G - Genética
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Browsing G - Genética by Subject "ARX"
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- Development and validation of a multiplex-PCR assay for X-linked intellectual disabilityPublication . Jorge, P.; Oliveira, B.; Marques, I.; Santos, R.BACKGROUND: X-linked intellectual disability is a common cause of inherited cognitive deficit affecting mostly males. There are several genetic causes implicated in this condition, which has hampered the establishment of an accurate diagnosis. We developed a multiplex-PCR assay for the mutational hotspot regions of the FMR1, AFF2 and ARX genes. METHODS: The multiplex-PCR was validated in a cohort of 100 males selected to include known alleles for the FMR1 repetitive region: five full mutations (250-650 CGGs), ten premutations (70-165 CGGs) and eighty-five in the normal range (19-42 CGGs). Sequencing or Southern blotting was used to confirm the results, depending on the allele class. In this cohort, with the exception of one sample showing an AFF2 intermediate-sized allele, all other samples were normal (8-34 CCGs). No ARX variant was found besides the c.429_452dup. The validated assay was applied to 5000 samples (64.4% males and 35.6% females). RESULTS: The normal-allelic range of both FMR1 and AFF2 genes as well as the nature of ARX variants identified was similar in both genders. The rate of homozygosity observed in female samples, 27.5% for FMR1 and 17.8% for AFF2 alleles, is comparable to that published by others. Two FMR1 premutations were identified, in a male (58 CGGs) and a female case [(CGG)(47)/(CGG)(61)], as well as several FMR1 or AFF2 intermediate-sized alleles. One AFF2 premutation (68 CCGs) and two putative full expansions were picked up in male subjects, which seems relevant considering the rarity of reported AFF2 mutations found in the absence of a family history. CONCLUSIONS: We developed a robust multiplex-PCR that can be used to screen the mutational hotspot regions of FMR1, AFF2 and ARX genes. Moreover, this strategy led to the identification of variants in all three genes, representing not only an improvement in allele-sizing but also in achieving a differential diagnosis. Although the distinction between females who are truly homozygous and those with a second pre- or full mutation sized allele, as well as a definitive diagnosis, requires a specific downstream technique, the use of this multiplex-PCR for initial screening is a cost-effective approach which widens the scope of detection.
- Unraveling the pathogenesis of ARX polyalanine tract variants using a clinical and molecular interfacing approachPublication . Marques, I.; Sá, J.; Soares, G.; Mota, M.; Pinheiro, C.; Aguiar, L.; Amado, M.; Soares, C.; Calado, A.; Dias, P.; Sousa, A.; Fortuna, A.; Santos, R.; Howell, K.; Ryan, M.; Leventer, R.; Sachdev, R.; Catford, R.; Friend, K; Mattiske, T.; Shoubridge, C.; Jorge, P.The Aristaless-related homeobox (ARX) gene is implicated in intellectual disability with the most frequent pathogenic mutations leading to expansions of the first two polyalanine tracts. Here, we describe analysis of the ARX gene outlining the approaches in the Australian and Portuguese setting, using an integrated clinical and molecular strategy. We report variants in the ARX gene detected in 19 patients belonging to 17 families. Seven pathogenic variants, being expansion mutations in both polyalanine tract 1 and tract 2, were identifyed, including a novel mutation in polyalanine tract 1 that expands the first tract to 20 alanines. This precise number of alanines is sufficient to cause pathogenicity when expanded in polyalanine tract 2. Five cases presented a probably non-pathogenic variant, including the novel HGVS: c.441_455del, classified as unlikely disease causing, consistent with reports that suggest that in frame deletions in polyalanine stretches of ARX rarely cause intellectual disability. In addition, we identified five cases with a variant of unclear pathogenic significance. Owing to the inconsistent ARX variants description, publications were reviewed and ARX variant classifications were standardized and detailed unambiguously according to recommendations of the Human Genome Variation Society. In the absence of a pathognomonic clinical feature, we propose that molecular analysis of the ARX gene should be included in routine diagnostic practice in individuals with either nonsyndromic or syndromic intellectual disability. A definitive diagnosis of ARX-related disorders is crucial for an adequate clinical follow-up and accurate genetic counseling of at-risk family members.