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Title: Isolation and characterization of candidate genes of the 5q13 region in spinal muscular atrophy

Abstract

Based on a fine genetic and physical map of the region deleted in spinal muscular atrophy, we defined the smallest rearrangements encompassing the SMA gene. This interval is entirely contained in the 903D1 YAC clone. Several approaches to identify candidate genes were applied, including the search for interspecies conservation, exon trapping amplification and direct cDNA selection. Combining these strategies, six different cDNA molecules mapping to the YAC contig were isolated. Four cDNA molecules were isolated using the exon trapping system. They map to chromosome 5p and to more than one locus within the 5q13 region. They are homologous to each other and share sequence homology with the {beta}-glucuronidase gene. Based on interspecies conservation, a fifth candidate gene was identified. Sequence analyses of the cDNAs revealed no homologies with any other described genes. This gene mapped to two loci within the 5q13 region. Two other cDNA molecules isolated by direct cDNA selection are also under investigation. Complete characterization and fine physical mapping of those genes with respect to the physical interval defined by the deletions of the SMA region will allow the identification of the disease gene (or genes).

Authors:
; ;  [1]
  1. Hopital Enfants-Malades, Paris (France) [and others
Publication Date:
OSTI Identifier:
134432
Report Number(s):
CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-1166
Resource Type:
Journal Article
Resource Relation:
Journal Name: American Journal of Human Genetics; Journal Volume: 55; Journal Issue: Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; GENES; GENE MUTATIONS; GENETIC MAPPING; DNA-CLONING; BIOLOGICAL EVOLUTION; HUMAN CHROMOSOME 5; DISEASES; MUSCLES; YEASTS; CONTIGS; EXONS; GENE AMPLIFICATION; DNA SEQUENCING

Citation Formats

Lefebvre, S., Reboullet, S., and Benichou, B.. Isolation and characterization of candidate genes of the 5q13 region in spinal muscular atrophy. United States: N. p., 1994. Web.
Lefebvre, S., Reboullet, S., & Benichou, B.. Isolation and characterization of candidate genes of the 5q13 region in spinal muscular atrophy. United States.
Lefebvre, S., Reboullet, S., and Benichou, B.. 1994. "Isolation and characterization of candidate genes of the 5q13 region in spinal muscular atrophy". United States. doi:.
@article{osti_134432,
title = {Isolation and characterization of candidate genes of the 5q13 region in spinal muscular atrophy},
author = {Lefebvre, S. and Reboullet, S. and Benichou, B.},
abstractNote = {Based on a fine genetic and physical map of the region deleted in spinal muscular atrophy, we defined the smallest rearrangements encompassing the SMA gene. This interval is entirely contained in the 903D1 YAC clone. Several approaches to identify candidate genes were applied, including the search for interspecies conservation, exon trapping amplification and direct cDNA selection. Combining these strategies, six different cDNA molecules mapping to the YAC contig were isolated. Four cDNA molecules were isolated using the exon trapping system. They map to chromosome 5p and to more than one locus within the 5q13 region. They are homologous to each other and share sequence homology with the {beta}-glucuronidase gene. Based on interspecies conservation, a fifth candidate gene was identified. Sequence analyses of the cDNAs revealed no homologies with any other described genes. This gene mapped to two loci within the 5q13 region. Two other cDNA molecules isolated by direct cDNA selection are also under investigation. Complete characterization and fine physical mapping of those genes with respect to the physical interval defined by the deletions of the SMA region will allow the identification of the disease gene (or genes).},
doi = {},
journal = {American Journal of Human Genetics},
number = Suppl.3,
volume = 55,
place = {United States},
year = 1994,
month = 9
}
  • Childhood-onset proximal spinal muscular atrophy (SMA) is a heritable neurological disorder, which has been mapped by genetic linkage analysis to chromosome 5q13, in the interval between markers D5S435 and D5S557. Here, we present gene sequences that have been isolated from this interval, several of which show sequence homologies to exons of {beta}-glucuronidase. These gene sequences are repeated several times across the candidate region and are also present on chromosome 5p. The arrangement of these repetitive gene motifs is polymorphic between individuals. The high degree of variability observed may have some influence on the expression of the genes in the region.more » Since SMA is not inherited as a classical autosomal recessive disease, novel genomic rearrangements arising from aberrant recombination events between the complex repeats may be associated with the phenotype observed.« less
  • We previously reported that the 400 kb interval flanked the polymorphic loci D5S435 and D5S557 contains blocks of a chromosome 5 specific repeat. This interval also defines the SMA candidate region by genetic analysis of recombinant families. A YAC contig of 2-3 Mb encompassing this area has been constructed and a 5.5 kb conserved fragment, isolated from a YAC end clone within the above interval, was used to obtain cDNAs from both fetal and adult brain libraries. We describe the identification of cDNAs with stretches of high DNA sequence homology to exons of {beta} glucuronidase on human chromosome 7. Themore » cDNAs map both to the candidate region and to an area of 5p using FISH and deletion hybrid analysis. Hybridization to bacteriophage and cosmid clones from the YACs localizes the {beta} glucuronidase related sequences within the 400 kb region of the YAC contig. The cDNAs show a polymorphic pattern on hybridization to genomic BamH1 fragments in the size range of 10-250 kb. Further analysis using YAC fragmentation vectors is being used to determine how these {beta} glucuronidase related cDNAs are distributed within 5q13. Dinucleotide repeats within the region are being investigated to determine linkage disequilibrium with the disease locus.« less
  • The SMA disease gene region on chromosome 5q13 has been characterized by the presence of low copy repeat element DNA and concomitant genomic instability. The identification of candidate genes has been complicated by the low-copy repeat sequences which include coding and non-coding DNA. To circumvent this problem, we have focused on the identification and characterization of exonic DNA sequence isolated from YAC-derived bacteriophage subclones. Contrary to hybridization-based protocols which identify all closely homologous genes, exons isolated in this manner are known to map the disease gene region. Exon trapping was limited to an approximately 400 kb {open_quotes}minimum genetic region{close_quotes} definedmore » by recombination mapping. Approximately six new genes have been identified by this method. One of the candidate genes is expressed predominantly in muscle and demonstrates sequence homology with known translational regulatory proteins. Physical mapping places the gene within the minimal genetic region and adjacent to a region characterized with genomic instability. We will report our progress in the identification of disease-specific mutations in this gene based upon comparison of normal and SMA cDNA sequence, amplification and sequencing of SMA DNA samples, and RT-PCR sequencing studies.« less
  • A high number of cosmids and phages were identified that contained di-, tri-, and tetranucleotide repeats. These clones were isolated from STSs that map to the SMA region as well as from three cosmid and one phage library that had been prepared from YACs which span the SMA candidate interval. We developed two new microsatellites, A31 (D5S823) and 95/23, which enabled us to define new borders of the SMA region and to reduce it to approximately 700 kb. Physically, the marker A31 maps to the overlapping region of the YACs y116, y55 and y122 at about 550 kb distal tomore » the locus D5S435. A recombination in one SMA type I family places A31 proximal to the SMA gene. The multicopy microsatellite, 95/23, developed from a cosmid including the STS y97U revealed in a consanguine SMA type I family different alleles, while all proximal markers were heterozygous. This suggests a location of 95/23 distal to the SMA gene. Furthermore, we tested 157 German SMA families (100 SMA type I, 50 SMA type II and 20 SMA type III) for linkage disequilibrium with the marker AG1-CA which reveals no recombination with the SMA gene. We found in SMA type I families strong allelic association between the 100 bp allele and the SMA gene. In 7 families we got deletions within the AG1-CA microsatellite. On the way to isolate the SMA gene, we hybridized whole cosmid and YAC inserts to cDNA libraries of spinal cord, brain and muscle. Seven cDNAs were initially identified and cDNA walking was used to begin the isolation of whole transcribed sequences.« less
  • The mutation that underlies the autosomal recessive disorder spinal muscular atrophy (SMA) is located on chromosome 5q13. Recent studies show that SMA patients frequently have deletions and rearrangements in this region compared to normal controls. During the isolation of candidate cDNAs for the disease, the authors identified a sequence that shows high homology to the THE-1 retrotransposon gene family. Using YAC fragmentation techniques, they have refined the localization of this sequence to the domain known to show instability in SMA patients. The implication of these results for the mechanism of the mutation in SMA is discussed. 20 refs., 1 fig.