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- Study of FRAXE-MR in intellectually disabled individuals referred for Fragile-X Syndrome testing in PortugalPublication . Jorge, Paula; Marques, Isabel; Gonçalves, Rui; Gonçalves-Rocha, Miguel; Santos, RosárioAmong the genetic causes involved in X-linked intellectual disability (XLID), pathogenic variations in FMR1 (Fragile Mental Retardation 1), AFF2 (AF4/ FMR2 family member 2) and ARX (Aristaless Related Homeobox) genes emerge as main causes. FMR1 and AFF2 genes contain (polymorphic repetitive regions) a repeat polymorphism which is susceptible to suffer dynamic mutation, a process that may induce pathogenic expansions. FRAXEassociated mental retardation (FRAXE-MR) is mainly a non-syndromic form of XLID and is due to AFF2 gene silencing as a consequence of 5’UTR-CCG expansion or gene mutations. A CCG triplet number up to 30 repeats is consider normal, while full expansion (>200 repeats) and hypermethylation of CCG cluster results in FRAXE-MR. AFF2 variants are not frequently sought. An implementation of a cost-effective strategy (co-amplification with other ID genes) represents an improvement in molecular diagnosis with consequent gains in clinical genetic diagnosis and counseling. Herein we present results of AFF2 molecular analysis in a subpopulation of 5000 intellectually-disabled individuals with primary referral for FRAXA screening, by a novel multiplex-PCR strategy. This approach accurately detected normal to pre-mutated alleles. A pre-mutated allele with 68 CCG was identified and further characterized by Southern blot analysis in order to exclude methylation and/or repeat number mosaics, as well as PCR failure. Possible phenotype-genotype correlations based on the clinical data of one previously diagnosed family with AFF2 full expansion, the newly characterized pre-mutation carrier and one case with a new variant of the AFF2 gene will be investigated and presented.
- Further contributions towards the molecular analysis of NIPBL and SMC1A genes in a cohort of patients with Cornelia de Lange SyndromePublication . Pinto da Costa, Eurico; Oliveira, Jorge; Silva, João; Santos, RosárioCornelia de Lange Syndrome [CdLS (MIM#122470)] is a rare multisystemic disorder, characterized by a typical phenotype that includes distinctive facial dysmorphism, hirsutism, growth and psychomotor developmental delay, abnormalities of the upper extremities, and relatively frequent gastrointestinal and congenital heart defects. CdLS is essentially caused by mutations in the NIPBL and SMC1A genes (~50% and 5% of cases, respectively), but mutations have been also described in other genes (PDS5A, PDS5B, SMC3) (http://www.lovd.nl/CDLS). This genetic heterogeneity in CdLS can however be explained by a close functional relationship at the cellular level, since all these proteins are involved in chromatid cohesion. The molecular and clinical characterization of 42 Portuguese CdLS patients has been previously described (Oliveira et al., 2010). In this work we conducted the molecular analysis of NIPBL gene and more recently expanded this study to other 20 patients. Subsequently, all the molecularly unresolved patients were screened for large deletions and duplications in NIPBL by MLPA, and point mutations in SMC1A by high resolution melting curve analysis and sequencing. Preliminary results allowed us to identify 4 previously known mutations (including a case with somatic mosaicism), 3 novel mutations (c.3316C>T, c.6983C>G and c.7307C>T) and 2 large deletions in the NIPBL gene. Mutation screening in SMC1A is still ongoing. Our results, at least for NIPBL gene analysis, suggest that the use of several different techniques is essential for attaining a high mutation detection rate. CdLS cases with somatic mosaicism are probably underestimated in the literature and may explain some degree of phenotypical variability.