Somatic mosaicism of p(CTG)n expansion in a case of myotonic dystrophy with parotid tumor

Myotonic dystrophy (MD) is an autosomal dominant systemic disorder with an unstable expansion of the CTG triplet repeat in the 3'-untranslated region of the gene encoding myotonine protein kinase (DMPK) which maps to chromosome 19q13.3. Somatic mosaicism of CTG repeats in MD has been reported; and it has been observed that CTG repeats in tumor tissues associated with MD are more expanded than the other tissues. It is not rare that parotid tumors are found in patients with MD. We performed Southern blot analysis for tissues from the parotid tumor, the normal parotid gland, the skeletal muscles, and the leukocyte from a 60-year-old patient with MD. CTG repeat was most expanded in the parotid tumor, and the normal parotid gland had longer expansion of CTG repeat than the skeletal muscles. The leukocyte had the shortest expansion of CTG repeat. The expansion of CTG repeat in the parotid tumor may be related to active cell division and may underlie the occurrence of tumors in MD.     

Myotonic dystrophy phenotype without expansion of (CTG)n repeat: an entity distinct from proximal myotonic myopathy (PROMM)?

Myotonic dystrophy (DM) is associated with an expansion of an unstable (CTG)n repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene on chromosome 19q13.3. We studied six patients from two families who showed no expansions of the repeat, in spite of their clinical diagnosis of DM. These patients had multi-systemic manifestations that were distinguishable from those seen in other myotonic disorders, including proximal myotonic myopathy (PROMM). In one additional family, two symptomatic members showed no expanded (CTG)n repeats, while their affected relatives had the expanded repeats. DM haplotype analysis failed to exclude the DMPK locus as a possible site of mutation in each family; however, DMPK mRNA levels were normal. We conclude that a mutation(s) other than the expanded (CTG)n repeat can cause the DM phenotype. The mutation(s) in these families remain(s) to be mapped and characterized.     

Characterization of large CTG repeat expansions in myotonic dystrophy alleles using PCR

BACKGROUND: Stigmatization is a central experience of patients with psoriasis with a broad psychic and social impact. OBJECTIVE: The purpose of this study is to identify the dimensions of stigma experience by a Questionnaire on Experience with Skin Complaints (QES; 'Fragebogen zum Erleben von Hautbeschwerden'). METHODS: 187 in-patients with psoriasis were examined with the QES, an adopted and extended (German) version of the Feeling of Stigmatization Questionnaire by Ginsburg and Link. A factor analysis of the questionnaire was conducted and the construct validity was evaluated. RESULTS: Five factors were found: 'self-esteem', 'retreat', 'rejection', 'composure' and 'concealment'. Self-esteem, retreat and rejection are mainly influenced by 'problematic regions' affected by psoriasis, rejection also by 'visible parts' and retreat also by 'invisible regions'. The influences of age, age at onset and sex on the stigma experience are small but significant. CONCLUSIONS: The QES proved to be an economical and reliable psychometric instrument to differentiate the stigma experience of psoriasis patients.     

Relative stability of a minimal CTG repeat expansion in a large kindred with myotonic dystrophy

The genetic basis for myotonic dystrophy (DM) is a CTG trinucleotide repeat expansion. The number of CTG repeats commonly increases in affected individuals of successive generations, in association with anticipation. We identified a large DM family in which multiple members had minimal CTG repeat expansions, and in which the number of CTG repeats remained in the minimally expanded range through at least three, and possibly four, generations. This relative stability of minimal CTG repeat expansions may help to maintain the DM mutation in the population.     

Abnormal calcium metabolism in myotonic dystrophy as shown by the Ellsworth-Howard test and its relation to CTG triplet repeat length

Myotonic dystrophy (DM) is an autosomal dominant disorder characterized by peculiar clinical features. Its molecular basis is the unstable expansion of a CTG triplet repeat in the gene encoding myotonin protein kinase (Mt-PK), the nucleotide sequence of which has extensive homology to the cyclic AMP (cAMP)-dependent protein kinase gene. Extensive efforts have been made to clarify the signal transduction pathway in which the responsible gene operates, but confirming evidence has yet to be obtained. Because some symptoms in DM are similar to those in hypoparathyroidism, we divided 24 DM patients into two groups on the basis of their serum calcium levels; Group 1, those with normocalcemia (11 patients), and group 2, those with hypocalcemia (13 patients). The highly sensitive parathyroid hormone (HS-PTH) plasma levels in group 1 were within normal limits, whereas those in group 2 were abnormally high. Laboratory findings for the group 2 patients resembled those for pseudohypoparathyroidism (PHP), whereas those for group 1 patients were normal. The Ellsworth-Howard (EH) test was used to determine which type of PHP the group 2 patients belonged to. Both the phosphaturic (delta P) and urinary cAMP (UcAMP) responses were estimated. The delta P responses in group 2 were significantly lower than those in group 1, but their UcAMP responses did not differ. This is evidence that group 2 patients had PHP type II, whereas group 1 patients were normal. We also investigated whether the disease severity differed between the groups. Cataracts, ectopic calcifications, and ossifications, which are associated with PHP, were more frequent in group 2. In addition, the mean IQ in that group was significantly lower. Clinically, the group 2 signs agreed well with those of PHP, whereas for group 1 there was only a slight similarity. These results are additional evidence that the patients in group 2 have abnormal calcium metabolism, the abnormality being in the postadenylate cyclase-cAMP pathway in the renal tubular cells. The degree of (CTG)n expansion, the so-called expanded DNA fragment (EF) size, was determined by standard Southern blot analysis. The allelic EF sizes in both groups were greater than in the healthy controls. Moreover, those in group 2 were significantly longer than those in group 1. We therefore investigated whether EF size is correlated with the serum calcium and plasma PTH levels, the delta P responses in the EH test, and IQ. All these items were significantly correlated with EF size. Our findings show that the expanded DNA fragment size in DM is correlated with the degree of abnormal calcium metabolism.     

Molecular genetics of myotonic dystrophy--population genetics of the CTG repeat expansion of the MTPK gene

The CTG repeat number in the 3'-untranslated region of the myotonin protein kinase (MTPK) gene varies between 5 and 37 in normal individuals, whereas myotonic dystrophy (DM) patients have expansions from 50 to 3000 copies. However little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. To explain the dynamic mutation mechanism and high prevalence in the population, slippage theory, multistep model and meiotic drive hypothesis have been proposed. Recent studies have shown that repeat expansion may affect neighboring genes (59 gene and DMAHP gene), or exert its effect at the RNA level by modulating the binding of (CUG)n-RNA binding proteins which are required for the maturation, stability and translation of specific mRNAs.     

Iraqi-Jewish kindreds with optic atrophy plus (3-methylglutaconic aciduria type 3) demonstrate linkage disequilibrium with the CTG repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene

Iraqi-Jewish optic atrophy plus is an autosomal recessive condition characterized by infantile optic atrophy, an early onset movement disorder, and 3-methylglutaconic aciduria. Other features include spastic paraplegia, mild ataxia, mild cognitive deficiency and dysarthria. This disorder was identified in inbred Iraqi-Jewish kindreds in which relationships between most of the affected individuals were unknown. In this study we identify linkage to chromosome 19q13.2-q13.3 by using a DNA pooling strategy to perform a genome wide screen followed by a high density search for shared segments among affected individuals in candidate regions identified in the initial genome wide screen. A significantly high positive lod score of 6.14 at zero recombination was obtained for the CTG repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene. The existence of multiple recombinant individuals indicates the disease interval can be further narrowed with additional markers. Linkage disequilibrium was seen in six polymorphic markers across a 1 Mb interval. This region is well characterized and contains several candidate genes.     

Altered phosphorylation and intracellular distribution of a (CUG)n triplet repeat RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice

Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3'-untranslated region of the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the (CUG)n repeats in the 3'-untranslated region of DMPK gene induces DM is unknown. We previously isolated a protein with specific binding to CUG repeat sequences (CUG-BP/hNab50) that possibly plays a role in mRNA processing and/or transport. Here we present evidence that the phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 protein (CUG-BP/hNab50), are altered in homozygous DM patient and that CUG-BP/hNab50 is a substrate for DMPK both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK, homozygous DM patient and DMPK knockout mice, show that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP/hNab50 protein. Decreased levels of DMPK observed in DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP/hNab50. Nuclear concentration of the hypophosphorylated CUG-BP/hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patient. DMPK also interacts with and phosphorylates CUG-BP/hNab50 protein in vitro. DMPK-mediated phosphorylation of CUG-BP/hNab50 results in dramatic reduction of the CUG-BP2, hypophosphorylated isoform, accumulation of which was observed in the nuclei of DMPK knockout mice. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter phosphorylation status of CUG-BP/hNab50, thus facilitating nuclear localization of CUG-BP/hNab50. Our results suggest that DM pathophysiology could be, in part, a result of sequestration of CUG-BP/hNab50 and, in part, of lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs.     

Instability of normal (CTG)n alleles in the DM kinase gene

We report on a myotonic dystrophy (DM) family exhibiting instability of normal sized (CTG)n alleles in the DM kinase gene on the non-DM chromosome. At least two mutational events involving normal DM alleles must have occurred in this family; one was characterised as a 34-35 (CTG)n repeat mutation. These findings represent a dissociation between (CTG)n repeat instability and myotonic dystrophy. Furthermore, this family highlights genetic counselling issues relating to the pathogenicity of alleles at the upper end of the normal size range and the risk of further expansion into the disease range.     

Relationship between parental trinucleotide GCT repeat length and severity of myotonic dystrophy in offspring

OBJECTIVE: To assess the relationship between the GCT repeat number in the myotonic dystrophy gene and the clinical phenotype and examine its predictive utility in prenatal testing. DESIGN: DNA from patients was examined for the length of the myotonic dystrophy GCT repeat region, using both Southern blot analysis and polymerase chain reaction. The results were compared with the clinical onset of disease, as well as with pregnancy outcomes. SETTING: Patient samples were referred to the Kleberg DNA Diagnostic Laboratory at the Baylor College of Medicine for DNA analysis by geneticists and genetic counselors (84%), neurologists (10%), and obstetricians and other specialists (6%). Clinical features including onset of disease and family pedigrees were determined by the referring centers. PATIENTS: A total of 241 patient samples from 118 families referred from primarily genetic or neurological centers for genetic linkage analysis or mutation analysis for myotonic dystrophy. This included 44 families referred for prenatal diagnosis. MAIN OUTCOME MEASURES: A relationship between myotonic dystrophy disease onset and length of the GCT repeat allele, parental origin of the disease allele, and results of prenatal diagnosis predictions of disease status were measured. RESULTS: There is a relationship between increasing repeat length and earlier clinical onset of disease. Essentially all (> 99%) myotonic mutations causing myotonic dystrophy are accounted for by GCT repeat amplification. Congenital myotonic dystrophy occurs with as few as 730 GCT repeats but only with alleles of maternal origin. Maternal GCT repeats were found as low as 75 (asymptomatic) that were amplified to result in a child with congenital myotonic dystrophy. Application of DNA diagnosis to 32 pregnancies provided an accurate method for identification of at-risk fetuses and allele enlargement. CONCLUSIONS: The GCT repeat in myotonic dystrophy is highly mutable. The triplet repeat amplification is highly specific for mutations involving the myotonin protein kinase gene accounting for myotonic dystrophy. The quantitation of triplet repeats can be more sensitive than physical, ophthalmologic, and electromyography examinations since the mutation can be detected in patients without evidence of myotonic dystrophy clinical findings. The length of the triplet expansion is influenced by the sex of the transmitting parent and is related to the clinical onset of disease features. Prenatal measurement of the GCT triplet repeat has utility for families with myotonic dystrophy risk since mutant and normal repeats are distinguishable and the length of mutant repeat alleles is associated with clinical severity. Thus, GCT triplet measurement provides a highly accurate means of detecting the myotonic dystrophy mutation in patients and offers a new reproductive option for families at risk for myotonic dystrophy.     

Features of the type of inheritance of myotonic dystrophy

The characteristic features of the genetics of myotonic dystrophy (MD) in the Bulgarian population were studied. Seventy-nine pedigrees were analyzed, comprising a total of 119 patients with MD. The following characteristic features of the MD genetics were revealed: (1) Different families exhibited different patterns of the disease transmission, including vertical (as in the autosomal dominant mode of inheritance), horizontal (as in the autosomal recessive mode of inheritance), and mixed (the horizontal transmission for the first generations and the vertical transmission for the subsequent ones); (2) All studied pedigrees were traced back to clinically healthy ancestors; (3) The symptoms of MD exhibited in anticipation, as well as a clinical heterogeneity in sibships with respect to the severity of the disease. The characteristic features of the revealed mode of inheritance may be explained by dynamic mutations.     

Cataract and myotonic dystrophy: the role of molecular diagnosis

Myotonic dystrophy (dystrophia myotonica), the commonest and most variable of the muscular dystrophies of adult life, has long been known to be associated with cataract, while slit-lamp examination for specific lens opacities has been one of the principal methods of presymptomatic detection of gene carriers. The recent discovery that the myotonic dystrophy mutation is an unstable DNA sequence, composed of varying numbers of CTG triplet repeats, now allows a specific molecular test for this disorder, as well as explaining the phenomenon of anticipation. A series of case reports is presented to illustrate the important practical applications of this development in relation to ophthalmic aspects of the disorder. Reassessment of the specificity of the ophthalmic changes may be required and it will be important for molecular analysis to be used alongside ophthalmic studies, when determining whether family members carry the mutation for myotonic dystrophy.     

Fluorescence in situ hybridization analysis of the replication properties of the myotonic dystrophy protein kinase (DMPK) gene region

Myotonic dystrophy (DM) is caused by an expansion of a CTG repeat sequence in the 3' noncoding region of a protein kinase gene (DMPK) at 19q13.3. We used in situ hybridization to analyse the replication timing of the genomic region containing DMPK in fibroblasts and myoblasts from controls and myotonic dystrophy patients. In this method the relative proportion of singlet to doublet hybridization signals is used to infer the relative time of replication of specific loci or regions. Our results show that in cells from normal individuals approximately 65% of signals appear as doublets, indicating early replication. In DM patients with a number of CTG repeats ranging from about 600-1800 we observed a significant increase of singlet-doublets compared to the background level. These results suggest the existence of replication alternations and/or structural differences between the normal and mutant alleles induced by the presence of the DM mutation.     

Mapping and characterization of novel (CAG)n repeat cDNAs from adult human brain derived by the oligo capture method

BACKGROUND: The role of chest CT scan in the assessment of patients with hemoptysis is uncertain. AIM: To evaluate the usefulness of CT scan in patients with non massive hemoptysis. PATIENTS AND METHODS: Ninety six patients, 60 male, aged 23 to 76 years old, who presented with hemoptysis to an University Hospital, were studied. All patients were studied with a chest radiograph, a fiberoptic bronchoscopy and a high resolution CT scan. RESULTS: The final causes of hemoptysis were bronchiectasis in 27 cases, bronchogenic carcinoma in 24 cases and lung infections in nine. The source of bleeding was not identified in 18 patients (19%). CT scan clarified abnormalities seen in the chest radiograph in 30 patients (31%) and provided new diagnostic information in 13 (14%). CT scan correctly localized the source of bleeding found by fiberoptic bronchoscopy in 35 of 43 patients (81%), whereas chest radiograph did so in 27 (77%). All patients with bronchogenic carcinoma were identified by chest radiograph or bronchoscopy. Twenty of the 27 patients with bronchiectasis had radiological abnormalities in the chest radiograph. In only two patients, with lung metastases and non conclusive chest radiograph and bronchoscopy, CT scan provided information that significantly modified clinical management. CONCLUSIONS: CT scan was useful to stage patients with bronchogenic carcinoma and to assess the extension of bronchiectasis, but its impact in the management and clinical evolution of patients was limited. Therefore we do not recommend the routine use of CT scan in the assessment of patients with hemoptysis.     

Expression of myotonic dystrophy protein kinase gene during in vivo and in vitro mouse myogenesis

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder characterized by a great variability in its clinical manifestations. The mutational basis underlying DM consists of an unstable (CTG)n trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene (DMPK). Conflicting results on DMPK gene expression in congenitally affected infants (CDM) have been published. Moreover, the prominence of satellite cells seen in muscle of CDM infants supports the notion that the congenital form is associated with an arrest in muscle development and suggests a role for the DMPK gene during differentiation and maturation of muscle. In order to clarify these findings, a comparative study of DMPK and myogenic factor mRNA levels was performed in developing mouse muscle tissues and cultured muscle cells at different developmental stages. Results show that DMPK gene expression is upregulated at a late stage of muscular development. This upregulation does not seem to depend on a given muscle specific bHLH factor.     

The role of extracellular calcium in the pathophysiology of myotonic dystrophy

Increased intracellular calcium levels in myotonic dystrophy have been repeatedly reported in many studies. In our recent investigations, the entrance of extracellular calcium ions through voltage sensitive calcium channels (VSCCS) during repetitive action potentials and late after potentials (LAPs) in tetanic responses were found. Since there is an increased amplitude of LAPs and after-discharges in muscle fibers of myotonic dystrophy, we suggest that this pathological increase in the electrical properties of the muscle could also be responsible for this elevated intracellular calcium level.     

Somatic genetic alterations in human malignant mesothelioma (review)

Rhodococcus sp. strain ECRD-1 was evaluated for its ability to desulfurize a 232 to 343 degrees C middle-distillate (diesel range) fraction of Oregon basin (OB) crude oil. OB oil was provided as the sole source of sulfur in batch cultures, and the extent of desulfurization and the chemical fate of the residual sulfur in the oil after treatment were determined. Gas chromatography (GC), flame ionization detection, and GC sulfur chemiluminesce detection analysis were used to qualitatively evaluate the effect of Rhodococcus sp. strain ECRD-1 treatment on the hydrocarbon and sulfur content of the oil, respectively. Total sulfur was determined by combustion of samples and measurement of released sulfur dioxide by infrared absorption. Up to 30% of the total sulfur in the middle distillate cut was removed, and compounds across the entire boiling range of the oil were affected. Sulfur K-edge X-ray absorption-edge spectroscopy was used to examine the chemical state of the sulfur remaining in the treated OB oil. Approximately equal amounts of thiophenic and sulfidic sulfur compounds were removed by ECRD-1 treatment, and over 50% of the sulfur remaining after treatment was in an oxidized form. The presence of partially oxidized sulfur compounds indicates that these compounds were en route to desulfurization. Overall, more than two-thirds of the sulfur had been removed or oxidized by the microbial treatment.     

Limited expansion of the (CAG)n repeat of the Huntington gene: a premutation (?)

Pantoprazole is a specific inhibitor of the H+/K(+)-ATPase of the gastric parietal cell. The dose-dependency of a range of pantoprazole pharmacokinetic characteristics was studied. Twelve healthy male subjects were given 10, 20, 40 and 80 mg pantoprazole intravenously according to a randomized, single blind, 4-period change-over scheme. The area under the concentration vs time curve (AUC) and the maximum serum concentration (Cmax) showed a linear increase in line with the dose. Apparent volume of distribution (Vd area), clearance (Cl) and terminal half-life (t1/2) were independent of the dose. The dose-independent elimination of pantoprazole was attributed to the lack of interaction of the drug with cytochrome P450. In clinical practice, a good predictable response, as well as a low potential for interaction with other drugs might be expected.     

A novel zinc finger cDNA with a polymorphic pentanucleotide repeat (ATTTT)n maps on human chromosome 19p

PURPOSE: To evaluate the therapeutic effectiveness of a combined chemoradiotherapy program, followed by surgery in selected cases, in Stage III non-small cell lung cancer. METHODS AND MATERIALS: Between August 1988 and February 1990, 43 patients Staged IIIa-b (UICC 1987, 58% IIIb) have been treated with concomitant chemotherapy (cisplatin 15 mg/m2 and VP16 75 mg/m2, 5 days a week on week 1 and 5) and radiotherapy (40 Gy split course, 2 Gy/day on week 1, 2, 5, and 6), followed by attempted curative thoracotomy or more cycles of full dose chemotherapy with the same two drugs. RESULTS: Planned chemoradiotherapy has been given to 91% of patients; 13/43 patients have been operated, with 12 complete resections and three (7%) pathological complete responses. Toxicity was significant, with two postoperative deaths and two fatal radiation pneumonitis. Crude progression-free survival rate is 21% at 30 months, with nine patients (21%) alive and free from progression at follow-up times ranging from 31 to 49 months. Subset survival analysis showed a possibly greater therapeutic effect for non-squamous histology as compared to squamous carcinoma. CONCLUSION: These results are encouraging in a cohort of patients with quite advanced disease (58% Stage IIIb).