RESUMO
Kabuki syndrome (KS) is a multiple congenital anomalies syndrome characterized by characteristic facial features and varying degrees of mental retardation, caused by mutations in KMT2D/MLL2 and KDM6A/UTX genes. In this study, we performed a mutational screening on 303 Kabuki patients by direct sequencing, MLPA, and quantitative PCR identifying 133 KMT2D, 62 never described before, and four KDM6A mutations, three of them are novel. We found that a number of KMT2D truncating mutations result in mRNA degradation through the nonsense-mediated mRNA decay, contributing to protein haploinsufficiency. Furthermore, we demonstrated that the reduction of KMT2D protein level in patients' lymphoblastoid and skin fibroblast cell lines carrying KMT2D-truncating mutations affects the expression levels of known KMT2D target genes. Finally, we hypothesized that the KS patients may benefit from a readthrough therapy to restore physiological levels of KMT2D and KDM6A proteins. To assess this, we performed a proof-of-principle study on 14 KMT2D and two KDM6A nonsense mutations using specific compounds that mediate translational readthrough and thereby stimulate the re-expression of full-length functional proteins. Our experimental data showed that both KMT2D and KDM6A nonsense mutations displayed high levels of readthrough in response to gentamicin treatment, paving the way to further studies aimed at eventually treating some Kabuki patients with readthrough inducers.
Assuntos
Anormalidades Múltiplas/genética , Face/anormalidades , Doenças Hematológicas/genética , Doenças Vestibulares/genética , Anormalidades Múltiplas/tratamento farmacológico , Linhagem Celular , Códon sem Sentido/efeitos dos fármacos , Estudos de Coortes , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Estudos de Associação Genética , Gentamicinas/farmacologia , Gentamicinas/uso terapêutico , Haploinsuficiência , Doenças Hematológicas/tratamento farmacológico , Histona Desmetilases/genética , Proteínas de Homeodomínio/genética , Humanos , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas Nucleares/genética , Sítios de Splice de RNA , Análise de Sequência de DNA , Transcrição Gênica , Doenças Vestibulares/tratamento farmacológicoRESUMO
Kabuki syndrome is a rare, multiple congenital anomaly/mental retardation syndrome caused by MLL2 point mutations and KDM6A microdeletions. We screened a large cohort of MLL2 mutation-negative patients for MLL2 and KDM6A exon(s) microdeletion and microduplication. Our assays failed to detect such rearrangements in MLL2 as well as in KDM6A gene. These results show that these genomic events are extremely rare in the Kabuki syndrome, substantiating its genetic heterogeneity and the search for additional causative gene(s).
Assuntos
Anormalidades Múltiplas/genética , Proteínas de Ligação a DNA/genética , Heterogeneidade Genética , Doenças Hematológicas/genética , Histona Desmetilases/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Doenças Vestibulares/genética , Anormalidades Múltiplas/diagnóstico , Adolescente , Criança , Pré-Escolar , Éxons , Face/anormalidades , Feminino , Deleção de Genes , Duplicação Gênica , Doenças Hematológicas/diagnóstico , Humanos , Lactente , Masculino , Fenótipo , Análise de Sequência de DNA , Doenças Vestibulares/diagnóstico , Adulto JovemRESUMO
BACKGROUND: Kabuki syndrome (Niikawa-Kuroki syndrome) is a rare, multiple congenital anomalies/mental retardation syndrome characterized by a peculiar face, short stature, skeletal, visceral and dermatoglyphic abnormalities, cardiac anomalies, and immunological defects. Recently mutations in the histone methyl transferase MLL2 gene have been identified as its underlying cause. METHODS: Genomic DNAs were extracted from 62 index patients clinically diagnosed as affected by Kabuki syndrome. Sanger sequencing was performed to analyze the whole coding region of the MLL2 gene including intron-exon junctions. The putative causal and possible functional effect of each nucleotide variant identified was estimated by in silico prediction tools. RESULTS: We identified 45 patients with MLL2 nucleotide variants. 38 out of the 42 variants were never described before. Consistently with previous reports, the majority are nonsense or frameshift mutations predicted to generate a truncated polypeptide. We also identified 3 indel, 7 missense and 3 splice site. CONCLUSIONS: This study emphasizes the relevance of mutational screening of the MLL2 gene among patients diagnosed with Kabuki syndrome. The identification of a large spectrum of MLL2 mutations possibly offers the opportunity to improve the actual knowledge on the clinical basis of this multiple congenital anomalies/mental retardation syndrome, design functional studies to understand the molecular mechanisms underlying this disease, establish genotype-phenotype correlations and improve clinical management.