Detection of new mutations and molecular pathology of mild and moderate haemophilia A patients from southern Brazil.

A total of 76 unrelated male patients with mild (n = 55) or moderate (n = 21) haemophilia A living in the southern Brazilian state of Rio Grande do Sul were studied by direct sequencing of all F8 26 exons, the 5' UTR and 3' UTR, intron-exon junctions and the promoter region. When no mutation was found, a multiplex ligation-dependent probe amplification analysis was performed. We identified the disease-causing mutations in 69 patients, who showed 33 different mutations: 27 missense, one small deletion, two small duplications and three splice site mutations. Seven missense and two splice site mutations were not previously reported in HAMSTeRS and were not identified in any current literature search. Nine recurrent mutations were found, one of them never described before (p.Tyr1786Phe). Haplotype analysis indicated that this mutation had originated in the Brazilian population as a single event in a common ancestor. The possible influence of these mutations in the determination of the disease was carefully considered, including bioinformatic tools. These data add to the general knowledge of the disease and can also be useful for HA diagnosis and detection of carriers in the southern Brazilian population.

Association between the 1858T allele of the protein tyrosine phosphatase nonreceptor type 22 and type 1 diabetes in a Brazilian population.

The 1858T allele of the protein tyrosine phosphatase nonreceptor 22 (PTPN22) gene has been associated to diabetes in different populations. We investigated a possible relationship between this polymorphism and type 1 diabetes in a cohort of Brazilian patients. A significantly higher frequency of the 1858T allele was observed in diabetic patients (n = 211) than in control individuals (n = 241). Additionally, the heterozygote genotype was also increased in the diabetic group. No association was observed between the PTPN22 T allele and gender, or between T carriers and age of onset of T1D. This work describes for the first time a strong association of the 1858T allele with type 1 diabetes in a Brazilian population, reinforcing the role of this variant as an important susceptibility factor for this disease.

Introns 1 and 22 inversions and factor VIII inhibitors in patients with severe haemophilia A in southern Brazil.

A total of 107 unrelated severe haemophilia A patients living in the southern Brazilian state of Rio Grande do Sul were studied in relation to the prevalence of inversions present in introns 22 and 1 and a subsample of them (95) tested for the presence of Factor VIII inhibitors. These data were then incorporated with those from 15 other countries and 3871 patients. The frequencies of these two inversions show a remarkable homogeneity in series collected in different continents, from people with diverse ethnic extraction. The prevalence of inhibitors among patients with inversion 22, on the other hand, varies widely (5-51%; seven countries, 1482 patients), the value observed by us being the highest. The importance of obtaining data from patients throughout the world to clarify the aetiology of this important complicating factor in the therapeutics of the disease is emphasized.

Polymorphisms in genes MTHFR, MTR and MTRR are not risk factors for cleft lip/palate in South Brazil.

Non-syndromic cleft lip and palate (CL/P) occurs due to interaction between genetic and environmental factors. Abnormalities in homocysteine metabolism may play a role in its etiology due to polymorphisms in genes involved in this pathway. Because of the involvement of MTHFR, MTR and MTRR genes with folate metabolism and the evidence that maternal use of folic acid in early pregnancy reduces the risk for CL/P, we evaluated the influence of their polymorphisms on the etiology of CL/P through a case-control study. The analyses involved 114 non-syndromic phenotypically white children with clefts (case) and 110 mothers, and 100 non-affected (control) children and their mothers. The polymorphisms 677C>T of MTHFR, 2756A>G of MTR, and 66A>G of MTRR genes were analyzed by PCR-RFLP. Allelic frequencies did not differ from other studies conducted on white populations for MTHFR 677T allele (0.35) and for MTR 2756G allele (0.17), but MTRR 66G allele frequency (0.35) was lower than observed elsewhere. The genotypic distribution of the 677C>T polymorphisms under study did not show significant differences between CL/P patients, their mothers and controls. These results suggest that the alterations of folate metabolism related to these polymorphisms are not involved in clefting in the population under study.

Polymorphisms in genes MTHFR, MTR and MTRR are not risk factors for cleft lip/palate in South Brazil

Non-syndromic cleft lip and palate (CL/P) occurs due to interaction between genetic and environmental factors. Abnormalities in homocysteine metabolism may play a role in its etiology due to polymorphisms in genes involved in this pathway. Because of the involvement of MTHFR, MTR and MTRR genes with folate metabolism and the evidence that maternal use of folic acid in early pregnancy reduces the risk for CL/P, we evaluated the influence of their polymorphisms on the etiology of CL/P through a case-control study. The analyses involved 114 non-syndromic phenotypically white children with clefts (case) and 110 mothers, and 100 non-affected (control) children and their mothers. The polymorphisms 677C>T of MTHFR, 2756A>G of MTR, and 66A>G of MTRR genes were analyzed by PCR-RFLP. Allelic frequencies did not differ from other studies conducted on white populations for MTHFR 677T allele (0.35) and for MTR 2756G allele (0.17), but MTRR 66G allele frequency (0.35) was lower than observed elsewhere. The genotypic distribution of the 677C>T polymorphisms under study did not show significant differences between CL/P patients, their mothers and controls. These results suggest that the alterations of folate metabolism related to these polymorphisms are not involved in clefting in the population under study.

Von Willebrand factor gene polymorphisms in three Brazilian ethnic groups.

Von Willebrand factor gene polymorphisms were studied in three Brazilian ethnic groups: Euro-Brazilians, Afro-Brazilians, and Amerindians. Six polymorphic sites were analyzed: RsaI (exon 18), NlaIV (exon 20), HphI, KpnI, D1472H, and V1565L (all four in exon 28). The allele frequencies were significantly different between Euro- and Afro-Brazilians for RsaI, HphI, D1472H, and V1565L, while in Amerindians NlaIV and HphI showed significant differences among tribes. This is the first report of these allele frequencies in Amerindians. Eighteen haplotypes were observed, and they showed significant differences between Euro- and Afro-Brazilians, among Amerindian tribes, and among the three ethnic groups. These results furnish important background data for evolutionary and anthropological investigations; in addition, they will be useful for establishing the origin and molecular characterization of the different forms of von Willebrand disease, as well as for detecting carriers and offering genetic counseling to those with this condition.

Doxorubicin plus lonidamine: in vivo metabolic effects on the rat heart.

Lonidamine (LND) is a new drug that interferes with mitochondrial functions, thereby inhibiting cellular oxygen consumption and energy metabolism in both normal and neoplastic cells. These metabolic actions of LND seem to increase the cytotoxic effect of antitumor agents such as doxorubicin (Dx). Dx is a widely used antitumor agent, but the specific cardiac toxicity which develops at a critical cumulative dose is the major limiting factor in its long term use. So far, nothing is known about a possible synergic action of LND and Dx on the metabolism of cardiac cells. The purpose of this study was to verify in an experimental model in vivo, whether LND could increase the toxicity of Dx on rat heart. Groups each consisting of 10 female Wistar rats (weighing 100-150 g) were injected ip with a single dose of Dx (10 mg/kg), LDN (50 mg/kg), or Dx plus LND and dimethylsulfoxide (DMSO) (ratio LND:DMSO = 1/10). After 24 h, oxygen uptake (QO2) and intracellular concentrations of ATP and GTP indices of cardiac metabolic impairment, were measured on heart slices in Warburg apparatus and by high-pressure liquid chromatography. Dx, significantly (p less than 0.01), reduced QO2 (34%) and intracellular concentration of ATP and GTP (32-57%). LND alone only partially reduced cardiac QO2 (23%) and intracellular ATP-GTP concentration (16-31%). By contrast, the combination of the two agents did not enhance Dx-related metabolic cardiac toxicity.

Inheritance and prevalence of von Willebrand's disease severe form in a Brazilian population.

Reviewed data suggest that the prevalence of severe von Willebrand's disease is influenced by ethnic and geographic factors. In the State of Rio Grande do Sul, Brazil, seven genealogies in which 11 individuals had a severe expression of von Willebrand's disease were localized. These affected subjects had similar laboratory results and all of them seemed to have resulted from double genetic defects, but the genealogic examination revealed that four of them probably resulted from combinations of autosomal recessive genes, while in the remaining the presence of dominant genes was likely and the involvement of genes for types I or II of von Willebrand's disease was possible. All of their examined relatives were asymptomatic but some of them presented unusual laboratory results, indicative of heterozygosis. The prevalence of severe cases in the surveyed population was higher than expected even when only the recessive forms were considered. It entered the expected values when it was presumed that these were all the cases currently living in the State. Genetic heterogeneity of the severe form was confirmed and it is suggested that the designations 'severe von Willebrand's disease' and 'type III von Willebrand's disease' should not be used as synonyms.