High frequencies of plexiform neurofibromas, mental retardation, learning difficulties, and scoliosis in Brazilian patients with neurofibromatosis type 1

A clinical study of Brazilian patients with neurofibromatosis type 1 (NF1) was performed in a multidisciplinary Neurofibromatosis Program called CEPAN (Center of Research and Service in Neurofibromatosis). Among 55 patients (60% females, 40% males) who met the NIH criteria for the diagnosis of NF1, 98% had more than six café-au-lait patches, 94.5% had axillary freckling, 45% had inguinal freckling, and 87.5% had Lisch nodules. Cutaneous neurofibromas were observed in 96%, and 40% presented plexiform neurofibromas. A positive family history of NF1 was found in 60%, and mental retardation occurred in 35%. Some degree of scoliosis was noted in 49%, 51% had macrocephaly, 40% had short stature, 76% had learning difficulties, and 2% had optic gliomas. Unexpectedly high frequencies of plexiform neurofibromas, mental retardation, learning difficulties, and scoliosis were observed, probably reflecting the detailed clinical analysis methods adopted by the Neurofibromatosis Program. These same patients were screened for mutations in the GAP-related domain/GRD (exons 20-27a) by single-strand conformation polymorphism. Four different mutations (Q1189X, 3525-3526delAA, E1356G, c.4111-1G>A) and four polymorphisms (c.3315-27G>A, V1146I, V1317A, c.4514+11C>G) were identified. These data were recently published.

Frequency of CCR5delta32 in Brazilian populations

A sample of 103 randomly chosen healthy individuals from Alegrete, RS, Brazil, was tested for the CCR5delta32 allele, which is known to influence susceptibility to HIV-1 infection. The CCR5delta32 allele was identified by PCR amplification using specific primers flanking the region of deletion, followed by electrophoresis on a 3% agarose gel. The data obtained were compared to those reported for other populations and interpreted in terms of Brazilian history. The individuals studied came from a highly admixed population. Most of them were identified as white (N = 59), while blacks and browns (mulattoes) were N = 13 and N = 31, respectively. The observed frequencies, considering the white, black and brown samples (6.8, 3.8, and 6.4%, respectively), suggest an important European parental contribution, even in populations identified as black and brown. However, in Brazil as a whole, this allele shows gradients indicating a relatively good correlation with the classification based on skin color and other physical traits, used here to define major Brazilian population groups.

Association of the 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene with unstable angina

The 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene consists of the substitution of a guanine base by a thymine at the 894th nucleotide of the gene. An association of this polymorphism with acute coronary syndromes has been described, only when in combination with other polymorphisms of this gene. The aim of the present study was to search for an association between this polymorphism and unstable angina in a southern Brazilian population. In a case-control study, 156 patients (group 1 (N = 83): unstable angina, group 2 (N = 73): stable angina) were genotyped by PCR and digestion of the product. Univariate analysis demonstrated that the minimal luminal diameter and the degree of stenosis of the culprit lesion differed between groups (P = 0.006 and 0.005, respectively). In addition, the frequencies of the T allele and of the T allele carriers (combined TT and TG genotypes) were significantly higher in the group with unstable angina (41.6 vs 28.8%; P = 0.025, Pearson chi-square test, and 73.5 vs 45.2%; P = 0.001, Pearson chi-square test, respectively). Multivariate logistic regression showed that the frequency of the T allele carriers was the only variable with a predictive value for unstable angina, when controlled for the other variables (6.1 (95% CI = 2.55-14.43); P < 0.001). Thus, in a homogenous group of patients, the endothelial constitutive nitric oxide synthase 894G>T polymorphism was associated with unstable angina. We suggest that this polymorphism may be a genetic risk factor for unstable angina.

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.

Apolipoprotein E polymorphism in Brazilian dyslipidemic individuals: Ouro Preto study

The influence of apolipoprotein E alleles and genotypes on plasma lipid levels was determined in 185 individuals of mixed ethnicity living in Ouro Preto, Brazil. DNA was obtained from blood samples and the genotypes were determined by an RFLP-PCR procedure. The *3 allele was the most frequent (72%), followed by *4 (20%) and *2 (8%); *4 frequency was higher and *2 frequency was lower in the dyslipidemic group than in the normal control group. The *2 carriers presented lower LDL and total cholesterol levels compared to the *3 and *4 carriers. All six expected genotypes were observed in the individuals genotyped: E2/2 (2.1%), E4/4 (2.7%), E2/4 (3.7%), E2/3 (8.0%), E3/3 (53.3%), E3/4 (29.9%); no difference in genotype frequencies was found between the normal and dyslipidemic groups. Compared with *2, the presence of *3 increases more than two times the risk for dyslipidemia (OR = 2.31; P = 0.025; 95% CI = 1.06-5.06) and the presence of *4 increases it three times (OR = 3.31; P = 0.006; 95% CI = 1.36-8.04). The only significant effect of genotype was an increased risk for dyslipidemia in the *4 genotype carriers (E3/4 + E4/4) compared with the *2 genotype carriers (E2/2 + E2/3) with OR = 3.69 (95% CI = 1.25-10.88). The present study indicates that in the Ouro Preto admixed population the presence of APOE *2 can confer a protective effect, whereas the presence of APOE *4 implies an enhanced risk for dyslipidemia.

ABCB1 C1236T, G2677T/A and C3435T polymorphisms in systemic lupus erythematosus patients

P-glycoprotein (Pgp), the ABCB1 gene product, acts as an efflux pump that transports a large variety of substrates and is a mechanism of cell protection against xenobiotics. An increasing number of studies have shown that some ABCB1 polymorphisms may affect Pgp expression and activity, as well as affecting the development and susceptibility to diseases and pharmacological response. High activity of Pgp has been detected in systemic lupus erythematosus (SLE) patients. The C1236T, G2677T/A, and C3435T are the most commonly studied single nucleotide polymorphisms in the ABCB1 gene. Therefore, their frequencies were determined in Brazilian individuals with European ancestry (N = 143) and in SLE patients (N = 137). Genotyping was performed by PCR-RFLP analysis using specific primers followed by incubation with the appropriate restriction enzymes. The resulting DNA fragments were visualized on agarose or polyacrylamide gels. No statistically significant differences were observed in allelic and genotypic frequencies between SLE and healthy subjects (Fisher exact test). Nevertheless, the 2677A allelic frequency was lower in SLE patients with malar rash (0.007) compared with patients without this feature (0.04; P = 0.0054), while the frequency of this variant was higher in SLE patients with pleuritis (0.07) compared with patients without this feature (0.01; P = 0.0156). We suggest that although the ABCB1 polymorphisms do not directly interfere in SLE susceptibility, their evaluation, especially the 2677A allele, in other immunological processes may be interesting since they can interfere in clinical features of this disease.

Distribution of the human leukocyte antigen class II alleles in Brazilian patients with chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection is a global medical problem. The current standard of treatment consists of the combination of peginterferon plus ribavirin. This regimen eradicates HCV in 55% of cases. The immune response to HCV is an important determinant of disease evolution and can be influenced by various host factors. HLA class II may play an important role in immune response against HCV. The objective of the present study was to determine the distribution of HLA class II (DRB1 and DQB1) alleles, their association with chronic HCV infection and their response to interferon therapy. One hundred and two unrelated white Brazilian patients with chronic HCV infection, 52 responders (45 males and 7 females) and 50 non-responders (43 males and 7 females) to antiviral treatment, were included in the study. Healthy Brazilian bone marrow donors of Caucasian origin from the same geographic area constituted the control group (HLA-DRB1, N = 99 and HLA-DQB1, N = 222 individuals). HLA class II genotyping was performed using a low-resolution DRB1, DQB1 sequence-specific primer amplification. There were higher frequencies of HLA-DRB1*13 (26.5 vs 14.1%) and HLA-DQB1*02 (52.9 vs 38.7%) in patients compared with controls; however, these were not significantly different after P correction (Pc = 0.39 and Pc = 0.082, respectively). There was no significant difference between the phenotypic frequencies of HLA-DRB1 (17.3 vs 14.0%) and HLA-DQB1 alleles in responder and non-responder HCV patients. The HLA-DRB1*07 allele was significantly more common in HCV patients (33.3 vs 12.1%) than in controls (Pc = 0.0039), suggesting that the HLA-DRB1*07 allele is associated with chronic HCV infection.

The effect of 677C>T and 1298A>C MTHFR polymorphisms on sulfasalazine treatment outcome in rheumatoid arthritis

Despite the availability of several new agents for the treatment of rheumatoid arthritis (RA), sulfasalazine remains the mainstay because of both cost and experience with its use. Methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism and several polymorphisms have been described in the MTHFR gene. Of these, the 677C>T and 1298A>C polymorphisms have been associated with altered enzyme activity. To examine the association between 677C>T and 1298A>C MTHFR polymorphisms and sulfasalazine efficacy for the treatment of RA, a total of 117 RA patients treated with sulfasalazine (1 g daily; duration of treatment 17 ± 5 months) were analyzed. The 677C>T and 1298 A>C polymorphisms were detected using a PCR-RFLP method. RA was diagnosed according to the criteria of the American College of Rheumatology (ACR). The remission of RA symptoms was evaluated according to the ACR 20% response criteria. Allele and genotype frequencies were compared by the two-sided Fisher exact test. The frequency of remission was 47.2% and 44.6% in carriers of 677T and 1298C alleles, compared to 40.7% and 42.0% in carriers of 677C and 1298A alleles, respectively. These differences were statistically non-significant. When the multivariate analysis was additionally adjusted for patients’ age, gender and RA duration, the association of the MTHFR 677T allele with increased frequency of remission was statistically significant. Although RA remission rate in carriers of the MTHFR 677T and 1298C alleles was more frequently observed, it does not seem that 677C>T and 1298A>C MTHFR polymorphisms have a major influence on treatment outcome in RA patients treated with sulfasalazine.

Do Caucasian and Asian clocks tick differently?

The Period 3 and Clock genes are important components of the mammalian molecular circadian system. Studies have shown association between polymorphisms in these clock genes and circadian phenotypes in different populations. Nevertheless, differences in the pattern of allele frequency and genotyping distribution are systematically observed in studies with different ethnic groups. To investigate and compare the pattern of distribution in a sample of Asian and Caucasian populations living in Brazil, we evaluated two well-studied polymorphisms in the clock genes: a variable number of tandem repeats (VNTR) in PER3 and a single nucleotide polymorphism (SNP) in CLOCK. The aim of this investigation was to search for clues about human evolutionary processes related to circadian rhythms. We selected 109 Asian and 135 Caucasian descendants. The frequencies of the shorter allele (4 repeats) in the PER3 gene and the T allele in the CLOCK gene among Asians (0.86 and 0.84, respectively) were significantly higher than among Caucasians (0.69 and 0.71, respectively). Our results directly confirmed the different distribution of these polymorphisms between the Asian and Caucasian ethnic groups. Given the genetic differences found between groups, two points became evident: first, ethnic variations may have implications for the interpretation of results in circadian rhythm association studies, and second, the question may be raised about which evolutionary conditions shaped these genetic clock variations.

Genetic variability of vascular endothelial growth factor and prognosis of head and neck cancer in a Brazilian population

Vascular endothelial growth factor (VEGF) is one of the most potent endothelial cell mitogens and plays a critical role in angiogenesis. Polymorphisms in this gene have been evaluated in patients with several types of cancer. The objectives of this study were to determine if there was an association of the -1154G/A polymorphism of the VEGF gene with head and neck cancer and the interaction of this polymorphism with lifestyle and demographic factors. Additionally, the distribution of the VEGF genotype was investigated with respect to the clinicopathological features of head and neck cancer patients. The study included 100 patients with histopathological diagnosis of head and neck squamous cell carcinoma. Patients with treated tumors were excluded. A total of 176 individuals 40 years or older were included in the control group and individuals with a family history of neoplasias were excluded. Analysis was performed after extraction of genomic DNA using the real-time PCR technique. No statistically significant differences between allelic and genotype frequencies of -1154G/A VEGF polymorphism were identified between healthy individuals and patients. The real-time PCR analyses showed a G allele frequency of 0.72 and 0.74 for patients and the control group, respectively. The A allele showed a frequency of 0.28 for head and neck cancer patients and 0.26 for the control group. However, analysis of the clinicopathological features showed a decreased frequency of the A allele polymorphism in patients with advanced (T3 and T4) tumors (OR = 0.36; 95%CI = 0.14-0.93; P = 0.0345). The -1154A allele of the VEGF gene may decrease the risk of tumor growth and be a possible biomarker for head and neck cancer. This polymorphism is associated with increased VEGF production and may have a prognostic importance.

Genetic polymorphism of alcohol-metabolizing enzyme and alcohol dependence in Polish men

Alcohol dependence poses a serious medical and sociological problem. It is influenced by multiple environmental and genetic factors, which may determine differences in alcohol metabolism. Genetic polymorphism of the enzymes involved in alcohol metabolism is highly ethnically and race dependent. The purpose of this study was to investigate the differences, if present, in the allele and genotype frequency of alcohol dehydrogenase 1B (ADH1B), ADH1C and the microsomal ethanol-oxidizing system (MEOS/CYP2E1) between alcohol-dependent individuals and controls and also to determine if these genotypes cause a difference in the age at which the patients become alcohol dependent. The allele and genotype frequencies of ADH1B, ADH1C, and CYP2E1 were determined in 204 alcohol dependent men and 172 healthy volunteers who do not drink alcohol (control group). Genotyping was performed by PCR-RFLP methods on white cell DNA. ADH1B*1 (99.3%) and ADH1C*1 (62.5%) alleles and ADH1B*1/*1 (N = 201) and ADH1C*1/*1 (N = 85) genotypes were statistically more frequent among alcohol-dependent subjects than among controls (99.3 and 62.5%, N = 201 and 85 vs 94.5 and 40.7%, N = 153 and 32, respectively). Differences in the CYP2E1 allele and genotype distribution between groups were not significant. The persons with ADH1C*1/*1 and CYP2E1*c1/*c2 genotypes became alcohol dependent at a considerably younger age than the subjects with ADH1C*1/*2, ADH1C*2/*2 and CYP2E1*c1/*c1 genotypes (28.08, 25.67 years vs 36.0, 45.05, 34.45 years, respectively). In the Polish men examined, ADH1C*1 and ADH1B*1 alleles and ADH1C*1/*1 and ADH1B*1/*1 genotypes favor alcohol dependence. The ADH1B*2 allele may protect from alcohol dependence. However, subjects with ADH1C*1/*1 and CYP2E1*c1/*c2 genotypes become alcohol dependent at a considerably younger age than the subjects with ADH1C*1/*2, ADH1C*2/*2 and CYP2E1*c1/*c1 genotypes.

Prevalence of the STK15 F31I polymorphism and its relationship with mammographic density

Several studies have identified the single nucleotide polymorphism STK15 F31I as a low-penetrance risk allele for breast cancer, but its prevalence and risk association in the Brazilian population have not been determined. The goal of this study was to identify the frequency of this polymorphism in the Brazilian setting. Considering the high degree of admixture of our population, it is of fundamental importance to validate the results already reported in the literature and also to verify the relationship between this variant and breast cancer risk. A total of 750 women without breast cancer were genotyped using the TaqMan PCR assay for STK15 F31I polymorphism. Clinical information was obtained from review of the medical records and mammographic density from the images obtained using the BI-RADS System. The estimated risk of developing cancer was calculated according to the Gail model. The genotypic frequencies observed in this study were 4.5, 38.7, and 56.6%, respectively, for the STK15 F31I AA, AT and TT genotypes. The AT and AA genotypes were encountered significantly more often in premenopausal women with moderately dense, dense and heterogeneously dense breast tissue (P = 0.023). In addition, the presence of the TT genotype was significantly associated with age at menarche ≥12 years (P = 0.023). High mammographic density, associated with increased breast cancer risk, was encountered more frequently in premenopausal women with the risk genotypes STK15 F31I AA and AT. The genotypic frequencies observed in our Brazilian sample were similar to those described in other predominantly European populations.

A high-carbohydrate diet enhances the adverse effect of the S2 allele of APOC3 SstI polymorphism on the TG/HDL-C ratio only in young Chinese females

Both genetic background and diet have profound effects on plasma lipid profiles. We hypothesized that a high-carbohydrate (high-CHO) diet may affect the ratios of serum lipids and apolipoproteins (apo) differently in subjects with different genotypes of the SstI polymorphism in the apoCIII gene (APOC3). Fifty-six healthy university students (27 males and 29 females, 22.89 ± 1.80 years) were given a washout diet of 54% carbohydrate for 7 days, followed by a high-CHO diet of 70% carbohydrate for 6 days without total energy restriction. Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apoB100, apoAI, and the APOC3 SstI polymorphism were analyzed. The ratios of serum lipids and apoB100/apoAI were calculated. At baseline, the TG/HDL-C ratio was significantly higher in females, but not in males, with the S2 allele. The differences in the TG/HDL-C ratio between genotypes remained the same after the washout and the high-CHO diet in females. When compared with those before the high-CHO diet, the TC/HDL-C (male S2 carriers: 3.13 ± 1.00 vs 2.36 ± 0.65, P = 0.000; male subjects with the S1S1 genotype: 2.97 ± 0.74 vs 2.09 ± 0.55, P = 0.000; female S2 carriers: 2.68 ± 0.36 vs 2.24 ± 0.37, P = 0.004; female subjects with the S1S1 genotype: 2.69 ± 0.41 vs 2.09 ± 0.31, P = 0.000) and LDL-C/HDL-C (male S2 carriers: 1.44 ± 0.71 vs 1.06 ± 0.26, P = 0.012; male subjects with the S1S1 genotype: 1.35 ± 0.61 vs 1.01 ± 0.29, P = 0.005; female S2 carriers: 1.18 ± 0.33 vs 1.00 ± 0.18, P = 0.049; female subjects with the S1S1 genotype: 1.18 ± 0.35 vs 1.04 ± 0.19, P = 0.026) ratios were significantly decreased after the high-CHO diet regardless of gender and of genotype of the APOC3 SstI polymorphism. However, in female S2 carriers, the TG/HDL-C (1.38 ± 0.46 vs 1.63 ± 0.70, P = 0.039) ratio was significantly increased after the high-CHO diet. In conclusion, the high-CHO diet has favorable effects on the TC/HDL-C and LDL-C/HDL-C ratios regardless of gender and of genotype of the APOC3 SstI polymorphism. Somehow, it enhanced the adverse effect of the S2 allele on the TG/HDL-C ratio only in females.

Protective effect of the APOE-e3 allele in Alzheimer’s disease

Although several alleles of susceptibility to Alzheimer’s disease (AD) have been studied in the last decades, few polymorphisms have been considered as risk factors for the disease. Among them, the APOE-e4 allele appears to be the major genetic risk factor for the onset of the disease. However, it is important to confirm the potential susceptibility of these genetic variants in different populations in order to establish a genetic profile for the disease in specific communities. This study analyzed the APOE polymorphisms regarding susceptibility to AD in a sample of 264 individuals (primarily Caucasians; 82 cases and 182 controls) in the population from Vitória, ES, Brazil, by PCR restriction fragment length polymorphism (PCR-RFLP) methods. The patients were selected according to clinical criteria for probable AD. Whereas the e4 allele showed statistically significant positive association with susceptibility to AD (OR = 3.01, 95%CI = 1.96-4.61; P < 0.0001), the e2 allele did not. The results of the e4 allele confirm the role of this polymorphism as a risk factor for AD in the sample studied as observed in other populations. Although the e3 allele has been considered neutral in several studies, our results suggest that it acts as a protective factor against AD in the population studied (OR = 0.46, 95%CI = 0.30-0.67; P < 0.0001). This study may provide a new insight into the role of the APOE-e3 allele in the etiology of AD and might help to estabilish a profile of risk for AD in the population from Vitória, ES.

MSX1 gene and nonsyndromic oral clefts in a Southern Brazilian population

Nonsyndromic oral clefts (NSOC) are the most common craniofacial birth defects in humans. The etiology of NSOC is complex, involving both genetic and environmental factors. Several genes that play a role in cellular proliferation, differentiation, and apoptosis have been associated with clefting. For example, variations in the homeobox gene family member MSX1, including a CA repeat located within its single intron, may play a role in clefting. The aim of this study was to investigate the association between MSX1CA repeat polymorphism and NSOC in a Southern Brazilian population using a case-parent triad design. We studied 182 nuclear families with NSOC recruited from the Hospital de Clínicas de Porto Alegre in Southern Brazil. The polymorphic region was amplified by the polymerase chain reaction and analyzed by using an automated sequencer. Among the 182 families studied, four different alleles were observed, at frequencies of 0.057 (175 bp), 0.169 (173 bp), 0.096 (171 bp) and 0.67 (169 bp). A transmission disequilibrium test with a family-based association test (FBAT) software program was used for analysis. FBAT analysis showed overtransmission of the 169 bp allele in NSOC (P=0.0005). These results suggest that the CA repeat polymorphism of theMSX1 gene may play a role in risk of NSOC in populations from Southern Brazil.