Molecular genetics of charcot-marie-tooth disease: from genes to genomes.

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of disorders of the peripheral nervous system, mainly characterized by distal muscle weakness and atrophy leading to motor handicap. With an estimated prevalence of 1 in 2,500, this condition is one of the most commonly inherited neurological disorders. Mutations in more than 30 genes affecting glial and/or neuronal functions have been associated with different forms of CMT leading to a substantial improvement in diagnostics of the disease and in the understanding of implicated pathophysiological mechanisms. However, recent data from systematic genetic screening performed in large cohorts of CMT patients indicated that molecular diagnosis could be established only in ∼50-70% of them, suggesting that additional genes are involved in this disease. In addition to providing an overview of genetic and functional data concerning various CMT forms, this review focuses on recent data generated through the use of highly parallel genetic technologies (SNP chips, sequence capture and next-generation DNA sequencing) in CMT families, and the current and future impact of these technologies on gene discovery and diagnostics of CMTs.

Differential gene expression studies to explore the molecular pathophysiology of Down syndrome.

Trisomy 21, which causes Down syndrome, is the model human disorder due to the presence of a supernumerary chromosome. The completion of the sequence of chromosome 21 and the development of appropriate animal models now provide the molecular infrastructure and the reagents to elucidate the molecular mechanisms of the different phenotypes of Down syndrome. The study of the overexpression of single genes, and the dysregulation of global gene expression will enhance the understanding of the pathogenesis of the cognitive impairment of this syndrome.

Insertion of beta-satellite repeats identifies a transmembrane protease causing both congenital and childhood onset autosomal recessive deafness.

Approximately 50% of childhood deafness is caused by mutations in specific genes. Autosomal recessive loci account for approximately 80% of nonsyndromic genetic deafness. Here we report the identification of a new transmembrane serine protease (TMPRSS3; also known as ECHOS1) expressed in many tissues, including fetal cochlea, which is mutated in the families used to describe both the DFNB10 and DFNB8 loci. An 8-bp deletion and insertion of 18 monomeric (approximately 68-bp) beta-satellite repeat units, normally present in tandem arrays of up to several hundred kilobases on the short arms of acrocentric chromosomes, causes congenital deafness (DFNB10). A mutation in a splice-acceptor site, resulting in a 4-bp insertion in the mRNA and a frameshift, was detected in childhood onset deafness (DFNB8). This is the first description of beta-satellite insertion into an active gene resulting in a pathogenic state, and the first description of a protease involved in hearing loss.

The mouse brain transcriptome by SAGE: differences in gene expression between P30 brains of the partial trisomy 16 mouse model of Down syndrome (Ts65Dn) and normals.

Trisomy 21, or Down syndrome (DS), is the most common genetic cause of mental retardation. Changes in the neuropathology, neurochemistry, neurophysiology, and neuropharmacology of DS patients' brains indicate that there is probably abnormal development and maintenance of central nervous system structure and function. The segmental trisomy mouse (Ts65Dn) is a model of DS that shows analogous neurobehavioral defects. We have studied the global gene expression profiles of normal and Ts65Dn male and normal female mice brains (P30) using the serial analysis of gene expression (SAGE) technique. From the combined sample we collected a total of 152,791 RNA tags and observed 45,856 unique tags in the mouse brain transcriptome. There are 14 ribosomal protein genes (nine under expressed) among the 330 statistically significant differences between normal male and Ts65Dn male brains, which possibly implies abnormal ribosomal biogenesis in the development and maintenance of DS phenotypes. This study contributes to the establishment of a mouse brain transcriptome and provides the first overall analysis of the differences in gene expression in aneuploid versus normal mammalian brain cells.

Mice trisomic for a bacterial artificial chromosome with the single-minded 2 gene (Sim2) show phenotypes similar to some of those present in the partial trisomy 16 mouse models of Down syndrome.

The Drosophila single-minded (sim) transcription factor, is a master regulator of fruitfly neurogenesis. Recently, we have cloned and mapped a human homolog of sim, SIM2, to chromosome 21 in the so-called 'Down syndrome chromosomal region'. Three copies of SIM2 may contribute to some Down syndrome (DS) phenotypes because of the mapping position function as transcriptional repressor, temporal and spatial expression pattern of mouse Sim2, and the potentially analogous role of human SIM2 to that of Drosophila sim during neurogenesis. In order to validate this hypothesis in vivo, we have created the first bacterial artificial chromosome transgenic mice overexpressing a gene possibly involved in DS with only one or two additional copies of mouse Sim2. The transgene was shown to be expressed in the same spatial pattern as the endogenous gene. The mice develop normally, are fertile and do not show detectable histopathological abnormalities. However, detailed analysis of their behavior revealed anxiety-related/reduced exploratory behaviour and sensitivity to pain, phenotypes similar to those also present in other partial trisomy 16 mouse models of DS. Our data therefore suggest that overexpression of SIM2 contributes to some of the complex DS phenotypes.

Linearization and purification of BAC DNA for the development of transgenic mice.

Bacterial artificial chromosome (BAC) vectors are increasingly used for generation of transgenic mice due to the relatively large size and the stability of their inserts compared to YACs. We have compared methods for purification and linearization of BACs, and describe an optimised protocol for preparation of high quality linear BAC DNA based on lambda terminase digestion, electroelution of linearized DNA together with simple preliminary multiplex PCR screening to detect transgenic mice. Linearized BAC DNA purified this way was successfully used for the development of transgenic mice containing 2-4 copies of the transgene.

Cloning of two human homologs of the Drosophila single-minded gene SIM1 on chromosome 6q and SIM2 on 21q within the Down syndrome chromosomal region.

As part of our effort to clone genes of human chromosome 21 that may contribute to Down syndrome, we have previously isolated four exons with homology to Drosophila single-minded (sim) gene, which encodes a transcription factor that is a master regulator of fruit fly neurogenesis. These exons were used to clone and characterize two human homologs of the Drosophila sim gene, SIM1 and SIM2, which map to chromosomes 6q16.3-q21 and 21q22.2, respectively; SIM2 maps within the so-called Down syndrome chromosomal region. Recently, two mouse homologs, Sim1 and Sim2, also have been identified. There is a high level of homology among human, mouse, and Drosophila sim genes in their amino-terminal half where the conserved bHLH, PAS1, PAS2, and HST domains are present. In contrast, the carboxy-terminal parts are only homologous between SIM1 and Sim1 and SIM2 and Sim2. Two isoforms (SIM2 and SIM2s) of human SIM2 have been detected that differ in their 3' ends. Northern blot analysis revealed one mRNA SIM1 species of approximately 9.5 kb and four different mRNA SIM2 species of 2.7, 3, 4.4, and 6 kb in human fetal kidney. The function of both human SIM1 and SIM2 is unknown. However, three copies of SIM2 may contribute to some specific Down syndrome phenotypes because of (1) mapping position, (2) potential function as transcriptional repressor, (3) likely dimerization with other transcription factors, (4) the temporal and spatial expression pattern of mouse Sim2, and (5) the potentially analogous role of human SIM2 to that of Drosophila sim during neurogenesis.

Cloning of 559 potential exons of genes of human chromosome 21 by exon trapping.

Chromosome 21 represents approximately 1% of the human genome, and its long arm has been estimated to contain 600-1000 genes. A dense linkage map and almost complete physical maps based on yeast artificial chromosomes (YACs) and cosmids have been developed. We have used exon trapping to identify portions of genes from randomly picked chromosome 21-specific cosmids, to contribute to the creation of the transcription (genic) map of this chromosome and the cloning of its genes. A total of 559 different sequences were identified after elimination of false-positive clones and repetitive elements. Among these, exons for 13 of the 30 known chromosome 21 genes have been "trapped." In addition, a considerable number of trapped sequences showed homologies to genes from other species and to human expressed sequence tags (ESTs). One hundred thirty-three trapped sequences were mapped, and every one mapped back to chromosome 21. We estimate that we have identified portions of up to approximately 40% of all genes on chromosome 21. The genic map of chromosome 21 provides a valuable tool for the elucidation of function of the genes and will enhance our understanding of the pathophysiology of Down syndrome and other disorders of chromosome 21 genes.

Localization of 102 exons to a 2.5 Mb region involved in Down syndrome.

Exon amplification has been applied to a 2.5 Mb region of chromosome 21 that has been associated with some features of Down syndrome (DS). Identification of the majority of genes from this region will facilitate the correlation of the over-expression of particular genes with specific phenotypes of DS. Over 100 gene fragments have been isolated from this 2.5 Mb segment. The exons have been characterized by sequence analysis, comparison with public databases and expansion to cDNA clones. Localization of the exons to chromosome 21 has been determined by hybridization to genomic Southern blots and to YAC and cosmid clones representing the region. This has resulted in a higher resolution physical map with a marker approximately every 25 kb. This integrated physical and transcript map will be valuable for fine mapping of DNA from individuals with partial aneuploidy of chromosome 21 as well as for assessing and ultimately generating a complete gene map of this segment of the genome.

Mapping of the human transcription factor GABPA (E4TF1-60) gene to chromosome 21.

Exon trapping/amplification was used to clone portions of genes from human chromosome 21. One trapped sequence showed complete homology with nucleotide sequence D13318 of GenBank, which corresponds to the gene for human transcription factor E4TF1-60 (HGMW-approved nomenclature GABPA). We mapped this gene to human chromosome 21 by FISH, somatic cell hybrids, and hybridization to chromosome 21-specific YACs. The GABPA gene localizes to YACs 816B7 and 848G1 of the Chumakov et al. (Nature 359: 380, 1992) YAC contig, near the APP gene in 21q21-q22.1. This transcription factor, which is an ETS-related DNA-binding protein and forms heterodimers with other polypeptides, may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome.