Manifesting carriers of DMD gene mutations may present diagnostic challenges, particularly in the absence of a family history of dystrophinopathy. We review the clinical and genetic features in fifteen manifesting carriers identified among 860 subjects within the United Dystrophinopathy Project, a large clinical dystrophinopathy cohort whose members undergo comprehensive DMD mutation analysis. We defined manifesting carriers as females with significant weakness, excluding those with only myalgias/cramps. DNA extracted from peripheral blood was used to study X chromosome inactivation patterns. Among these manifesting carriers, age at symptom onset ranged from 2 to 47 years. Seven had no family history and eight had male relatives with Duchene muscular dystrophy (DMD). Clinical severity among the manifesting carriers varied from a DMD-like progression to a very mild Becker muscular dystrophy-like phenotype. Eight had exonic deletions or duplications and six had point mutations. One patient had two mutations (an exonic deletion and a splice site mutation), consistent with a heterozygous compound state. The X chromosome inactivation pattern was skewed toward nonrandom in four out of seven informative deletions or duplications but was random in all cases with nonsense mutations. We present the results of DMD mutation analysis in this manifesting carrier cohort, including the first example of a presumably compound heterozygous DMD mutation. Our results demonstrate that improved molecular diagnostic methods facilitate the identification of DMD mutations in manifesting carriers, and confirm the heterogeneity of mutational mechanisms as well as the wide spectrum of phenotypes.

It is generally believed that signi®cant ribosomal frameshifting during translation does not occur without a functional purpose. The distribution of two frameshift-prone sequences, A_AAA_AAG and CCC_TGA, in coding regions of Escherichia coli has been analyzed. Although a moderate level of selection against the ®rst sequence is evident, 68 genes contain A_AAA_AAG and 19 contain CCC_TGA. The majority of those tested in their genomic context showed >1% frameshifting. Comparative sequence analysis was employed to assess a potential biological role for frameshifting in decoding these genes. Two new candidates, in pheL and ydaY, for utilized frameshifting have been identi®ed in addition to those previously known in dnaX and nine insertion sequence elements. For the majority of the shift-prone sequences no functional role can be attributed to them, and the frameshifting is likely erroneous. However, none of frameshift sequences is in the 306 most highly expressed genes. The unexpected conclusion is that moderate frameshifting during expression of at least some other genes is not suf®ciently harmful for cells to trigger strong negative evolutionary pressure.

Objective: The degenerative muscle diseases Duchenne (DMD) and Becker muscular dystrophy result from mutations in the DMD gene, which encodes the dystrophin protein. Recent improvements in mutational analysis techniques have resulted in the increasing identification of deep intronic point mutations, which alter splicing such that intronic sequences are included in the messenger RNA as "pseudoexons." We sought to test the hypothesis that the clinical phenotype correlates with splicing efficiency of these mutations, and to test the feasibility of antisense oligonucleotide (AON)-mediated pseudoexon skipping. Methods: We identified three pseudoexon insertion mutations in dystrophinopathy patients, two of whom had tissue available for further analysis. For these two out-of-frame pseudoexon mutations (one associated with Becker muscular dystrophy and one with DMD), mutation-induced splicing was tested by quantitative reverse transcription polymerase chain reaction; pseudoexon skipping was tested using AONs composed of 2Ј-O-methyl-modified bases on a phosphorothioate backbone to treat cultured primary myoblasts. Results: Variable amounts of pseudoexon inclusion correlates with the severity of the dystrophinopathy phenotype in these two patients. AON treatment directed at the pseudoexon results in the expression of full-length dystrophin in a DMD myoblast line. Interpretation: Both DMD and Becker muscular dystrophy can result from out-of-frame pseudoexons, with the difference in phenotype being due to variable efficiency of the newly generated splicing signal. AON-mediated pseudoexon skipping therapy is a viable approach to these patients and would be predicted to result in increased expression of wild-type dystrophin protein.

Mutations in the DMD gene result in two common phenotypes associated with progressive muscle weakness: the more severe Duchenne Muscular Dystrophy (DMD) and the milder Becker Muscular Dystrophy (BMD). We have previously identified a nonsense mutation (c.9G>A; p.Trp3X) within the first exon of the DMD gene, encoding the unique N-terminus of the 427 kDa muscle isoform of the dystrophin protein. Although this mutation would be expected to result in severe disease, the clinical phenotype is very mild BMD, with ambulation preserved into the 7 th decade. We identify the molecular mechanism responsible for the amelioration of disease severity to be initiation of translation at two proximate AUG codons within exon 6. Analysis of large mutational data sets suggests that this may be a general mechanism of phenotypic rescue for point mutations within at least the first two exons of the DMD gene. Our results directly demonstrate, for the first time, the use of alternate translational initiation codons within the DMD gene, and suggest that dystrophin protein lacking amino acids encoded by the first five exons retains significant function.

The enthalpy of solution of C60 in toluene, carbon disulfide, decalin, and N,N-diethylaniline and of C70 in CS2 has been measured using an isoperibol solution calorimeter. Exothermic heats of solution are observed in all cases. The heat of solution of C60 in toluene is −8.6 ± 0.7 kJ mol-1; in carbon disulfide it is −20 ± 1 kJ mol-1. In decalin, slow dissolution prevents an accurate determination, but the heat of solution is less than −5 kJ mol-1. In N,N-diethylaniline, a fast exothermic step comparable to that observed in other systems is followed by a much larger heat evolution, implying that a chemical reaction is occurring. The heat of solution of C70 in CS2 is −9 ± 1 kJ mol-1. A thermodynamic theory of the temperature dependence of fullerene solubility is presented. The central idea is that the unusual temperature dependence of the solubilities of fullerenes is caused by the formation of solvated crystals. Conditions are derived under which a temperature maximum of solubility, caused by an incongruent melting point of a solvated crystal, will occur. The calorimetric results suggest that the negative temperature dependence of the solubility of fullerenes may be quite general if the solid phase in equilibrium with the saturated solution is unsolvated fullerene.