Profound misregulation of muscle-specific gene expression in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder characterized by an insidious onset and progressive course. The disease has a frequency of about 1 in 20,000 and is transmitted in an autosomal dominant fashion with almost complete penetrance. Deletion of an integral number of tandemly arrayed 3.3-kb repeat units (D4Z4) on chromosome 4q35 is associated with FSHD but otherwise the molecular basis of the disease and its pathophysiology remain obscure. Comparison of mRNA populations between appropriate cell types can facilitate identification of genes relevant to a particular biological or pathological process. In this report, we have compared mRNA populations of FSHD and normal muscle. Unexpectedly, the dystrophic muscle displayed profound alterations in gene expression characterized by severe underexpression or overexpression of specific mRNAs. Intriguingly, many of the deregulated mRNAs are muscle specific. Our results suggest that a global misregulation of gene expression is the underlying basis for FSHD, distinguishing it from other forms of muscular dystrophy. The experimental approach used here is applicable to any genetic disorder whose pathogenic mechanism is incompletely understood.

Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos

Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes—survival motor neuron (SMN) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the SMN gene product in vivo we generated SMN-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one SMN gene. Mice with homozygous SMN disruption display massive cell death during early embryonic development, indicating that the SMN gene product is necessary for cellular survival and function.

A second promoter provides an alternative target for therapeutic up-regulation of utrophin in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an inherited muscle-wasting disease caused by the absence of a muscle cytoskeletal protein, dystrophin. We have previously shown that utrophin, the autosomal homologue of dystrophin, is able to compensate for the absence of dystrophin in a mouse model of DMD; we have therefore undertaken a detailed study of the transcriptional regulation of utrophin to identify means of effecting its up-regulation in DMD muscle. We have previously isolated a promoter element lying within the CpG island at the 5′ end of the gene and have shown it to be synaptically regulated in vivo. In this paper, we show that there is an alternative promoter lying within the large second intron of the utrophin gene, 50 kb 3′ to exon 2. The promoter is highly regulated and drives transcription of a widely expressed unique first exon that splices into a common full-length mRNA at exon 3. The two utrophin promoters are independently regulated, and we predict that they respond to discrete sets of cellular signals. These findings significantly contribute to understanding the molecular physiology of utrophin expression and are important because the promoter reported here provides an alternative target for transcriptional activation of utrophin in DMD muscle. This promoter does not contain synaptic regulatory elements and might, therefore, be a more suitable target for pharmacological manipulation than the previously described promoter.

Defining Extracellular Integrin α-Chain Sites That Affect Cell Adhesion and Adhesion Strengthening without Altering Soluble Ligand Binding

It was previously shown that mutations of integrin α4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in α4β1-dependent cell adhesion. Some reports have suggested that α-chain “EF-hand” sites may interact directly with ligands. However, we show here that mutations of three different α4 “EF-hand” sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on α4β1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin α4 “EF-hand-like” sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.

A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy

SMN1 and SMN2 (survival motor neuron) encode identical proteins. A critical question is why only the homozygous loss of SMN1, and not SMN2, results in spinal muscular atrophy (SMA). Analysis of transcripts from SMN1/SMN2 hybrid genes and a new SMN1 mutation showed a direct relationship between presence of disease and exon 7 skipping. We have reported previously that the exon-skipped product SMNΔ7 is partially defective for self-association and SMN self-oligomerization correlated with clinical severity. To evaluate systematically which of the five nucleotides that differ between SMN1 and SMN2 effect alternative splicing of exon 7, a series of SMN minigenes was engineered and transfected into cultured cells, and their transcripts were characterized. Of these nucleotide differences, the exon 7 C-to-T transition at codon 280, a translationally silent variance, was necessary and sufficient to dictate exon 7 alternative splicing. Thus, the failure of SMN2 to fully compensate for SMN1 and protect from SMA is due to a nucleotide exchange (C/T) that attenuates activity of an exonic enhancer. These findings demonstrate the molecular genetic basis for the nature and pathogenesis of SMA and illustrate a novel disease mechanism. Because individuals with SMA retain the SMN2 allele, therapy targeted at preventing exon 7 skipping could modify clinical outcome.

Ullrich scleroatonic muscular dystrophy is caused by recessive mutations in collagen type VI

Ullrich syndrome is a recessive congenital muscular dystrophy affecting connective tissue and muscle. The molecular basis is unknown. Reverse transcription–PCR amplification performed on RNA extracted from fibroblasts or muscle of three Ullrich patients followed by heteroduplex analysis displayed heteroduplexes in one of the three genes coding for collagen type VI (COL6). In patient A, we detected a homozygous insertion of a C leading to a premature termination codon in the triple-helical domain of COL6A2 mRNA. Both healthy consanguineous parents were carriers. In patient B, we found a deletion of 28 nucleotides because of an A → G substitution at nucleotide −2 of intron 17 causing the activation of a cryptic acceptor site inside exon 18. The second mutation was an exon skipping because of a G → A substitution at nucleotide −1 of intron 23. Both mutations are present in an affected brother. The first mutation is also present in the healthy mother, whereas the second mutation is carried by their healthy father. In patient C, we found only one mutation so far—the same deletion of 28 nucleotides found in patient B. In this case, it was a de novo mutation, as it is absent in her parents. mRNA and protein analysis of patient B showed very low amounts of COL6A2 mRNA and of COL6. A near total absence of COL6 was demonstrated by immunofluorescence in fibroblasts and muscle. Our results demonstrate that Ullrich syndrome is caused by recessive mutations leading to a severe reduction of COL6.

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Expressed cadherin pseudogenes are localized to the critical region of the spinal muscular atrophy gene.

Low-copy repeats have been associated with genomic rearrangements and have been implicated in the generation of mutations in several diseases. Here we characterize a subset of low-copy repeats in the spinal muscular atrophy (SMA) region in human chromosome 5q13. We show that this repeated sequence, named c41-cad, is a highly expressed pseudogene derived from an intact neuronal cadherin gene, Br-cadherin, situated on 5p13-14. Br-cadherin is expressed specifically in the brain, whereas the c41-cad transcripts are 10-15 times more abundant and are present in all tissues examined. We speculate that the c41-cad repeats, separately or in concert with other repeats in the SMA region, are involved in the pathogenesis of SMA by promoting rearrangements and deletions.