Genetic Polymorphisms in Replication-related Genes and Risk of Breast Cancer Among European and East Asian Women

Background: The role of common, low to intermediate risk alleles in breast cancer need to be examined due to their relatively high prevalence. Among many cellular pathways, replication has a pivotal role in cell division and frequently targeted during carcinogenesis. Replication is governed by a host of genes involved in a number of different pathways. This study investigates the effects of replication-gene variants in relation to breast cancer and how this relationship is affected by ethnicity, menopausal status and breast tumour subtype. Methods: Data from a case-control study with 997 incident breast cancer cases and 1,050 age frequency matched controls in Vancouver, British Columbia and Kingston, Ontario were used. Unconditional logistic regression was used to calculate odds ratios between 45 replication gene variants and breast cancer risk, assuming an additive genetic model adjusted for age and centre, presented for Europeans and East Asians separately. Polytomous logistic regression was used to assess odds ratios between each SNP and four breast cancer subtypes defined by hormone receptor status among Europeans. All analyses were stratified by menopausal status. The Benjamini–Hochberg false discovery rate (FDR) was used to address multiple comparisons. Results: Among Europeans, the SNPs in FGFR2, TOX3 and 11q13 loci were associated with breast cancer after controlling for multiple comparisons. Test of heterogeneity showed the SNPs rs1045185, rs4973768, rs672888, rs1219648, rs2420946 among Europeans and rs889312 among East Asians conferred differential risk across the tumour subtypes. Conclusions: Specific SNPs in replication genes were associated with breast cancer, and the risk level differed by tumour subtype defined by ER/PR/Her2 status and ethnicity.

Comparison of sequence variants in the PDK4 and COX4I2 genes between racing and cutting lines of quarter horses and associations with the speed index.

Different selection objectives within the Quarter Horse breed led to the formation of groups with distinct skills, including the racing and cutting lines. With a smaller population size in Brazil, but of great economic representativeness, the racing line is characterized by animals that can reach high speeds over short distances and within a short period of time. The cutting line is destined for functional tests, exploring skills such as agility and obedience. Although the athletic performance of horses is likely to be influenced by a large number of genes, few genetic variants have so far been related to this trait and this was done exclusively in Thoroughbreds, including the g.38973231G>A singlenucleotide polymorphism in the PDK4 gene and the g.22684390C>T single-nucleotide polymorphism in the COX4I2 gene. The results of the present study demonstrate the presence of polymorphic PDK4 and COX4I2 genes in Quarter Horses. The analysis of 296 racing animals and 68 cutting animals revealed significant differences in allele and genotype frequencies between the two lines. The same was not observed when these frequencies were compared between extreme racing performance phenotypes. There were also no significant associations between alleles of the two polymorphisms and the speed index. These results suggest that the alleles of the PDK4 and COX4I2 genes, which are related to better racecourse performance in Thoroughbreds, are probably associated with beneficial adaptations in aerobic metabolism and therefore play secondary roles in sprint racing performance in Quarter Horses, which is mainly anaerobic.

Marcador alelo-específico associado com níveis de expressão do gene ZmMATE1 em milho.

2016

Valutazione statistica della ripetibilità di esperimenti di sequenziamento del DNA in specie batteriche

I progressi della biologia molecolare assieme alle nuove tecnologie di sequenziamento applicate su scala genomica alla genetica molecolare, hanno notevolmente elevato la conoscenza sulle componenti di base della biologia e delle patologie umane. All’interno di questo contesto, prende piede lo studio delle sequenze genetiche dei batteri, consentendo dunque, una migliore comprensione di ciò che si nasconde dietro le malattie legate all’uomo. Il seguente lavoro di tesi si propone come obiettivo l’analisi del DNA del batterio Listeria monocytogenes, un microrganismo presente nel suolo e in grado di contaminare l’acqua e gli alimenti. Lo scopo principale è quello di confrontare la variabilità tecnica e biologica, al fine di capire quali siano gli SNPs reali (Single Nucleotide Polymorphism) e quali artefatti tecnici. La prima parte, quindi, comprende una descrizione del processo di individuazione degli SNPs presenti nel DNA dei campioni in esame, in particolare di tre isolati diversi e tre copie. Nella seconda parte, invece, sono effettuate delle indagini statistiche sui parametri relativi agli SNPs individuati, ad esempio il coverage o il punteggio di qualità assegnato alle basi. Il fine ultimo è quello di andare a verificare se sussistano particolari differenze tra gli SNPs dei vari isolati batterici.

Nucleotide, cytogenetic and expression impact of the human chromosome 8p23.1 inversion polymorphism

Background: The human chromosome 8p23.1 region contains a 3.8–4.5 Mb segment which can be found in different orientations (defined as genomic inversion) among individuals. The identification of single nucleotide polymorphisms (SNPs) tightly linked to the genomic orientation of a given region should be useful to indirectly evaluate the genotypes of large genomic orientations in the individuals. Results: We have identified 16 SNPs, which are in linkage disequilibrium (LD) with the 8p23.1 inversion as detected by fluorescent in situ hybridization (FISH). The variability of the 8p23.1 orientation in 150 HapMap samples was predicted using this set of SNPs and was verified by FISH in a subset of samples. Four genes (NEIL2, MSRA, CTSB and BLK) were found differentially expressed (p<0.0005) according to the orientation of the 8p23.1 region. Finally, we have found variable levels of mosaicism for the orientation of the 8p23.1 as determined by FISH. Conclusion: By means of dense SNP genotyping of the region, haplotype-based computational analyses and FISH experiments we could infer and verify the orientation status of alleles in the 8p23.1 region by detecting two short haplotype stretches at both ends of the inverted region, which are likely the relic of the chromosome in which the original inversion occurred. Moreover, an impact of 8p23.1 inversion on gene expression levels cannot be ruled out, since four genes from this region have statistically significant different expression levels depending on the inversion status. FISH results in lymphoblastoid cell lines suggest the presence of mosaicism regarding the 8p23.1 inversion.

High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species

Background: High-throughput SNP genotyping has become an essential requirement for molecular breeding and population genomics studies in plant species. Large scale SNP developments have been reported for several mainstream crops. A growing interest now exists to expand the speed and resolution of genetic analysis to outbred species with highly heterozygous genomes. When nucleotide diversity is high, a refined diagnosis of the target SNP sequence context is needed to convert queried SNPs into high-quality genotypes using the Golden Gate Genotyping Technology (GGGT). This issue becomes exacerbated when attempting to transfer SNPs across species, a scarcely explored topic in plants, and likely to become significant for population genomics and inter specific breeding applications in less domesticated and less funded plant genera. Results: We have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of Eucalyptus from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for E. grandis. A systematic assessment of in silico SNP filtering requirements showed that stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. SNP assay success was high for the 288 SNPs selected with more rigorous in silico constraints; 93% of them provided high quality genotype calls and 71% of them were polymorphic in a diverse panel of 96 individuals of five different species. SNP reliability was high across nine Eucalyptus species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased. Conclusions: This study indicates that the GGGT performs well both within and across species of Eucalyptus notwithstanding its nucleotide diversity >= 2%. The development of a much larger array of informative SNPs across multiple Eucalyptus species is feasible, although strongly dependent on having a representative and sufficiently deep collection of sequences from many individuals of each target species. A higher density SNP platform will be instrumental to undertake genome-wide phylogenetic and population genomics studies and to implement molecular breeding by Genomic Selection in Eucalyptus.

Detection of the TLR4 1196C > T Polymorphism by Mismatched-Polymerase Chain Reaction Using Plasmid DNA as Internal Control in Restriction Fragment Length Polymorphism Assays

Background: Restriction fragment length polymorphism (RFLP) is a common molecular assay used for genotyping, and it requires validated quality control procedures to prevent mistyping caused by impaired endonuclease activity. We have evaluated the usefulness of a plasmid-based internal control in RFLP assays. Results: Blood samples were collected from 102 individuals with acute myocardial infarction (AMI) and 108 non-AMI individuals (controls) for DNA extraction and laboratory analyses. The 1196C> T polymorphism in the toll-like receptor 4 (TLR4) gene was amplified by mismatched-polymerase chain reaction (PCR). Amplicons and pBluescript II SK-plasmid were simultaneously digested with endonuclease HincII. Fragments were separated on 2% agarose gels. Plasmid was completely digested using up to 55.2 nmL/L DNA solutions and 1 mu L PCR product. Nevertheless, plasmid DNA with 41.4 nM or higher concentrations was incompletely digested in the presence of 7 mL PCR product. In standardized conditions, TLR4 1196C> T variant was accurately genotyped. TLR4 1196T allele frequency was similar between AMI (3.1%) and controls (2.0%, p = 0.948). TLR4 SNP was not associated with AMI in this sample population. In conclusion, the plasmid-based control is a useful approach to prevent mistyping in RFLP assays, and it is validate for genetic association studies such as TLR4 1196C> T.

Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild-type, and B, C or D also known as O) were evaluated using real-time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme-linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0.022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0.04) and to the control group (P = 0.011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non-responsiveness to pginterferon and ribavirin treatment (P = 0.023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.

ERCC1 protein, mRNA expression and T19007C polymorphism as prognostic markers in head and neck squamous cell carcinoma patients treated with surgery and adjuvant cisplatin-based chemoradiation

Adjuvant cisplatin-based chemoradiation improves survival in HNSCC patients presenting with risk features. ERCC1 (excision repair cross-complementation group 1) is associated with resistance to chemo- and radiation therapy and may have a prognostic value in HNSCC patients. Here we studied ERCC1 expression and the polymorphism T19007C as prognostic markers in these patients. This is a retrospective and translational analysis, where ERCC1 protein expression was evaluated by immunohistochemistry, using an H-score, and mRNA expression was determined by RT-PCR. T 19007C genotypes were detected by PCR-RFLP carried out using DNA template extracted from normal lymph nodes. A high H-score was seen in 32 patients (54%), who presented better 5-year overall survival (5-y OS: 50% vs. 18%, HR 0.43, p=0.026). Fifteen out of 45 patients (33%), with high mRNA expression, presented better 5-year overall survival (OS) (86% vs. 30%, HR 0.26, p=0.052). No OS difference was detected among T 19007C genotypes. High H-score and mRNA expression remained significant as favorable prognostic factors in a multivariate analysis. Collectively, our results suggest that high ERCC1 expression seems to be associated with better OS rates in HNSCC patients submitted to adjuvant cisplatin-based chemoradiation.

Correlation between beta-2-glycoprotein I gene polymorphism and anti-beta-2 glycoprotein I antibodies in patients with multibacillary leprosy

Antiphospholipid antibodies, such as anti-beta 2-glycoprotein I (beta 2GPI), are present in multibacillary leprosy (MB) patients; however, MB patients do not usually present with antiphospholipid antibody syndrome (APS), which is characterized by thromboembolic phenomena (TEP). Rare cases of TEP occur in leprosy patients, but the physiopathology of this condition remains unclear. In this case-control study, we examined whether single-nucleotide polymorphisms (SNPs) of the beta 2GPI gene contributed to the risk of leprosy and APS co-morbidity. SNPs Ser88Asn, Leu247Val, Cys306Gly and Trp316Ser were identified in 113 Brazilian leprosy patients. Additionally, anti-beta 2GPI antibodies and plasma concentrations of beta 2GPI were quantified. The Ser88Asn, Cys306Gly and Trp316Ser SNPs were not risk factors for APS in leprosy. A higher frequency of Val/Val homozygosity was observed in leprosy patients compared to controls (36 vs. 5%; P < 0.001). Forty-two percent of MB and 17% of paucibacillary leprosy patients were positive for anti-beta 2GPI IgM (P = 0.014). There was no correlation between SNP Ser88Asn or Cys306Gly and anti-beta 2GPI antibody levels. In MB patients with positive anti-beta 2GPI IgM, the frequency of Val/Val homozygosity was higher than in controls (32 vs. 15%; P = 0.042). The frequency of the mutant allele Ser316 was higher in MB patients with positive rather than negative anti-beta 2GPI IgM levels (6 vs. 0%; P = 0.040) and was greater than in the control group (6 vs. 1%; P = 0.034). The studied polymorphisms did not influence the plasma concentrations of beta 2GPI. These results suggest that Leu247Val and Trp316Ser SNPs may represent genetic risk factors for anti-beta 2GPI antibody production in MB patients.

Polymorphisms at GLUT1 Gene Are Not Associated With the Development of TSP/HAM in Brazilian HTLV-1 Infected Individuals and the Discovery of a New Polymorphism at GLUT1 Gene

The development of HTLV-1 associated clinical manifestations, such as TSP/HAM and ATLL, occur in 2-4% of the infected population and it is still unclear why this infection remains asymptomatic in most infected carriers. Recently, it has been demonstrated that HTLV uses the Glucose transporter type 1 (GLUT1) to infect T-CD4(+) lymphocytes and that single nucleotide polymorphisms (SNP) in the GLUT1 gene are associated with diabetic nephropathy in patients with diabetes mellitus in different populations. These polymorphisms could contribute to a higher GLUT1 protein expression on cellular membrane, facilitating the entry of HTLV and its transmission cell by cell. This could result in a higher provirus load and consequently in the development of TSP/HAM. To evaluate the role of GLUT1 gene polymorphisms in the development of TSP/HAM in HTLV-1 infected individuals, the g.22999G > T, g.15339T > C and c.-2841A > T sites were analyzed by PCR/RFLP or sequencing in 244 infected individuals and 102 normal controls. The proviral load of the HTLV-1 infected patients was also analyzed using Real Time Quantitative PCR. Genotypic and allelic frequencies of the three sites did not differ significantly between controls and HTLV-1 infected individuals. There was no difference in genotypic and allelic distributions among patients as to the presence or absence of HTLV-1 associated clinic manifestations. As regards the quantification of the provirus load, we observed a significant reduction in the asymptomatic individuals compared with the oligosymptomatic and TSP/HAM individuals. These results suggest that g.22999G > T, g.15339T > C, and c.-2841A > T SNP do not contribute to HTLV-1 infection nor to the genetic susceptibility of TSP/HAM in Brazilian HTLV-1 infected individuals. J. Med. Virol. 81:552557, 2009. (C) 2009 Wiley-Liss, Inc.

Q192R polymorphism of the paraoxonase-1 gene as a risk factor for obesity in Portuguese women

Introduction - Obesity became a major public health problem as a result of its increasing prevalence worldwide. Paraoxonase-1 (PON1) is an esterase able to protect membranes and lipoproteins from oxidative modifications. At the PON1 gene, several polymorphisms in the promoter and coding regions have been identified. The aims of this study were i) to assess PON1 L55M and Q192R polymorphisms as a risk factor for obesity in women; ii) to compare PON1 activity according to the expression of each allele in L55M and Q192R polymorphisms; iii) to compare PON1 activity between obese and normal-weight women. Materials and methods - We studied 75 healthy (35.9±8.2 years) and 81 obese women (34.3±8.2 years). Inclusion criteria for obese subjects were body mass index ≥30 kg/m2 and absence of inflammatory/neoplasic conditions or kidney/hepatic dysfunction. The two PON1 polymorphisms were assessed by real-time PCR with TaqMan probes. PON1 enzymatic activity was assessed by spectrophotometric methods, using paraoxon as a substrate. Results - No significant differences were found for PON1 activity between normal and obese women. Nevertheless, PON1 activity was greater (P<0.01) for the RR genotype (in Q192R polymorphism) and for the LL genotype (in L55M polymorphism). The frequency of allele R of Q192R polymorphism was significantly higher in obese women (P<0.05) and was associated with an increased risk of obesity (odds ratio=2.0 – 95% confidence interval (1.04; 3.87)). Conclusion - 55M and Q192R polymorphisms influence PON1 activity. The allele R of the Q192R polymorphism is associated with an increased risk for development of obesity among Portuguese Caucasian premenopausal women.

PRELIMINARY REPORT ON THE PUTATIVE ASSOCIATION OF IL10 -3575 T/A GENETIC POLYMORPHISM WITH MALARIA SYMPTOMS

Only a small percentage of individuals living in endemic areas develop severe malaria suggesting that host genetic factors may play a key role. This study has determined the frequency of single nucleotide polymorphisms (SNPs) in some pro and anti-inflammatory cytokine gene sequences: IL6 (-174; rs1800795), IL12p40 (+1188; rs3212227), IL4 (+33; rs2070874), IL10 (-3575; rs1800890) and TGFb1 (+869; rs1800470), by means of PCR-RFLP. Blood samples were collected from 104 symptomatic and 37 asymptomatic subjects. Laboratory diagnosis was assessed by the thick blood smear test and nested-PCR. No association was found between IL6 (-174), IL12p40 (+1188), IL4 (+33), IL10 (- 3575), TGFb1 (+869) SNPs and malaria symptoms. However, regarding the IL10 -3575 T/A SNP, there were significantly more AA and AT subjects, carrying the polymorphic allele A, in the symptomatic group (c2 = 4.54, p = 0.01, OR = 0.40 [95% CI - 0.17- 0.94]). When the analysis was performed by allele, the frequency of the polymorphic allele A was also significantly higher in the symptomatic group (c2 = 4.50, p = 0.01, OR = 0.45 [95% CI - 0.21-0.95]). In conclusion, this study has suggested the possibility that the IL10 - 3575 T/A SNP might be associated with the presence and maintenance of malaria symptoms in individuals living in endemic areas. Taking into account that this polymorphism is related to decreased IL10 production, a possible role of this SNP in the pathophysiology of malaria is also suggested, but replication studies with a higher number of patients and evaluation of IL10 levels are needed for confirmation.

IL28B polymorphism is significantly correlated with ifn anti-viral effectiveness already on first day of pegylated interferon-alpha and ribavirin therapy of chronic HCV infection

Background and Aims: Recently, single nucleotide polymorphisms (SNPs) in IL28B were shown to correlate with response to pegylated interferon-a (IFN) and ribavirin therapy of chronic HCV infection. However, the cause for the SNPs effect on therapy response and its application for direct anti-viral (DAV) treatment are not clear. Here, we analyze early HCV kinetics as function of IL28B SNPs to determine its specific effect on viral dynamics. Methods: IL28B SNPs rs8099917, rs12979860 and rs12980275 were genotyped in 252 chronically HCV infected Caucasian naïve patients (67% HCV genotype 1, 28% genotype 2-3) receiving peginterferonalfa- 2a (180 mg/qw) plus ribavirin (1000-1200 mg/qd) in the DITTO study. HCV-RNA was measured (LD = 50 IU/ml) frequently during first 28 days. Results: RVR was achieved in 33% of genotype 1 patients with genotype CC at rs12979860 versus 12-16% for genotypes TT and CT (P < 0.03). Significant (P < 0.001) difference in viral decline was observed already at day 1 (see Figure). First phase decline was significantly (P < 0.001) larger in patients with genotype CC (2.0 log) than for TT and CT genotypes (0.6 and 0.8), indicating IFN anti-viral effectiveness in blocking virion production of 99% versus 75-84%. There was no significant association between second phase slope and rs12979860 genotype in patients with a first phase decline larger than 1 log. HCV kinetics as function of IL28b SNP. The same trend (not shown) was observed for HCV genotype 2-3 patients with different SNP genotype distribution that may indicate differential selection pressure as function of HCV genotype. Similar results were observed for SNPs rs8099917 and rs12980275, with a strong linkage disequilibrium among the 3 loci allowing to define the composite haplotype best associated with IFN effectiveness. Conclusions: IFN effectiveness in blocking virion production/ release is strongly affected by IL28B SNPs, but not other viral dynamic properties such as infected cell loss rate. Thus, IFN based therapy, as standard-of-care or in combination with DAV, should consider IL28B SNPs for prediction and personalized treatment, while response to pure DAV treatment may be less affected by IL28B SNPs. Additional analyses are undergoing to pinpoint the SNP effect on IFN anti-viral effectiveness.

Analysis of CXCL12 3 ' UTR G > A polymorphism in colorectal cancer

Colorectal cancer is one of the most prevalent cancers in developed countries. However, the genetic factors influencing its appearance remain far from being fully characterized. Recently, a G>A functional transition mapping the 3' untranslated region of the CXCL12 gene (rs1801157) has been found to be under-represented among rectal cancer patients when compared to colon cancer patients from a Swedish series. Here we present the results from an independent analysis of CXCL12 rs1801157 in a larger CRC series of Spanish origin in order to analyse the robustness of this association within a different European population. No significant difference was observed between controls and colon or rectal cancer patients. We were also unable to find a correlation between rs1801157 and different prognostic markers such as metastasis development or disease-free survival time. The epidemiologic data involving CXCL12 rs1801157 in colorectal cancer risk are discussed.