Deciphering the contribution of genetic instability in somatic cells is critical to our understanding of many human disorders. Myotonic dystrophy type 1 (DM1) is one such disorder that is caused by the expansion of a CTG repeat that shows extremely high levels of somatic instability. This somatic instability has compromised attempts to measure intergenerational repeat dynamics and infer genotype-phenotype relationships. Using single-molecule PCR, we have characterized more than 17 000 de novo somatic mutations from a large cohort of DM1 patients. These data reveal that the estimated progenitor allele length is the major modifier of age of onset. We find no evidence for a threshold above which repeat length does not contribute toward age at onset, suggesting pathogenesis is not constrained to a simple molecular switch such as nuclear retention of the DMPK transcript or haploinsufficiency for DMPK and/or SIX5. Importantly, we also show that age at onset is further modified by the level of somatic instability; patients in whom the repeat expands more rapidly, develop the symptoms earlier. These data establish a primary role for somatic instability in DM1 severity, further highlighting it as a therapeutic target. In addition, we show that the level of instability is highly heritable, implying a role for individual-specific trans-acting genetic modifiers. Identifying these trans-acting genetic modifiers will facilitate the formulation of novel therapies that curtail the accumulation of somatic expansions and may provide clues to the role these factors play in the development of cancer, aging and inherited disease in the general population.

Myotonic dystrophy type 1 (DM1) is one of the most variable inherited human disorders. It is characterized by the involvement of multiple tissues and is caused by the expansion of a highly unstable CTG repeat. Variation in disease severity is partially accounted for by the number of CTG repeats inherited. However, the basis of the variable tissue-specific symptoms is unknown. We have determined that an unusual Dutch family co-segregating DM1, Charcot-Marie-Tooth neuropathy, encephalopathic attacks and early hearing loss, carries a complex variant repeat at the DM1 locus. The mutation comprises an expanded CTG tract at the 5'-end and a complex array of CTG repeats interspersed with multiple GGC and CCG repeats at the 3'-end. The complex variant repeat tract at the 3'-end of the array is relatively stable in both blood DNA and the maternal germ line, although the 5'-CTG tract remains genetically unstable and prone to expansion. Surprisingly though, even the pure 5'-CTG tract is more stable in blood DNA and the maternal germ line than archetypal DM1 alleles of a similar size. Complex variant repeats were also identified at the 3'-end of the CTG array of approximately 3-4% of unrelated DM1 patients. The observed polarity and the stabilizing effect of the variant repeats implicate a cis-acting modifier of mutational dynamics in the 3'-flanking DNA. The presence of such variant repeats very likely contributes toward the unusual symptoms in the Dutch family and additional symptomatic variation in DM1 via affects on both RNA toxicity and somatic instability.

OBJECTIVE:To report new disease components in a unique myotonic dystrophy type 1 (DM1) family previously described by us in which all affected members also had a sensorimotor neuropathy that co-segregated with markers flanking the DM1 locus.METHODS:Clinical observations, electrophysiology, audiometry, DNA studies.RESULTS:During a follow-up period of over 25 years, the following were observed: (i) co-segregation of a striking new encephalopathic phenotype. In middle age, five patients were admitted on multiple occasions with attacks of impaired consciousness, psychomotor agitation, fever and, in about half of the cases, focal neurological signs, including unilateral weakness, sensory deficits and dysphasia. Reported onset phenomena consisted of confusion, headache, focal neurological symptoms and nausea; (ii) many patients show an early and severe sensorineural hearing loss; (iii) although they have mothers with the adult onset type, the four affected subjects from the youngest generation do not show any signs or symptoms of childhood or congenital myotonic dystrophy; (iv) the neuropathy meets the criteria of an intermediate type Charcot-Marie-Tooth (CMT), and is more severe in males; and (v) patients presented with an expanded fragment at the DM1 CTG repeat but this allele was refractory to PCR amplification and triplet repeat primed PCR at the 3' end of the array, indicating the existence of an additional lesion at the 3' end.CONCLUSIONS:The phenotype in this unique family extends beyond myotonic dystrophy and CMT to include encephalopathic attacks and early hearing loss, and is associated with an atypical mutation at the DM1 locus.

The dominant oculo-pharyngeal muscular dystrophy mutation consists of an expanded (GCN)(12-17) in the coding region of the PolyA Binding Protein Nuclear 1 gene. A founder effect has been demonstrated in Canadian and Bukhara Jewish populations with relatively high prevalence of this disease. Since the oculo-pharyngeal muscular dystrophy prevalence was remarkably high in Southern Uruguay, a founder effect was hypothesized. To identify the ancestral haplotype we determined the (GCN) repeat number and the variants of four intragenic SNPs in Uruguayan OPMD families and a control sample. All families carrying the mutation (GCG)(11)(GCA)(3)(GCG) shared a common ancestral haplotype and the age of the mutation was estimated in 37-53 generations by a composite likelihood method. One family carrying the (GCG)(9)(GCA)(3)(GCG) allele had a different haplotype. The genealogical and molecular data suggested that the common ancestors were Canary Islands' settlers that arrived in Uruguay in the XIX century.

The relaxant effect of the vasodilator drug, nicorandil, was studied in circular strips of bovine coronary arteries. To differentiate between relaxation caused by cyclic GMP (cGMP) and by hyperpolarization, the influence of cGMP was blocked with methylene blue and that of hyperpolarization with the inhibitor of ATP-dependent K+ channels, glibenclamide. Methylene blue and glibenclamide inhibited nicorandil-induced relaxation to similar extents. Cromakalim-induced relaxation but not that due to sodium nitroprusside (nitroprusside-Na) was inhibited by glibenclamide. Methylene blue inhibited the relaxation caused by nitroprusside-Na but not that due to cromakalim. The different modes of action of the two components of relaxation caused by nicorandil were studied in agonist-agonist interaction experiments. The interaction between nicorandil and nitroprusside-Na or 3-morpholino-sydnonimine (SIN-1) was overadditive in the absence of glibenclamide but additive, i.e. competitive, in the presence of glibenclamide. The interaction of nicorandil with cromakalim or pinacidil was overadditive in the absence of methylene blue but additive, i.e. competitive, in the presence of methylene blue. The results show that nicorandil relaxes smooth muscle through two independent mechanisms: ATP-dependent activation of K+ channels and stimulation of guanylyl cyclase resulting in increases in cGMP.

In addition to previous results from our laboratory showing that micorandil relayed vascular smooth muscle by increasing evelic GMP levels it was shown to activate K-channel, as well. an effect that also leads to relavation. In the present study. we attempted to differentiate quantitatively between these two elieels in isolated bovine coronary artery strips with simultaneous isotonic measurement of length and radioimmunoassay (RIA) determination of cyclie GMP. When the strips were contracted by the thromhovane A analogue U 46619 (1 μM) with 10 μM methylene blue added. nieorandil produced 30–50 relavation without significant changes in cyclic GMP. When in U 46619-contracted strips the hyperpolarizing effect of nicorandil was suppressed by increasing extracellular K to 80.4 mM (30-fold), nieorandil caused only 52 relaxation. whereas cyclic GMP increases were not significantly suppressed. Quantitative separation of both mechantsms f relaxation H nicorandil was further achicved through calculation of the cyclic GMP mediated component from a correlation between increases in cyclie GMP and percentage of relayation as, produced by mcorandil under condition, of inhibited hyperpolarization. i.e., in strips contracted with 1μM U 46619 or 26.8 mM K (10 fold) and exposed to either 30-fold K or 10 mM Ba Under both condition,. similar correlations between cylic GMP and relayation were ohtained. Because U 46619, in Addition to its contractile effect. partially antugonized the relaxation by nicorandil without changing cyclic GMP. the correlation was corrected for this effect and indicated a participation of cyclie GMP In the overall reluvunl response of 30–40 at low and 80–90 at high concentrations of nicorandil.