Zuzana Musova

Highly unstable sequence interruptions of the CTG repeat in the myotonic dystrophy gene.

Myotonic dystrophy type 1 is caused by the expansion of a CTG repeat in the 3′ UTR of the DMPK gene. A length exceeding 50 CTG triplets is pathogenic. Intermediate alleles with 35–49 triplets are not disease‐causing but show instability in intergenerational transmissions. We report on the identification of multiple patients with different patterns of CCG and CTC interruptions in the DMPK CTG repeat tract that display unique intergenerational instability. In patients bearing interrupted expanded alleles, the location of the interruptions changed dramatically between generations and the repeats tended to contract. The phenotype for these patients corresponded to the classical form of the disease, but in some cases without muscular dystrophy and possibly with a later onset than expected. Symptomatic patients bearing interrupted intermediate length repeat tracts were also identified, although the role of the interruptions in their phenotype remains unclear. The identification of interruptions in the DMPK repeat has important consequences for molecular genetic testing where they can lead to false negative conclusions.

CLCN1 Mutations in Czech Patients with Myotonia Congenita, In Silico Analysis of Novel and Known Mutations in the Human Dimeric Skeletal Muscle Chloride Channel

Myotonia congenita (MC) is a genetic disease caused by mutations in the skeletal muscle chloride channel gene (CLCN1) encoding the skeletal muscle chloride channel (ClC-1). Mutations of CLCN1 result in either autosomal dominant MC (Thomsen disease) or autosomal recessive MC (Becker disease). The ClC-1 protein is a homodimer with a separate ion pore within each monomer. Mutations causing recessive myotonia most likely affect properties of only the mutant monomer in the heterodimer, leaving the wild type monomer unaffected, while mutations causing dominant myotonia affect properties of both subunits in the heterodimer. Our study addresses two points: 1) molecular genetic diagnostics of MC by analysis of the CLCN1 gene and 2) structural analysis of mutations in the homology model of the human dimeric ClC-1 protein. In the first part, 34 different types of CLCN1 mutations were identified in 51 MC probands (14 mutations were new). In the second part, on the basis of the homology model we identified the amino acids which forming the dimer interface and those which form the Cl- ion pathway. In the literature, we searched for mutations of these amino acids for which functional analyses were performed to assess the correlation between localisation of a mutation and occurrence of a dominant-negative effect (corresponding to dominant MC). This revealed that both types of mutations, with and without a dominant-negative effect, are localised at the dimer interface while solely mutations without a dominant-negative effect occur inside the chloride channel. This work is complemented by structural analysis of the homology model which provides elucidation of the effects of mutations, including a description of impacts of newly detected missense mutations.

CpG Methylation, a Parent-of-Origin Effect for Maternal-Biased Transmission of Congenital Myotonic Dystrophy

CTG repeat expansions in DMPK cause myotonic dystrophy (DM1) with a continuum of severity and ages of onset. Congenital DM1 (CDM1), the most severe form, presents distinct clinical features, large expansions, and almost exclusive maternal transmission. The correlation between CDM1 and expansion size is not absolute, suggesting contributions of other factors. We determined CpG methylation flanking the CTG repeat in 79 blood samples from 20 CDM1-affected individuals; 21, 27, and 11 individuals with DM1 but not CDM1 (henceforth non-CDM1) with maternal, paternal, and unknown inheritance; and collections of maternally and paternally derived chorionic villus samples (7 CVSs) and human embryonic stem cells (4 hESCs). All but two CDM1-affected individuals showed high levels of methylation upstream and downstream of the repeat, greater than non-CDM1 individuals (p = 7.04958 × 10−12). Most non-CDM1 individuals were devoid of methylation, where one in six showed downstream methylation. Only two non-CDM1 individuals showed upstream methylation, and these were maternally derived childhood onset, suggesting a continuum of methylation with age of onset. Only maternally derived hESCs and CVSs showed upstream methylation. In contrast, paternally derived samples (27 blood samples, 3 CVSs, and 2 hESCs) never showed upstream methylation. CTG tract length did not strictly correlate with CDM1 or methylation. Thus, methylation patterns flanking the CTG repeat are stronger indicators of CDM1 than repeat size. Spermatogonia with upstream methylation may not survive due to methylation-induced reduced expression of the adjacent SIX5, thereby protecting DM1-affected fathers from having CDM1-affected children. Thus, DMPK methylation may account for the maternal bias for CDM1 transmission, larger maternal CTG expansions, age of onset, and clinical continuum, and may serve as a diagnostic indicator.

Spinocerebellar Ataxias Type 8, 12, and 17 and Dentatorubro-Pallidoluysian Atrophy in Czech Ataxic Patients

Spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders currently associated with 27 genes. The most frequent types are caused by expansions in coding CAG repeats. The frequency of SCA subtypes varies among populations. We examined the occurrence of rare SCAs, SCA8, SCA12, SCA17 and dentatorubro-pallidoluysian atrophy (DRPLA), in the Czech population from where the data were missing. We analyzed causal gene expansions in 515 familial and sporadic ataxic patients negatively tested for SCA1–3 and SCA6–7. Pathogenic SCA8 and SCA17 expansions were identified in eight and five patients, respectively. Tay–Sachs disease was later diagnosed in one patient with an SCA8 expansion and the diagnosis of multiple sclerosis (MS) was suspected in two other patients with SCA8 expansions. These findings are probably coincidental, although the participation of SCA8 expansions in the susceptibility to MS and disease progression cannot be fully excluded. None of the patients had pathogenic SCA12 or DRPLA expansions. However, three patients had intermediate SCA12 alleles out of the normal range with 36 and 43 CAGs. Amyotrophic lateral sclerosis (ALS) was probable in the patient with 43 CAGs. This coincidence is remarkable, especially in the context with the recently identified predisposing role of longer SCA2 alleles in ALS. Five families with SCA17 represent a significant portion of ataxic patients and this should be reflected in the diagnostics of SCAs in the Czech population. SCA8 expansions must be considered after careful clinical evaluation.

FMR1 gene expansion, large deletion of Xp, and skewed X-inactivation in a girl with mental retardation and autism.

We describe a girl with mild facial anomalies, mild mental retardation, and atypical autism with a remarkable behavioral phenotype of persistent anger, aggression, and dysphoria. The occurrence of late‐onset tremor and premature ovarian failure in the maternal branch of the family pointed to a possible defect in the FMR1 gene. Indeed, the patient carried a full FMR1 mutation. Unexpectedly, both alleles of the gene were almost completely methylated. Cytogenetic examination of the patient revealed in addition a large de novo deletion in band Xp22 on one of her X chromosomes. The deletion was fine mapped using oligonucleotide array CGH, and its breakpoints were localized using sequencing. The size of the deletion was about 17.4 Mb, and it contained more than 90 protein‐coding genes. Microsatellite analysis indicated paternal origin of the aberrant chromosome. The large rearrangement was the most probable cause of the X‐inactivation skewing, thus explaining the methylation of not only the expanded (maternal) but also the normal (paternal) FMR1 alleles. This pattern of skewed X‐inactivation was confirmed using the analysis of methylation at the AR locus. The relatively mild phenotype of the patient resulted most likely from unmasking of the FMR1 defect. Although the deleted region contained many important genes, the phenotypic contribution of the rearranged X chromosome was probably limited by its almost complete inactivation. However, reduced dose of several genes escaping X‐inactivation might also play a role in the phenotype of the patient.

Myotonic dystrophy type 2 and multiple sclerosis: case report.

Myotonic dystrophy type 2 (DM2) is an autosomal dominant ultisystem disorder that manifests itself as myotonia, weakness, ataracts, impaired testicular function, impaired glucose tolerance, ypogammaglobulinemia, and heart conduction defects. When ompared with myotonic dystrophy type 1 (DM1), cerebral sympoms develop much less frequently. These cerebral symptoms nclude seizures, and the symptoms that occur during attacks of ocal (stroke-like) symptoms, parkinsonian symptoms, and hyperomnia. MRI imaging typically shows white matter abnormalities, nd SPECT shows areas of hypoperfusion in the frontal and parietoccipital areas. Compared with DM1, the clinical course of DM2 s less severe. The concurrent development of another brain or pine disorder is rare. To date, we have found only one case report n the literature describing the development of multiple sclerosis MS) in a 37-year-old man who had been monitored for dystrophic yotonia since the age of 13. In our neuromuscular clinic, a large amily with DM2 has been followed. A 31-year-old woman from his family experienced an attack of central symptoms and was ubsequently diagnosed with MS.

Novel mutations in the NF1 gene in Czech patients with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is one of the most common inherited human disorders, with an estimated incidence of 1 per 3500 births. In most cases, the disease is caused either by mutation in the NF1 gene, or by a particular or complete deletion of the NF1 gene. The NF1 gene exhibits one of the highest mutation rates of any human disorder. In this experimental study of the NF1 gene, we screened the mutational spectrum of 22 unrelated patients from the Czech Republic using the denaturing high-performance liquid chromatography (DHPLC) and multiplex ligation-dependent probe amplification (MLPA) methods. We found NF1 mutations in 17 patients: 15 causal mutations were detected with the use of the DHPLC method (15/20, 75%). With the MPLA method, we also confirmed and specified two large deletions that were previously genotyped by microsatellite markers. Twelve of the above mentioned mutations were newly found:c.1_2delATinsCC, c.1185+1G>C, c.1757_1760delCTAG, c.1642-7A>G, c.2329 T>G, c.2816delA, c.3738_3741delGTTT, c.4733 C>T, c.5220delT, c.6473_6474insGAAG, ex 14_49del, ex28_49del. We present this study as a first effectual step in the routine diagnosis of the NF1 in patients from the Czech Republic.

Expanded DMPK repeats in dizygotic twins referred for diagnosis of autism versus absence of expanded DMPK repeats at screening of 330 children with autism.

Myotonic dystrophy type 1 (DM1) belongs to the broad spectrum of genetic disorders associated with autism spectrum disorders (ASD). ASD were reported predominantly in congenital and early childhood forms of DM1. We describe dizygotic twin boys with ASD who were referred for routine laboratory genetic testing and in whom karyotyping, FMR1 gene testing, and single nucleotide polymorphism array analysis yielded negative results. The father of the boys was later diagnosed with suspected DM1, and testing revealed characteristic DMPK gene expansions in his genome as well as in the genomes of both twins and their elder brother, who also suffered from ASD. In accord with previous reports on childhood forms of DM1, our patients showed prominent neuropsychiatric phenotypes characterized especially by hypotonia, developmental and language delay, emotional and affective lability, lowered adaptability, and social withdrawal. The experience with this family and multiple literature reports of ASD in DM1 on the one side but the lack of literature data on the frequency of DMPK gene expansions in ASD patients on the other side prompted us to screen the DMPK gene in a sample of 330 patients with ASD who were first seen by a geneticist before they were 10 years of age, before the muscular weakness, which may signal DM1, usually becomes obvious. The absence of any DMPK gene expansions in this cohort indicates that targeted DMPK gene testing can be recommended only in ASD patients with specific symptoms or family history suggestive of DM1.