Intranuclear RNA-foci



Premutation differences mother/offspring



Premutation differences father/offspring



aDisease despite normal allele. Assumed.

aDisease despite normal allele. Assumed.

classified as protomutation with a mild or asymptomatic phenotype.[3] Alleles between 38 and 49 repeats, designated as premutation, do not manifest clinically, but may expand to the full mutation range in subsequent generations. In the normal population the repeat length ranges between 4 and 37. The normal allele is usually stable upon transmission. Occasionally mutations occur, causing a transition from a large normal allele to a small, expanded allele (transition mutation). One factor that prevents DM1 from dying out is segregation distortion (alleles with 19-37 repeats are preferentially transmitted to offsprings than alleles with 4-18 repeats). The expanded DMPK allele is transcribed but not translated. It produces RNA transcripts containing long tracts of CUG repeats, but no dysfunctional protein. Mutant mRNA is not exported to the cytoplasm but retained within multiple nuclear foci. The amount of DMPK in the cytoplasm is presumably reduced, as it can be translated only from the normal allele (haploinsufficiency).

A hallmark of the CTG expansion is its high intra-individual, interindividual and intergenerational variability in somatic and germline cells. Expansion instability is thought to arise from its capacity to form long hairpin loops during replication, leading to folded structures, and the involvement of various DNA-repair mechanisms. Several models try to explain how an encounter between folded structures and the replication apparatus leads to expansion, including the following: Rad52 and Rad54, involved in DNA strand break repair (homologous recombination), strongly destabilize long CTG repeats, enabling expansion. Msh2 (major component of the mismatch repair system) stabilizes anomalous slipped strand or loop end structures thereby promoting incorporation of further repeats.[4] DNA Pkcs is involved in nonhomologous end joining.[4] So far, however, it remains unknown how these mechanisms affect repeat instabili-ty.[4] Once in the disease-associated range, the expansion size progressively increases in subsequent generations (anticipation), although reversion back to a normal size may also occur (retraction). The CTG expansion continues to expand throughout life, causing age-dependent somatic mosaicism. During embryogenesis the CTG expansion remains fairly equal in various tissues, but postnatally marked variability emerges in different tissues. CTG expansions appear to continue to increase even in non-dividing cells. The repeat instability is tissue specific, with muscle cells and myocardiocytes containing larger expansions than blood leukocytes. Premutation alleles are generally more stable during transmission through the female than the male germline.[3] Small protomutations are very unstable in the male germline and highly biased toward expansion.[3] Congenital DM patients have CTG expansions >1000 and inherit the disease almost invariably from their mother, reflecting a sex difference in the dynamics of the repeat instability during gametogenesis. Rarely, fathers transmit congenital DM1. When the repeat size varies between sibs, differences tend to be greater when the affected parent is the father.

Immunohistological investigations localize DMPK, an 80-kDa protein kinase, most highly expressed in the skeletal muscle and heart, to the neuromuscular junction, terminal cisternae of the sarcoplasmic reticulum, and gap junctions. DMPK consists of a leucine-rich repeat, a catalytic domain, an a-helical coil-coiled region, and a transmembrane-spanning tail.[5] The DMPK function is not yet fully understood.1-6-1 DMPK has been implicated in modulating calcium homeostasis and initial events concerning excitation-contraction coupling and was associated with the heat shock protein myotonic dystrophy protein kinase-binding protein.[6] Additionally, the specific regulation of DMPK expression and activity during myocyte differentiation suggests a functional implication of DMPK in the generation or maintenance of myo-tubes.[6] DMPK is reduced in the DM1 muscle.

Mechanisms to explain the deleterious effect of mutant DMPK are the following:

1. Chromatin conformation alteration in the vicinity of the DMPK gene, resulting in partial suppression of neighboring genes, like the dystrophia myotonica-associated homeobox protein (DMAHP or SIX5) or immunodominant peptide N59. Suppression of DMAHP may contribute to the development of

cataracts. ]

2. Haploinsufficiency, resulting in shortage of the functionally available enzyme.

3. Altered cellular DMPK location.

4. Disturbed distribution and transport of the expanded mRNA transcripts.

5. Toxic effect of mutant mRNAs by sequestering essential RNA-CUG-binding proteins.There is evidence that rather a gain of function for RNA than haploinsufficiency plays the prominent pathogenetic role.[7] Gain of function involves a transdominant effect (both alleles are poorly processed) on RNA-CUG-binding proteins, such as CUG-binding protein 1 (CUGBP1), elav-like RNA-binding protein 3 (ETR3), muscleblind proteins (MBNL, MBLL, and MBXL), and PKR.[7] Gain of function is substantiated by recruitment of muscleblind proteins into ribonuclear inclusions in fibroblasts and muscle cells from DM1 and DM2 patients, which clearly interact with expanded CUG repeats. mRNA accumulation presumably skews alternative splicing also of unrelated specific pre-RNAs,[8] because RNA CUG induces reiteration of and reversion to embryonic splicing patterns, mediated by CELF proteins. CUGBP1 does not extensively bind expanded CUG repeats in vitro, although some studies detected CUGBP1 within ribo-nuclear inclusions.

Targets of abnormal splicing are mRNAs for:

1. The insulin receptor. Insulin resistance may be due to missplicing of exon 11 skipping isoform pre-mRNA.

2. Cardiac troponine-T. Splicing regulation of troponine transcripts is abnormal.

3. Tau protein. Altered stoichiometry of the exon 2 skipping tau protein isoform could explain behavioral and cognitive alterations.

4. Myotubularin-1. Muscle-specific isoform is reduced and an abnormal transcript appears in differentiated DM1 myocytes.

5. Muscle-specific chloride channel ClC-1. Loss of ClC-1 mRNA and protein after inappropriate regulation of ClC-1 alternative splicing because of CUGBP-1 overactivity results in hyperexcitability as a result of reduced transmembrane chloride conduction.[7]

6. MyoD. This transcription factor, required for the differentiation of myoblasts during muscle regeneration, by activating differentiation-specific genes via binding E-boxes, is reduced in cells containing mutant DMPK transcripts.[8] All these findings suggest abnormal regulation of alternative splicing to be fundamentally involved in the pathogenesis of DM1. There is also evidence for a delay in muscle maturation, possibly because of a retarded rate of myoblast fusion to multinucleated myotubes.[6]

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