Effect of the myotonic dystrophy (DM) mutation on mRNA levels of the DM gene.

Myotonic dystrophy (DM) results from the amplification of an unstable CTG repeat in the 3' untranslated region of a transcript encoding a putative serine/threonine kinase. We have analysed the amplification of the repeat and the steady state levels of the DM kinase (DMK) mRNA in tissues and cell lines from normal and congenital DM individuals. Southern blot analysis of DNA samples from a severely affected neonate shows somatic heterogeneity of the repeat in all tissues studied. RNA analyses on these tissues show a marked increase in DMK steady state mRNA levels. We demonstrate that the mutant DMK allele is expressed regardless of the number of CTG repeats and that the increase in DMK mRNA levels is due to elevated mutant mRNA levels. We postulate that elevated DMK levels explains the dominant inheritance pattern of DM.

Reduction in size of the myotonic dystrophy trinucleotide repeat mutation during transmission.

Myotonic dystrophy (DM) is an autosomal-dominant disorder that affects 1 in 8000 individuals. Amplification of an unstable trinucleotide CTG repeat, located within the 3' untranslated region of a gene, correlates with a more severe DM phenotype. In three cases, the number of CTG repeats was reduced during the transmission of the DM allele; in one of these cases, the number was reduced to within the normal range and correlated at least with a delay in the onset of clinical signs of DM. Haplotype data of six polymorphic markers in the DM gene region indicate that, in this latter case, two stretches of the affected chromosome had been exchanged with that region of the wild-type chromosome.

Overexpression of MBNL1 fetal isoforms and modified splicing of Tau in the DM1 brain: two individual consequences of CUG trinucleotide repeats.

Neurofibrillary degeneration is often observed in the brain of patients with type 1 myotonic dystrophy (DM1). It consists principally of the aggregation of Tau isoforms that lack exon 2/3 encoded sequences, and is the consequence of the modified splicing of Tau pre-mRNA. In experimental models of DM1, the splicing of several transcripts is modified due to the loss of Muscleblind-like 1 (MBNL1) function. In the present study, we demonstrate that the MBNL1 protein is also present in the human brain, and consists of several isoforms, as shown by RT-PCR and sequencing. In comparison with controls, we show that the adult DM1 brain exhibits modifications in the splicing of MBNL1, with the preferential expression of long MBNL1 isoforms--a splicing pattern similar to that seen in the fetal human brain. In cultured HeLa cells, the presence of long CUG repeats, such as those found in the DM1 mutation, leads to similar changes in the splicing pattern of MBNL1, and the localization of MBNL1 in nuclear RNA foci. Long CUG repeats also reproduce the repression of Tau exon 2/3 inclusion, as in the human disease, suggesting that their effect on MBNL1 expression may lead to changes in Tau splicing. However, while an overall reduction in the expression of MBNL1 mimics the effect of the DM1 mutation, none of the MBNL1 isoforms tested so far modulates the endogenous splicing of Tau. The modified splicing of Tau thus results from a possibly CUG-mediated loss of function of MBNL1, but not from changes in the MBNL1 expression pattern.

Myotonic dystrophy: the role of the CUG triplet repeats in splicing of a novel DMPK exon and altered cytoplasmic DMPK mRNA isoform ratios.

The mechanism by which (CTG)n expansion in the 3' UTR of the DMPK gene causes myotonic dystrophy (DM) is unknown. We identified four RNA splicing factors--hnRNP C, U2AF (U2 auxiliary factor), PTB (polypyrimidine tract binding protein), and PSF (PTB associated splicing factor)--that bind to two short regions 3' of the (CUG)n, and found a novel 3' DMPK exon resulting in an mRNA lacking the repeats. We propose that the (CUG)n is an essential cis acting element for this splicing event. In contrast to (CUG)n containing mRNAs, the novel isoform is not retained in the nucleus in DM cells, resulting in imbalances in relative levels of cytoplasmic DMPK mRNA isoforms and a new dominant effect of the mutation on DMPK.

The myotonic dystrophy expanded CUG repeat tract is necessary but not sufficient to disrupt C2C12 myoblast differentiation.

Myotonic dystrophy type 1 (DM1) is a dominant neuromuscular disorder caused by a trinucleotide (CTG) repeat expansion. Mutant DMPK 3'-untranslated region (3'-UTR) transcripts aggregate in nuclear foci and are thought to impose dominant-negative effects by interacting with RNA binding proteins. We demonstrated previously that the mutant 3'-UTR RNA disrupted C2C12 myoblast differentiation, and that the CUG expansion was necessary for this effect. Several proteins are known to interact with the CUG tract or the region 3' (distal) to it. Here, using a library of transfected C2C12 clones, we show that although transcripts containing a CUG expansion alone or a CUG expansion plus the distal region of the DMPK 3'-UTR accumulate into RNA foci, neither of these RNAs affect C2C12 myogenesis. Thus, RNA foci formation, and perturbation of any RNA binding factors involved in this process, are not sufficient to block myoblast differentiation. Interestingly, we found that transcripts containing expanded CUG tracts can form both nuclear and cytoplasmic RNA foci, demonstrating that factors involved in foci formation are present in the nucleus and cytoplasm. RNA analysis of myogenic markers revealed that the mutant DMPK 3'-UTR mRNA does not affect myoblast determination factors MyoD or Myf5, but significantly impedes upregulation of the differentiation factors myogenin and p21. C2C12 provide a good model to study adult muscle regeneration. Our observations in this system may be relevant to the lack of a regenerative response to continued muscle wasting in DM, and point to defects in early events in the myogenic response to muscle damage.

Cis and trans effects of the myotonic dystrophy (DM) mutation in a cell culture model.

The mutation causing myotonic dystrophy (DM) has been identified as a CTG expansion in the 3'-untranslated region (3'-UTR) of the DM protein kinase gene ( DMPK ), but the mechanism(s) of pathogenesis remain unknown. Studies using DM patient materials have often produced confusing results. Therefore, to study the effects of the DM mutation in a controlled environment, we have established a cell culture model system using C2C12 mouse myoblasts. By expressing chimeric reporter constructs containing a reporter gene fused to a human DMPK 3'-UTR, we identified both cis and trans effects that are mediated by the DM mutation. Our data show that a mutant DMPK 3'-UTR, with as few as 57 CTGs, had a negative cis effect on protein expression and resulted in the aggregation of reporter transcripts into discrete nuclear foci. We determined by deletion analysis that an expanded (CTG) (n) tract alone was sufficient to mediate these cis effects. Furthermore, in contrast to the normal DMPK 3'-UTR mRNA, a mutant DMPK 3'-UTR mRNA with (CUG)(200)selectively inhibited myogenic differentiation of C2C12 myoblasts. Genetic analysis and the Cre- loxP system were used to clearly demonstrate that the myoblast fusion defect could be rescued by eliminating the expression of the mutant DMPK 3'-UTR transcript. Characterization of spontaneous deletion events mapped the inhibitory effect to the (CTG) (n) expansion and/or the 3' end of the DMPK 3'-UTR. These results provide evidence that the DM mutation acts in cis to reduce protein production (consistent with DMPK haploinsufficiency) and in trans as a 'riboregulator' to inhibit myogenesis.

High resolution genetic analysis suggests one ancestral predisposing haplotype for the origin of the myotonic dystrophy mutation.

The mutation causing myotonic dystrophy (DM) has been identified as an amplification of an unstable trinucleotide (CTG)n repeat in over 99% of the global DM population. It is in complete linkage disequilibrium with an Alu element polymorphism within the DM kinase gene, suggesting that DM is a consequence of one or few ancestral mutations. A recent analysis utilizing this polymorphism as well as a flanking dinucleotide marker, suggested that similar to Fragile X syndrome, DM exhibited a founder effect (Imbert et al., 1993 Nature Genet. 4, 72-76). In contrast, the low reproductive fitness of individuals with congenital DM (the endpoint of genetic anticipation in myotonic dystrophy) suggests a higher rate of new mutations. We present a high resolution genetic analysis of the DM locus using PCR based assays of nine polymorphisms, spanning a physical distance of 30 kb, within and immediately flanking the DM kinase gene. The persistent complete allelic association of the DM mutation with all these polymorphisms provides further support to previous observations and suggests more strongly that the DM mutation occurred on the background of a particular haplotype in which the (CTG)n repeat became inherently unstable and therefore predisposed to amplification.

Intergenerational stability of the myotonic dystrophy protomutation.

The amplification of the CTG trinucleotide repeat in myotonic dystrophy (DM) correlates with increasingly severe phenotypes. We designate its minimal amplification the 'protomutation' since it is the mutation itself at an early stage of intergenerational evolution and is associated with very mild clinical signs. From the study of 536 DM mutation carriers (from 158 affected families), a total of 60 DM-parent/DM-offspring pairings were identified in which the parent had the protomutation. We found a strong correlation between the protomutation length and the amplification observed in the next generation. We also observed the stable transmission of the protomutation through successive generations. This stability may explain the maintenance in the population of this autosomal dominant disease despite the low reproductive fitness of severe DM phenotypes.

Characterization and polymerase chain reaction (PCR) detection of an Alu deletion polymorphism in total linkage disequilibrium with myotonic dystrophy.

The mutation causing myotonic dystrophy has been identified as an unstable trinucleotide CTG repeat located in the 3' untranslated region of a gene putatively encoding a serine-threonine protein kinase. The mutation has been reported to be in total linkage disequilibrium with an insertion/deletion polymorphism located within the kinase gene. To determine the nature of this polymorphism, we have sequenced this genomic fragment and have found that the sequence of this region consists of five consecutive Alu repeats. Further analysis suggests that the smaller of two alleles is actually due to a proposed deletion event that resulted in the loss of an equivalent of three Alu repeats. We have developed a PCR-based assay to detect this polymorphism, the closest, distal marker to the DM mutation.

Evaluation of four commercially available assays for free thyroxin.

We assessed two two-step and two analog assays for measuring free thyroxin (FT4) in serum: Clinical Assays' "GammaCoat Free/Total T4" (CA), Vitek's "KinetiCount Phase II Free T4" (VTK), Diagnostic Products Corporation's "Coat-a-Count Free T4" (DPC) kit (June 1987 version), and Amersham's "Amerlex-M Free T4" (AMX). The VTK assay is automated except for the initial pipetting step. Interassay results correlated well except for samples with abnormal serum albumin concentrations. FT4 values for hypoalbuminemic samples showed a highly significant (P less than 0.0001) correlation with serum albumin concentration in the DPC and AMX assays. The relationships are described by the equations y = 0.382albumin (g/L) + 0.81 pmol/L and y = 0.450albumin (g/L) - 3.20 pmol/L, respectively. When we used an equation derived from the Law of Mass Action to adjust FT4 values to values expected at an ideal albumin concentration, the observed correlation of albumin and FT4 was abolished completely in the DPC assay, and partly so in the AMX assay. The precision of CA was comparable with that of the analog assays; the CV for the VTK assay was approximately twice that for the other three assays.

Structure and genomic sequence of the myotonic dystrophy (DM kinase) gene.

The mutation causing myotonic dystrophy (DM) has recently been identified as an unstable CTG trinucleotide repeat located in the 3' untranslated region of a gene encoding for a protein with putative serine-threonine protein kinase activity. In this report we present the genomic sequences of the human and murine DM kinase gene. A comparison of these sequences with each other and with known cDNA sequences from both species, led us to predict a translation initiation codon, as well as determine the organization of the DM kinase gene. Several polymorphisms within the human DM kinase gene have been identified, and PCR assays to detect two of these are described. The complete sequence and characterization of the structure of the DM kinase gene, as well as the identification of novel polymorphisms within the gene, represent an important step in a further understanding of the genetics of myotonic dystrophy and the molecular biology of the gene.

The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy.

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.

Isolation of a novel G protein-coupled receptor (GPR4) localized to chromosome 19q13.3.

We present the cloning and sequencing of the human gene for a novel G-protein coupled receptor (GPR4), from the critical myotonic dystrophy (DM) region on chromosome 19q13.3. The homologous porcine gene was isolated and sequenced as well. The genes of both species are intronless and contain an open reading frame encoding a protein of 362 amino acids. In human, two isoforms of GPR4 are expressed, differing in their 3' untranslated region due to the use of alternate polyadenylation signals and measuring approximately 2.8 and 1.8 kb, respectively. Northern blot analysis showed that GPR4 is widely expressed, with higher levels in kidney, heart, and especially lung, where it is at least fivefold greater than in other tissues. Sequence analysis suggests that GPR4 is a peptide receptor and shares strongest homologies with purinergic receptors and receptors for angiotensin II, platelet activating factor, thrombin, and bradykinin.

FISH detection of chromosome polymorphism and deletions in the spinal muscular atrophy (SMA) region of 5q13.

The search for the SMA defect has culminated in the identification of two candidate 5q13.1 SMA genes, NAIP and SMN both of which are deleted in individuals with SMA. It was postulated that the intact and degenerate versions of NAIP are present in variable and frequently high copy numbers in this region while SMN was proposed to be present in only two copies. In order to assess the copy number of NAIP and SMN we have conducted interphase FISH analysis using NAIP and SMN gene-containing cosmid and plasmid probes. Our results confirm the variability in the number of NAIP signals in non-SMA chromosomes (2-6) and show that SMN is present on average twice per chromosome although in one chromosome 4-5 signals for the SMN-containing cosmid probe were detected. Our analysis reveals that one of four and three of six type I SMA chromosomes had a lower than normal number of NAIP and SMN signals, respectively. In two of six SMA type I chromosomes, complete loss of hybridization signal was observed on one chromosome 5 with our SMN cosmid probe possibly reflecting a large scale deletion. Large scale deletions were not detectable when metaphase chromosomes of an SMA type II and III patient were analyzed.