ABSTRACT
OBJECTIVE: Mutations in the skeletal muscle gene dysferlin cause two autosomal recessive forms of muscular dystrophy: Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B). The purpose of this study was to define the genomic organization of the dysferlin gene and conduct mutational screening and a survey of clinical features in 21 patients with defined molecular defects in the dysferlin gene. METHODS: Genomic organization of the gene was determined by comparing the dysferlin cDNA and genomic sequence in P1-derived artificial chromosomes (PACs) containing the gene. Mutational screening entailed conformational analysis and sequencing of genomic DNA and cDNA. Clinical records of patients with defined dysferlin gene defects were reviewed retrospectively. RESULTS: The dysferlin gene encompasses 55 exons spanning over 150 kb of genomic DNA. Mutational screening revealed nine novel mutations associated with MM. The range of onset in this patient group was narrow with a mean of 19.0 +/- 3.9 years. CONCLUSION: This study confirms that the dysferlin gene is mutated in MM and LGMD2B and extends understanding of the timing of onset of the disease. Knowledge of the genomic organization of the gene will facilitate mutation detection and investigations of the molecular biologic properties of the dysferlin gene.
Subject(s)
Membrane Proteins , Muscle Proteins/genetics , Muscular Dystrophies/genetics , Mutation/genetics , Adolescent , Adult , Child , Chromosome Mapping , Dysferlin , Exons , Female , Genotype , Humans , Introns , Male , Polymorphism, Single-Stranded ConformationalABSTRACT
Miyoshi myopathy (MM) is an adult onset, recessive inherited distal muscular dystrophy that we have mapped to human chromosome 2p13. We recently constructed a 3-Mb P1-derived artificial chromosome (PAC) contig spanning the MM candidate region. This clarified the order of genetic markers across the MM locus, provided five new polymorphic markers within it and narrowed the locus to approximately 2 Mb. Five skeletal muscle expressed sequence tags (ESTs) map in this region. We report that one of these is located in a novel, full-length 6.9-kb muscle cDNA, and we designate the corresponding protein 'dysferlin'. We describe nine mutations in the dysferlin gene in nine families; five are predicted to prevent dysferlin expression. Identical mutations in the dysferlin gene can produce more than one myopathy phenotype (MM, limb girdle dystrophy, distal myopathy with anterior tibial onset).
Subject(s)
Membrane Proteins , Muscle Proteins/genetics , Muscular Dystrophies/genetics , Mutation , Adult , Amino Acid Sequence , Blotting, Northern , Cloning, Molecular , Dysferlin , Female , Gene Expression , Genetic Markers , Humans , Male , Molecular Sequence Data , Muscle Proteins/chemistry , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Polymorphism, Genetic , Polymorphism, Single-Stranded ConformationalABSTRACT
The Human Genome Project has created a formidable challenge: the extraction of biological information from extensive amounts of raw sequence. With the increasing availability of genomic sequence from other species, one approach to extracting coding and regulatory element information is through cross-species sequence comparison. To assess the strengths and weaknesses of this methodology for large-scale sequence analysis, 227 kb of mouse sequence syntenic to a gene-rich cluster on human chromosome 12p13 was obtained. Primarily through percent identity plots (PIPs) of SIM comparative sequence alignments, the sequence of coding regions, putative alternative exons, conserved noncoding regions, and correlation in repetitive element insertions were easily determined. The analysis demonstrated that the number, order, and orientation of all 17 genes are conserved between the two species, whereas two human pseudogenes are absent in mouse. In addition, apart from MIRs, no direct correlation of distribution or position of the majority of repetitive elements between the two species is seen. Finally, in examining the synonymous and nonsynonymous substitution rates in the conserved genes, a large variation in nonsynonymous rates is observed indicating that the genes in this region are diverging at different rates. This study indicates the utility and strength of large-scale cross-species sequence comparisons in the extraction of biological information from raw sequence, especially when combined with other computational tools such as GRAIL and BLAST.
Subject(s)
Chromosomes, Human, Pair 12/genetics , Chromosomes/genetics , Multigene Family , Amino Acid Sequence/genetics , Animals , Chromosome Mapping , Conserved Sequence , Humans , Mice , Molecular Sequence Data , Repetitive Sequences, Nucleic Acid , Sequence Alignment , Sequence Analysis, DNASubject(s)
DNA Helicases , Hominidae/genetics , Mice/genetics , Regulatory Sequences, Nucleic Acid , Sequence Alignment , Transcription Factors , Agammaglobulinaemia Tyrosine Kinase , Animals , Base Sequence , DNA-Binding Proteins/genetics , Gene Expression Regulation , Genome , Globins/genetics , Humans , Kinesins , Microtubule-Associated Proteins/genetics , Models, Genetic , Molecular Sequence Data , Myosins/genetics , Protein-Tyrosine Kinases/genetics , Proteins/genetics , Receptors, Antigen, T-Cell/genetics , X-ray Repair Cross Complementing Protein 1 , Xeroderma Pigmentosum Group D ProteinABSTRACT
Large-scale genomic DNA sequencing of orthologous and paralogous loci in different species should contribute to a basic understanding of the evolution of both the protein-coding regions and noncoding regulatory elements. We compared 93 kb of human sequence to 89 kb of mouse sequence in the Bruton's tyrosine kinase (BTK) region. In addition to showing the conservation of both position and orientation of the five functionally unrelated genes in the region (BTK, alpha-D-galactosidase A, L44L, FTP-3, and FCI-12), the comparison revealed conservation of clusters of noncoding sequence flanking the first exon of each gene. Furthermore, in the sequence comparison at the BTK locus, the conservation of clusters of noncoding sequence extends throughout the locus; the noncoding sequence is more highly conserved in the BTK locus in comparison to the flanking loci. This suggests a correlation with the complex developmental regulation of expression of btk. To determine whether a highly conserved 3.5-kb segment flanking the first exon of BTK contains transcriptional regulatory signals, we tested various portions of the segment for promoter and expression activity in several appropriate cell lines. The results demonstrate the contribution of the conserved region flanking the first exon to the cell lineage-specific expression pattern of btk. These data show the usefulness of large scale sequence comparisons to focus investigation on regions of noncoding sequence that play essential roles in complex gene regulation.
Subject(s)
Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Regulatory Sequences, Nucleic Acid , Sequence Analysis, DNA/methods , Sequence Homology, Nucleic Acid , Agammaglobulinaemia Tyrosine Kinase , Animals , Base Sequence , Conserved Sequence , Enhancer Elements, Genetic , Genetic Variation , Humans , Mice , Models, Genetic , Molecular Sequence Data , Promoter Regions, Genetic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repetitive Sequences, Nucleic Acid , Sequence Alignment , Transcription Factors/metabolism , Transcription, Genetic , Transfection , alpha-Galactosidase/geneticsABSTRACT
Several disease loci have been mapped to the Xq21.3-Xq22 region of the human X Chromosome (Chr) including X-linked agammaglobulinemia (XLA), Fabry disease, Alport syndrome, and Pelizaeus Merzbacher disease. Upon cloning of the XLA gene, Bruton's tyrosine kinase (btk), both Fabry disease and XLA were mapped within the same 50- to 70-kb interval. In order to investigate the genomic organization of the region surrounding btk and the Fabry disease gene, alpha-galactosidase A (gla), we constructed a 6-cosmid contig spanning the region from 5' of gla to 3' of btk. Two of these cosmids spanning most of the coding sequence and the upstream region of btk and gla, U237D10 and U230D1, were sequenced by a random shotgun strategy combined with automated sequencing, resulting in 69 kb of contiguous genomic sequence. Sequencing of U237D10 showed btk to be comprised of 19 exons spanning over 35 kb. Sequencing of U230D1 showed that the 3' end of gla is 9 kb from the 5' end of btk and also demonstrated the presence of two additional genes in the region immediately 5' to btk. The surprisingly high gene density is similar to that seen previously only in the human major histocompatibility locus.
