RESUMO
Loss-of-function mutations in the Src homology 3 (SH3) domain and tetratricopeptide repeats 2 (SH3TC2) gene cause autosomal recessive demyelinating Charcot-Marie-Tooth neuropathy. The SH3TC2 protein has been implicated in promyelination signaling through axonal neuregulin-1 and the ERBB2 Schwann cell receptor. However, little is known about the transcriptional regulation of the SH3TC2 gene. We performed computational and functional analyses that revealed two cis-acting regulatory elements at SH3TC2-one at the promoter and one â¼150 kb downstream of the transcription start site. Both elements direct reporter gene expression in Schwann cells and are responsive to the transcription factor SOX10, which is essential for peripheral nervous system myelination. The downstream enhancer harbors a single-nucleotide polymorphism (SNP) that causes an â¼80% reduction in enhancer activity. The SNP resides directly within a predicted binding site for the transcription factor cAMP response element binding protein (CREB), and we demonstrate that this regulatory element binds to CREB and is activated by CREB expression. Finally, forskolin induces Sh3tc2 expression in rat primary Schwann cells, indicating that SH3TC2 is a CREB target gene. These findings prompted us to determine if SNP genotypes at SH3TC2 are associated with differential phenotypes in the most common demyelinating peripheral neuropathy, CMT1A. Interestingly, this revealed several associations between SNP alleles and disease severity. In summary, our data indicate that SH3TC2 is regulated by the transcription factors CREB and SOX10, define a regulatory SNP at this disease-associated locus and reveal SH3TC2 as a candidate modifier locus of CMT disease phenotypes.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Haplótipos , Proteínas/genética , Elementos de Resposta , Fatores de Transcrição SOXE/metabolismo , Alelos , Animais , Sequência de Bases , Sítios de Ligação , Doença de Charcot-Marie-Tooth/diagnóstico , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Colforsina/farmacologia , Biologia Computacional , Sequência Conservada , Bases de Dados Genéticas , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Loci Gênicos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Neurônios Motores/metabolismo , Motivos de Nucleotídeos , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Sequências Reguladoras de Ácido Nucleico , Células de Schwann/metabolismo , Alinhamento de Sequência , Índice de Gravidade de Doença , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
INTRODUCTION: Charcot-Marie-Tooth (CMT) disease is a group of peripheral neuropathies affecting both motor and sensory nerves. CMTX3 is an X-linked CMT locus, which maps to chromosome Xq26.3-q27.3. Initially, CMTX3 was mapped to a 31.2-Mb region in 2 American families. We have reexamined 1 of the original families (US-PED2) by next generation sequencing. METHODS: Three members of the family underwent exome sequencing. Candidate variants were validated by PCR and Sanger sequencing analysis. CONCLUSION: No pathogenic coding variants localizing to the CMTX3 region were identified. However, exome sequencing identified a known BSCL2 mutation (N88S). This study demonstrates the power of exome sequencing as a tool to identify gene mutations for a small family in the absence of statistically significant linkage data.