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.

Relationship between polymorphisms in thrombophilic genes and preeclampsia in a Brazilian population.

The purpose of this study was to evaluate the thrombophilic genes in pregnant women with and without preeclampsia independently or in combination. In a prospective case-control study, we investigated four polymorphisms in thrombophilic genes in 75 women with mild or severe preeclampsia and 145 women with normal pregnancy. The genotype frequencies were assessed and the odds ratio (OR) calculated. When we analyzed the polymorphisms independently and the development of preeclampsia, no association was observed [methylenetetrahydrofolate reductase (MTHFR) 677TT genotype, OR 2.07, 95% confidence interval (CI) 0.99-4.30; prothrombin mutation (F II) (GA or AA genotypes) OR 8.11, 95% CI 0.89-73.92; factor V Leiden (FV Leiden) OR 3.94, 95% CI 0.35-44.23; plasminogen activator inhibitor (PAI-1) 4G/4G genotype, OR 1.63, 95% CI 0.87-3.05] not even with severe preeclampsia subgroup analysis. However, when we investigated a possible interaction among these polymorphisms on the development of the preeclampsia, the OR for having one risk genotype, one or two genotype risk factors and two genotype risk factors compared to those without genotype risk factors were 1.97 (95% CI 1.08-3.59), 2.21 (95% CI 1.25-3.92) and 4.27 (95% CI 1.3-13.9), respectively. In conclusion, in the population analyzed, the presence of the genotype risk factors alone does not seem to be associated with the development of preeclampsia even in the severe presentation form. However, an interaction among the MTHFR, F II, FV and PAI-1 gene polymorphisms on the development of the preeclampsia was indicated.

The European-specific mitochondrial cluster J/T could confer an increased risk of insulin-resistance and type 2 diabetes: an analysis of the m.4216T > C and m.4917A > G variants.

The aims of this study were to investigate the contributions of the mitochondrial DNA m.4216T > C and m.4917A > G variants, and also of the European-specific mitochondrial cluster J/T, to the development of type 2 diabetes mellitus in Caucasian-Brazilian patients from Southern Brazil. We analyzed 347 type 2 diabetes patients and 350 control subjects. Variant frequencies in patients and control subjects were compared using chi2 tests or odds ratio. We also compared clinical and laboratory characteristics among patients with and without the variants. We found that the frequencies of the m.4216T > C and m.4917A > G variants are higher in diabetic patients than in control subjects. Moreover, haplogroups J (partially defined by the presence of the m.4216T > C variant only) and T (partially defined by the presence of both m.4216T > C and m.4917A > G variants) are more frequent in the type 2 diabetic group than in the control group. Patients belonging to the cluster J/T are more insulin resistant than patients of other haplogroups. In conclusion, our results indicate the association of the cluster J/T (as suggested by analyses of the m.4216T > C and m.4917A > G variants) with insulin resistance and type 2 diabetes.

The presence of allele D of angiotensin-converting enzyme polymorphism is associated with diabetic nephropathy in patients with less than 10 years duration of Type 2 diabetes.

AIM: To investigate the association between angiotensin-converting enzyme gene I/D polymorphism and diabetic nephropathy (DN) in patients with Type 2 diabetes mellitus (DM) taking into consideration the known duration of DM. METHODS: Cross-sectional study with 982 patients categorized according to urinary albumin excretion (UAE) into normoalbuminuria (UAE < 20 microg/min or < 17 mg/l, 24-h timed urine or spot random sterile urine, respectively), incipient DN (UAE 20-199 microg/min or 17-174 mg/l) and overt DN (UAE > 200 microg/min or > 174 mg/l or dialysis). Patients were further grouped regarding presence of the D allele (DD/ID vs. II) and DM duration (< or = 10 years or > 10 years). RESULTS: Incipient DN was diagnosed in 17.3% (n = 170), and 20.7% (n = 203) had overt DN (macroalbuminuria, n = 129; dialysis, n = 74). Genotype distribution (DD/ID/II) was similar in patients with incipient (49/92/29) or overt DN (77/89/37) if compared with patients without DN (181/308/120, P = 0.172). In patients with DM < or = 10 years, having the D allele (DD/ID) resulted in an odds ratio (OR) of 2.66 (95% CI: 1.12-6.58, P = 0.015) for incipient DN, and 3.19 (95% CI: 1.18-9.30, P = 0.012) for overt DN. In patients with longer DM duration, the D allele did not increase the risk for incipient (OR 0.68, 95% CI 0.36-1.29, P = 0.206) or overt DN (OR 0.67, 95% CI 0.39-1.17, P = 0.138). CONCLUSION: The DD/ID genotypes were associated with incipient or overt DN in patients with DM < or = 10 years.

Prevalence of retinopathy in Caucasian type 2 diabetic patients from the South of Brazil and relationship with clinical and metabolic factors.

Diabetic retinopathy (DR) is a sight-threatening chronic complication of diabetes mellitus and is the leading cause of acquired blindness in adults. In this cross-sectional study, we investigated the prevalence of and the factors associated with DR in an analysis of 210 consecutive and unrelated Brazilian Caucasians with type 2 diabetes mellitus. Retinopathy was evaluated by ophthalmoscopy and/or biomicroscopy through dilated pupils. The relationship between clinical and metabolic variables and the presence of DR was assessed by logistic regression analysis. DR was detected in 99 of the 210 patients (47%). In the univariate logistic regression analyses, male sex, duration of diabetes, body mass index, glycated hemoglobin, C-peptide, LDL cholesterol, smoking, and albumin excretion rate were found to be associated with the presence of DR. However, the multiple logistic regression analysis showed that only duration of diabetes (odds ratio (OR) = 1.15, 95% CI = 1.09-1.22; P < 0.001), glycated hemoglobin (OR = 1.21, 95% CI = 1.01-1.46; P = 0.047) and albumin excretion rate > 100 microg/min (OR = 12.72, 95% CI = 3.89-41.56; P < 0.001) were independently associated with DR. Although DR was found to be frequent among Brazilian type 2 diabetic patients, its prevalence was within the range observed in other Caucasian populations. Our findings emphasize the need for good glycemic control in order to prevent or delay the onset of DR, since the most well-known risk factors for the development of this complication in type 2 diabetes mellitus, such as duration of diabetes, glycated hemoglobin and albumin excretion rate were independently related to DR.

Prevalence of retinopathy in Caucasian type 2 diabetic patients from the South of Brazil and relationship with clinical and metabolic factors

Diabetic retinopathy (DR) is a sight-threatening chronic complication of diabetes mellitus and is the leading cause of acquired blindness in adults. In this cross-sectional study, we investigated the prevalence of and the factors associated with DR in an analysis of 210 consecutive and unrelated Brazilian Caucasians with type 2 diabetes mellitus. Retinopathy was evaluated by ophthalmoscopy and/or biomicroscopy through dilated pupils. The relationship between clinical and metabolic variables and the presence of DR was assessed by logistic regression analysis. DR was detected in 99 of the 210 patients (47 percent). In the univariate logistic regression analyses, male sex, duration of diabetes, body mass index, glycated hemoglobin, C-peptide, LDL cholesterol, smoking, and albumin excretion rate were found to be associated with the presence of DR. However, the multiple logistic regression analysis showed that only duration of diabetes (odds ratio (OR) = 1.15, 95 percent CI = 1.09-1.22; P < 0.001), glycated hemoglobin (OR = 1.21, 95 percent CI = 1.01-1.46; P = 0.047) and albumin excretion rate >100 µg/min (OR = 12.72, 95 percent CI = 3.89-41.56; P < 0.001) were independently associated with DR. Although DR was found to be frequent among Brazilian type 2 diabetic patients, its prevalence was within the range observed in other Caucasian populations. Our findings emphasize the need for good glycemic control in order to prevent or delay the onset of DR, since the most well-known risk factors for the development of this complication in type 2 diabetes mellitus, such as duration of diabetes, glycated hemoglobin and albumin excretion rate were independently related to DR.

The G1888A variant in the mitochondrial 16S rRNA gene may be associated with Type 2 diabetes in Caucasian-Brazilian patients from southern Brazil.

AIM: To compare the frequencies of the G1888A variant in the mitochondrial 16S rRNA gene between patients with Type 2 diabetes and non-diabetic control subjects from southern Brazil. METHODS: We analysed 520 Type 2 diabetic patients (389 Caucasian- and 131 African-Brazilians) and 530 control subjects (400 Caucasian- and 130 African-Brazilians). DNA samples were amplified by polymerase chain reaction and digested with the RsaI enzyme. Variant frequency in patients and control subjects was compared using chi2 test, Fisher's exact test or odds ratio test. We also investigated the frequency of the G1888A variant in a subgroup of the patients with a maternal history of Type 2 diabetes plus two or more features of maternally inherited diabetes and deafness. RESULTS: The 1888A allele does not seem to be associated with Type 2 diabetes in African-Brazilians (frequency of 3.8% in patients and 0.8% in control subjects; PFisher=0.213). However, in Caucasian-Brazilians, the 1888A allele was significantly associated with diabetes (12.3% in patients vs. 3.5% in control subjects; OR=3.881; 95% CI 2.106-7.164; P<0.001) and also with higher levels of insulin resistance. The majority of the patients carrying the 1888A allele did not have clinical features of maternally inherited diabetes and deafness. CONCLUSION: The present study indicates the association of the mitochondrial G1888A variant with Type 2 diabetes and insulin resistance in Caucasian-Brazilian patients from southern Brazil. However, further studies are required to confirm its effects on mitochondrial function and the role of these effects on the pathogenesis of Type 2 diabetes.

Association studies between -1185A/G von Willebrand factor gene polymorphism and coronary artery disease.

High levels of von Willebrand factor (vWF) have been associated with cardiovascular disease. The A allele of the -1185A/G polymorphism in the 5'-regulatory region of the vWF gene was associated with the highest plasma vWF levels in a normal population. To examine the association between -1185A/G polymorphism and coronary artery disease (CAD), 173 Brazilian Caucasian subjects submitted to coronary angiography were studied. Of these, 57 (33%) had normal coronary arteries (control group) and 116 (67%) had CAD (patient group). Plasma vWF levels were higher in patients (145 U/dl) than in controls (130 U/dl), but the differences were significant only for O blood group subjects. Polymerase chain reaction amplification of the 864-bp vWF promoter region followed by AccII restriction digestion was used to identify the -1185A/G genotypes. The -1185A allele frequency was 43.1% in patients and 44.7% in controls. Allele and genotype frequencies were not significantly different between patients and controls. No association was observed between the -1185A/G genotypes and plasma vWF levels in patients or controls. These results suggest that -1185A/G polymorphism is not an independent risk factor for CAD.

Association studies between -1185A/G von Willebrand factor gene polymorphism and coronary artery disease

High levels of von Willebrand factor (vWF) have been associated with cardiovascular disease. The A allele of the -1185A/G polymorphism in the 5'-regulatory region of the vWF gene was associated with the highest plasma vWF levels in a normal population. To examine the association between -1185A/G polymorphism and coronary artery disease (CAD), 173 Brazilian Caucasian subjects submitted to coronary angiography were studied. Of these, 57 (33 percent) had normal coronary arteries (control group) and 116 (67 percent) had CAD (patient group). Plasma vWF levels were higher in patients (145 U/dl) than in controls (130 U/dl), but the differences were significant only for O blood group subjects. Polymerase chain reaction amplification of the 864-bp vWF promoter region followed by AccII restriction digestion was used to identify the -1185A/G genotypes. The -1185A allele frequency was 43.1 percent in patients and 44.7 percent in controls. Allele and genotype frequencies were not significantly different between patients and controls. No association was observed between the -1185A/G genotypes and plasma vWF levels in patients or controls. These results suggest that -1185A/G polymorphism is not an independent risk factor for CAD

von Willebrand factor antigen levels in plasma of patients with malignant breast disease

von Willebrand factor (vWF) is a protein that mediates platelet adherence to the subendothelium during primary hemostasis. High plasma vWF concentrations have been reported in patients with various types of cancer, such as head and neck, laryngeal and prostatic cancer, probably representing an acute phase reactant. In the present study we determined the plasma levels of vWF antigen (vWF:Ag) by quantitative immunoelectrophoresis in 128 female patients with breast cancer as well as in 47 women with benign breast disease and in 27 healthy female controls. The levels of vWF:Ag were 170.7 + or - 78 U/dl in patients with cancer, 148.4 + or - 59 U/dl in patients with benign disease and 130.6 + or - 45 U/dl in controls (P<0.005). We also detected a significant increase in the levels of vWF:Ag (P<0.0001) in patients with advanced stages of the disease (stage IV = 263.3 + or - 113 U/dl, stage IIIB = 194.0 + or - 44 U/dl) as compared to those with earlier stages of the disease (stage I = 155.3 + or - 65 U/dl, stage IIA = 146.9 + or - 75 U/dl). In conclusion, vWF levels were increased in plasma of patients with malignant breast disease, and these levels correlated with tumor progression

von Willebrand factor antigen levels in plasma of patients with malignant breast disease.

von Willebrand factor (vWF) is a protein that mediates platelet adherence to the subendothelium during primary hemostasis. High plasma vWF concentrations have been reported in patients with various types of cancer, such as head and neck, laryngeal and prostatic cancer, probably representing an acute phase reactant. In the present study we determined the plasma levels of vWF antigen (vWF:Ag) by quantitative immunoelectrophoresis in 128 female patients with breast cancer as well as in 47 women with benign breast disease and in 27 healthy female controls. The levels of vWF:Ag were 170.7 +/- 78 U/dl in patients with cancer, 148.4 +/- 59 U/dl in patients with benign disease and 130.6 +/- 45 U/dl in controls (P<0.005). We also detected a significant increase in the levels of vWF:Ag (P<0.0001) in patients with advanced stages of the disease (stage IV = 263.3 +/- 113 U/dl, stage IIIB = 194.0 +/- 44 U/dl) as compared to those with earlier stages of the disease (stage I = 155.3 +/- 65 U/dl, stage IIA = 146.9 +/- 75 U/dl). In conclusion, vWF levels were increased in plasma of patients with malignant breast disease, and these levels correlated with tumor progression.

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.

Preparation of a heterologous antiserum for the determination of von Willebrand factor in human plasma.

A simple method for the preparation of rabbit antiserum against human von Willebrand factor (vWF) from commercial lyophilized factor VIII concentrate is described. vWF antigen (vWFAg)-like protein was obtained by gel filtration of the concentrate on Sepharose 4B. A combination of measurements of protein content by absorbance at 280 nm, and of vWFAg by electroimmunoassay using a commercial antibody, provided the data needed to select the Sepharose-filtered fractions with the highest concentrations of vWFAg-like protein. The immunization scheme used induced high antibody titers from the 45th to the 126th day after the first immunization. The resulting antiserum showed a performance similar to that of a commercial preparation in terms of vWFAg determination by electroimmunoassay and two-dimensional crossed-immunoelectrophoresis.

Preparation of a heterologus antiserum for the determination of von Willebrand factor in human plasma

A simple method for the preparation of rabbit antiserum against human von Willebrand factor (vWF) from commercial lyophilized factor VIII concentrate is described. vWF antigen (vWFAg)-like protein was obtained by gel filtration of the concentrate on Sepharose 4BTM. A combination of measurements of protein content by absorbance at 280 nm, and of vWFAg by electroimmunoassay using a commercial antibody, provided the data needed to select the Sepharosefiltered fractions with the highest concentrations of vWFAg-like protein. The immunization scheme used induced high antibody titers from the 45th to the 126th day after the first immunization. The resulting antiserum showed a performance similar to that of a commercial preparation in terms of vWFAg determination by electroimmunoassay and two-dimensional crossed-immunoelectrophoresis.