Dušan Keckarević

Frequency of the hemochromatosis gene mutations in the population of Serbia and Montenegro

To the Editor: The most common form of hereditary hemochromatosis (HH) is HH type 1, which affects between 1 in 200 and 1 in 400 individuals of northern European descent (1, 2). It is an autosomal recessive iron-overload disorder frequently associated with two mutations in the hemochromatosis gene (HFE) resulting in the substitution of cysteine with tyrosine at amino acid position 282 (C282Y) and the substitution of histidine with aspartate at amino acid position 63 (H63D) (3). In the original work by Feder et al., 83% of HH type 1 patients were found to be homozygous for the C282Y mutation and 4% were compound heterozygotes (C282Y/H63D) (3). Numerous other genotyping studies have confirmed this finding (4, 5). One more mutation, the substitution of serine with cysteine at amino acid position 65 (S65C), has been identified in the HFE gene. Some C282Y/S65C individuals have mild iron loading (6). The high frequency of the C282Y mutation has been shown in the northern European populations (7–9), whereas lower mutation frequencies were detected in different populations from southern Europe (10–12). However, no HFE mutations frequency data have been published on the population of Serbia and Montenegro. In addition, there is a limited knowledge of those frequencies in populations of the Balkan Peninsula. Therefore, the aim of this study was to determine the frequency of the C282Y, H63D and S65C mutations in the healthy population of Serbia and Montenegro and compare it with some other European populations. The study was performed on 318 unrelated healthy individuals (181 females and 137 males) from Serbia and Montenegro. Informed consent was obtained from all participants of the study. The analysis of HFE gene mutations was performed using modified method described by Milman et al. (13). Chi-square test or Fischer’s exact test was used to compare proportions of HFE genotypes between males and females and allelic frequencies among different populations. The distribution ofHFE genotypes in the population of Serbia and Montenegro are shown in Table 1. The C282Y and S65C allele frequencies in the analyzed population were the same, 1.6% (95% confidence interval (CI): 0.9–2.9), and the H63D allele frequency was 15.7% (95% CI: 13.1–18.7) (Table 1). The frequency of C282Y homozygotes was estimated on approximately 1:3900 indicating the prevalence of hereditary hemochromatosis type 1 in Serbia and Montenegro. The frequencies of the HFE mutations in the population of Serbia and Montenegro were compared to already published data for other Slavic populations (14–16), populations that are geographically close to Serbia and Montenegro (11, 12, 17–19) and the Danish population (13), as a representative of northwestern European population (Table 2). The frequency of the C282Y mutation in the analyzed population is significantly lower than the frequency found in Austria, Czech Republic, Denmark, Hungary, Northern Italy, Poland and Slovenia. A statistically significant difference was found also in the frequency of H63D mutation between the population of Serbia and Montenegro and the population of Austria and Bulgaria. The distribution of the C282Y mutation decreases going from northwest to southeast Europe (20), with maximal frequency in Ireland (9.7%) and minimal in Bulgaria (0%) (21). According to the geographical position in Europe, the frequency of the C282Y mutation in Serbia and Montenegro (1.6%) fits in the observed north/south gradient. The frequency of the C282Y mutation in the population of Serbia and Montenegro is similar to that in the Croatian population, but, at the same time, it differs from that found in other populations of Slavic origin. In addition, there is similarity between the studied population and

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.

Lafora disease: Severe phenotype associated with homozygous deletion of the NHLRC1 gene

Lafora disease (LD) is a severe, autosomal recessive, latechildhood- to teenage-onset, progressive myoclonic epilepsy. It is due to either EPM2A or NHLRC1 mutations. We describe a patient with homozygous deletion encompassing the entire NHLRC1 gene, not previously reported, and with clinical course more progressive than in the most patients with NHLRC1 mutations. The diagnosis of LD in our patient was based on the typical clinic, neurophysiological presentation, as well as skin biopsy followed by molecular genetics findings. She developed normally until the age of 15, when she had her first occipital and generalized seizures. Four years after the first seizure the patient became bedridden, demented and presented with severe clinical condition. She died of pneumonia at age 20. This report is the first case of homozygosity for NHLRC1 deletion and thus adds to mutational heterogeneity of LD. Besides, it widens the spectrum of LD patients with severe phenotype and NHLRC1 mutations.

Mutational analysis of GJB1, MPZ, PMP22, EGR2, and LITAF/SIMPLE in Serbian Charcot-Marie-Tooth patients

We report the results of mutational analysis in the following genes: GJB1, MPZ, PMP22, EGR2, and LITAF/SIMPLE in 57 Charcot‐Marie‐Tooth (CMT) patients of Serbian origin without the PMP22 duplication. We found 10 different mutations in 14 CMT patients: 6 mutations in GJB1, 3 in MPZ, and 1 in PMP22. Five of six GJB1 mutations are reported for the first time, and the most frequent one appears to be a founder mutation in the Serbian population. No mutations were found in EGR2 or LITAF. Thus, GJB1 mutation analysis should be done in patients without the PMP22 duplication and male‐to‐male transmission of CMT.

HD phenocopies--possible role of Saitohin gene.

Saitohin (STH) is located in the intron of the human gene for microtubule-associated protein tau. Q7R polymorphism has been identified in the STH gene. Some neurodegenerative disorders were found to be associated with the presence of certain STH allele. This study genotyped 37 subjects with diagnosis of Huntingtons disease, but lacking mutations in HD, PRNP, JPH-3, and FTL genes for STH polymorphism. It was determined that Q allele of STH gene was over-represented in a tested group of patients (P > Pt). Over-representation of Q allele in a group of patients might be considered as genetic risk factor for HD like diseases.

Genetics of Lafora progressive myoclonic epilepsy: current perspectives

Lafora disease (LD) is a fatal neurodegenerative disorder caused by loss-of-function mutations in either laforin glycogen phosphatase gene (EPM2A) or malin E3 ubiquitin ligase gene (NHLRC1). LD is associated with gradual accumulation of Lafora bodies (LBs). LBs are aggregates of polyglucosan, a long, linear, poorly branched, hyperphosphorylated, insoluble form of glycogen. Loss-of-function mutations either in the EPM2A or in the NHLRC1 gene lead to polyglucosan formation. One hypothesis on LB formation is based on findings that laforin–malin complex downregulates glycogen synthase (GS) through malin-mediated ubiquitination, and the other one is based on findings that laforin dephosphorylates glycogen. According to the first hypothesis, polyglucosan formation is a result of increased GS activity, and according to the second, an increased glycogen phosphate leads to glycogen conformational change, unfolding, precipitation, and conversion to polyglucosan, while GS remains bound to the precipitating glycogen. In this review, we summarize all the recent findings that have important implications for the treatment of LD, all of them showing that partial inhibition of GS activity may be sufficient to prevent the progression of the disease. The current perspective in LD is high-throughput screening for small molecules that act on the disease pathway, that is, partial inhibitors of GS, which opens a therapeutic window for potential treatment of this fatal disease.

A novel P66S mutation in exon 3 of the SOD1 gene with early onset and rapid progression

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease in adults of unknown origin in most cases. Here we report a novel P66S mutation in exon 3 of the SOD1 gene in an apparently sporadic ALS patient with unusual early onset and rapid disease progression. Our data widen the spectrum of SOD1 mutations and clinical presentations of ALS.

A Shared Haplotype Indicates a Founder Event in Unverricht–Lundborg Disease Patients from Serbia

Unverricht–Lundborg disease (ULD) is an autosomal recessive disorder caused by dodecamer repeat expansion in the promoter region of the cystatin B (CSTB) gene in approximately 90% of the disease alleles worldwide. This study presents results of genetic findings in four Serbian unrelated patients with clinical and molecular diagnosis of ULD. Using newly established PCR protocol with betaine, we detected a homozygous expansion of dodecamer repeats in the CSTB gene in four patients with clinical diagnosis of ULD. Our results are in agreement with previous studies showing that dodecamer repeats expansion is the most common mutation associated with ULD. Haplotype analysis of eight unrelated ULD chromosomes was performed using seven markers flanking CSTB gene and one intragenic variant. We demonstrated the existence of a founder effect, strongly supported by LD calculations. Size of the minimal common haplotype implies that the most recent common ancestor of the Serbian ULD patients lived about 110 generations ago. We showed that Serbian ULD patients share the same common ancestor with patients from Baltic countries and North Africa. In the light of our data, we proposed extended minimal common haplotype, which could be considered as initial haplotype of the founder event common for Serbian, Baltic, and North African ULD patients.

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.

Clinical and genetic data on Lafora disease patients of Serbian/Montenegrin origin

Lafora disease (LD) is an autosomal recessive, progressive disorder characterized by myoclonus and seizures, inexorable neurologic deterioration, cognitive decline and poor prognosis. LD is caused by mutations either in the EPM2A or in NHLRC1 genes. Here we report clinical and genetic findings on 14 LD patients from 10 families of Serbian/Montenegrin origin. Molecular diagnostics was performed by sequencing the coding regions of the EPM2A and NHLRC1 genes. In addition, haplotype analysis of the chromosomes carrying the two most frequent mutations (c.1048‐1049delGA and deletion of the whole NHLRC1 gene) using eight different markers flanking the NHLRC1 gene was conducted. We identified one new mutation (c.1028T>C) along with the 3 previously reported mutations (c.1048‐1049delGA, c.990delG, deletion of the whole NHLRC1 gene), all of which were located on the NHLRC1 gene. The two predominant mutations (c.1048‐1049delGA and complete NHLRC1 gene deletion) appear to be founder mutations. In addition to documenting the genetic heterogeneity observed for LD, our study suggests that mutations in the NHLRC1 gene may be a common cause of LD in the Serbian/Montenegrin population, primarily because of a founder effect.