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The minisequencing technique offers accuracy and robustness to genotyping of polymorphic DNA variants, being an excellent option for the identification and analyses of prognostic/susceptibility markers in human diseases. Two multiplex minisequencing assays were designed and standardized to screen 23 candidate SNPs in cytokine, chemokine receptor and ligand genes previously associated with susceptibility to cancer and autoimmune disorders as well as to infectious diseases outcome. The SNPs were displayed in two separate panels (panel 1-IL2 rs2069762, TNFα rs1800629, rs361525; IL4 rs2243250; IL6 rs1800795; IL10 rs1800896, rs1800872; IL17A rs8193036, rs2275913 and panel 2-CCR3 rs309125, CCR4 rs6770096, rs2228428; CCR6 rs968334; CCR8 rs2853699; CXCR3 rs34334103, rs2280964;CXCR6 rs223435, rs2234358; CCL20 rs13034664, rs6749704; CCL22 rs4359426; CXCL10/IP-10 rs3921, rs56061981). A total of 305 DNA samples from healthy individuals were genotyped by minisequencing. To validate the minisequencing technique and to encompass the majority of the potential genotypes for all 23 SNPs, 20 of these samples were genotyped by Sanger sequencing. The results of both techniques were 100% in agreement. The technique of minisequencing showed high accuracy and robustness, avoiding the need for high quantities of DNA template samples. It was easily to be conducted in bulk samples derived from a highly admixed human population, being therefore an excellent option for immunogenetic studies.

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Background Systemic lupus erythematosus (SLE) is an autoimmune disease marked by the disruption of the immune homeostasis. Patients exhibit a wide range of clinical manifestations, and environmental and genetic factors are involved in SLE pathogenesis. Evidence suggests that abnormalities in the cellular and molecular events that coordinate apoptosis may favour the generation of autoantigens involved in autoimmunity. In this way, the apoptotic deregulation may be affected by polymorphic variants in apoptotic-related genes. Methods We analyzed FAS, FASL, BCL-2 and BAX polymorphisms in order to correlate to SLE susceptibility and clinical features. A total of 427 SLE patients from the Hospital de Clínicas de Porto Alegre and 543 controls from southern Brazil were evaluated. Results We observed higher frequencies of the FASL -844CC genotype and -844C allele, as well as of the FASL-844C/IVS2nt-124A haplotype in African-derived SLE patients when compared to controls ( P < 0.001). FASL -844C, which is related to high FasL expression, could contribute to increased apoptosis and to the breakdown of immunological tolerance, favouring autoantibody production and inflammation. On the other hand, the BAX -248GA genotype and the -248A allele , related to low protein expression, were observed as a protective factor against SLE in this same population. The rate of apoptosis and cell death was evaluated in peripheral lymphocytes, and SLE patients presented a higher percentage of dead lymphocytes (CD3+Annexin V+ 7-AAD+) compared to the control group. Conclusion Our data support a role for apoptosis in SLE susceptibility.

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The major pathological hallmark of the systemic sclerosis (SSc) is skin and internal organ fibrosis, which results from normal tissue architecture alterations and extracellular matrix (ECM) protein deposition. ECM components are degraded by matrix metalloproteinases (MMP). Promoter region polymorphisms in MMP genes may influence gene expression, resulting in an imbalance between ECM protein production and degradation. Here, we analyzed MMP1 -1607 1G/2G (rs1799750), MMP3 -1171 5A/6A (rs3025058), and MMP9 -1562 C/T (rs3918242) polymorphisms in relation to susceptibility to SSc and its clinical features. The patient group included 98 individuals with longstanding or recently diagnosed disease, meeting the American College of Rheumatology or LeRoy and Medsger criteria for SSc; the control group included 100 healthy blood donors. All participants were of European descent. Genotyping was performed by polymerase chain reaction followed by restriction digestion. Genotype and allele frequencies of MMP polymorphisms were similar between the two groups. In secondary analyses, significantly higher frequency of 1G/2G genotype from MMP1 polymorphism was observed for patients testing positive for antinuclear autoantibodies (P = 0.007), while 1G/1G genotype was associated with interstitial lung disease development (P = 0.018). The 6A/6A genotype from MMP3 polymorphism was absent in patients with calcinosis (P = 0.011), while the MMP3 5A/5A genotype correlated with the presence of anti-topoisomerase I antibodies (P = 0.009) and reduced diffusing capacity for carbon monoxide (P = 0.024). These results suggest that MMP polymorphisms are not associated with SSc susceptibility, although MMP1 and MMP3 variants are associated with specific SSc clinical and laboratory features.

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Human leukocyte antigen G (HLA-G) is an immunomodulatory molecule with important roles both physiologically as well as an escape mechanism of cancer cells. In this study, we evaluated the impact of eight polymorphisms at the 3' untranslated region (3'UTR) of the HLA-G gene in the development of prostate cancer (PCa) and benign prostatic hyperplasia (BPH). A total of 468 DNA samples of Brazilian men predominantly Euro-descendant with PCa (N = 187), BPH (N = 152) and healthy control individuals (N = 129) were evaluated. The HLA-G 3'UTR region was amplified by polymerase chain reaction (PCR), sequenced and genotyped to identify the 14 bp insertion/deletion (rs371194629), +3003T/C (rs1707), +3010C/G (rs1710), +3027A/C (rs17179101), +3035C/T (rs17179108), +3142G/C (rs1063320), +3187A/G (rs9380142) and +3196C/G (rs1610696) polymorphisms. Regression logistic and chi-square tests were performed to verify the influence of single nucleotide polymorphisms (SNPs) in PCa and/or BPH susceptibility, as well as in PCa progression (clinicopathological status). Our data showed the UTR-4 haplotype as a risk factor to PCa in comparison with control [odds ratio (OR) 2.35, 95% confidence interval (CI) 1.39-3.96, P adjusted = 0.003) and BPH groups (OR 1.82, 95% CI 1.15-2.86, P adjusted = 0.030). Further, the 'non-14bp Ins_ + 3142G_+3187A' haplotype (OR 1.56, 95% CI 1.10-2.20, P adjusted = 0.036), the +3003CT genotype (OR 4.44, 95% CI 1.33-4.50, P adjusted = 0.032) and the +3003C allele (OR 2.33, 95% CI 1.38-3.92, P adjusted = 0.016) also conferred susceptibility to PCa. Our data suggest an important influence of HLA-G 3'UTR polymorphisms in PCa susceptibility and support the use of the +3003 variant as a tag SNP for PCa risk.

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In this study, we sought to investigate the genetic influence of two HLA-G 3'-untranslated region (3'-UTR) polymorphisms - 14 bp (rs66554220) and +3142C>G (rs1063320) and their compounding haplotypes in susceptibility to rheumatoid arthritis (RA) in a two-region Brazilian study comprising of 539 patients and 489 controls. All subjects were polymerase chain reaction (PCR) genotyped for the referred polymorphisms and logistic regression models controlling for sex, city and age were performed. Homozygozity for the +3142G allele was associated with an increased risk of RA [odds ratio (OR) = 1.45, 95% confidence interval (CI) = 1.075-1.959, P(Bonf) = 0.030], whereas no association was observed for the 14 bp polymorphism. Haplotype comparisons between patients and controls showed a decreased frequency of the delC haplotype in patients (OR = 0.70, 95% CI = 0.521-0.946, P(Bonf) = 0.040), which remained significant in the rheumatoid factor (RF)-positive group (OR = 0.66, 95% CI = 0.482-0.900, P(Bonf) = 0.018), but not in the RF-negative group. These results corroborate the hypothesis of an involvement of HLA-G in the susceptibility of RA. The +3142G allele is associated with haplotype lineages that share high identity and are regarded as low producers. The presence of the G allele in homozygosis could be responsible for a low HLA-G expression profile that could favor the triggering of RA.

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The aim of this study was to analyze the allelic and genotypic frequencies of the CCR5delta32 polymorphism in systemic lupus erythematosus (SLE) patients and to investigate a possible association of this allele with SLE susceptibility and clinical outcome. A total of 367 SLE patients and 435 healthy controls were genotyped for the CCR5delta32 polymorphism. We observed that, in European-derived individuals, the frequency of the CCR5delta32 allele was smaller in patients than in controls (2.7% vs. 7.5%, OR 0.34, 95% CI 0.17-0.65, p Bonf=0.002), suggesting that this allele could be considered a protective factor for the disease. Regarding clinical manifestations, we observed that CCR5delta32 female African-derived carrier patients presented a higher predisposition to class IV nephritis when compared with absent nephritis/other class group (13.8% vs. 3.8%, OR 37.1, 95% CI 2.8-1854.7, p Bonf=0.030). A multivariate analysis including all female patients and controlling for the presence or absence of anti-dsDNA antibodies, ethnicity and age at diagnosis showed an increased relative risk of 3.9 times for patients carrying the CCR5delta32 allele to develop class IV nephritis as compared with noncarriers. Our data suggest that the CCR5delta32 allele is a protective factor for the disease in European-derived patients and a susceptibility factor to class IV nephritis in African-derived female patients.

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Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrotic, immunological and vascular abnormalities. Nuclear factor-kB (NFKB), as a key transcription factor involved in the regulation of immune responses, appears to be a good candidate for studies on the pathogenesis of autoimmune diseases, as well as the interleukin-10 (IL-10) polymorphism, which other studies have suggested an association with SSc. Our objective was to study the association of NFKB and IL-10 gene polymorphisms with SSc. One hundred and fifty-one SSc patients and 147 healthy bone marrow donors were enrolled in a case-control study. Blood was collected for DNA extraction; typing of IL-10 genes was made by polymerase chain reaction with sequence-specific primers (PCR-SSP), and NFKB gene typing was made by restriction fragment length polymorphism (RFLP). Patients underwent clinical evaluation, serology, Doppler echocardiography and chest high-resolution computed tomography. The frequency of IL-10 (-1082) GG genotype was found to be significantly higher in SSc patients (36.4%) as compared to healthy controls (22.4%) (P = 0.012). The frequency of heterozygous genotype GA was significantly lower (P = 0.004) in patients (38.4%) in comparison with control subjects (55.8%). A predominance of the high-producing IL-10 phenotype (GCC(+) /GCC(+) ) was observed in SSc patients compared with healthy controls (37.7% versus 24.5%, respectively; OR: 1.87, 95% CI: 1.10-3.19, P = 0.019). No significant difference was found in the allelic and genotype distribution of the NFKB promoter polymorphism between patients and controls. No statistically significant associations were found between IL-10 or NFKB polymorphisms clinical and laboratory features of SSc. Our results confirmed the association of the high-producing phenotype (GCC(+) /GCC(+) ) with increased risk for SSc, but found no correlation with NFKB polymorphisms.

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Vitamin D deficiency has been described in systemic lupus erythematosus (SLE). BsmI VDR (vitamin D receptor) gene polymorphism was associated with SLE in Asian patients. Studies in Brazilian populations have not been realized. A case-control study with 195 SLE patients and 201 healthy controls was conducted to investigate the influence of BsmI and FokI VDR gene polymorphisms on susceptibility to SLE. In addition, 25-hydroxyvitamin D [25(OH)D] was measured in SLE patients to evaluate possible associations with VDR polymorphic variants and clinical and laboratory expressions of disease. Genotyping was performed by RFLP-PCR. The measurement of 25(OH)D was performed by chemiluminescence. There was no statistically significant difference in genotype and allelic frequencies of BsmI and FokI polymorphisms between European-derived cases and controls. The mean serum levels of 25(OH)D were 25.51 ± 11.43 ng/ml in SLE patients. According to genotype distribution, 25(OH)D concentrations were significantly higher in patients carrying the FokI f/f genotype compared with patients carrying the F/F genotype (31.6 ± 14.1 ng/ml versus 23.0 ± 9.2 ng/ml, p = 0.004), reinforcing its role in the functional activity of VDR. This feature may be considered in future clinical and experimental studies involving vitamin D measurements. Therefore, genetic-specific definitions of ideal levels of vitamin D in SLE need to be established in future studies.

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