Myotonic dystrophy (DM) is the most common inherited neuromuscular disease in adults, with a global incidence of 1 in 8000 individuals. DM is an autosomal dominant, multisystemic disorder characterized primarily by myotonia and progressive muscle weakness. Genomic and complementary DNA probes that map to a 10-kilobase Eco RI genomic fragment from human chromosome 19q13.3 have been used to detect a variable length polymorphism in individuals with DM. Increases in the size of the allele in patients with DM are now shown to be due to an increased number of trinucleotide CTG repeats in the 3' untranslated region of a DM candidate gene. An increase in the severity of the disease in successive generations (genetic anticipation) is accompanied by an increase in the number of trinucleotide repeats. Nearly all cases of DM (98 percent or 253 of 258 individuals) displayed expansion of the CTG repeat region. These results suggest that DM is primarily caused by mutations that generate an amplification of a specific CTG repeat.

The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.

Myotonic dystrophy (DM1), the most common muscular dystrophy in adults, is caused by an expanded (CTG) n tract in the 3′ UTR of the gene encoding myotonic dystrophy protein kinase (DMPK) 1 , which results in nuclear entrapment of the 'toxic' mutant RNA and interacting RNAbinding proteins (such as MBNL1) in ribonuclear inclusions 2 . It is unclear if therapy aimed at eliminating the toxin would be beneficial. To address this, we generated transgenic mice expressing the DMPK 3′ UTR as part of an inducible RNA transcript encoding green fluorescent protein (GFP). We were surprised to find that mice overexpressing a normal DMPK 3′ UTR mRNA reproduced cardinal features of myotonic dystrophy, including myotonia, cardiac conduction abnormalities, histopathology and RNA splicing defects in the absence of detectable nuclear inclusions. However, we observed increased levels of CUG-binding protein (CUG-BP1) in skeletal muscle, as seen in individuals with DM1. Notably, these effects were reversible in both mature skeletal and cardiac muscles by silencing transgene expression. These results represent the first in vivo proof of principle for a therapeutic strategy for treatment of myotonic dystrophy by ablating or silencing expression of the toxic RNA molecules.Common features of adult-onset DM1 include myotonia, progressive skeletal muscle loss, cardiac conduction defects, smooth muscle dysfunction, cataracts and insulin resistance 2 . The normal number of CTG repeats (n = 5 to ~30) is higher (n = 50 to >3,000) in individuals with DM1 (ref. 1 ). Unlike the wild-type transcript, mutant DMPK mRNA forms nuclear aggregates 3,4 and is thought to trigger dominant effects by aberrant interactions with or altered activity of RNA splicing factors, principally members of the muscleblind-like (MBNL) family (such as MBNL1) and the CUG-BP and ETR3-like factor (CELF) family (such as CUG-BP1), leading to abnormal splicing of specific RNAs such as chloride channel (Clcn1), insulin Correspondence should be addressed to M.S.M. (mahadevan@virginia.edu). 4 These authors contributed equally to this work. AUTHOR CONTRIBUTIONSM.S.M., R.S.Y., Q.Y., C.D.F.-M., T.D.B. and L.H.P. performed experimental work and data analysis. S.B. generated the transgene constructs. M.S.M. was responsible for conceptual design and execution. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. One potential therapeutic approach in DM1 is to get rid of the toxic RNA from cells. However, it is unclear if this will alleviate the effects of the disease. We used the tetracycline (Tet) inducible system with the reverse tetracycline transactivator (rtTA) to generate double transgenic mice harboring (i) a Tet-responsive, DMPK promoter 10,11 -driven transgene (named GFP-DMPK 3′ UTR) expressing the DMPK 3′ UTR mRNA as part of a GFP transcript, and (ii) a constitutively expressed rtTA transgene (Fig. 1a) Fig. 1). Notably, RNA blots of skeletal muscle RNA showed two major species due to alternative use of polyadenylation signal...

Myotonic dystrophy is a common dominant disorder (global incidence of 1:8,000) with variable onset and a protean nature of symptoms mainly involving progressive muscle wasting, myotonia and cataracts. To define the molecular defect, we have cloned the essential region of chromosome 19q13.3, including proximal and distal markers in a 700-kilobase contig formed by overlapping cosmids and yeast artificial chromosomes (YACs). The central part of the contig bridges an area of about 350 kilobases between two new flanking crossover borders. This segment has been extensively characterized through the isolation of five YAC clones and the subsequent subcloning in cosmids from which a detailed EcoRI, HindIII, MluI and NotI restriction map has been derived. Two genomic probes and two homologous complementary DNA probes were isolated using the cosmids. These probes are all situated within approximately 10 kilobases of genomic DNA and detect an unstable genomic segment in myotonic dystrophy patients. The length variation in this segment shows similarities to the instability seen at the fragile X locus. The physical map location and the genetic characteristics of the length polymorphism is compatible with a direct role in the pathogenesis of myotonic dystrophy.