D. Savić

Survival and mortality of myotonic dystrophy type 1 (Steinert's disease) in the population of Belgrade

The purpose of this investigation was to determine survival and mortality in patients with myotonic dystrophy type 1 (DM1) in the Belgrade population within the period from 1983 to 2002. Data of a number of diagnosed DM1 patients with their demographic, clinical and genetic characteristics were gathered from hospital records in all neurologic institutions in Belgrade for the period 1983–2002. Death certificates were reviewed to determine the cause of death. Survival analysis by life table method and Cox proportional hazard model was performed. Within the observed period, in the population of Belgrade, 15 fatal outcomes among 101 patients with DM1 were registered. Average DM1 mortality rate was 0.5/1 000 000 (95% CI 0.3–0.8), and standardized mortality ratio (SMR) was 5.3. A significant inverse correlation was found between age at onset of DM1 and CTG repeats (P = 0.023). The cumulative probability of 15‐year survival for DM1 patients in Belgrade was 49 ± 5% (48 ± 2% for males and 50 ± 7% for females). Younger age at onset was a significant unfavorable prognostic factor (hazard ratio = 4.2; P = 0.012).

CTG repeat polymorphism in DMPK gene in healthy Yugoslav population.

Objectives– Myotonic dystrophy type 1 (DM1) is caused by large expansions of cytosine‐thymine‐guanine (CTG)‐repeats in myotonic dystrophy protein kinase (DMPK)‐gene. This gene is highly polymorphic in healthy individuals. It has been proposed that expanded alleles originated from the group of large sized normal alleles. If this is correct, one should expect a positive correlation between the frequency of large sized normal alleles and a prevalence of this disorder in a population. In this paper we determined the distribution of alleles of DMPK gene in healthy Yugoslav population. Material and methods– A sample of 235 healthy individuals of Yugoslav origin have been genotyped for the alleles of DMPK locus. Results– We found 22 different alleles, ranging in size from 5 to 29 repeats. Among 470 chromosomes studied, 41 chromosomes had more than 18 repeats (8.72%). Conclusions– Relatively high frequency of large sized normal alleles found in our population, suggest that prevalence of DM1 in Yugoslavia should not be different from the prevalence in other European populations.

Yugoslav HD phenocopies analyzed on the presence of mutations in PrP, ferritin, and Jp-3 genes.

Huntington disease (HD) is a well-defined autosomal dominant neurodegenerative disease caused by CAG repeat expansions in HD gene. There are a significant number of HD cases where this mutation was not found and such cases are named HD-like phenotype (HDL). This article reports 48 patients with HDL phenotype. Patients were analyzed on the presence of mutations in prion (PrP), ferritin and junctophilin-3 (JP-3) genes. None of the patients showed the presence of the mutation in analyzed genes. This could suggest that there is some other gene/genes where the mutation can cause the disease with clinical features of HD.

Intergenerational changes of CTG repeat depending on the sex of the transmitting parent in myotonic dystrophy type 1.

Sir, Myotonic dystrophy type 1 (DM1) is an autosomal dominant, multisystemic disease caused by expansion of a CTG repeat, located in the 3¢ untranslated region of dystrophia myotonica protein kinase (DMPK) gene in the chromosome region 19q13.3. Normal DMPK alleles contain between five and 37 copies of the CTG repeat, but this number is greatly increased in DM1 patients. DM1 is the locus that appears to show the most dramatic instability, often with very high levels of somatic mosaicism and very large intergenerational differences. To date, most studies have concentrated on the comparison of blood DNA from patients within families, and the parent–child differences observed defined as intergenerational differences (Harley et al., 1992; Ashizawa et al., 1993; O’Hoy et al., 1993; Martorell et al., 2000). Size of CTG repeat expansion usually increases upon intergeneration transmission and underlies the phenomena of genetic anticipation. However, the reduction in size of the DM1 trinucleotide repeat mutation during transmission is relatively rare. The aim of this study was to analyse the role of parents sex in intergenerational changes of CTG repeats in patients with DM1. Fifty-four DM1 patients (31 males and 23 females), aged between 19 and 51 years (mean ± SD: 36 ± 15), were studied. Diagnosis was based on clinical, electromyographic and genetic examination. Genomic DNA was isolated from white blood cells using proteinase K/SDS digestion and phenol-chloroform extraction. All subjects were studied by both polymerase chain reaction (PCR) and southern blot analyses. All samples showed a series of discrete bands representing somatic mosaicism of diseaseassociated alleles seen in the blood of DM1 patients. The size of each discrete band (expanded allele) was determined according to DNA molecular weight marker, using scatter plot on which band size of DNA marker was plotted against the length to which it migrated in the gel. For each patient the progenitor, the average, and the largest allele was estimated from the lower boundary of the series of discrete bands observed in allele distribution in lymphocytes beyond which rare alleles were detected in repeated analyses. PCR, as previously described (Brook et al., 1992), was performed for precise determination of wild type (wt) DMPK alleles size. Products were resolved on 6% denaturing polyacrylamide gels, detected by silver staining, and a number of CTG repeats was determined using a DNA sequencing ladder. Out of 54 investigated DM1 patients, 30 inherited the DM1 mutation from their mothers and 24 from their affected fathers. Analysis of variance showed that the mean of the smallest CTG expansion (progenitor allele) was smaller in patients with paternal DM1 mutation inheritance (194 ± 113 CTG repeats) than in those with maternal inheritance (292 ± 123 CTG repeats), P < 0.05. The same was found when comparing the means of the average expansions. Conversely, there was no significant influence of parents sex on the value of the largest CTG expansion (P > 0.05), Table 1. We analysed intergenerational changes of the smallest CTG repeats (progenitor alleles) in 20 parent– child pairs (16 mother–child and four father–child pairs). All 16 mother–child pairs exhibited increased CTG repeat expansion in the children (from 63 to 159 CTG repeats in the mothers and 129–596 CTG repeats in the children). A 22-yearold woman exhibited typical clinical signs for juvenile-adult type of DM1 and DMPK expansion range in blood cells from 429 to 900 repeats. As she has a risk of having children affected with congenital type of DM1, she was referred for prenatal diagnosis. CVS sample was taken during gestation week 10, and isolated DNA was tested for DM1 mutation using SP/LR PCR. The CVS sample was contaminated with maternal DNA. Repeated prenatal diagnosis from DNA isolated from amniotic fluid cells revealed the presence of expansion in the range of 2000–3000 CTG repeats. This pregnancy was terminated. From four father–child pairs, we found increased CTG repeat expansion in three pairs, but in one paternal transmission we found reduction (contraction) in size of the CTG repeats (from 113 in the father to 96 in the child). This study shows that maternal transmission of the DM1 mutation results in a greater number of CTG repeats than does paternal transmission. This suggests a greater instability of mutant alleles in female meiosis and predominantly

Epidemiology of myotonic dystrophy type 1 (Steinert disease) in Belgrade (Serbia)

The aim of this study was to estimate the incidence and prevalence of myotonic dystrophy type 1 (DM1) in Belgrade during the period 1983-2002. The patients who had DM1 were ascertained through hospital records from all neurological departments in Belgrade during 1983-2002. The molecular genetic analysis was performed in all patents included in the study. We identified 101 DM1 patients (52 males and 49 females). The average annual incidence rate of DM1 in Belgrade for the period observed was 2.0/1,000,000 (95% confidence interval (CI), 0.3-8.3), 2.1/1,000,000 (95% CI, 0.3-8.3) for males and 2.0/1,000,000 (95% CI, 0.3-8.3) for females. The highest age-specific DM1 incidence was registered in the age group 20-49: 3.4/1,000,000 (95% CI, 0.5-7.6), 4.0/1,000,000 (95% CI, 1.1-10.2) in males and 2.5/1,000,000 (95% CI, 0.5-7.6) in females. In the population of Belgrade, a cumulative probability of acquiring DM1 was 1 per 8621 for men and 1 per 9259 for women (1 per 8940 of the population for both sexes). The prevalence of DM1 in Belgrade on 31 December 2002 was 5.3/100,000 (95% CI, 4.2-6.6).

The Status of SCA1, MJD/SCA3, FRDA, DRPLA and MD Triplet Containing Genes in Patients with Huntington Disease and Healthy Controls

A number of human hereditary neuromuscular and neurodegenerative disorders are caused by the expansion of trinucleotide repeats within certain genes. Here we report the results of the analysis of five trinucleotide repeats containing genes (SCA1, MJD/SCA3, DRPLA, FRDA and MD) in HD patients and in a group of healthy controls. Allelic frequency distributions for SCA1 and FRDA genes were shifted toward larger alleles in the group of unrelated HD patients, compared to healthy controls. This linkage disequilibrium suggests a possible existance of a common mechanism of trinucleotide repeats expansion in these loci.

Haplotype analysis of the DM1 locus in the Serbian population

Objectives – Analysis of the CTG‐repeat number and three biallelic markers, Alu(+/−), HinfI(+/−), and TaqI(+/−), in the DMPK gene in healthy and myotonic dystrophy type 1 (DM1) Serbian individuals. Also, the consideration of haplotypes in the light of the proposed models of CTG‐repeat evolution and origin of the DM1 mutation.

The coexistence of myasthenia gravis and myotonic dystrophy type 2 in a single patient.

Background Myasthenia gravis (MG) and myotonic dystrophy type 2 (DM2) are rare disorders individually, and their coexistence in the same patient is very rare. We present a patient in which these two diseases coexisted. Case Report The patient complained of diplopia, fluctuating limb weakness, and difficulties in swallowing and speaking. A neurological examination revealed diplopia, facial, weakness of the neck and proximal limb muscles, dysphagia, dysphonia, and myotonia. The patients mother had DM2 and her maternal grandfather had cataracts. MG was confirmed in our patient by positive results for neostigmine and a repetitive nerve stimulation test, and elevated serum anti-acetylcholine-receptor antibodies, while DM2 was confirmed by electromyography and genetic testing. The patient improved remarkably after treatment with anticholinesterases, corticosteroids, and azathioprine. Conclusions This is the second reported case of the coexistence of DM2 and MG in the same patient. Since the symptoms of these two diseases overlap it is very important to keep in mind the possibility of their coexistence, so that MG is not overlooked in patients with a family history of myotonic dystrophy.

SCA2 and SCA3 mutations in young‐onset dopa‐responsive parkinsonism

In this study no one of our 85 patients of Serbian origin with young‐onset (≤ 45 years) dopa‐responsive parkinsonism (YOP), previously proved negative for PARK1 and PARK2 mutations, had either spinocerebellar ataxia type 2 (SCA2) or SCA3 mutation. These data do not prove the significance of these two mutations in either sporadic or familial YOP suggestive of Parkinsons disease.

Is the 31 CAG repeat allele of the spinocerebellar ataxia 1 (SCA1) gene locus non-specifically associated with trinucleotide expansion diseases?

A number of human hereditary neuromuscular and neurodegenerative disorders are caused by the expansion of trinucleotide repeats within certain genes. The molecular mechanisms that underlie these expansions are not yet known. We have analyzed six trinucleotide repeat-containing loci [spinocerebellar ataxias (SCA1, SCA3, SCA8), dentatorubral-pallidoluysian atrophy (DRPLA), Huntington chorea (HD) and fragile X syndrome (FRAXA)] in myotonic dystrophy type 1 (DM1) patients (n = 52). As controls, we analyzed two groups of subjects: healthy control subjects (n =133), and a group of patients with non-triplet neuromuscular diseases (n = 68) caused by point mutations, deletions or duplications (spinal muscular atrophy, Charcot–Marie–Tooth disease, type 1A, hereditary neuropathy with liability to pressure palsies, and Duchenne and Becker muscular dystrophy). Allele frequency distributions for all tested loci were similar in these three groups with the exception of the SCA1 locus. In DM1 patients, the SCA1 allele with 31 CAG repeats account for 40.4% of all chromosomes tested, which is significantly higher than in two other groups (11.3% in healthy controls and 6.6% in the group of non-triplet diseased patients;P < 0.001, Fishers exact test). This is consistent with our previous findings in HD patients. The absence of this association in non-triplet diseases as well as in healthy controls could indicate a possible role of this SCA1 allele with 31 repeats in triplet diseases. Here we discuss a possible role of the SCA1 region in pathological trinucleotide repeat expansions.