986C affected a genomic region encoding the beta subunit. To address this issue, the entire coding region of the wild-type GNPTAB was cloned into the pcDNAHisMax TOPO vector and the c.440delC (A147AfsX5), c.2956T>C (R986C) and c.242G>T (W81L) were introduced on this vector using the QuikChange Site-directed Mutagenesis kit. The presence of additional mutations, resulting from possible enzymatic misincorporation, was excluded by sequencing all constructs. COS7 cells were transfected with control and mutant plasmids using Lipofectamine 2000 reagent. Protein expression levels and subcellular location were determined through Western Blot and Immunofluorescence, respectively. Results and Conclusions: We analyzed the protein expression levels of three GNPTAB mutations: c.242G>T (W81L), c.2956T>C (R986C) andc.440delC (A147AfsX5). The frameshift c.440delC (A147AfsX5), predicting to introduce a premature stop codon, was used as negative control. and, as expected, no GNPTAB protein product was detectable. Instead, the analysis of both missense mutations, c.2956T>C (R986C) and c.242G>T (W81L), revealed a decrease in GNPTAB protein expression, compared to the control wild type. This concurs with a previous computational assessment by the Polyphen and SIFT algorithms, predicting that the 2 mutations were likely to be potentially damaging. In addition, computational analysis (http://www.ensembl.org/) revealed that both missense mutations occurred at evolutionarily conserved amino acid residues. The results of all these approaches correlate with the severe ML II phenotype of the patients.
- Mucolipidosis type II α/β with a homozygous missense mutation in the GNPTAB genePublication . Coutinho, Maria Francisca; da Silva Santos, Liliana; Girisha, K.M.; Satyamoorthy, K.; Lacerda, Lúcia; Prata, Maria João; Alves, Sandra
- Sortilin/Neurotensin Receptor 3: Three-dimensional Insights on its Coding VariantsPublication . Coutinho, Maria Francisca; da Silva Santos, Liliana; Lacerda, Lúcia; Macedo-Ribeiro, Sandra; Prata, Maria João; Alves, SandraIntroduction: Sortilin is a multifunctional sorting receptor required for Golgi-to-lysosome protein transport by a M-6-P independent pathway. It presents a unique structural organization with a 10-bladed β-propeller domain forming a tunnel where several ligands bind and is able to mediate the lysosomal targeting of sphingolipid activator proteins (SAPs), acid sphingomyelinase (AS) and cathepsins D and H. For some of these proteins no alternative receptor is known. The inner rim of the tunnel, along with several patches which function as additional binding sites have particularly high sequence conservation. Such structural organization is a key element for regulation of sortilin’s function and any sequence alterations changing it may potentially result in impaired lysosomal transport. As a result from the “1000Genomes Project”, several coding variants were identified for this protein with no known individuals carrying two of those variant alleles ever identified. Methods: In silico mutagenesis of sortilin was performed with SwissModelServer and the effect of each variant on its 3-dimensional structure was evaluated. The results were cross-linked with the potential pathogenicity predictions of PolyPhen and SIFT. Results/Discussion: Here we present the complete results of this in silico analysis, highlighting the potential of each one to disrupt sortilin’s structure and, consequently, impair its biological function and, ultimately, cause lysosomal storage and disease.