Primary gamma-sarcoglycanopathy (LGMD 2C): broadening of the mutational spectrum guided by the immunohistochemical profile.

An important step in the diagnostic evaluation of a patient with recessive limb-girdle muscular dystrophy is the immunohistochemical analysis of the components of the sarcoglycan complex in a muscle biopsy specimen. Even though a primary mutation in any of the four sarcoglycan genes (alpha-, beta-,gamma-, delta-sarcoglycan) may cause secondary deficiencies in all the other sarcoglycan proteins, more specific immunohistochemical patterns have emerged with the potential to guide and abbreviate the necessary molecular genetic investigations. In gamma-sarcoglycan mutations, the pattern consists of absent or prominently reduced gamma-sarcoglycan immunoreactivity in combination with reduced but detectable immunoreactivity for the other components, with preservation of delta-sarcoglycan. In five consecutive patients, this pattern was able to predict primary gamma-sarcoglycan mutations. Five different mutations were found, including a recurrent novel splice mutation, a large deletion of the entire gene and a novel missense mutation (Leu90Ser). The mutation Cys283Tyr, previously restricted to Gypsy populations was found in compound heterozygosity with del521T, common in north Africa. The variety of known and novel mutations found indicates that the immunohistochemical profile of gamma-sarcoglycan mutations is not restricted to a particular mutation or type of mutation, but rather is a general reflection of the effect of gamma-sarcoglycan mutations on the composition of the sarcoglycan complex. Complete immunohistochemical analysis with all available sarcoglycan antibodies, therefore, is a useful tool to guide the molecular genetic investigations that are necessary to arrive at the correct genetic diagnosis in a given case.

Intrafamilial phenotypic variation in limb-girdle muscular dystrophy type 2C with compound heterozygous mutations.

Two Japanese-Brazilian siblings with type 2C limb girdle muscular dystrophy showed a maternal 521-T deletion in exon 6 and a larger paternal deletion of exon 6 in the gamma-sarcoglycan gene. One sib was ambulant at 29 years of age, whereas the other sib was confined to a wheelchair at the age of 12. Sarcoglycan staining of the muscle was reduced in both siblings but it did not correlate with the observed variability of the clinical severity.

A mutation in the alpha 3 chain of type IX collagen causes autosomal dominant multiple epiphyseal dysplasia with mild myopathy.

Multiple epiphyseal dysplasia (MED) is a degenerative cartilage condition shown in some cases to be caused by mutations in genes encoding cartilage oligomeric matrix protein or type IX collagen. We studied a family with autosomal dominant MED affecting predominantly the knee joints and a mild proximal myopathy. Genetic linkage to the COL9A3 locus on chromosome 20q13.3 was established with a peak log(10) odds ratio for linkage score of 3.87 for markers D20S93 and D20S164. Reverse transcription-PCR performed on the muscle biopsy revealed aberrant mRNA lacking exon 3, which predicted a protein lacking 12 amino acids from the COL3 domain of alpha3(IX) collagen. Direct sequencing of genomic DNA confirmed the presence of a splice acceptor mutation in intron 2 of the COL9A3 gene (intervening sequence 2, G-A, -1) only in affected family members. By electron microscopy, chondrocytes from epiphyseal cartilage exhibited dilated rough endoplasmic reticulum containing linear lamellae of alternating electron-dense and electron-lucent material, reflecting abnormal processing of mutant protein. Type IX collagen chains appeared normal in size and quantity but showed defective cross-linking by Western blotting. The novel phenotype of MED and mild myopathy is likely caused by a dominant-negative effect of the exon 3-skipping mutation in the COL9A3 gene. Patients with MED and a waddling gait but minimal radiographic hip involvement should be evaluated for a primary myopathy and a mutation in type IX collagen.

The genomic organization of human dystrobrevin.

Dystrophin-related and dystrophin-associated proteins (DAPs) are thought to play an important role in the stability and maintenance of the plasma membrane during muscle contraction and relaxation. Studies conducted on the electric organ of Torpedo californica have shown that some of the DAPs are also involved in the formation and maintenance of neuromuscular junctions (NMJs). In addition, dystrophin and several DAPs have been shown to be the primary genetic defect in a number of phenotypically similar muscular dystrophies. We previously reported the identification and characterization of human dystrobrevin, a protein which is unique in being both homologous to dystrophin and a dystrophin-associated protein. Here we describe the genomic organization of the human dystrobrevin gene. It is encoded by 23 exons spanning at least 180 kb of chromosome 18q12. Three different C-termini of dystrobrevin are generated by the mutually exclusive mRNA splicing of three exons. Two alternatively spliced exons (exons 11A and 12) are utilized exclusively in striated muscles. A comparison between the genomic organization of dystrophin and human dystrobrevin shows that the two genes have significant similarities in their genomic structure, implying an ancestral or evolutionary relationship. Based on intronic sequence, a primer set was designed to specifically amplify each exon of dystrobrevin to screen for mutations by SSCP in patients with neuromuscular diseases for which dystrobrevin could be a candidate.

Cloning of human microtubule-associated protein 1B and the identification of a related gene on chromosome 15.

We report here the complete cloning and sequencing of human microtubule associated protein 1B (MAP1B). Comparisons to mouse and partial rat MAP1B sequence indicate that this gene is extremely well conserved, with 91 and 90% identity, respectively. The entire human MAP1B genomic region has been isolated and the genomic organization determined. The gene includes seven exons, and the third exon contains sequence not represented in mouse or rat MAP1B. This sequence, labeled 3A, is present at the 5' end of an alternative transcript that is expressed at approximately 1/10th the level of the full-length transcript. By comparisons of human MAP1B with the sequence databases, we have identified a MAP1B-related gene that is probably the human homologue of rat MAP1A. This gene is expressed at high levels in brain and spinal cord and much lower levels in muscle and maps to the long arm of human chromosome 15.

Cloning of human basic A1, a distinct 59-kDa dystrophin-associated protein encoded on chromosome 8q23-24.

Duchenne and Becker muscular dystrophies are caused by defects of dystrophin, which forms a part of the membrane cytoskeleton of specialized cells such as muscle. It has been previously shown that the dystrophin-associated protein A1 (59-kDa DAP) is actually a heterogeneous group of phosphorylated proteins consisting of an acidic (alpha-A1) and a distinct basic (beta-A1) component. Partial peptide sequence of the A1 complex purified from rabbit muscle permitted the design of oligonucleotide probes that were used to isolate a cDNA for one human isoform of A1. This cDNA encodes a basic A1 isoform that is distinct from the recently described syntrophins in Torpedo and mouse and is expressed in many tissues with at least five distinct mRNA species of 5.9, 4.8, 4.3, 3.1, and 1.5 kb. A comparison of our human cDNA sequence with the GenBank expressed sequence tag (EST) data base has identified a relative from human skeletal muscle, EST25263, which is probably a human homologue of the published mouse syntrophin 2. We have mapped the human basic component of A1 and EST25263 genes to chromosomes 8q23-24 and 16, respectively.

Rapid detection of CA polymorphisms in cloned DNA: application to the 5' region of the dystrophin gene.

To identify CA repeats in genomic sequences which had been previously subcloned into plasmids, we performed PCR using a (CA)n primer and a flanking vector primer on the genomic inserts. By incorporation of a restriction enzyme site into the (CA)n primer, we have been able to subclone the genomic DNA so that the sequence flanking the CA repeat is readily determined. Primers can then be designed to amplify across the CA repeat in patient DNA samples. Application of this technique to genomic DNAs surrounding the upstream "brain" promoter of the dystrophin gene has led to the discovery of four new CA repeats. Three of these repeats are highly polymorphic, with PICs ranging from .586 to .768. The location of these markers at the extreme 5' terminus of the dystrophin gene, together with their high degree of polymorphism and ease of assay, makes them ideal for linkage analysis in families with Duchenne muscular dystrophy.

Dystrophin is transcribed in brain from a distant upstream promoter.

Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is expressed in brain as well as muscle. The role of dystrophin in the brain is not clear, though one-third of Duchenne muscular dystrophy patients exhibit some degree of mental retardation. We have isolated the genomic region encoding the alternative 5' terminus of dystrophin used in the brain. Primer extension and polymerase chain reaction assays on RNA demonstrate that this region contains an alternative promoter for dystrophin used in the brain. Physical mapping of this region indicates that this brain promoter is located greater than 90 kilobases 5' to the promoter used in muscle and 400 kilobases from exon 2 to which it is spliced. The large physical distance between the promoters, taken together with their known tissue selectivities, suggests that in certain patients a deletion of either dystrophin promoter might give rise to reduced dystrophin expression selective to brain or muscle. We have identified one such individual with specific deletion of the dystrophin muscle promoter, giving rise to Becker muscular dystrophy, and we predict that specific loss of the brain promoter may be one cause of X chromosome-linked mental retardation.

Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus.

Dystrophin is the protein product of the Duchenne/Becker muscular dystrophy locus. It has a relative molecular mass of 427,000 and is encoded by a large RNA transcript processed from more than 65 exons spread over two million base pairs of the human X chromosome. We have used the polymerase chain reaction to see whether any of these exons are used alternatively in the different tissues that express dystrophin. As reported for rat dystrophin, we find that the first exons of the human dystrophin transcript is different in brain and muscle, indicating that dystrophin expression could be differentially regulated in these tissues by usage of distinct promoters. The 3' end of the dystrophin transcript can be alternatively spliced to create numerous isoforms differing at their carboxyl domains; this is the only domain of dystrophin that does not share any similarity with the related cytoskeletal alpha-actinins. These alternative transcripts yield dystrophin molecules which may interact with different proteins of the tissues expressing dystrophin.

Expression of the Duchenne's muscular dystrophy gene in cultured muscle cells.

A partial cDNA clone for the Duchenne's muscular dystrophy (DMD) locus was used to investigate the expression of this locus in human muscle in vitro. Hybridization to a 14-kilobase RNA transcript was demonstrated in both fetal and mature human skeletal muscle and four lines of human muscle cells in culture. The DMD transcript was not detected in cultured cells outside the muscle lineage. In cultured muscle cells, gene expression was evident only in myotubes both before and after innervation with mouse spinal cord. Primary cultures of human myoblasts did not show the presence of the DMD transcript prior to fusion to form myotubes. An in vitro model is potentially an excellent system in which to investigate factors controlling expression of the DMD gene in normal muscle and how this expression is altered in cultured DMD muscle.

Conservation of the Duchenne muscular dystrophy gene in mice and humans.

A portion of the Duchenne muscular dystrophy (DMD) gene transcript from human fetal skeletal muscle and mouse adult heart was sequenced, representing approximately 25 percent of the total, 14-kb DMD transcript. The nucleic acid and predicted amino acid sequences from the two species are nearly 90 percent homologous. The amino acid sequence that is predicted from this portion of the DMD gene indicates that the protein product might serve a structural role in muscle, but the abundance and tissue distribution of the messenger RNA suggests that the DMD protein is not nebulin.

Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals.

The 14 kb human Duchenne muscular dystrophy (DMD) cDNA corresponding to a complete representation of the fetal skeletal muscle transcript has been cloned. The DMD transcript is formed by at least 60 exons which have been mapped relative to various reference points within Xp21. The first half of the DMD transcript is formed by a minimum of 33 exons spanning nearly 1000 kb, and the remaining portion has at least 27 exons that may spread over a similar distance. The DNA isolated from 104 DMD boys was tested with the cDNA for detection of deletions and 53 patients exhibit deletion mutations. The majority of deletions are concentrated in a single genomic segment corresponding to only 2 kb of the transcript.

Localization and cloning of Xp21 deletion breakpoints involved in muscular dystrophy.

Twenty-nine deletion breakpoints were mapped in 220 kb of the DXS164 locus relative to potential exons of the Duchenne and Becker muscular dystrophy gene. Four deletion junction fragments were isolated to acquire outlying Xp21 loci on both the terminal and centromere side of the DXS164 locus. The junction loci were used for chromosome walking, searches for DNA polymorphisms, and mapping against deletion and translocation breakpoints. Forty-four unrelated deletions were analyzed using the junction loci as hybridization probes to map the endpoints between cloned Xp21 loci. DNA polymorphisms from the DXS164 and junction loci were used to follow the segregation of a mutation in a family that represents a recombinant. Both the physical and genetic data point to a very large size for this X-linked muscular dystrophy locus.

Rapid cloning of multiple amplified nucleotide sequences from human neuroblastoma cell lines by phenol emulsion competitive DNA reassociation.

A protocol for the rapid cloning of many DNA fragments from an amplified genomic region is described. The procedure is based on a modification of the phenol-emulsion reassociation technique (PERT) previously used to clone DNA fragments missing from a chromosomal deletion [Kunkel et al., Proc. Natl. Acad. Sci. USA 82 (1985) 4778-4782]. The procedure was used to construct recombinant libraries in the plasmid pBR322 which were highly enriched for amplified sequences from two neuroblastoma cell lines, CHP-126 and IMR-32. Many new amplified DNA fragments were isolated from these libraries, indicating that the PERT methodology should be of general use in isolating amplified DNA from other cell lines and tumors.

Molecular studies of progressive muscular dystrophy (Duchenne).

The Duchenne muscular dystrophy gene cloning is presented along with evidence which indicates that the gene locus encoding the Duchenne muscular dystrophy (DMD) transcript is extremely large. Partial nucleotide sequence of the cDNA from mouse and humans has been used to predict the amino acid sequence for a part of the DMD protein. The protein is highly conserved between the two mammals and might have structural characteristics similar to other muscle-specific proteins.

Localisation of Xp21 meiotic exchange points in Duchenne muscular dystrophy families.

The inheritance of Duchenne muscular dystrophy in 25 families was studied with 13 X chromosome specific cloned DNA fragments from 10 loci in and surrounding Xp21. When multiple probes were informative, the meiotic exchange points for each meiosis were located in individual families. Neither genetic nor physical evidence indicates an unusually high recombination rate across Xp21 in these 25 families.

Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene.

Duchenne muscular dystrophy (DMD) and the less severe Becker muscular dystrophy (BMD) are human X-linked muscle-wasting disorders that have been localized to the band Xp21 by genetic linkage analysis and cytologically detectable abnormalities. A cloned DNA segment, DXS164 (or pERT87), has been shown to detect deletions in the DNA of unrelated DMD and BMD males. Here we present the nucleotide sequence of two highly conserved DNA fragments from the DXS164 locus and their homologous sequences from the mouse X chromosome. One of the human conserved segments hybridized to a large transcript in RNA isolated from human fetal skeletal muscle and was used to isolate cDNA clones which cover approximately 10% of this transcript. The cDNA clones map to Xp21 and hybridize with a minimum of eight small regions that span 130 kilobases (kb) of the DXS164 locus. These expressed sequences are candidates for portions of the gene responsible for both DMD and BMD.