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- Usher syndrome and Nebulin‐associated myopathy in a single patient due to variants in MYO7A and NEBPublication . Maia, N; Soares, Ana Rita; Fortuna, Ana; Marques, Isabel; Gonçalves, Ana; Santos, Rosário; Pires, Manuel; De Brouwer, Arjan; Jorge, PaulaIn a patient with Usher syndrome and atypical muscle complaints, we have identified two separate variants in MYO7A andNEB genes by exome sequencing. The homozygous variants in these two recessive genes could explain the full phenotype of our patient.
- Arx-related disorders: several distinct phenotypes, one mutated genePublication . Sá, M.J.; Soares, G.; Silva, J.; Fortuna, A.; Santos, R.; Marques, I.; Jorge, P.
- Development and Validation of a Mathematical Model to Predict the Complexity of FMR1 Allele CombinationsPublication . Rodrigues, Bárbara; Vale-Fernandes, Emídio; Maia, N; Santos, Flávia; Marques, Isabel; Santos, Rosário; Nogueira, António J. A.; Jorge, PaulaThe polymorphic trinucleotide repetitive region in the FMR1 gene 5'UTR contains AGG interspersions, particularly in normal-sized alleles (CGG < 45). In this range repetitive stretches are typically interrupted once or twice, although alleles without or with three or more AGG interspersions can also be observed. AGG interspersions together with the total length of the repetitive region confer stability and hinder expansion to pathogenic ranges: either premutation (55 < CGG < 200) or full mutation (CGG > 200). The AGG interspersions have long been identified as one of the most important features of FMR1 repeat stability, being particularly important to determine expansion risk estimates in female premutation carriers. We sought to compute the combined AGG interspersion numbers and patterns, aiming to define FMR1 repetitive tract complexity combinations. A mathematical model, the first to compute this cumulative effect, was developed and validated using data from 131 young and healthy females. Plotting of their allelic complexity enabled the identification of two statistically distinct groups - equivalent and dissimilar allelic combinations. The outcome, a numerical parameter designated allelic score, depicts the repeat substructure of each allele, measuring the allelic complexity of the FMR1 gene including the AGGs burden, thus allowing new behavioral scrutiny of normal-sized alleles in females.
- A multiplex assay for x‐linked intellectual disability assessmentPublication . Jorge, P.; Marques, I.; Oliveira, B.; Santos, R.
- 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.
- Advances in fragile-x testingPublication . Maia, N.; Marques, Isabel; Jorge, P.; Santos, Rosário
- A 26-Year Experience in Chorionic Villus Sampling Prenatal Genetic DiagnosisPublication . Jorge, P.; Mota-Freitas, M.; Santos, R.; Silva, M.; Soares, G.; Fortuna, A.Abstract: This report describes the trends of chorionic villus sampling (CVS) referred for prenatal genetic diagnosis in the past two and a half decades in a Portuguese Center. Our cohort of 491 CVS was mostly performed by the transcervical method at the 12th gestational week. Data collected within the framework of this study relate to the following: sampling method, referral reason versus abnormality and incidence of procedure-related pregnancy loss, that declined to about 0.5% over the last 15 years. The year 2000 represented a change in referral reasons for chorionic tissue collection, shifting from almost exclusively for cytogenetic testing to an increasing number of molecular tests for monogenic disorders. Herein, success rates as well as cytogenetic and/or molecular DNA results are presented. These latter include not only tests for several monogenic disorders, but also aneuploidy and maternal cell contamination screening. This retrospective analysis reiterates that CVS is a safe and reliable first trimester technique for prenatal diagnosis in high genetic risk pregnancies.
- 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.
- Sindrome de X frágil: pessoas, contextos & percursosPublication . Franco, V.; Martins, M.; Jorge, P.; Ferreira, F.; Bertão, A.; Apolónio, A.; Pires, H.; Melo, M.; Albuquerque, C.; Cunha, M.; Carmona, C.; Costa, T.; Reis, S.; Jiménez, S.
- Can the Synergic Contribution of Multigenic Variants Explain the Clinical and Cellular Phenotypes of a Neurodevelopmental Disorder?Publication . Maia, N; Nabais Sá, Maria João; Oliveira, Cláudia; Santos, Flávia; Soares, Celia A; Prior, Catarina; Tkachenko, Nataliya; Santos, Rosário; de Brouwer, Arjan P. M.; Jacome, Ariana; Porto, Beatriz; Jorge, PaulaWe describe an infant female with a syndromic neurodevelopmental clinical phenotype and increased chromosome instability as cellular phenotype. Genotype characterization revealed heterozygous variants in genes directly or indirectly linked to DNA repair: a de novo X-linked HDAC8 pathogenic variant, a paternally inherited FANCG pathogenic variant and a maternally inherited BRCA2 variant of uncertain significance. The full spectrum of the phenotype cannot be explained by any of the heterozygous variants on their own; thus, a synergic contribution is proposed. Complementation studies showed that the FANCG gene from the Fanconi Anaemia/BRCA (FA/BRCA) DNA repair pathway was impaired, indicating that the variant in FANCG contributes to the cellular phenotype. The patient's chromosome instability represents the first report where heterozygous variant(s) in the FA/BRCA pathway are implicated in the cellular phenotype. We propose that a multigenic contribution of heterozygous variants in HDAC8 and the FA/BRCA pathway might have a role in the phenotype of this neurodevelopmental disorder. The importance of these findings may have repercussion in the clinical management of other cases with a similar synergic contribution of heterozygous variants, allowing the establishment of new genotype-phenotype correlations and motivating the biochemical study of the underlying mechanisms.