The study of MED12 gene in Lujan-Fryns and Opitz-Kaveggia syndromes
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2015
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O défice intelectual consiste numa das formas mais comuns de alterações do desenvolvimento neurológico e é caracterizado por limitações não só a nível da função cognitiva, como também do comportamento adaptativo. As síndromes de Lujan-Fryns (LF; OMIM#309520) e Opitz-Kaveggia (FG; OMIM#305450) são formas raras de défice intelectual ligado ao cromossoma X (XLID). Indivíduos com a síndrome de LF podem ser reconhecidos, clinicamente, por determinados traços faciais e pela presença de algumas das seguintes características: estatura marfanóide, agenesia do corpo caloso, macrocefalia, mãos longas com dedos hiperextensíveis e hipotonia ligeira. Relativamente aos indivíduos diagnosticados com a síndrome de FG, estes apresentam uma aparência facial e características morfológicas distintas, incluindo orelhas pequenas e salientes com padrão helicoidal simples, zona frontal do cabelo em curva ascendente, estatura baixa, dedos pequenos com polegares largos, hipertelorismo, hipotonia e obstipação. Ambas as síndromes são comumente caracterizadas por défice intelectual ligeiro ou moderado e por problemas comportamentais, como por exemplo, personalidade hiperativa.
O gene MED12 está localizado no braço longo do cromossoma X (Xq13) e codifica a proteína MED12, uma subunidade fundamental do complexo Mediador. A proteína MED12 (OMIM#300188) está envolvida em diferentes vias do desenvolvimento e na regulação da expressão de genes neuronais. Alterações na sequência do gene MED12 podem causar XLID. As síndromes de LF, FG e Ohdo foram previamente descritas como sendo frequentemente associadas a variantes neste gene. Algumas mutações no gene MED12 foram consideradas como a causa das síndromes de LF e FG. Na síndrome de LF apenas a mutação c.3020A>G no exão 22 foi descrita, enquanto que na síndrome de FG duas mutações localizadas no exão 21 foram descritas, nomeadamente c.2873G>A e c.2881C>T.
O principal objetivo deste trabalho foi a análise de variantes no gene MED12 em 43 pacientes do sexo masculino, por PCR seguido de sequenciação de Sanger. Para determinar o impacto das variantes encontradas, estudos in silico foram realizados utilizando ferramentas bioinformáticas. Os resultados revelaram a presença de 34 variantes MED12 distintas. A maioria das variantes encontradas localizavam-se em regiões intrónicas, sendo apenas quatro alterações exónicas. Nenhuma das mutações publicadas foi encontrada mas, por sua vez, nove das 34 variantes foram consideradas como alterações de significado desconhecido (VUS). O estudo de familiares foi realizado para quatro das alterações VUS identificadas. Os estudos de co-segregação sugeriram a variante c.397-39G>A como uma potencial causa para a síndrome de LF. Estudos preliminares, incluindo a amplificação de cDNA, foram realizados para esta alteração e os resultados obtidos não foram conclusivos, sendo, por isso, ainda considerada como VUS. Outros estudos devem ser realizados para todas as VUS encontradas, nomeadamente estudos de co-segregação, análise de RNA e investigação complementar relativa ao impacto na expressão proteica.
O contínuo estudo do gene MED12 e a análise de todas as suas variantes desempenham um papel fundamental na compreensão da relação genótipo-fenótipo associado a XLID. O gene MED12 é um grande candidato a ser considerado como causa genética das síndromes de XLID, incluindo as síndromes de FG e LF, mas a análise de outros genes também deve ser considerada.
Intellectual disability is one of the most common neurodevelopmental disorders and is characterized by limitations not only in cognitive function but also in adaptative behaviour. Lujan-Fryns syndrome (LF; OMIM#309520) and Opitz-Kaveggia syndrome (FG; OMIM#305450) are rare forms of X-linked Intellectual Disability (XLID). Patients with LF syndrome can be clinically recognized by a peculiar facies in addition to the presentation of some of the following features: tall marfanoid stature, dysgenesis of the corpus callosum, macrocephaly, long hands with hyperextensible digits and mild general hypotonia. Additionally, patients with the diagnosis of FG syndrome show a distinctive facial appearance and morphological features, including small prominent ears with simplified helical pattern, a frontal hair upsweep, short stature, relatively short fingers with broad thumbs, hypertelorism, hypotonia and constipation. Mild to moderate intellectual disability and several behavioural problems, e.g. hyperactivity personality, are commonly present in both syndromes. MED12 gene is localized at long arm of X chromosome (Xq13) and encodes for MED12 protein, an essential subunit of the Mediator complex. MED12 protein (OMIM#300188) is implicated in different developmental pathways and is involved in the regulation of neuronal gene expression. Alterations on MED12 gene sequence can lead to XLID disorders. LF, FG and Ohdo syndromes have been previously described as being frequently associated with MED12 variants. Few MED12 mutations were found to be the cause of LF and FG syndromes. In LF syndrome a single mutation c.3020A>G in exon 22 has been reported, while in FG syndrome two mutations both in exon 21, have been described: c.2873G>A and c.2881C>T. The aim of this work was the screening for MED12 gene variants in a cohort of 43 male patients by PCR-amplification followed by Sanger sequencing. In order to determine the impact of the variants found, in silico studies using bioinformatics tools were carried out. Results revealed the presence of 34 different MED12 variants. The majority of these variants were located in intronic regions, being only four the exonic variants. None of the already published mutations were found in the cohort but in turn nine of the 34 variants were considered as variants of uncertain significance (VUS). Family members study was performed for four of the VUS identified. The co-segregation studies indicated the c.397-39G>A variant as a potential cause for LF syndrome. Preliminary studies, including amplification of cDNA, were performed for this variant and not conclusive results were achieved, still being considered as VUS. Further investigation must be performed for all VUS found in this work, namely co-segregation studies, RNA analysis and additional research for the impact on protein expression. The continuous study of MED12 gene and the analysis of all its variants have a major role for the understanding of the relation between genotype and phenotype associated with XLID. MED12 gene is a great candidate as cause of XLID syndromes, like FG and LF syndromes, but the study of other genes must not be ruled-out.
Intellectual disability is one of the most common neurodevelopmental disorders and is characterized by limitations not only in cognitive function but also in adaptative behaviour. Lujan-Fryns syndrome (LF; OMIM#309520) and Opitz-Kaveggia syndrome (FG; OMIM#305450) are rare forms of X-linked Intellectual Disability (XLID). Patients with LF syndrome can be clinically recognized by a peculiar facies in addition to the presentation of some of the following features: tall marfanoid stature, dysgenesis of the corpus callosum, macrocephaly, long hands with hyperextensible digits and mild general hypotonia. Additionally, patients with the diagnosis of FG syndrome show a distinctive facial appearance and morphological features, including small prominent ears with simplified helical pattern, a frontal hair upsweep, short stature, relatively short fingers with broad thumbs, hypertelorism, hypotonia and constipation. Mild to moderate intellectual disability and several behavioural problems, e.g. hyperactivity personality, are commonly present in both syndromes. MED12 gene is localized at long arm of X chromosome (Xq13) and encodes for MED12 protein, an essential subunit of the Mediator complex. MED12 protein (OMIM#300188) is implicated in different developmental pathways and is involved in the regulation of neuronal gene expression. Alterations on MED12 gene sequence can lead to XLID disorders. LF, FG and Ohdo syndromes have been previously described as being frequently associated with MED12 variants. Few MED12 mutations were found to be the cause of LF and FG syndromes. In LF syndrome a single mutation c.3020A>G in exon 22 has been reported, while in FG syndrome two mutations both in exon 21, have been described: c.2873G>A and c.2881C>T. The aim of this work was the screening for MED12 gene variants in a cohort of 43 male patients by PCR-amplification followed by Sanger sequencing. In order to determine the impact of the variants found, in silico studies using bioinformatics tools were carried out. Results revealed the presence of 34 different MED12 variants. The majority of these variants were located in intronic regions, being only four the exonic variants. None of the already published mutations were found in the cohort but in turn nine of the 34 variants were considered as variants of uncertain significance (VUS). Family members study was performed for four of the VUS identified. The co-segregation studies indicated the c.397-39G>A variant as a potential cause for LF syndrome. Preliminary studies, including amplification of cDNA, were performed for this variant and not conclusive results were achieved, still being considered as VUS. Further investigation must be performed for all VUS found in this work, namely co-segregation studies, RNA analysis and additional research for the impact on protein expression. The continuous study of MED12 gene and the analysis of all its variants have a major role for the understanding of the relation between genotype and phenotype associated with XLID. MED12 gene is a great candidate as cause of XLID syndromes, like FG and LF syndromes, but the study of other genes must not be ruled-out.
Descrição
Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Gene MED12 , Mutação , Genética molecular , Deficiência intelectual , Processamento alternativo , Síndrome de Lujan-Fryns , Síndrome de Opitz-Kaveggia