Manuela C. Koch

A polymorphic microsatellite that mediates induction of PIG3 by p53

The gene PIG3 is induced by the tumor suppressor p53 but not by p53 mutants unable to induce apoptosis, suggesting its involvement in p53-mediated cell death. Here we show that p53 directly binds and activates the PIG3 promoter, but not through the previously described DNA element. Instead, p53 interacts with a pentanucleotide microsatellite sequence within the PIG3 promoter (TGYCC)n where Y=C or T. Despite its limited similarity to the p53-binding consensus, this sequence is necessary and sufficient for transcriptional activation of the PIG3 promoter by p53 and binds specifically to p53 in vitro and in vivo. In a population of 117 healthy donors from Germany, the microsatellite was found to be polymorphic, the number of pentanucleotide repeats being 10, 15, 16 or 17, and the frequency of alleles 5.1%, 62.0%, 21.4% and 11.5%, respectively. The number of repeats directly correlated with the extent of transcriptional activation by p53. This is the first time that a microsatellite has been shown to mediate the induction of a promoter through direct interaction with a transcription factor. Moreover, this sequence of PIG3 is the first p53-responsive element found to be polymorphic. Inheritance of this microsatellite may affect an individuals susceptibility to cancer.

Proximal myotonic myopathy: A new dominant disorder with myotonia, muscle weakness, and cataracts

We describe three families with a dominantly inherited disorder. Affected individuals have myotonia, proximal muscle weakness, and cataracts. There was no abnormal CTG repeat expansion of the myotonic dystrophy (DM) gene in DNA from blood and muscle. The structure of the three families permitted linkage analysis, and there is no linkage to the gene loci for DM or to the loci for the muscle chloride channel disorders or muscle sodium channel disorders. The collection of symptoms in these three families seems to represent a new disorder.

Multimeric structure of ClC-1 chloride channel revealed by mutations in dominant myotonia congenita (Thomsen).

Voltage‐gated ClC chloride channels play important roles in cell volume regulation, control of muscle excitability, and probably transepithelial transport. ClC channels can be functionally expressed without other subunits, but it is unknown whether they function as monomers. We now exploit the properties of human mutations in the muscle chloride channel, ClC‐1, to explore its multimeric structure. This is based on analysis of the dominant negative effects of ClC‐1 mutations causing myotonia congenita (MC, Thomsens disease), including a newly identified mutation (P480L) in Thomsens own family. In a co‐expression assay, Thomsens mutation dramatically inhibits normal ClC‐1 function. A mutation found in Canadian MC families (G230E) has a less pronounced dominant negative effect, which can be explained by functional WT/G230E heterooligomeric channels with altered kinetics and selectivity. Analysis of both mutants shows independently that ClC‐1 functions as a homooligomer with most likely four subunits.

Mutations in dominant human myotonia congenita drastically alter the voltage dependence of the CIC-1 chloride channel

Autosomal dominant myotonia congenita (Thomsens disease) is caused by mutations in the muscle chloride channel CIC-1. Several point mutations found in affected families (I29OM, R317Q, P480L, and Q552R) dramatically shift gating to positive voltages in mutant/WT heterooligomeric channels, and when measurable, even more so in mutant homooligomers. These channels can no longer contribute to the repolarization of action potentials, fully explaining why they cause dominant myotonia. Most replacements of the isoleucine at position 290 shift gating toward positive voltages. Mutant/WT heterooligomers can be partially activated by repetitive depolarizations, suggesting a role in shortening myotonic runs. Remarkably, a human mutation affecting an adjacent residue (E291K) is fully recessive. Large shifts in the voltage dependence of gating may be common to many mutations in dominant myotonia congenita.

Linkage disequilibrium of MTHFR genotypes 677C/T‐1298A/C in the German population and association studies in probands with neural tube defects(NTD)

A number of studies have demonstrated that the common polymorphism 677C-->T in the gene encoding 5, 10-methylenetetrahydrofolate reductase (MTHFR) leads to a thermolabile variant with decreased enzyme activity and to mildly elevated plasma homocysteine. 677TT homozygosity was shown to be more frequent in NTD probands compared with controls in some studies. Recently, another polymorphism, 1298A-->C, in the MTHFR gene was described and combined heterozygosity 677CT/1298AC was suggested to be an additional risk factor for NTD. The present study examines the genotype and haplotype distribution of the two polymorphisms in the German population and evaluates the impact on NTD individuals and their relatives. To determine the haplotype of all individuals tested, we developed an easy-to-perform ARMS-RFLP test. Our data show that the two polymorphisms are in linkage disequilibrium in the general population and in NTD individuals. There was no statistically significant difference in allele and genotype frequency between probands (patients, fetuses) and controls (P > 0.10) and between observed and expected values for mother-child pairs (P > 0.80). Taking into account gender, an increased rate of 677CT heterozygotes was found in affected and unaffected males compared to affected and unaffected females. A family-based association study using a multiallelic transmission disequilibrium test (TDT) also shows that transmission rates do not deviate significantly from equilibrium (P > 0.50). Thus, our data provide no evidence for an association between NTD phenotype and MTHFR 677C/T-1298A/C genotypes and haplotypes.

Evaluation of the MTHFR C677T allele and the MTHFR gene locus in a German spina bifida population

Abstract A number of recent studies have demonstrated that the occurrence and recurrence risk of neural tube defects (NTD) is reduced by folic acid supplementation before and during pregnancy. Epidemiological studies have shown low plasma folate and raised plasma homocysteine in women with spina bifida aperta (SB) children suggesting an abnormal folate metabolism. The 5,10-methylenetetrahydrofolate reductase (MTHFR) variant C677T, resulting in a decreased activity of the enzyme, has been associated with the development of NTD. Several studies demonstrated that homozygosity for the C677T mutation occurs at a higher frequency in patients with SB phenotype than in control individuals. The SB risk is strongest if both the mother and her child have the mutation in the homozygous state. In the present study we compared the frequency of the C- and T-alleles in healthy German individuals (n=153) with German SB patients (n=137). Our study groups reveal no significant difference in C/T-allele frequencies and genotype distributions. A family based association study, the transmission disequilibrium test, confirms the absence of an association between T-allele and SB. In 9 of 40 families we were able to exclude linkage to the MTHFR locus (1p36.3) employing different inheritance models. Conclusion Our data show no evidence for an association between the C677T mutation and the occurrence of the SB phenotype. Therefore we cannot support the hypothesis that the MTHFR variant does account for a significant genetic predisposition to the SB phenotype in the studied German patients.

Muscle pathology in 57 patients with myotonic dystrophy type 2

We evaluated muscle biopsies from 57 patients with genetically confirmed myotonic dystrophy type 2/proximal myotonic myopathy (DM2/PROMM). Light microscopy showed myopathic together with “denervation‐like” changes in almost all biopsies obtained from four different muscles: increased fiber size variation, internal nuclei, small angulated fibers, pyknotic nuclear clumps, and predominant type 2 fiber atrophy. Quantitative morphometry in 18 biopsies that were immunostained for myosin heavy chain confirmed a predominance of nonselective type 2 fiber atrophy. These histological changes were similar in all patients regardless of the site of biopsy, the predominant clinical symptoms and signs, and the clinical course. It is likely that, in a number of undiagnosed patients, DM2 is the underlying disorder. With a better understanding of the histopathological pattern in DM2, biopsies from patients with undiagnosed neuromuscular disorders can now be reevaluated. Muscle Nerve 29: 275–281, 2004

Interaction of folate and homocysteine pathway genotypes evaluated in susceptibility to neural tube defects (NTD) in a German population

AbstractNeural tube defects (NTD) are likely to result from an interaction of several genes and environmental factors. Because periconceptional folate intake reduces the NTD risk in the fetus, and because mothers of children with NTD showed elevated plasma homocysteine levels, gene polymorphisms of the folate and homocysteine pathway, such as 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C→T, MTHFR 1298A→C and cystathionine β-synthase (CBS) 844ins68, have been implicated in the etiology of NTD. Several studies have demonstrated that these polymorphisms may indeed be associated with NTD in some populations. In order to evaluate the role of these polymorphisms and their interaction in NTD, we genotyped 417 individuals for case-control studies and 129 families for transmission disequilibrium tests. We are the first to present detailed data on MTHFR haploid genotypes in combination with CBS 844ins68. The MTHFR risk genotype 677CT/1298AC, known to be associated with decreased enzyme activity and increased homocysteine, was found significantly more often in patients than in controls (P = 0.02). A CBS insertion allele in addition to MTHFR 677CT/1298AC heterozygosity or MTHFR 677TT/1298AA homozygosity did not result in an increased risk for NTD. This is in agreement with the recently reported homocysteine-lowering effect of the CBS 844ins68 allele in carriers of MTHFR variants.

Linkage of proximal myotonic myopathy to chromosome 3q.

Article abstract We performed genetic linkage analysis in nine German proximal myotonic myopathy (PROMM) families using DNA-markers D3S1541 and D3S1589 from the region of the recently discovered gene locus of myotonic dystrophy type 2 (DM2) on chromosome 3q. Two-point analysis supplied an lod score of 5.9. We conclude that a gene causing PROMM is located on chromosome 3q. PROMM and DM2 may be allelic disorders or may be caused by closely linked genes.

Proximal Myotonic Myopathy with MRI White Matter Abnormalities of the Brain

Proximal myotonic myopathy (PROMM) is an autosomal dominantly inherited multisystemic disorder characterized by myotonia, proximal muscle weakness, and cataracts.This disorder is not linked to the gene locus of myotonic dystrophy (DM). We describe three new families with PROMM. In all patients, CTG repeats of the DM gene in DNA from blood leukocytes were normal. MRI of the brain revealed a consistent pattern of marked white matter hyperintensity on T2-weighted images in four patients; two additional patients had similar but mild to moderate MRI abnormalities. The morphology of these abnormalities is unknown. Clinical symptoms of brain disease were not consistent and included mental changes with hypersomnia, parkinsonian features, stroke-like episodes, and seizures. The causative relationship of these clinical features with the MRI white matter abnormalities remains to be established. Our observations suggest that PROMM may involve the brain. NEUROLOGY 1997;48: 33-37