Vanessa Erichsen Emmel

Evaluation of genetic damage in a Brazilian population occupationally exposed to pesticides and its correlation with polymorphisms in metabolizing genes.

Cytogenetic damage in individuals occupationally exposed to pesticides has received the attention of investigators in several countries, but no definitive conclusions can yet be made. The present study aimed at assessing if prolonged exposure to complex mixtures of pesticides leads to an increase in cytogenetic damage. Vineyard workers exposed to pesticides in Caxias do Sul (Brazil) were evaluated using the micronucleus (MN) test in binucleated lymphocytes and the comet assay in peripheral leukocytes. In order to evaluate if genetically determined individual variations in xenobiotic metabolizing capacity could modify individual susceptibility to the possible genotoxic effects of pesticides, the subjects were genotyped for several genes: GSTT1, GSTM1, GSTP1, CYP1A1, CYP2E1 and PON. The study involved a total number of 173 men: 108 were agricultural workers exposed to pesticides and 65 were controls. The present study showed a high rate of MN and DNA damage in pesticide-exposed individuals (P <or= 0.001; Mann-Whitney U-test). In addition, some effects of genetic polymorphisms in PON in the modulation of MN results were observed in the exposed group, and an association between GSTM1, GSTT1 and CYP2E1 polymorphisms was suggested.

Common origin of pure and interrupted repeat expansions in spinocerebellar ataxia type 2 (SCA2)

The spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disease characterized by gait and limb ataxia. This disease is caused by the expansion of a (CAG)n located in the ATXN2, that encodes a polyglutamine tract of more than 34 repeats. Lately, alleles with 32–33 CAGs have been associated to late‐onset disease cases. Repeat interruptions by CAA triplets are common in normal alleles, while expanded alleles usually contain a pure repeat tract. To investigate the mutational origin and the instability associated to the ATXN2 repeat, we performed an extensive haplotype study and sequencing of the CAG/CAA repeat, in a cohort of families of different geographic origins and phenotypes. Our results showed (1) CAA interruptions also in expanded ATXN2 alleles; (2) that pathological CAA interrupted alleles shared an ancestral haplotype with pure expanded alleles; and (3) higher genetic diversity in European SCA2 families, suggesting an older European ancestry of SCA2. In conclusion, we found instability towards expansion in interrupted ATXN2 alleles and a shared ancestral ATXN2 haplotype for pure and interrupted expanded alleles; this finding has strong implications in mutation diagnosis and counseling. Our results indicate that interrupted alleles, below the pathological threshold, may be a reservoir of mutable alleles, prone to expansion in subsequent generations, leading to full disease mutation.

Diphyllobothrium latum: relato de caso no Brasil

Diphyllobothriasis is caused in humans by infection with adult tapeworms of the genus Diphyllobothrium acquired by consuming raw or undercooked freshwater fish. Diphyllobothrium latum was confirmed by examination of the gravid proglottids and typical operculated eggs in the stool. The patient had a history of eating crustaceans and fish. This is the case report of the first Brazilian infected.

Normal ATXN3 Allele but Not CHIP Polymorphisms Modulates Age at Onset in Machado–Joseph Disease

Background: Age at onset (AO) in Machado–Joseph disease (MJD) is closely associated with the length of the CAG repeat at the mutant ATXN3 allele, but there are other intervening factors. Experimental evidence indicates that the normal ATXN3 allele and the C-terminal heat shock protein 70 (Hsp70)-interacting protein (CHIP) may be genetic modifiers of AO in MJD. Methods: To investigate this hypothesis, we determined the length of normal and expanded CAG repeats at the ATXN3 gene in 210 unrelated patients with MJD. In addition, we genotyped five single nucleotide polymorphisms (SNPs) within the CHIP gene. We first compared the frequencies of the different genotypes in two subgroups of patients who were highly discordant for AO after correction for the length of the expanded CAG allele. The possible modifier effect of each gene was then evaluated in a stepwise multiple linear regression model. Results: AO was associated with the length of the expanded CAG allele (r2 = 0.596, p < 0.001). Frequencies of the normal CAG repeats at the ATXN3 gene and of CHIP polymorphisms did not differ significantly between groups with highly discordant ages at onset. However, addition of the normal allele improved the model fit for prediction of AO (r2 = 0.604, p = 0.014). Indeed, we found that the normal CAG allele at ATXN3 had a positive independent effect on AO. Conclusion: The normal CAG repeat at the ATXN3 gene has a small but significant influence on AO of MJD.

Does DNA methylation in the promoter region of the ATXN3 gene modify age at onset in MJD (SCA3) patients

To the Editor : Machado–Joseph disease (MJD/SCA3) is a neurodegenerative disease caused by an unstable CAG repeat expansion in ATXN3 gene, leading to an expanded polyglutamine tract in ataxin-3, the corresponding protein (1). The distribution of age at onset (AO) is inversely correlated with CAG expansion size; however, repeat length is responsible only for 45–60% of AO variation in MJD (2, 3), indicating other still unidentified factors (genetic, environmental or others). The degree of neurodegeneration induced by the polyQ protein is correlated with protein storage levels (4). We therefore hypothesized that DNA methylation, specifically targeting the mutant allele and leading to transcriptional deregulation, might influence the levels of mutant ataxin-3 in affected cells and thus AO in MJD patients. Our aim was to assess the methylation degree at six CpG dinucleotides at the ATXN3 promoter and explore their role as potential modifiers for AO in MJD. One hundred and twenty-three Brazilian patients from 62 families with MJD were ascertained in Rio Grande do Sul, Brazil. The inclusion criterion was molecular confirmation in a symptomatic patient. AO was defined as the age at which the patient, or a close person, noticed the first symptoms (usually gait unbalance); 35 healthy individuals were also neurologically examined to be used as controls. This study was approved by the Hospital Ethics Committee. DNA was isolated from leukocytes, as described (5). Evaluation of the (CAG)n tract was performed by fluorescently labeled polymerase chain reaction (PCR) and capillary electrophoresis. Methylationspecific multiplex ligation-dependent probe amplification (MS-MLPA) analysis was carried out according to manufacturer’s instructions (MRC Holland, Amsterdam) (6). We designed four specific probes for the ATXN3 promoter, which were able to detect the methylation degree at six CpG dinucleotides, through restriction enzyme recognition. We have also designed three control probes, lacking a restriction enzyme recognition site, in genes located outside this region. Data analysis was performed by exporting peak areas to an Excel-based analysis program. Methylation differences between patients and controls, for each probe, were evaluated using the Student’s unpaired t-test. Generalized linear models (GLMs) by generalized estimating equations (GEEs) were used to test the effect of the methylation degree in AO variation. Controlled variables were the family and (CAG)n length (normal and expanded). Statistical analyses were performed using spss for Windows v.16. Length distribution of (CAG)n with AO is shown in Table 1. A significant inverse correlation was found between AO and repeat length (r2 = 0.57, p < 0.001). Probe 1 (containing one CpG) was predominantly methylated (average 94.8%). Probe 2, also containing one CpG dinucleotide, had an average methylation degree of 40.1%. Probes 3 and 4, both containing two CpGs, were predominantly non-methylated (averages of 6.7% and 3.6%) (Fig. 1). No statistically significant differences were found when comparing methylation status between the whole patients’ cohort and controls (p > 0.05). A stepwise analysis of each probe was performed. Given the factor effect under study being the family, a trend toward a direct relation between methylation degree for probe 1 and AO was suggested (p = 0.055), when GLM analyzed methylation status against AO only (Fig. 1a). The regression coefficient relating probe 1 and AO was 24.0; i.e. each 10% decrease in probe 1 methylation status was related to a 2.4-year reduction in AO. The effect of the methylation status was small; however, when compared to the effect of the expanded repeat: when (CAG)n

Spinocerebellar ataxia type 10: common haplotype and disease progression rate in Peru and Brazil

Spinocerebellar ataxia type 10 is a neurodegenerative disorder that is due to an expanded ATTCT repeat tract in the ATXN10 gene. Our aim was to describe clinical characteristics and intragenic haplotypes of patients with spinocerebellar ataxia type 10 from Brazil and Peru.