We continue to study the underlying mechanisms, diagnosis, and treatment of the muscular dystrophies, and we have recently expanded our studies to include the genetic basis of human longevity, interstitial cystitis, and autism.

To expand our knowledge of normal muscle membrane function and understand how abnormalities in these proteins cause myofiber degeneration and the pathogenesis of disease, we continue to study the dystrophin-associated protein complex and its function in normal muscle. In previous reports we described our analysis and cloning of some of these proteins, including the sarcoglycans. We and others have shown that sarcoglycans are mutated or abnormal in some forms of autosomal-recessive muscular dystrophies, and that if one of these proteins is abnormal, the expression of other members at the membrane is compromised. Our work has also led to improved diagnosis of the muscular dystrophies, a new understanding of the common pathogenesis underlying these disorders, and testable ideas for therapeutic intervention.

In past reports we have outlined our mRNA expression studies of normal and diseased muscle isolated from both mice and humans. We have shown that the mRNA expression patterns reflect what is seen on immunohistochemical analysis of muscle. We have expanded our analysis of RNA expressed in muscle to include microRNAs (miRNAs), which are likely a widely used mechanism for post-transcriptional regulation of important cellular pathways, including muscle development and disease.

To determine if changes in miRNA populations might be linked to muscular dystrophies, we have carried out a comparative miRNA expression profiling of muscle samples obtained from patients with 10 different groups of muscle diseases. Among 428 human miRNAs surveyed, a subset of 185 human miRNAs was found to be differentially expressed at a significant level (p < 0.05, false discovery rate < 0.05) in at least 1 of the 10 muscle conditions compared with the control panel. Among this set of differentially expressed miRNAs, the expression profile in human tissues has been previously established for 145. Of these, 60 percent are known to be expressed in adult muscle (as well as in other tissues). Two miRNAs differentially expressed just in muscle biopsies taken from Duchenne muscular dystrophy (DMD) patients were chosen for further validation of their normal muscle function and how the changed expression influences disease. Changes in expression of the two miRNAs were validated by RT-PCR, and both are currently being increased or decreased in expression levels in muscle cell culture. Those genes predicted to be targets for these miRNAs were indeed found to change mRNA expression in these cell culture experiments. We are currently looking at how these miRNAs influence gene expression in muscle cells isolated from DMD patients.

Zebrafish have rapidly emerged as an excellent animal model to study the genetics of complex tissues. Fish muscle is very similar to mammalian muscle, and we have shown that the entire dystrophin-associated protein complex is present at the membrane of zebrafish muscle. We have obtained or generated a dozen zebrafish strains with muscle weakness, including the sapje strain, which has been shown recently by others to have a stop codon mutation in the zebrafish dystrophin gene. One of our newly generated strains has a second mutation at the dystrophin locus. We have paralleled our studies of mutant alleles in these zebrafish to create gene knockdowns by introducing morpholinos during development. Knockdown of the Fkrp gene in fish recapitulated the effect of mutations in the human FKRP gene. Introduction of the normal human mRNA for FKRP corrects the phenotype of the fish knockdown, yet human mutant alleles fail to correct the phenotype.

Therapeutics for Muscular Dystrophy
We have previously described our work on myoblast transplantation into diseased muscle and the expression of dystrophin in the injected muscles in the muscular dystrophy mouse model. In previous reports we detailed the ability of a population of muscle progenitor cells to reconstitute the bone marrow of lethally irradiated mice and to migrate subsequently into the muscle and contribute to its regeneration, albeit at low levels. We have shown that the degenerative process in the disease is sufficient to emit signals that recruit these cells from the circulation. We have expanded our studies of these cells and have shown that they can also be introduced into the circulation via the femoral artery. In collaboration with Jeffrey Chamberlain (University of Washington), we have transfected the muscle progenitor cells with lentiviruses expressing human microdystrophin and enhanced green fluorescent protein (GFP). The transfected muscle progenitor cells are found in the muscle of these mice following intra-arterial delivery. We also identified fibers expressing human microdystrophin and GFP and have documented that these cells follow myogenic differentiation pathways and are not just simply fusing to existing fibers. We are currently looking to improve the engraftment of these cells into mouse muscle.

Genetics of Complex Human Traits
In past reports, we have outlined our studies of the genetic basis of human longevity and have expanded these studies to include other genetic disorders with complex traits. During the interval since our first linkage study of centenarians we have doubled the number of centenarian sibling pairs we have for analysis. At the same time, genotyping technology has improved tremendously, and the new Affymetrix 10K SNP (single-nucleotide polymorphism) chip allows the simultaneous genotyping of more than 10,000 SNPs spaced over the human genome. We have finished genotyping all 750 centenarian sibling pair DNA samples on these chips and have found a new locus in the human genome that might have genetic variants under- or overrepresented in centenarians.

We have continued our genetic studies of interstitial cystitis (IC) and autism. IC is a complex human genetic disorder affecting nearly 1/2,000 individuals in the United States. There is an increased clustering of the disorder in families, indicating a clear genetic component to the disease. In collaboration with Jordan Dimitrakov (Children's Hospital Boston), we have identified six families where IC is segregating as an apparent autosomal-dominant trait. We have used the Affymetrix 10K SNP chips to map in these families five different loci that are significantly or suggestively linked to the disease. We are currently sequencing genes under the most significant linkage peak to find the causative mutations in two of the IC families. Identification of the gene under the linkage peak should highlight potential disease pathways and might reveal insights into therapy and identify possible candidate genes under the other linkage peaks. We are also recruiting additional families.

Our laboratory has also entered into a multidisciplinary collaborative study of autism. This behavioral problem of children has been extensively studied, yet the underlying basis in most cases has remained elusive. Autism is a child developmental disorder that most likely involves neuronally expressed genes and probably has genetic as well as environmental causes. We are working on the assumption that we can use peripheral blood mRNA expression profiles as a surrogate to mRNA expression in the brain. Given that many genes expressed in the central nervous system are also expressed in whole blood, we might be able to find signatures of gene expression that will allow us to categorize patients with autism and possibly identify causative genes. We have now performed expression array on more than 100 blood samples isolated from autistic patients and a similar number of control samples. Using part of the data set to create a predictor and the other part to test the predictor, we can now be predictive that an mRNA profile derives from a blood sample taken from an autistic individual versus a control. We are expanding these studies to many more patients and controls, in the hopes that these types of studies could form a diagnostic test for autism.