Genetic background of Huntington disease in Croatia: Molecular analysis of CAG, CCG, and Delta2642 (E2642del) polymorphisms.

This study presents the first molecular data on the basis and the origin of Huntington disease in Croatia and is the first such analysis performed among a Slavic population. We analyzed three trinucleotide polymorphisms in the HD gene: CAG, CCG and GAG Delta2642 (E2642del) triplets. Analysis of the CAG repeat size among 44 Huntington patients (39-66 CAGs) and 51 normal individuals (9-34 CAGs) showed that the range of the repeats was similar to previous findings. The frequency of the CCG and Delta2642 polymorphic alleles on N and HD chromosomes was found to correlate well with earlier reports for Western European populations. We found significance for both the CCG7 allele (p=0.004) and the Delta2642 allele (p<0.001) among HD chromosomes. The CCG7 allele was overpresented among affected chromosomes (94.6%), but was also the most frequent CCG allele among normal chromosomes (66.7%). Interestingly, the Delta2642 allele was present on 40.5% HD chromosomes compared to only 9.8% of control chromosomes. Our results indicate that HD mutations in Croatia could be of the same origin as in Western populations and also support the multi-step hypothesis for generating new HD alleles. Similar frequencies and distributions of both the CCG and the Delta2642 polymorphisms in Croatia and Western European normal chromosomes indicate that the prevalence rate of HD in Croatia may be as high as in Western populations. Since we estimated a lower prevalence rate (1 : 100,000), we assume that there are still many misdiagnosed and/or unrecognized cases of Huntington disease in Croatia.

Calpainopathy (LGMD2A) in Croatia: molecular and haplotype analysis.

AIM: To determine types and frequency of CAPN3 mutations in 29 unrelated Croatian families, analyzed during 6-year prospective and ongoing genetic and epidemiological study of muscular dystrophies in Croatia. METHODS: Mutation analysis included allele-specific polymerase chain reaction (PCR) or combination of PCR and restriction fragment length polymorphisms (RFLP) methods. Haplotype analysis was performed by PCR and DNA electrophoresis using 5 highly polymorphic markers flanking CAPN3 gene locus. RESULTS: Mutation analysis revealed the presence of 6 different CAPN3 mutations (550delA, R541W, P82L, delFWSAL, R49H, Y537X), accounting for 94.8% of CAPN3 chromosomes in the studied population. 550delA was the most frequent mutation, found in 43/58 (74%) CAPN3 chromosomes, whereas the frequency of other five mutations ranged from 2-9%. Haplotype analysis of 38 chromosomes carrying 550delA mutation showed the presence of the same haplotype on 66% of analyzed chromosomes. CONCLUSIONS: The present data, together with our previously published results, explain the frequency and the distribution of the 550delA mutation in Croatia by founder effect and genetic drift. Results of haplotype study are in favor of the hypothesis that 550delA is an old, rather than a recurrent mutation. The findings are important for effective diagnostic screening of CAPN3 gene in Croatia and neighboring countries, as well as for accurate genetic counseling.

Prevalence of the 550delA mutation in calpainopathy (LGMD 2A) in Croatia.

Mutations in the calpain 3 (CAPN3) gene are responsible for limb-girdle muscular dystrophy (LGMD) type 2A. We report five causal mutations: 550delA, DeltaFWSAL, R541W, Y357X and R49H found on 45/50 of alleles studied in 25 unrelated families from Croatia. The 550delA mutation was present on 76% of CAPN3 chromosomes that led us to screen general population for this mutation; 532 random blood samples from three different regions were analyzed using allele-specific PCR. Four healthy 550delA heterozygous were found suggesting a frequency of 1 in 133. All four carriers detected originated from an island and mountain region close to the Adriatic Sea. These findings combined with haplotype analysis confirm that our general population is rather "closed" with a probable founder effect in some parts of the country. In addition, the high frequency of 550delA mutation found in some neighboring European countries together with the easy detection of the 550delA mutation should streamline genetic analysis, especially bearing in mind the geographic and ethnic origin of the patients. Our results, combined with published haplotype studies suggest that 550delA originated in the Eastern Mediterranean from which it has probably spread widely across Europe. Extending this study to other areas would help to address this epidemiological question. Our data are relevant to accurate genetic counseling and patient testing since we lack sensitive and specific biopsy screening methods for detecting patients with calpainopathy. Thus, detection of patients relies on the direct detection of gene mutation and our findings may be helpful in establishing diagnostic screening strategy.