Evaluation of HPFH and δβ-thalassemia mutations in a Brazilian group with high Hb F levels.

Fetal hemoglobin (Hb F) is characteristic of the fetal development period. However, in some genetic conditions, such as hereditary persistence of fetal hemoglobin (HPFH) and delta-beta thalassemia (δβ-thalassemia), Hb F continues to be produced in adulthood. We evaluated the frequency of two mutations of HPFH, HPFH-1 and HPFH-2 African, and two mutations in δβ-thalassemia, Sicilian and Spanish, in a Brazilian population. Peripheral blood samples were collected from adults from hospitals and blood centers in southeast and northeast Brazil. These individuals were healthy and without complaints of anemia, but had increased Hb F. Samples were submitted to electrophoretic and chromatographic analyses to quantify Hb F values and, subsequently, to molecular analyses to verify the mutations. In the molecular analysis, 16 of the 60 samples showed a heterozygous profile for the HPFH mutations, two for HPFH-1 and 14 for HPFH-2. In the same sample set, three were heterozygous for Spanish δβ-thalassemia and none were heterozygous for Sicilian δβ- thalassemia. The Hb F values in the HPFH-2 heterozygotes differed from those previously reported for this mutation. In this group, the HPFH mutations were more frequent than the δβ-thalassemia mutations. The finding of these mutations in this Brazilian population reflects the mixing process that occurred during its formation.

Could contaminant induced mutations lead to a genetic diversity overestimation?

Contaminant driven genetic erosion reported through the inspection of selectable traits can be underestimated using neutral markers. This divergence was previously reported in the aquatic system of an abandoned pyrite mine. The most sensitive genotypes of the microcrustacean cladoceran Daphnia longispina were found to be lacking in the impacted reservoir near the entrance of the metal rich acid mine drainage (AMD). Since that divergence could be, at least partially, accounted for by mutagenicity and genotoxicity of the AMD, the present study aimed at providing such a characterization. The Allium cepa chromosomal aberration assay, using root meristematic cells, was carried out, by exposing seeds to 100, 10, 1, and 0.1 % of the local AMD. Chromosomal aberrations, cell division phases and cell death were quantified after the AMD exposure and after 24 and 48 h recovery periods. The AMD revealed to be mutagenic and genotoxic, even after diluting it to 1 and 0.1 %. Dilutions within this range were previously found to be below the lethality threshold and to elicit sublethal effects on reproduction of locally collected D. longispina clonal lineages Significant mutagenic effects (micronuclei and chromosomal breaks) were also found at 0.1 % AMD, supporting that exposure may induce permanent genetic alterations. Recovery tests showed that AMD genotoxic effects persisted after the exposure. © 2013 Springer Science+Business Media New York.

Expanding the spectrum of PTH1R mutations in patients with primary failure of tooth eruption

Objectives: Primary failure of tooth eruption (PFE) is a rare autosomal-dominant disease characterized by severe lateral open bite as a consequence of incomplete eruption of posterior teeth. Heterozygous mutations in the parathyroid hormone 1 receptor (PTH1R) gene have been shown to cause PFE likely due to protein haploinsufficiency. To further expand on the mutational spectrum of PFE-associated mutations, we report here on the sequencing results of the PTH1R gene in 70 index PFE cases. Materials and methods: Sanger sequencing of the PTH1R coding exons and their immediate flanking intronic sequences was performed with DNA samples from 70 index PFE cases. Results: We identified a total of 30 unique variants, of which 12 were classified as pathogenic based on their deleterious consequences on PTH1R protein while 16 changes were characterized as unclassified variants with as yet unknown effects on disease pathology. The remaining two variants represent common polymorphisms. Conclusions: Our data significantly increase the number of presently known unique PFE-causing PTH1R mutations and provide a series of variants with unclear pathogenicity which will require further in vitro assaying to determine their effects on protein structure and function. Clinical relevance: Management of PTH1R-associated PFE is problematic, in particular when teeth are exposed to orthodontic force. Therefore, upon clinical suspicion of PFE, molecular DNA testing is indicated to support decision making for further treatment options. © 2013 Springer-Verlag Berlin Heidelberg.

Análise mutacional da região dos exons 5 a 8 do gene supressor de tumor p53 em neoplasias mamárias caninas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Characterization of 23S rRNA domain V mutations in gastric biopsy patients from the eastern Amazon

Resistance of Helicobacter pylori to clarithromycin is characterised by simple point mutations in the 23S ribosomal RNA (rRNA) gene and is responsible for the majority of cases of failure to eradicate this bacterium. In this paper, we characterised the variability of the 23S rRNA gene in biopsies of patients with gastric pathologies in the eastern Amazon (Northern Region of Brazil) using PCR and sequencing. A total of 49 sequences of H. pylori strains were analysed and of those, 75.6% presented nucleotide substitutions: A2142G (3.3%), T2182C (12.9%), G2224A (6.45%), T2215C (61.3%), A2192G (3.3%), G2204C (6.4%) and T2221C (6.4%). Of the mutations identified, four are known mutations related to cases of resistance and 16.1% are not yet described, revealing a high prevalence of mutations in the H. pylori 23S rRNA gene among the strains circulating in the in the eastern Amazon. The high prevalence in individuals with gastric pathologies in the Northern Region of Brazil demonstrates the need for characterising the profile of these strains to provide correct therapy for patients, considering that mutations in this gene are normally associated with resistance to the primary medication used in controlling H. pylori infection.

Human aging and somatic point mutations in mtDNA: a comparative study of generational differences (grandparents and grandchildren)

The accumulation of somatic mutations in mtDNA is correlated with aging. In this work, we sought to identify somatic mutations in the HVS-1 region (D-loop) of mtDNA that might be associated with aging. For this, we compared 31 grandmothers (mean age: 63 ± 2.3 years) and their 62 grandchildren (mean age: 15 ± 4.1 years), the offspring of their daughters. Direct DNA sequencing showed that mutations absent in the grandchildren were detected in a presumably homoplasmic state in three grandmothers and in a heteroplasmic state in an additional 13 grandmothers; no mutations were detected in the remaining 15 grandmothers. However, cloning followed by DNA sequencing in 12 grandmothers confirmed homoplasia in only one of the three mutations previously considered to be homoplasmic and did not confirm heteroplasmy in three out of nine grandmothers found to be heteroplasmic by direct sequencing. Thus, of 12 grandmothers in whom mtDNA was analyzed by cloning, eight were heteroplasmic for mutations not detected in their grandchildren. In this study, the use of genetically related subjects allowed us to demonstrate the occurrence of age-related (> 60 years old) mutations (homoplasia and heteroplasmy). It is possible that both of these situations (homoplasia and heteroplasmy) were a long-term consequence of mitochondrial oxidative phosphorylation that can lead to the accumulation of mtDNA mutations throughout life.

Prevalence of ∆F508, G551D, G542X, and R553X mutations among cystic fibrosis patients in the North of Brazil

ABSTRACT: Cystic fibrosis (CF) is the most common genetic disease among Caucasians and is rare among sub-Saharan Africans. The Brazilian population is not ethnically homogeneous but it is the result of three-way ethnic admixture of Europeans, Africans and Amerindians in varying proportions, depending on the region. In the present study, we investigated 33 patients who had been diagnosed and are currently under treatment for CF at the University Hospital João de Barros Barreto, Belém, Pará State. The molecular analysis for G542X, G551D and R553X mutations was performed by PCR followed by RFLP using BstNI, HincII and MboI, respectively, in polyacrylamide gel eletrophoresis and stained with AgNO3. The DF508 mutation (a deletion of 3 bp) was only analyzed by polyacrylamide gel electrophoresis and stained with AgNO3. Each sample was analyzed for regions of interest in the CFTR gene using amplified by PCR and specific primers. The DF508 and G551D mutations presented frequencies of 22.7 and 3%, respectively. In 74.3% of the remaining patients, none of the mutations investigated was found. The present study characterized in a sample of patients with an established clinical diagnosis of CF (asthma, repeated bronchopneumonia, disorders of nutritional status, etc.) the most frequent mutation ( DF508) in the North region of Brazil and is also the first report of the G551D mutation. In spite of the wide spectrum of CF mutations and the heterogeneous ethnic origin of the Amazon population, the molecular diagnosis is a helpful additional tool for the diagnosis and treatment of CF patients.

Frequencies of CCR5-D32, CCR2-64I and SDF1-3'A mutations in Human Immunodeficiency Virus (HIV) seropositive subjects and seronegative individuals from the state of Pará in Brazilian Amazonia

ABSTRACT: The distribution of genetic polymorphisms of chemokine receptors CCR5-D32, CCR2-64I and chemokine (SDF1-3 A) mutations were studied in 110 Human Immunodeficiency Virus type 1 (HIV-1) seropositive individuals (seropositive group) and 139 seronegative individuals (seronegative group) from the population of the northern Brazilian city of Belém which is the capital of the state of Pará in the Brazilian Amazon. The CCR5-D32 mutation was found in the two groups at similar frequencies, i.e. 2.2% for the seronegative group and 2.7% for the seropositive group. The frequencies of the SDF1-3 A mutation were 21.0% for the seronegative group and 15.4% for the seropositive group, and the CCR2-64I allele was found at frequencies of 12.5% for the seronegative group and 5.4% for the seropositive group. Genotype distributions were consistent with Hardy-Weinberg expectations in both groups, suggesting that none of the three mutations has a detectable selective effect. Difference in the allelic and genotypic frequencies was statistically significant for the CCR2 locus, the frequency in the seronegative group being twice that found in the seropositive group. This finding may indicate a protective effect of the CCR2-64I mutation in relation to HIV transmission. However, considering that the CCR2-64I mutation has been more strongly associated with a decreased risk for progression for AIDS than to the resistance to the HIV infection, this could reflect an aspect of population structure or a Type I error.

CYP21 gene mutations in Brazilian patients with 21-hydroxylase deficiency from the Amazon region

ABSTRACT: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (P450c21, CYP21) accounts for about 95% of all CAH cases. The incidence of CYP21 gene mutations has been extensively studied in the last years, but in Brazil it has been investigated only in Southeast Brazilian patients. This study is the first report on the distribution of CYP21 mutations in patients from the Amazon region. Direct sequencing of the CYP21 gene identified at least one mutation in 96% of the studied chromosomes. The most common mutations found were IVS2-13A/C > G (36%), Q318X (12%), V281L (12%), 1760_1761insT (9%), Cluster E6 (7%), and P30L (7%). The worldwide most common mutations were identified among patients from the Amazon region at frequencies that may be expected for a population resulting from the admixture of Europeans (predominantly Portuguese), African Blacks and Amerindians, in proportions that differ from those estimated for South Brazilian populations. Interethnic mixture may explain the differences in the frequencies of some mutations between Brazilian patients from the Amazon and from the Southeast of the country. However, the differences found may also be due to variation in the number of patients with the different clinical forms of 21-hydroxylase deficiency in the studies carried out so far.

KRAS insertions in colorectal cancer: What do we know about unusual KRAS mutations?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Non-synonymous mutations mapped to chromosome X associated with andrological and growth traits in beef cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)