Efficient identification of novel mutations in patients with limb girdle muscular dystrophy.

Limb girdle muscular dystrophy type 2 (LGMD2) is a genetically heterogeneous autosomal recessive disorder caused by mutations in 15 known genes. DNA sequencing of all candidate genes can be expensive and laborious, whereas a selective sequencing approach often fails to provide a molecular diagnosis. We aimed to efficiently identify pathogenic mutations via homozygosity mapping in a population in which the genetics of LGMD2 has not been well characterized. Thirteen consanguineous families containing a proband with LGMD2 were recruited from Saudi Arabia, and for 11 of these families, selected individuals were genotyped at 10,204 single nucleotide polymorphisms. Linkage analysis excluded all but one or two known genes in ten of 11 genotyped families, and haplotype comparisons between families allowed further reduction in the number of candidate genes that were screened. Mutations were identified by DNA sequencing in all 13 families, including five novel mutations in four genes, by sequencing at most two genes per family. One family was reclassified as having a different myopathy based on genetic and clinical data after linkage analysis excluded all known LGMD2 genes. LGMD2 subtypes A and B were notably absent from our sample of patients, indicating that the distribution of LGMD2 mutations in Saudi Arabian families may be different than in other populations. Our data demonstrate that homozygosity mapping in consanguineous pedigrees offers a more efficient means of discovering mutations that cause heterogeneous disorders than comprehensive sequencing of known candidate genes.

Inefficient dystrophin expression after cord blood transplantation in Duchenne muscular dystrophy.

We report a boy who received two allogeneic stem cell transplantations from umbilical cord donors to treat chronic granulomatous disease (CGD). The CGD was cured after the second transplantation, but 2.5 years later he was diagnosed with Duchenne muscular dystrophy (DMD). Examinations of his DNA, muscle tissue, and myoblast cultures derived from muscle tissue were performed to determine whether any donor dystrophin was being expressed. The boy was found to have a large-scale deletion on the X chromosome that spanned the loci for CYBB and DMD. The absence of dystrophin led to muscle histology characteristic of DMD. Analysis of myofibers demonstrated no definite donor cell engraftment. This case suggests that umbilical cord-derived hematopoietic stem cell transplantation will not be efficacious in the therapy of DMD without additional interventions that induce engraftment of donor cells in skeletal muscle.

Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes.

BACKGROUND: One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive.These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels.The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. RESULTS: An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper. CONCLUSION: This automated process allows laboratories to discover DNA variations in a short time and at low cost.

Long-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation.

Duchenne muscular dystrophy (DMD) is a severe progressive muscle-wasting disorder caused by mutations in the dystrophin gene. Studies have shown that bone marrow cells transplanted into lethally irradiated mdx mice, the mouse model of DMD, can become part of skeletal muscle myofibers. Whether human marrow cells also have this ability is unknown. Here we report the analysis of muscle biopsies from a DMD patient (DMD-BMT1) who received bone marrow transplantation at age 1 year for X-linked severe combined immune deficiency and who was diagnosed with DMD at age 12 years. Analysis of muscle biopsies from DMD-BMT1 revealed the presence of donor nuclei within a small number of muscle myofibers (0.5-0.9%). The majority of the myofibers produce a truncated, in-frame isoform of dystrophin lacking exons 44 and 45 (not wild-type). The presence of bone marrow-derived donor nuclei in the muscle of this patient documents the ability of exogenous human bone marrow cells to fuse into skeletal muscle and persist up to 13 years after transplantation.

Dystrophin is a tumor suppressor in human cancers with myogenic programs.

Many common human mesenchymal tumors, including gastrointestinal stromal tumor (GIST), rhabdomyosarcoma (RMS) and leiomyosarcoma (LMS), feature myogenic differentiation. Here we report that intragenic deletion of the dystrophin-encoding and muscular dystrophy-associated DMD gene is a frequent mechanism by which myogenic tumors progress to high-grade, lethal sarcomas. Dystrophin is expressed in the non-neoplastic and benign counterparts of GIST, RMS and LMS tumors, and DMD deletions inactivate larger dystrophin isoforms, including 427-kDa dystrophin, while preserving the expression of an essential 71-kDa isoform. Dystrophin inhibits myogenic sarcoma cell migration, invasion, anchorage independence and invadopodia formation, and dystrophin inactivation was found in 96%, 100% and 62% of metastatic GIST, embryonal RMS and LMS samples, respectively. These findings validate dystrophin as a tumor suppressor and likely anti-metastatic factor, suggesting that therapies in development for muscular dystrophies may also have relevance in the treatment of cancer.

Diagnosis and cell-based therapy for Duchenne muscular dystrophy in humans, mice, and zebrafish.

The muscular dystrophies are a heterogeneous group of genetically caused muscle degenerative disorders. The Kunkel laboratory has had a longstanding research program into the pathogenesis and treatment of these diseases. Starting with our identification of dystrophin as the defective protein in Duchenne muscular dystrophy (DMD), we have continued our work on normal dystrophin function and how it is altered in muscular dystrophy. Our work has led to the identification of the defective genes in three forms of limb girdle muscular dystrophy (LGMD) and a better understanding of how muscle degenerates in many of the different dystrophies. The identification of mutations causing human forms of dystrophy has lead to improved diagnosis for patients with the disease. We are continuing to improve the molecular diagnosis of the dystrophies and have developed a high-throughput sequencing approach for the low-cost rapid diagnosis of all known forms of dystrophy. In addition, we are continuing to work on therapies using available animal models. Currently, there are a number of mouse models of the human dystrophies, the most notable being the mdx mouse with dystrophin deficiency. These mice are being used to test possible therapies, including stem-cell-based approaches. We have been able to systemically deliver human dystrophin to these mice via the arterial circulation and convert 8% of dystrophin-deficient fibers to fibers expressing human dystrophin. We are now expanding our research to identify new forms of LGMD by analyzing zebrafish models of muscular dystrophy. Currently, we have 14 different zebrafish mutants exhibiting various phenotypes of muscular dystrophy, including muscle weakness and inactivity. One of these mutants carries a stop codon mutation in dystrophin, and we have recently identified another carrying a mutation in titin. We are currently positionally cloning the disease-causative mutation in the remaining 12 mutant strains. We hope that one of these new mutant strains of fish will have a mutation in a gene not previously implicated in human muscular dystrophy. This gene would become a candidate gene to be analyzed in patients which do not carry a mutation in any of the known dystrophy-associated genes. By studying both disease pathology and investigating potential therapies, we hope to make a positive difference in the lives of people living with muscular dystrophy.

Gene expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal skeletal muscle.

The primary cause of Duchenne muscular dystrophy (DMD) is a mutation in the dystrophin gene leading to the absence of the corresponding RNA transcript and protein. Absence of dystrophin leads to disruption of the dystrophin-associated protein complex and substantial changes in skeletal muscle pathology. Although the histological pathology of dystrophic tissue has been well documented, the underlying molecular pathways remain poorly understood. To examine the pathogenic pathways and identify new or modifying factors involved in muscular dystrophy, expression microarrays were used to compare individual gene expression profiles of skeletal muscle biopsies from 12 DMD patients and 12 unaffected control patients. Two separate statistical analysis methods were used to interpret the resulting data: t test analysis to determine the statistical significance of differential expression and geometric fold change analysis to determine the extent of differential expression. These analyses identified 105 genes that differ significantly in expression level between unaffected and DMD muscle. Many of the differentially expressed genes reflect changes in histological pathology. For instance, immune response signals and extracellular matrix genes are overexpressed in DMD muscle, an indication of the infiltration of inflammatory cells and connective tissue. Significantly more genes are overexpressed than are underexpressed in dystrophic muscle, with dystrophin underexpressed, whereas other genes encoding muscle structure and regeneration processes are overexpressed, reflecting the regenerative nature of the disease.

Gene expression profiling of Duchenne muscular dystrophy skeletal muscle.

The primary cause of Duchenne muscular dystrophy (DMD) is a mutation in the dystrophin gene, leading to absence of the corresponding protein, disruption of the dystrophin-associated protein complex, and substantial changes in skeletal muscle pathology. Although the primary defect is known and the histological pathology well documented, the underlying molecular pathways remain in question. To clarify these pathways, we used expression microarrays to compare individual gene expression profiles for skeletal muscle biopsies from DMD patients and unaffected controls. We have previously published expression data for the 12,500 known genes and full-length expressed sequence tags (ESTs) on the Affymetrix HG-U95Av2 chips. Here we present comparative expression analysis of the 50,000 EST clusters represented on the remainder of the Affymetrix HG-U95 set. Individual expression profiles were generated for biopsies from 10 DMD patients and 10 unaffected control patients. Two methods of statistical analysis were used to interpret the resulting data (t-test analysis to determine the statistical significance of differential expression and geometric fold change analysis to determine the extent of differential expression). These analyses identified 183 probe sets (59 of which represent known genes) that differ significantly in expression level between unaffected and disease muscle. This study adds to our knowledge of the molecular pathways that are altered in the dystrophic state. In particular, it suggests that signaling pathways might be substantially involved in the disease process. It also highlights a large number of unknown genes whose expression is altered and whose identity therefore becomes important in understanding the pathogenesis of muscular dystrophy.