(CTG)n expansion at DMPK locus seen only in muscle tissue: a novel case

Triplet repeat expansion in 3 untranslated region of myotonic dystrophy protein kinase (DMPK) gene has been implicated as causative in myotonic dystrophy (DM). In cases of DM, high levels of somatic instability have been reported, in which inter-tissue repeat length differences as large as 3000 repeats have been observed. This study highlights the inter-tissue (CTG)n expansion variability at the DMPK locus. Molecular analysis of DMPK gene, encompassing the triplet repeat expansion, was carried out in 31 individuals (11 clinically identified DM patients, 20 controls). All controls showed a 2.1kb band (upto 35 CTG repeats), while four cases exhibited an expansion (>50 repeats). A novel observation was made in one case, wherein the DNA from lymphocytes showed a normal 2.1kb band while the muscle tissue DNA from the same patient was heterozygous for normal and 4.3 kb band (>700 repeats). Our results suggested that because inter-tissue variability existed in the (CTG)n repeat number at DMPK locus, an attempt should be made to evaluate affected tissue along with blood wherever possible prior to making a final diagnosis. This is important not only for diagnosis and prenatal analysis, but also while providing genetic counseling to families.     

CTG trinucleotide repeat "big jumps": large expansions, small mice

Trinucleotide repeat expansions are the genetic cause of numerous human diseases, including fragile X mental retardation, Huntington disease, and myotonic dystrophy type 1. Disease severity and age of onset are critically linked to expansion size. Previous mouse models of repeat instability have not recreated large intergenerational expansions ("big jumps"), observed when the repeat is transmitted from one generation to the next, and have never attained the very large tract lengths possible in humans. Here, we describe dramatic intergenerational CTG*CAG repeat expansions of several hundred repeats in a transgenic mouse model of myotonic dystrophy type 1, resulting in increasingly severe phenotypic and molecular abnormalities. Homozygous mice carrying over 700 trinucleotide repeats on both alleles display severely reduced body size and splicing abnormalities, notably in the central nervous system. Our findings demonstrate that large intergenerational trinucleotide repeat expansions can be recreated in mice, and endorse the use of transgenic mouse models to refine our understanding of triplet repeat expansion and the resulting pathogenesis.     

Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member.

Using positional cloning strategies, we have identified a CTG triplet repeat that undergoes expansion in myotonic dystrophy patients. This sequence is highly variable in the normal population. PCR analysis of the interval containing this repeat indicates that unaffected individuals have been 5 and 27 copies. Myotonic dystrophy patients who are minimally affected have at least 50 repeats, while more severely affected patients have expansion of the repeat containing segment up to several kilobase pairs. The CTG repeat is transcribed and is located in the 3' untranslated region of an mRNA that is expressed in tissues affected by myotonic dystrophy. This mRNA encodes a polypeptide that is a member of the protein kinase family.     

Somatic heterogeneity of the CTG repeat in myotonic dystrophy is age and size dependent.

The most common form of adult muscular dystrophy, myotonic dystrophy (DM), is caused by the abnormal expansion of the CTG repeat, located in the 3' UTR of the DM gene. The expanded-CTG allele often presents as a diffused band on Southern blot analysis, suggesting somatic mosaicism. In order to study the somatic instability of the CTG repeat, we have investigated the dynamics of the size heterogeneity of the CTG expansion. Size heterogeneity is shown as a smear on Southern blot and is measured by the midpeak-width ratio of the expanded allele to the normal sized allele. The ratio is also corrected for compression in the higher-molecular-weight region. It is found that the size heterogeneity of the expanded-CTG repeats, of 173 DM patients, correlates well with the age of the patient (r = .81, P << .001). The older patients show larger size variation. This correlation is independent of the sex of either the patient or the transmitting parent. The size heterogeneity of the expansion, based on age groups, is also dependent on the size of the expanded trinucleotide repeat. However, obvious size heterogeneity is not observed in congenital cases, regardless of the size of expansion. Comparison of individual patient samples collected at two different times has confirmed that the degree of size heterogeneity increases with age and has revealed a subtle but definite upward shift in the size of the expanded-CTG allele. The progression of the CTG repeat toward larger expansion with age is further confirmed by small-pool PCR assay that resolved the heterogeneous fragments into discrete bands.(ABSTRACT TRUNCATED AT 250 WORDS)     

CTG Trinucleotide Repeat “Big Jumps”: Large Expansions, Small Mice

Trinucleotide repeat expansions are the genetic cause of numerous human diseases, including fragile X mental retardation, Huntington disease, and myotonic dystrophy type 1. Disease severity and age of onset are critically linked to expansion size. Previous mouse models of repeat instability have not recreated large intergenerational expansions (“big jumps”), observed when the repeat is transmitted from one generation to the next, and have never attained the very large tract lengths possible in humans. Here, we describe dramatic intergenerational CTG•CAG repeat expansions of several hundred repeats in a transgenic mouse model of myotonic dystrophy type 1, resulting in increasingly severe phenotypic and molecular abnormalities. Homozygous mice carrying over 700 trinucleotide repeats on both alleles display severely reduced body size and splicing abnormalities, notably in the central nervous system. Our findings demonstrate that large intergenerational trinucleotide repeat expansions can be recreated in mice, and endorse the use of transgenic mouse models to refine our understanding of triplet repeat expansion and the resulting pathogenesis.     

Haploinsuffciency for Znf9 in Znf9+/- mice is associated with multiorgan abnormalities resembling myotonic dystrophy

Myotonic dystrophy type 2 is caused by a (CCTG)/(CCUG)n repeat expansion in the first intron of the ZNF9 gene. The pathomechanism for the myotonic dystrophies is not well understood and the role of ZNF9 in myotonic dystrophy type 2 pathogenesis has not been fully clarified. We characterized Znf9+/- mice, in which the expression of Znf9 was significantly decreased, and found that their phenotype reflects many of the features of myotonic dystrophy, including muscle histological morphology, and myotonic discharges and heart conduction abnormalities, shown by electromyography and electrocardiogram analysis, respectively. Znf9 is normally highly expressed in heart and skeletal muscle, where skeletal muscle chloride channel 1 (Clc1) plays an important role. Clc1 expression was dramatically decreased in Znf9+/- mice. Znf9 transgenic mice raised Znf9 and Clc1 expression and rescued the myotonic dystrophy phenotype in Znf9+/- mice. Our results suggest that the Znf9 haploinsufficiency contributes to the myotonic dystrophy phenotype in Znf9+/- mice.     

Genetic anticipation in Portuguese kindreds with familial amyloidotic polyneuropathy is unlikely to be caused by triplet repeat expansions

Familial amyloidotic polyneuropathy (FAP) is a lethal autosomal dominant type of amyloidosis resulting from the deposition of transthyretin (ATTR) variants in the peripheral and autonomic nervous systems. ATTR V30M-associated FAP exhibits marked genetic anticipation in some families, with clinical symptoms developing at an earlier age in successive generations. The genetic basis of this phenomenon in FAP is unknown. Anticipation has been associated with the dynamic expansion of trinucleotide repeats in several neurodegenerative disorders, such as Huntington disease, myotonic dystrophy, and fragile X syndrome. We have used the repeat expansion detection (RED) assay to screen affected members of Portuguese FAP kindreds for expansion of any of the ten possible trinucleotide repeats. Nine generational pairs with differences in their age of onset greater than 12 years and a control pair with identical ages of onset were tested. No major differences were found in the lengths of the ten trinucleotide repeats analyzed. The distribution of the maximal repeat sizes was consistent with reported studies in unrelated individuals with no known genetic disease. The present data do not support a role for trinucleotide repeat expansions as the molecular mechanism underlying anticipation in Portuguese FAP. Received: 13 December 1998 / Accepted: 23 March 1999     

Dentatorubral-pallidoluysian atrophy (DRPLA): Close correlation of CAG repeat expansions with the wide spectrum of clinical presentations and prominent anticipation

Dentatorubral-pallidoluysian atrophy (DRPLA) is a rare autosomal dominant neurodegenerative disease characterized by various combinations of ataxia, choreoathetosis, myoclonus, epilepsy and dementia as well as various ages of onset. We have identified a specific unstable trinucleotide repeat expansion in a gene on the short arm of chromosome 12 as the pathogenic mutation for DRPLA. We investigated how the degree of the expansion of the CAG repeat affects the clinical manifestations of DRPLA. The sizes of the expanded alleles were well correlated with the ages of onset (r = −0.6955, P < 0.001). Patients with progressive myoclonus epilepsy (PME) phenotype had larger expansions (62–79 repeats) and earlier ages of onset (onset before age 20). Furthermore, most of the patients with PME phenotype inherited their expanded alleles from their affected fathers. On the other hand, patients with non-PME phenotype showed later ages of onset (onset after age 20) and smaller expansions (54–67 repeats). When ages of onset of each clinical symptom are compared with sizes of the CAG repeat, there is again a remarkably high correlation of the sizes of CAG repeat with each of the clinical symptoms. Thus the wide variation in clinical manifestations of DRPLA can now be clearly explained based on the degree of CAG repeat expansion, which strongly indicates that the expanded alleles are intimately involved in the neuronal degeneration in dentatofugal and pallidofugal systems.     

De novo myotonic dystrophy mutation in a Nigerian kindred.

An expansion of an unstable (CTG)n trinucleotide repeat in the 3' UTR of a gene encoding a putative serine/threonine protein kinase (DMPK) on human chromosome 19q13.3 has been shown to be specific for the myotonic dystrophy (DM) disease phenotype. In addition, a single haplotype composed of nine alleles within and flanking DMPK over a physical distance of 30 kb has been shown to be in complete linkage disequilibrium with DM. This has led to two hypotheses: (1) predisposition for (CTG)n instability results from a founder effect that occurred only once or a few times in human evolution; and (2) elements within the disease haplotype may predispose the (CTG)n repeat to instability. A detailed haplotype analysis of the DM region was conducted on a Nigerian (Yoruba) DM family, the only indigenous sub-Saharan DM case reported to date. Each affected member of this family had an expanded (CTG)n repeat in one of his or her DMPK alleles. However, unlike all other DM populations studied thus far, disassociation of the (CTG)n repeat expansion from other alleles of the putative predisposing haplotype was found. We conclude that the expanded (CTG)n repeat in this family is the result of an independent mutational event. Consequently, the origin of DM is unlikely to be a single mutational event, and the hypothesis that a single ancestral haplotype predisposes to repeat expansion is not compelling.     

Anticipation and Instability of IT-15 (CAG)N Repeats in Parent-Offspring Pairs with Huntington Disease

Huntington disease (HD) is an autosomal dominant degenerative disorder caused by an expanded and unstable trinucleotide repeat (CAG)n in a gene (IT-15) on chromosome 4. HD exhibits genetic anticipation—earlier onset in successive generations within a pedigree. From a population-based clinical sample, we ascertained parent-offspring pairs with expanded alleles, to examine the intergenerational behavior of the trinucleotide repeat and its relationship to anticipation. We find that the change in repeat length with paternal transmission is significantly correlated with the change in age at onset between the father and offspring. When expanded triplet repeats of affected parents are separated by median repeat length, we find that the longer paternal and maternal repeats are both more unstable on transmission. However, unlike in paternal transmission, in which longer expanded repeats display greater net expansion than do shorter expanded repeats, in maternal transmission there is no mean change in repeat length for either longer or shorter expanded repeats. We also confirmed the inverse relationship between repeat length and age at onset, the higher frequency of juvenile-onset cases arising from paternal transmission, anticipation as a phenomenon of paternal transmission, and greater expansion of the trinucleotide repeat with paternal transmission. Stepwise multiple regression indicates that, in addition to repeat length of offspring, age at onset of affected parent and sex of affected parent contribute significantly to the variance in age at onset of the offspring. Thus, in addition to triplet repeat length, other factors, which could act as environmental factors, genetic factors, or both, contribute to age at onset. Our data establish that further expansion of paternal repeats within the affected range provides a biological basis of anticipation in HD.     

Application of a closely linked polymorphism of restriction fragment length to counselling and prenatal testing in families with myotonic dystrophy.

The close genetic linkage between the loci for apolipoprotein CII (ApoC2) and myotonic dystrophy makes ApoC2 the closest fully validated marker for prediction of myotonic dystrophy. Application to genetic counselling and presymptomatic and prenatal prediction is reported in seven families with myotonic dystrophy, including one case in which the disorder was excluded prenatally. Only one of the families did not have members with ApoC2 genotypes that allowed prediction, but careful clinical study of older family members was found to be an important factor. ApoC2 typing of families with myotonic dystrophy should be of practical help both in prediction for asymptomatic relatives and for prenatal diagnosis in pregnancies of an affected parent.     

Dominantly inherited,non-coding microsatellite expansion disorders

Dominantly inherited diseases are generally caused by mutations resulting in gain of function protein alterations. However, a CTG expansion located in the 3' untranslated portion of a kinase gene was found to cause myotonic dystrophy type 1, a multisystemic dominantly inherited disorder. The recent discovery that an untranslated CCTG expansion causes the same constellation of clinical features in myotonic dystrophy type 2 (DM2), along with other recent discoveries on DM1 pathogenesis, have led to the understanding that both DM1 and DM2 mutations are pathogenic at the RNA level. These findings indicate the existence of a new category of disease wherein repeat expansions in RNA alter cellular function. Pathogenic repeat expansions in RNA may also be involved in spinocerebellar ataxia types 8, 10 and 12, and Huntington's disease-like type 2.     

Häufige Erkrankungen mit Repeatexpansionen

Expansions of unstable DNA repeats are causes of a growing number of human developmental and degenerative disorders. The repetitive sequences vary in copy number even in normal individuals. In affected individuals the repeat is expanded beyond a disease-specific threshold. There is some relationship between the copy number of the repeat and the severity and/or age at onset of symptoms. An earlier age of onset and a more severe clinical phenotype in subsequent generations (anticipation) is usually correlated with larger repeat size. Expanded alleles show both somatic and germ-line instability and, usually, expand rather than contract upon transmission from parent to offspring. In doing so, the sex of the transmitting parent can influence the size of the disease allele in the affected child. The unstable repeat may be located in non-coding regions or within the open reading frame of the disease gene. Depending on the intragenic location and the mode of inheritance the pathogenetic mechanisms involve loss of protein function as well as gain of function at protein or RNA level.     

Somatic and germline instability of the ATTCT repeat in spinocerebellar ataxia type 10

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant disorder characterized by ataxia, seizures, and anticipation. It is caused by an expanded ATTCT pentanucleotide repeat in intron 9 of a novel gene, designated "SCA10." The ATTCT expansion in SCA10 represents a novel class of microsatellite repeat and is one of the largest found to cause human diseases. The expanded ATTCT repeat is unstably transmitted from generation to generation, and an inverse correlation has been observed between size of repeat and age at onset. In this multifamily study, we investigated the intergenerational instability, somatic and germline mosaicism, and age-dependent repeat-size changes of the expanded ATTCT repeat. Our results showed that (1) the expanded ATTCT repeats are highly unstable when paternally transmitted, whereas maternal transmission resulted in significantly smaller changes in repeat size; (2) blood leukocytes, lymphoblastoid cells, buccal cells, and sperm have a variable degree of mosaicism in ATTCT expansion; (3) the length of the expanded repeat was not observed to change in individuals over a 5-year period; and (4) clinically determined anticipation is sometimes associated with intergenerational contraction rather than expansion of the ATTCT repeat.     

Pathogenic RNA repeats: an expanding role in genetic disease

Fragile X mental retardation and Friedreich's ataxia were among the first pathogenic trinucleotide repeat disorders to be described in which noncoding repeat expansions interfere with gene expression and cause a loss of protein production. Invoking a similar loss-of-function hypothesis for the CTG expansion causing myotonic dystrophy type 1 (DM1) located in the 3' noncoding portion of a kinase gene was more difficult because DM is a dominantly inherited multisystemic disorder in which the second copy of the gene is unaffected. However, the discovery that a transcribed but untranslated CCTG expansion causes myotonic dystrophy type 2 (DM2), along with other discoveries on DM1 and DM2 pathogenesis, indicate that the CTG and CCTG expansions are pathogenic at the RNA level. This review will detail recent developments on the molecular mechanisms of RNA pathogenesis in DM, and the growing number of expansion disorders that might involve similar pathogenic RNA mechanisms.     

Molecular genetics of the fragile-X syndrome: a novel type of unstable mutation.

Fragile-X syndrome, the most common inherited form of mental retardation, has a very unusual mode of inheritance. The disease is caused by a multistep expansion, in successive generations, of a polymorphic CGG repeat localized in a 5' exon of FMR-1, a gene of unknown function. Two main mutation types have been categorized. Premutations are moderate expansions of the repeat and do not cause mental retardation. Full mutations are found in affected individuals and involve larger expansions of the repeat, with abnormal methylation of the neighboring CpG island. The full mutations demonstrate striking somatic instability and extinguish expression of FMR-1. Premutations are changed to full mutation only when transmitted by a female with a frequency that increases up to 100% as a function of the initial size of the premutation. Direct detection of the mutations provides an accurate test for pre- and postnatal diagnosis of the disease, and for carrier detection. A similar unstable expansion of a trinucleotide repeat occurs in myotonic dystrophy.     

Myotonic dystrophy type 1 (DM1): A triplet repeat expansion disorder

Myotonic dystrophy is a progressive multisystem genetic disorder affecting about 1 in 8000 people worldwide. The unstable repeat expansions of (CTG)n or (CCTG)n in the DMPK and ZNF9 genes cause the two known subtypes of myotonic dystrophy: (i) myotonic dystrophy type 1 (DM1) and (ii) myotonic dystrophy type 2 (DM2) respectively. There is currently no cure but supportive management helps equally to reduce the morbidity and mortality and patients need close follow up to pay attention to their clinical problems. This review will focus on the clinical features, molecular view and genetics, diagnosis and management of DM1.     

The likelihood of being affected with Huntington disease by a particular age, for a specific CAG size.

Prior studies describing the relationship between CAG size and the age at onset of Huntington disease (HD) have focused on affected persons. To further define the relationship between CAG repeat size and age at onset of HD, we now have analyzed a large cohort of affected and asymptomatic at-risk persons with CAG expansion. This cohort numbered 1,049 persons, including 321 at-risk and 728 affected individuals with a CAG size of 29-121 repeats. Kaplan-Meier analysis has provided curves for determining the likelihood of onset at a given age, for each CAG repeat length in the 39-50 range. The curves were significantly different (P < .0005), with relatively narrow 95% confidence intervals (95% CI) (+/-10%). Penetrance of the mutation for HD also was examined. Although complete penetrance of HD was observed for CAG sizes of > or = 42, only a proportion of those with a CAG repeat length of 36-41 showed signs or symptoms of HD within a normal life span. These data provide information concerning the likelihood of being affected, by a specific age, with a particular CAG size, and they may be useful in predictive-testing programs and for the design of clinical trials for persons at increased risk for HD.     

Characteristics of Intergenerational Contractions of the CTG Repeat in Myotonic Dystrophy

In myotonic dystrophy (DM), the size of a CTG repeat in the DM kinase gene generally increases in successive generations with clinical evidence of anticipation. However, there have also been cases with an intergenerational contraction of the repeat. We examined 1,489 DM parent-offspring pairs, of which 95 (6.4%) showed such contractions in peripheral blood leukocytes (PBL). In 56 of the 95 pairs, clinical data allowed an analysis of their anticipation status. It is surprising that anticipation occurred in 27 (48%) of these 56 pairs, while none clearly showed a later onset of DM in the symptomatic offspring. The contraction occurred in 76 (10%) of 753 paternal transmissions and in 19 (3%) of 736 maternal transmissions. Anticipation was observed more frequently in maternal (85%) than in paternal (37%) transmissions (P < .001). The parental repeat size correlated with the size of intergenerational contraction (r² = .50, P « .001), and the slope of linear regression was steeper in paternal (–.62) than in maternal (–.30) transmissions (P « .001). Sixteen DM parents had multiple DM offspring with the CTG repeat contractions. This frequency was higher than the frequency expected from the probability of the repeat contractions (6.4%) and the size of DM sib population (1.54 DM offspring per DM parent, in 968 DM parents). We conclude that (1) intergenerational contraction of the CTG repeat in leukocyte DNA frequently accompanies apparent anticipation, especially when DM is maternally transmitted, and (2) the paternal origin of the repeat and the presence of the repeat contraction in a sibling increase the probability of the CTG repeat contraction.