Single-Nucleotide Polymorphism and Copy Number Variation of the Multidrug Resistance-1 Locus of Plasmodium vivax: Local and Global Patterns

Emerging resistance to chloroquine (CQ) poses a major challenge for Plasmodium vivax malaria control, and nucleotide substitutions and copy number variation in the P. vivax multidrug resistance 1 (pvmdr-1) locus, which encodes a digestive vacuole membrane transporter, may modulate this phenotype. We describe patterns of genetic variation in pvmdr-1 alleles from Acre and Amazonas in northwestern Brazil, and compare then with those reported in other malaria-endemic regions. The pvmdr-1 mutation Y976F, which is associated with CQ resistance in Southeast Asia and Oceania, remains rare in northwestern Brazil (1.8%) and its prevalence mirrors that of CO resistance worldwide. Gene amplification of pvmdr-1, which is associated with mefloquine resistance but increased susceptibility to CO, remains relatively rare in northwestern Brazil (0.9%) and globally (< 4%), but became common (> 10%) in Tak Province, Thailand, possibly because of drug-mediated selection. The global database we have assembled provides a baseline for further studies of genetic variation in pvmdr-1 and drug resistance in P. vivax malaria.

Frequency of LCT -13910C>T single nucleotide polymorphism associated with adult-type hypolactasia/lactase persistence among Brazilians of different ethnic groups

Abstract Background Adult-type hypolactasia, the physiological decline of lactase some time after weaning, was previously associated with the LCT -13910C>T polymorphism worldwide except in Africa. Lactase non-persistence is the most common phenotype in humans, except in northwestern Europe with its long history of pastoralism and milking. We had previously shown association of LCT -13910C>T polymorphism with adult-type hypolactasia in Brazilians; thus, we assessed its frequency among different Brazilian ethnic groups. Methods We investigated the ethnicity-related frequency of this polymorphism in 567 Brazilians [mean age, 42.1 ± 16.8 years; 157 (27.7%) men]; 399 (70.4%) White, 50 (8.8%) Black, 65 (11.5%) Brown, and 53 (9.3%) Japanese-Brazilian. DNA was extracted from leukocytes; LCT -13910C>T polymorphism was analyzed by PCR-restriction fragment length polymorphism. Results Prevalence of the CC genotype associated with hypolactasia was similar (57%) among White and Brown groups; however, prevalence was higher among Blacks (80%) and those of Japanese descent (100%). Only 2 (4%) Blacks had TT genotype, and 8 (16%) had the CT genotype. Assuming an association between CC genotype and hypolactasia, and CT and TT genotypes with lactase persistence, 356 (62.8%) individuals had hypolactasia and 211 (37.2%) had lactase persistence. The White and Brown groups had the same hypolactasia prevalence (~57%); nevertheless, was 80% among Black individuals and 100% among Japanese-Brazilians (P < 0.01). Conclusion The lactase persistence allele, LCT -13910T, was found in about 43% of both White and Brown and 20% of the Black Brazilians, but was absent among all Japanese Brazilians studied.

Toll-like receptor 1 N248S single-nucleotide polymorphism is associated with leprosy risk and regulates immune activation during mycobacterial infection

Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.

Functional single-nucleotide polymorphisms in the DEFB1 gene are associated with systemic lupus erythematosus in Southern Brazilians

Systemic lupus erythematosus (SLE) is an autoimmune disease that results in inflammation and tissue damage. The etiology of SLE remains unknown, but recent studies have shown that the innate immune system may have a role in SLE pathogenesis through the secretion of small cationic peptides named defensins. The aim of the study was to determine the possible involvement in SLE of three functional single nucleotide polymorphisms (SNPs) (c.-52G>A, c.-44C>G and c.-20G>A) in the 5'UTR region of DEFB1 gene, by analyzing them in a population of 139 SLE patients and 288 healthy controls. The c.-52G>A SNP showed significant differences in allele and genotype frequency distribution between SLE patients and controls (p = 0.01 and p = 0.02 respectively) indicating protection against SLE (A allele, OR = 0.68, AA genotype OR = 0.51). Significant differences were also observed for c.-44C>G SNP, the C/G genotype being associated with susceptibility to SLE (OR = 1.60, p = 0.04). Moreover, statistically significant differences between patients and controls were found for two DEFB1 haplotypes (GCA and GGG, p = 0.01 and p = 0.02 respectively). When considering DEFB1 SNPs and SLE clinical and laboratory manifestations, significant association was found with neuropsychiatric disorders, immunological alterations and anti-DNA antibodies. In conclusion, our results evidence a possible role for the c.-52G>A and c.-44C>G DEFB1 polymorphisms in SLE pathogenesis, that can be considered as possible risk factors for development of disease and disease-related clinical manifestations. Additional studies are needed, to corroborate these results as well as functional studies to understand the biological role of these SNPs in the pathogenesis of SLE. Lupus (2012) 21, 625-631.

Association of single nucleotide polymorphisms in the bovine leptin and leptin receptor genes with growth and ultrasound carcass traits in Nellore cattle

Given the important role of leptin in metabolism, we looked for a possible association of leptin and leptin receptor polymorphisms with carcass and growth traits in Nellore cattle. We examined associations of leptin and leptin receptor SNPs with ultrasound carcass (longissimus dorsi muscle area (ribeye area), backfat thickness and rump fat thickness and growth traits (weaning weight adjusted to 210 days of age, yearling weight adjusted to 550 days of age, weight gain of weaning to yearling and scrotal circumference adjusted to 550 days of age) of 2162 Bos primigenius indicus (Nellore) animals. Allele and genotypic frequencies were calculated for each marker. Allele substitution, additive and dominance effects of the polymorphisms were also evaluated. Some alleles of the molecular markers had low frequencies, lower than 1%, in the sample analyzed, although the same polymorphisms described for B. p. taurus cattle were found. Due to very low allelic frequencies, the E2JW, A59V and UASMS2 markers were not included in the analysis, because they were almost fixed. E2FB was found to be significantly associated with weight gain, ribeye area and backfat thickness. The promoter region markers, C963T and UASMS1, were also found to be significantly associated with ribeye area. T945M was significantly associated with weight gain. We conclude that the leptin and receptor gene markers would be useful for marker-assisted selection.

IL28B polymorphisms are markers of therapy response and are influenced by genetic ancestry in chronic hepatitis C patients from an admixed population

Background: IL28B polymorphisms are predictors of therapy response in hepatitis C virus (HCV) patients. We do not know whether they are markers of treatment response in admixed populations or not. Aims: To determine whether IL28B polymorphisms are predictors of therapy response in patients with HCV from an admixed population and are influenced by genetic ancestry. Methods: rs12979860 and rs8099917 were genotyped in 222 HCV patients treated with pegylated interferon and ribavirin. Ancestry was determined using genetic markers. Results: IL28B rs12979860 C/C was associated with sustained virological response (SVR), whereas C/T and T/T were associated with failure to therapy (P = 1.12 x 10(-5)). IL28B rs8099917 T/T was associated with SVR, and G/G and G/T were associated with nonresponse/ relapse (NR/R) (P = 8.00 x 10(-3)). Among HCV genotype 1 patients with C/C genotype, genomic ancestry did not interfere with therapy response. Among patients with rs12979860 T/T genotype, African genetic contribution was greater in the NR/R group (P = 1.51 x 10(-3)), whereas Amerindian and European genetic ancestry contribution were higher in the SVR group (P = 3.77 x 10(-3) and P = 2.16 x 10(-2) respectively). Among HCV type 1 patients with rs8099917 T/T, African genetic contribution was significantly greater in the NR/R group (P = 5.0 x 10(-3)); Amerindian and European ancestry genetic contribution were greater in the SVR group. Conclusion: IL28B rs12979860 and rs8099917 polymorphisms were predictors of therapy response in HCV genotypes 1, 2 and 3 subjects from an admixed population. Genomic ancestry did not interfere with response to therapy in patients with rs12979860 C/C, whereas it interfered in patients with C/T and T/T genotypes. Among HCV genotype 1 rs8099917 T/T patients, genomic ancestry interfered with response to therapy.

CYBA C242T polymorphism is associated with obesity and diabetes mellitus in Brazilian hypertensive patients

Diabet. Med. 29, e55e61 (2012) Abstract Aims The CYBA C242T polymorphism has been associated with cardiovascular phenotypes such as hypertension and atherosclerosis, but available data are conflicting. This report investigated the impact of this variant on hypertension and metabolic determinants of cardiovascular risk in a large Brazilian sample. Methods We cross-sectionally evaluated 1856 subjects (826 normotensive subjects and 1030 hypertensive patients) by clinical history, anthropometry, laboratory analysis and genotyping of the CYBA C242T polymorphism. Results Genotype frequencies in the whole population were consistent with the HardyWeinberg equilibrium and genotype distributions were not different between hypertensive and normotensive subjects. Hypertensive patients with the CC genotype presented lower fasting plasma glucose levels (5.9 +/- 0.1 vs. 6.2 +/- 0.1 mmol/l, P = 0.020) and waist circumference (94.5 +/- 0.6 vs. 96.3 +/- 0.6 cm, P = 0.028) than CT + TT ones. Similarly, the prevalence of diabetes mellitus and obesity was also lower in hypertensive patients carrying the CC genotype (16% vs. 21%, P = 0.041; 36% vs. 43%, P = 0.029, respectively). In addition, multiple and logistic regression analysis demonstrated that the CYBA C242T polymorphism was associated with glucose levels, waist circumference, obesity and diabetes mellitus in hypertensive patients independently of potential confounders. Conversely, in normotensive subjects, no significant difference in studied variables was detected between the genotype groups. Conclusions These data suggest that the T allele of the CYBA C242T polymorphism may be used as a marker for adverse metabolic features in Brazilian subjects with systemic hypertension.

Evaluation of MLH1 I219V Polymorphism in Unrelated South American Individuals Suspected of Having Lynch Syndrome

Background: Some single-nucleotide polymorphisms are associated with higher risk of colorectal cancer development and are suggested to explain part of the genetic contribution to Lynch syndrome. Aim: To evaluate the mutL homolog 1 (MLH1) I219V polymorphism in 124 unrelated South American individuals suspected of having Lynch syndrome, based on frequency, association with pathogenic MLH1 and mutS homolog 2 (MSH2) mutation and clinical features. Materials and Methods: DNA was obtained from peripheral blood and polymerase chain reaction (PCR) was performed, followed by direct sequencing. Results: The Val allelic of the I219V polymorphism was found in 51.61% (64/124) of the individuals, with an allelic frequency of 0.3. MLH1 or MHS2 pathogenic mutations were found in 32.81% (21/64) and in 23.33% (14/60) of Val-carriers and non-carriers, respectively. Conclusion: The Val-carrying genotype was frequent in the studied population; however, it does not appear to exert any modifier effect on MLH1 or MSH2 pathogenic mutations and the development of colorectal cancer.

Geographic differentiation of polymorphism in the Plasmodium falciparum malaria vaccine candidate gene SERA5

SERA5 is regarded as a promising malaria vaccine candidate of the most virulent human malaria parasite Plasmodium falciparum. SERA5 is a 120 kDa abundantly expressed blood-stage protein containing a papain-like protease. Since substantial polymorphism in blood-stage vaccine candidates may potentially limit their efficacy, it is imperative to fully investigate polymorphism of the SERA5 gene (sera5). In this study, we performed evolutionary and population genetic analysis of sera5. The level of inter-species divergence (kS = 0.076) between P. falciparum and Plasmodium reichenowi, a closely related chimpanzee malaria parasite is comparable to that of housekeeping protein genes. A signature of purifying selection was detected in the proenzyme and enzyme domains. Analysis of 445 near full-length P. falciparum sera5 sequences from nine countries in Africa, Southeast Asia, Oceania and South America revealed extensive variations in the number of octamer repeat (OR) and serine repeat (SR) regions as well as substantial level of single nucleotide polymorphism (SNP) in non-repeat regions (2562 bp). Remarkably, a 14 amino acid sequence of SERA5 (amino acids 59-72) that is known to be the in vitro target of parasite growth inhibitory antibodies was found to be perfectly conserved in all 445 worldwide isolates of P. falciparum evaluated. Unlike other major vaccine target antigen genes such as merozoite surface protein-1, apical membrane antigen-1 or circumsporozoite protein, no strong evidence for positive selection was detected for SNPs in the non-repeat regions of sera5. A biased geographical distribution was observed in SNPs as well as in the haplotypes of the sera5 OR and SR regions. In Africa, OR- and SR-haplotypes with low frequency (<5%) and SNPs with minor allele frequency (<5%) were abundant and were mostly continent-specific. Consistently, significant genetic differentiation, assessed by the Wright's fixation index (FST) of inter-population variance in allele frequencies, was detected for SNPs and both OR- and SR-haplotypes among almost all parasite populations. The exception was parasite populations between Tanzania and Ghana, suggesting frequent gene flow in Africa. The present study points to the importance of investigating whether biased geographical distribution for SNPs and repeat variants in the OR and SR regions affect the reactivity of human serum antibodies to variants. (C) 2011 Elsevier Ltd. All rights reserved.

Somatotroph pituitary adenoma with acromegaly and autosomal dominant polycystic kidney disease: SSTR5 polymorphism and PKD1 mutation

A 39-year-old woman with autosomal dominant polycystic kidney disease (ADPKD) presented with acromegaly and a pituitary macroadenoma. There was a family history of this renal disorder. She had undergone surgery for pituitary adenoma 6 years prior. Physical examination disclosed bitemporal hemianopsia and elevation of both basal growth hormone (GH) 106 ng/mL (normal 0-5) and insulin-like growth factor (IGF-1) 811 ng/mL (normal 48-255) blood levels. A magnetic resonance imaging scan disclosed a 3.0 cm sellar and suprasellar mass with both optic chiasm compression and left cavernous sinus invasion. Pathologic, cytogenetic, molecular and in silico analysis was undertaken. Histologic, immunohistochemical and ultrastructural studies of the lesion disclosed a sparsely granulated somatotroph adenoma. Standard chromosome analysis on the blood sample showed no abnormality. Sequence analysis of the coding regions of PKD1 and PKD2 employing DNA from both peripheral leukocytes and the tumor revealed the most common PKD1 mutation, 5014_5015delAG. Analysis of the entire SSTR5 gene disclosed the variant c.142C > A (p.L48M, rs4988483) in the heterozygous state in both blood and tumor, while no pathogenic mutations were noted in the MEN1, AIP, p27Kip1 and SSTR2 genes. To our knowledge, this is the fourth reported case of a GH-producing pituitary adenoma associated with ADPKD, but the first subjected to extensive morphological, ultrastructural, cytogenetic and molecular studies. The physical proximity of the PKD1 and SSTR5 genes on chromosome 16 suggests a causal relationship between ADPKD and somatotroph adenoma.

DNA repair gene excision repair cross complementing-group 1 (ERCC1) in head and neck squamous cell carcinoma: analysis of methylation and polymorphism (G19007A), protein expression and association with epidemiological and clinicopathological factors

Aims: To evaluate the associations of excision repair cross complementing-group 1 (ERCC1) (DNA repair protein) (G19007A) polymorphism, methylation and immunohistochemical expression with epidemiological and clinicopathological factors and with overall survival in head and neck squamous cell carcinoma (HNSCC) patients. Methods and results: The study group comprised 84 patients with HNSCC who underwent surgery and adjuvant radiotherapy without chemotherapy. Bivariate and multivariate analyses were used. The allele A genotype variant was observed in 79.8% of the samples, GG in 20.2%, GA in 28.6% and AA in 51.2%. Individuals aged more than 45 years had a higher prevalence of the allelic A variant and a high (83.3%) immunohistochemical expression of ERCC1 protein [odds ratio (OR) = 4.86, 95% confidence interval (CI): 1.2-19.7, P = 0.027], which was also high in patients with advanced stage (OR= 5.04, 95% CI: 1.07-23.7, P = 0.041). Methylated status was found in 51.2% of the samples, and was higher in patients who did not present distant metastasis (OR = 6.67, 95% CI: 1.40-33.33, P = 0.019) and in patients with advanced stage (OR = 5.04, 95% CI: 1.07-23.7, P = 0.041). At 2 and 5 years, overall survival was 55% and 36%, respectively (median = 30 months). Conclusion: Our findings may reflect a high rate of DNA repair due to frequent tissue injury during the lifetime of these individuals, and also more advanced disease presentation in this population with worse prognosis.

Increased interleukin-10 and interferon-γ levels in Plasmodium vivax malaria suggest a reciprocal regulation which is not altered by IL-10 gene promoter polymorphism

Background In human malaria, the naturally-acquired immune response can result in either the elimination of the parasite or a persistent response mediated by cytokines that leads to immunopathology. The cytokines are responsible for all the symptoms, pathological alterations and the outcome of the infection depends on the reciprocal regulation of the pro and anti-inflammatory cytokines. IL-10 and IFN-gamma are able to mediate this process and their production can be affected by single nucleotide polymorphisms (SNPs) on gene of these cytokines. In this study, the relationship between cytokine IL-10/IFN-gamma levels, parasitaemia, and their gene polymorphisms was examined and the participation of pro-inflammatory and regulatory balance during a natural immune response in Plasmodium vivax-infected individuals was observed. Methods The serum levels of the cytokines IL-4, IL-12, IFN-gamma and IL-10 from 132 patients were evaluated by indirect enzyme-linked immunosorbent assays (ELISA). The polymorphism at position +874 of the IFN-gamma gene was identified by allele-specific polymerase chain reaction (ASO-PCR) method, and the polymorphism at position -1082 of the IL-10 gene was analysed by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism). Results The levels of a pro- (IFN-gamma) and an anti-inflammatory cytokine (IL-10) were significantly higher in P. vivax-infected individuals as compared to healthy controls. The IFN-gamma levels in primoinfected patients were significantly higher than in patients who had suffered only one and more than one previous episode. The mutant alleles of both IFN-gamma and IL-10 genes were more frequent than the wild allele. In the case of the IFNG+874 polymorphism (IFN-gamma) the frequencies of the mutant (A) and wild (T) alleles were 70.13% and 29.87%, respectively. Similar frequencies were recorded in IL-10-1082, with the mutant (A) allele returning a frequency of 70.78%, and the wild (G) allele a frequency of 29.22%. The frequencies of the alleles associated with reduced production of both IFN-gamma and IL-10 were high, but this effect was only observed in the production of IFN-gamma. Conclusions This study has shown evidence of reciprocal regulation of the levels of IL-10 and IFN-gamma cytokines in P. vivax malaria, which is not altered by the presence of polymorphism in the IL-10 gene.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2011-31 January 2012

This article documents the addition of 473 microsatellite marker loci and 71 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Barteria fistulosa, Bombus morio, Galaxias platei, Hematodinium perezi, Macrocentrus cingulum Brischke (a.k.a. M.abdominalis Fab., M.grandii Goidanich or M.gifuensis Ashmead), Micropogonias furnieri, Nerita melanotragus, Nilaparvata lugens Stal, Sciaenops ocellatus, Scomber scombrus, Spodoptera frugiperda and Turdus lherminieri. These loci were cross-tested on the following species: Barteria dewevrei, Barteria nigritana, Barteria solida, Cynoscion acoupa, Cynoscion jamaicensis, Cynoscion leiarchus, Cynoscion nebulosus, Cynoscion striatus, Cynoscion virescens, Macrodon ancylodon, Menticirrhus americanus, Nilaparvata muiri and Umbrina canosai. This article also documents the addition of 116 sequencing primer pairs for Dicentrarchus labrax.

FGFR4 Profile as a Prognostic Marker in Squamous Cell Carcinoma of the Mouth and Oropharynx

Background: Fibroblast growth factor receptor 4 (FGFR4) is a member of a receptor tyrosine kinase family of enzymes involved in cell cycle control and proliferation. A common single nucleotide polymorphism (SNP) Gly388Arg variant has been associated with increased tumor cell motility and progression of breast cancer, head and neck cancer and soft tissue sarcomas. The present study evaluated the prognostic significance of FGFR4 in oral and oropharynx carcinomas, finding an association of FGFR4 expression and Gly388Arg genotype with tumor onset and prognosis. Patients and Methods: DNA from peripheral blood of 122 patients with oral and oropharyngeal squamous cell carcinomas was used to determine FGFR4 genotype by PCR-RFLP. Protein expression was assessed by immunohistochemistry (IHC) on paraffin-embedded tissue microarrays. Results: Presence of allele Arg388 was associated with lymphatic embolization and with disease related premature death. In addition, FGFR4 low expression was related with lymph node positivity and premature relapse of disease, as well as disease related death. Conclusion: Our results propose FGFR4 profile, measured by the Gly388Arg genotype and expression, as a novel marker of prognosis in squamous cell carcinoma of the mouth and oropharynx.

MMP3 and TIMP1 variants contribute to chronic periodontitis and may be implicated in disease progression

Aim Matrix metalloproteinases (MMPs) play a key role in the tissue destruction characteristic of chronic periodontitis. The purpose of this study was to investigate the association of MMP and TIMP polymorphisms with chronic periodontitis in two populations. Material and Methods A total of 34 polymorphisms spanning 12 MMP and 2 TIMP genes were genotyped in 401 individuals from Brazil (99 cases with chronic periodontitis and 302 controls), and 274 individuals from the US (70 cases and 204 controls). Individuals were considered cases if presenting at least three teeth exhibiting sites of clinical attachment loss =5 mm in two different quadrants. Controls were characterized by absence of clinical attachment loss and no sites with probing depth >3 mm. MMP3 and TIMP1 mRNA expression was evaluated in healthy and diseased periodontal tissues. Results TIMP1 showed association with chronic periodontitis in the Brazilian population (for rs5906435, p = 0.0004), whereas MMP3 showed association in the US population (for rs679620, p = 0.0003; and rs650108, p = 0.002) and in the Brazilian population (for rs639752, p = 0.005). MMP3 and TIMP1 mRNA expression was significantly higher in diseased tissues when compared to control tissues. Conclusions Our results further support a role for variations in MMP3 in chronic periodontitis and report a novel association with TIMP1. These genes may be considered additional candidate genes for chronic periodontitis.