Expression array analyses promises to greatly expedite our understanding of the molecular pathophysiology of inherited muscle disease, and to assist in the identification of new disease genes through locus-specific decreases in loss-of-function (recessive) inherited dystrophies. Toward this end, we have initiated a series of expression array analyses of human muscular dystrophy patient muscle biopsies. We have chosen Affymetrix GeneChip technologies for the following reasons: (i) muscle contains large numbers of related, yet functionally diverse, genes (for example >10 myosin heavy chain genes), which are not distinguished by cDNA array methods; (ii) muscle is a relatively simple and homogeneous tissue which seems particularly amenable to expression array approaches and (iii) excellent non-normalized human muscle EST database has been produced by the Telethon EST project in Italy, with approximately 1,000 genes, 1,500 EST clusters and 1,500 EST singletons currently characterized.

Duchenne muscular dystrophy is the most common inherited lethal childhood disease in the world, and is equally frequent in animals. The gene is approximately 2.5 megabases, and has a new mutation rate of 1 in 10,000 gametes. Dystrophin deficiency is the biochemical cause, and this deficiency is expressed from fetal life (16 weeks gestation) onward, yet clinical symptoms are not obvious until 4–5 years of age. The reasons underlying the progressive pathophysiology are not understood. Here, we describe initial experiments using human fetal muscle samples from a normal fetus, and a DMD fetus, using Affymetrix stock human 6,800 chips. DMD comparison of signals from fluorescent cRNA samples was done using Affymetrix data analysis for significant differences. Ninety-nine percent (7,062/7,129) of the probe sets tested showed similar levels of cRNA between the control and DMD samples. Seventeen (0.24%) of the probe sets showed significant decreases in expression. These decreases included an important dystrophin-associated protein, beta-dystroglycan, and proteins involved in immune recognition of abnormal cells (tenascin X and macrophage migration inhibitory factor [MIF]). Our data show that secondary transcriptional changes occur soon after dystrophin deficiency is biochemically evident, long before clinical symptoms appear. This data will permit the delineation of the molecular pathophysiological progression of this important disorder, and provide insight into the development of the membrane cytoskeleton of muscle.

Our long-term goals are to construct an extensive database of expression array changes in muscular dystrophy patients, with known variables of patient age at time of muscle biopsy, and molecular diagnosis (gene and gene mutation). By constructing a gene- and age-specific expression array database, we anticipate that novel muscular dystrophy genes can be identified through specific changes in single RNAs in unknown samples. Toward this end, we have under production an Affymetrix human muscle chip, which uses the non-normalized muscle EST database of the University of Padova, Italy. The chip will have 3,000 genes/clusters/singletons ranked from most to least abundant on the chip, with the usual 40-fold redundancy (120,000 features). The informatics should ensure the ability to distinguish between closely homologous muscle genes, which would be impossible using cDNA array technology.