Comparison Of Allele Frequencies Research Articles

Association of Polymorphisms in Connective Tissue Growth Factor and Epidermal Growth Factor Receptor Genes With Human Longevity.

Growth pathways play key roles in longevity. The present study tested single-nucleotide polymorphisms (SNPs) in the connective tissue growth factor gene (CTGF) and the epidermal growth factor receptor gene (EGFR) for association with longevity. Comparison of allele and genotype frequencies of 12 CTGF SNPs and 41 EGFR SNPs between 440 American men of Japanese ancestry aged ≥95 years and 374 men of average life span revealed association with longevity at the p < .05 level for 2 SNPs in CTGF and 7 in EGFR. Two in CTGF and two in EGFR remained significant after Bonferroni correction. The SNPs of both CTGF and EGFR were in a haplotype block in each respective gene. Haplotype analysis confirmed the suggestive association found by χ2 analysis. We noted an excess of heterozygotes among the longevity cases, consistent with heterozygote advantage in living to extreme old age. No associations of the most significant SNPs were observed in whites or Koreans. In conclusion, the present findings indicate that genetic variation in CTGF and EGFR may contribute to the attainment of extreme old age in Japanese. More research is needed to confirm that genetic variation in CTGF and EGFR contributes to the attainment of extreme old age across human populations.

Association of single nucleotide polymorphisms in the IL2RA-RBM17 region with vitiligo in the Chinese Mongolian population

Objective To investigate the association between single nucleotide polymorphisms(SNPs)in the IL2RA-RBM17 region and vitiligo in the Chinese Mongolian population. Methods Five milliliters of venous blood samples were collected from 425 patients with vitiligo(patient group)and 503 healthy human controls(control group)of Mongolian nationality after informed consent, and genomic DNA was extracted with the AxyPrep DNA extraction kit(AP-MX-BL-GDNA-25). Nine SNPs were selected across the IL2RA-RBM17 region, including rs706779, rs3134883, rs7090530, rs12251307, rs4750005, rs3920615, rs4747887, rs4750012 and rs7099083. Ligase detection reaction(LDR)was performed for SNP genotyping. With the PLINK 1.07 and SPSS 11.0 packages, statistical analysis was carried out by the chi-square test for comparisons of allele and genotype frequencies between the patient group and control group. Linkage disequilibrium analysis was performed for 5 SNPs by calculating the r2 and D' values. Haplotype analysis of 5 related SNPs was conducted to investigate differences in haplotype frequencies between the patient group and control group. Results There were significant differences in allele frequencies of 5 SNPs, including rs4750005, rs3920615, rs4747887, rs4750012 and rs7099083, between the patient group and control group(all P < 0.05). Under a dominant mode of inheritance, a significant decrease was observed in the frequencies of GG/GC genotypes of rs3920615, CT/CC genotypes of rs4747887, CT/CC genotypes of rs4750005, TC/TT genotypes of rs4750012 and AG/AA genotypes of rs7099083 in the patient group compared with the control group(all P < 0.005 6). Moderate to strong linkage disequilibrium was observed between the 5 SNPs(D' = 0.424-1, r2 = 0.137-0.985). Haplotype analysis showed that the frequency of a haplotype(H2: CGCTA)was significantly lower in the patient group than in the control group, and the difference reached statistical significance after Bonferroni adjustment(P= 0.001 6, OR = 0.674). Conclusion SNPs in the IL2RA-RBM17 region are associated with vitiligo in the Chinese Mongolian population. Key words: Vitiligo; Polymorphism, single nucleotide; Mongolia; Genetic association studies; Genes, IL2RA-RBM17; China

Associations of Human Leukocyte Antigen-DRB1 Alleles with Nasopharyngeal Carcinoma and Its Clinical Significance in Xinjiang Uyghur Autonomous Region of China.

Background:Genetic susceptibility is one of the major etiological factors for nasopharyngeal carcinoma (NPC). Among the genetic predisposing factors, human leukocyte antigen (HLA) genes have been reported to be associated with NPC. This study aimed to investigate the associations of HLA-DRB1 alleles with NPC and the clinical significance of HLA-DRB1 alleles in NPC.Methods:From January 2009 to December 2013, 140 NPC patients (118 Han patients and 22 Uyghur patients) and 158 healthy controls (81 Han individuals and 77 Uyghur individuals) from Xinjiang Province were genotyped for HLA-DRB1 using the polymerase chain reaction-sequence specific primer technique. Chi-square analysis was used when comparing allele frequencies between groups. The clinical outcomes were evaluated by Kaplan-Meier method and Cox regression model.Results:Compared with healthy controls, the allele frequency of HLA-DRB1*0701 was increased in the Uyghur patients (P = 0.008) but not in the Han patients (P = 0.869). HLA-DRB1*0101 allele was presented with higher frequency in clinical Stage I + II group compared with clinical Stage III + IV group in the Han patients (P = 0.015) but not in the Uyghur patients (P = 1.000). Higher frequency of HLA-DRB1*1501 allele was observed in patients aged <45 years compared with those in patients aged ≥45 years (P = 0.002). Neither HLA-DRB1*0701 nor HLA-DRB1*0101 had a statistically significant association with 3-year survival.Conclusions:This study found HLA-DRB1*0701 in Uyghur population was associated with an increased risk of developing NPC. In Han population, we found HLA-DRB1*0101 was associated with protection from disease progression, and HLA-DRB1*1501 was associated with early age of onset. HLA-DRB1 could not be identified as a prognostic indicator for NPC in either Han or Uyghur patients.

Cytochrome P4502C19*3 allelic variant frequency in Iranian healthy Azeri Turkish population

Introduction: Cytochrome P4502C19 (CYP2C19) is well-known to be one of the determinants responsible for the pronounced interethnic and individual differences in response and character of clinically important drugs. CYP2C19*3 arises from a G to A transition at position 636 in exon 4 of CYP2C19. These individuals be situated poor metabolizers (PMs) of a wide range of medications including omeprazole (OMP). In this study, we determined genotypes of CYP2C19*3 in Iranian Azeri Turkish population to compare allele frequencies with previous findings in other ethnic groups. Methods: CYP2C19*3 allelic variant was determined in 200 unrelated healthy Iranian volunteers by polymerase chain reaction-restriction fragment length polymorphism assays (PCR-RFLP). Results: The frequencies of CYP2C19*3 in healthy volunteers reported in this study (P = 0.569, χ2 = 2.35). This is not higher than that would be predicted from the genotypic status of these cases in CYP2C19*3 allelic variants. Our results revealed that 95.46% had wild type allele, they did not carry any of the tested mutations and 9 (4.54%) had mutant alleles. Conclusion: Our data recommend that genotyping for CYP2C19*3 is interest in using pharmacokinetics to individualize medicine, but results of this study demonstrated that CYP2C19*3 genetic polymorphism is not important determinant of the efficacy of PM of drugs, such as OMP, which may be metabolized by this enzyme.

The Contribution of GWAS Loci in Familial Dyslipidemias.

Familial combined hyperlipidemia (FCH) is a complex and common familial dyslipidemia characterized by elevated total cholesterol and/or triglyceride levels with over five-fold risk of coronary heart disease. The genetic architecture and contribution of rare Mendelian and common variants to FCH susceptibility is unknown. In 53 Finnish FCH families, we genotyped and imputed nine million variants in 715 family members with DNA available. We studied the enrichment of variants previously implicated with monogenic dyslipidemias and/or lipid levels in the general population by comparing allele frequencies between the FCH families and population samples. We also constructed weighted polygenic scores using 212 lipid-associated SNPs and estimated the relative contributions of Mendelian variants and polygenic scores to the risk of FCH in the families. We identified, across the whole allele frequency spectrum, an enrichment of variants known to elevate, and a deficiency of variants known to lower LDL-C and/or TG levels among both probands and affected FCH individuals. The score based on TG associated SNPs was particularly high among affected individuals compared to non-affected family members. Out of 234 affected FCH individuals across the families, seven (3%) carried Mendelian variants and 83 (35%) showed high accumulation of either known LDL-C or TG elevating variants by having either polygenic score over the 90th percentile in the population. The positive predictive value of high score was much higher for affected FCH individuals than for similar sporadic cases in the population. FCH is highly polygenic, supporting the hypothesis that variants across the whole allele frequency spectrum contribute to this complex familial trait. Polygenic SNP panels improve identification of individuals affected with FCH, but their clinical utility remains to be defined.

Investigation of Genetic Variation Underlying Central Obesity amongst South Asians.

South Asians are 1/4 of the world’s population and have increased susceptibility to central obesity and related cardiometabolic disease. Knowledge of genetic variants affecting risk of central obesity is largely based on genome-wide association studies of common SNPs in Europeans. To evaluate the contribution of DNA sequence variation to the higher levels of central obesity (defined as waist hip ratio adjusted for body mass index, WHR) among South Asians compared to Europeans we carried out: i) a genome-wide association analysis of >6M genetic variants in 10,318 South Asians with focused analysis of population-specific SNPs; ii) an exome-wide association analysis of ~250K SNPs in protein-coding regions in 2,637 South Asians; iii) a comparison of risk allele frequencies and effect sizes of 48 known WHR SNPs in 12,240 South Asians compared to Europeans. In genome-wide analyses, we found no novel associations between common genetic variants and WHR in South Asians at P<5x10-8; variants showing equivocal association with WHR (P<1x10-5) did not replicate at P<0.05 in an independent cohort of South Asians (N = 1,922) or in published, predominantly European meta-analysis data. In the targeted analyses of 122,391 population-specific SNPs we also found no associations with WHR in South Asians at P<0.05 after multiple testing correction. Exome-wide analyses showed no new associations between genetic variants and WHR in South Asians, either individually at P<1.5x10-6 or grouped by gene locus at P<2.5x10−6. At known WHR loci, risk allele frequencies were not higher in South Asians compared to Europeans (P = 0.77), while effect sizes were unexpectedly smaller in South Asians than Europeans (P<5.0x10-8). Our findings argue against an important contribution for population-specific or cosmopolitan genetic variants underlying the increased risk of central obesity in South Asians compared to Europeans.

Comparison of allele frequencies of Plasmodium falciparum merozoite antigens in malaria infections sampled in different years in a Kenyan population.

BackgroundPlasmodium falciparum merozoite antigens elicit antibody responses in malaria-endemic populations, some of which are clinically protective, which is one of the reasons why merozoite antigens are the focus of malaria vaccine development efforts. Polymorphisms in several merozoite antigen-encoding genes are thought to arise as a result of selection by the human immune system.MethodsThe allele frequency distribution of 15 merozoite antigens over a two-year period, 2007 and 2008, was examined in parasites obtained from children with uncomplicated malaria. In the same population, allele frequency changes pre- and post-anti-malarial treatment were also examined. Any gene which showed a significant shift in allele frequencies was also assessed longitudinally in asymptomatic and complicated malaria infections.ResultsFluctuating allele frequencies were identified in codons 147 and 148 of reticulocyte-binding homologue (Rh) 5, with a shift from HD to YH haplotypes over the two-year period in uncomplicated malaria infections. However, in both the asymptomatic and complicated malaria infections YH was the dominant and stable haplotype over the two-year and ten-year periods, respectively. A logistic regression analysis of all three malaria infection populations between 2007 and 2009 revealed, that the chance of being infected with the HD haplotype decreased with time from 2007 to 2009 and increased in the uncomplicated and asymptomatic infections.ConclusionRh5 codons 147 and 148 showed heterogeneity at both an individual and population level and may be under some degree of immune selection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1304-8) contains supplementary material, which is available to authorized users.

Association of rs27044, rs30187 and rs26653 single nucleotide polymorphisms in the endoplasmic reticulum aminopeptidase 1 gene with psoriasis vulgaris in a Chinese Han population

Objective To investigate the association between endoplasmic reticulum aminopeptidase 1 (ERAP1) gene polymorphisms and psoriasis vulgaris (PsV) in a Chinese Han population. Methods Five milliliters of venous blood samples were collected from 289 patients with PsV and 292 human controls of Han nationality after informed consent. Three single nucleotide polymorphisms (SNPs) in the encoding area of the ERAP1 gene, including rs27044, rs30187 and rs26653, were genotyped by ligase detection reaction (LDR). With the PLINK 1.07 package, statistical analysis was carried out by using the chi-square test for comparisons of allele and genotype frequencies between the patient group and control group. The allelic odds ratio (OR) and its 95% confidence interval (CI) were calculated. In addition, haplotype analysis was conducted with the Haploview software. Results The frequencies of rs30187_C and rs26653_G alleles were significantly lower in the patient group (0.460 2 and 0.430 8 respectively), especially in patients with early-onset PsV (0.448 5 and 0.422 7 respectively), than in the control group (0.534 2 and 0.501 7 respectively, all P< 0.05). The SNPs rs27044, rs30187 and rs26653 showed strong linkage disequilibrium with each other (r2 ≥ 0.717, D' ≥ 0.962). Genotype analysis showed that the frequency of the rs30187 CC genotype was significantly lower in the patient group, especially in patients with early-onset PsV, than in the control group (P< 0.05 and 0.016 7 respectively) under a recessive mode of inheritance. Haplotype analysis revealed that the frequency of the haplotype H4: CTC was significantly increased in the patient group (0.050), especially in patients with early-onset PsV (0.052), compared with the control group (0.022, P< 0.05 and 0.016 7 respectively). Conclusions ERAP1 gene polymorphisms are associated with PsV, especially with early-onset PsV in Chinese Han population. The risk haplotype H4: CTC may be a susceptible factor for PsV. Key words: Psoriasis; Polymorphism, single nucleotide; Genome-wide association study; Haplotypes; Gene frequency; Genotype; Genes, ERAP1

The Effects of Both Recent and Long-Term Selection and Genetic Drift Are Readily Evident in North American Barley Breeding Populations.

Barley was introduced to North America ∼400 yr ago but adaptation to modern production environments is more recent. Comparisons of allele frequencies among growth habits and spike (inflorescence) types in North America indicate that significant genetic differentiation has accumulated in a relatively short evolutionary time span. Allele frequency differentiation is greatest among barley with two-row vs. six-row spikes, followed by spring vs. winter growth habit. Large changes in allele frequency among breeding programs suggest a major contribution of genetic drift and linked selection on genetic variation. Despite this, comparisons of 3613 modern North American cultivated barley breeding lines that differ for spike-type and growth habit permit the discovery of 142 single nucleotide polymorphism (SNP) outliers putatively linked to targets of selection. For example, SNPs within the Cbf4, Ppd-H1, and Vrn-H1 loci, which have previously been associated with agronomically adaptive phenotypes, are identified as outliers. Analysis of extended haplotype sharing identifies genomic regions shared within and among breeding populations, suggestive of a number of genomic regions subject to recent selection. Finally, we are able to identify recent bouts of gene flow between breeding populations that could point to the sharing of agronomically adaptive variation. These results are supported by pedigrees and breeders’ understanding of germplasm sharing.

Abstract P6-05-03: Genomic diversity of ductal carcinoma in situ (DCIS) as a driver of invasion and metastasis

Abstract Background: Recent evidence indicates that breast cancers may have high levels of heterogeneity. Based on principles of tumor evolution, we are investigating whether DCIS with higher levels of intra-tumoral genetic heterogeneity are more likely to progress to invasive and/or metastatic disease. Methods: Cases (DCIS with co-existing invasive or metastatic cancer) and controls (pure DCIS) are identified from Duke Pathology archives. From cases and controls, we are analyzing two areas of DCIS separated by &amp;gt;1cm with germ line DNA from the same subject to measure allele frequencies of somatic mutations and copy number variation (CNV). Small amounts of FFPE derived DNA are made into NGS libraries for full exome sequencing and hybridization to a 4.2 million element SNP array. Comparison of allele frequencies and CNV is made between specimens from the same cancer to assess heterogeneity. Results: We identified a series of pure DCIS (controls for this study) and generated high coverage sequencing data from 20ng of FFPE DNA from 12 samples (4 subjects, germline + 2 DCIS containing areas from each subject) as proof of principle. We compared the occurrence of deletions, insertions and SNP's after a 20X coverage filtration. From these data, we identified between 50 to greater than 600 sequence changes in these DCIS compared to normal DNA. Present/absent calls and allele frequencies indicate both significant and variable degrees of heterogeneity even in these pure DCIS samples. Additional data is now being gathered and analyzed based on this established technical approach. Conclusion: We have demonstrated the feasibility of acquiring high quality NGS data from archival DCIS specimens allowing us to test the hypothesis that genetic heterogeneity is a driving force in breast cancer progression. The degree and nature of this heterogeneity will be assessed in a panel of pure DCIS and DCIS co-existing with invasive and/or metastatic cancer. We are now generating and analyzing these data for Symposium presentation. Citation Format: King LM, Marks JR, Hall AH, Temko D, Graham TA, Mardis ER, Maley CC, Hwang E. Genomic diversity of ductal carcinoma in situ (DCIS) as a driver of invasion and metastasis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-05-03.

Association of MDR1 single-nucleotide polymorphisms and haplotype variants with multiple myeloma in Chinese Jiangsu Han population.

Multidrug resistance 1 (MDR1) gene encodes P-glycoprotein (P-gp), which acts as an efflux pump and provides cell protection against various substances, and its single-nucleotide polymorphisms (SNPs) are associated with the development of malignant hematologic diseases. The present study aimed at investigating whether the MDR1 SNPs and haplotype variants were correlated with the susceptibility to multiple myeloma (MM). A total of 115 MM patients and 153 healthy controls from Jiangsu Han population were enrolled and genotyped by polymerase chain reaction-allele-specific primer (PCR-ASP) method or DNA direct sequencing at MDR1 loci of C1236T, G2677T/A, and C3435T. No significance was found in the distribution of alleles and genotypes in MDR1 three loci. Diplotype analysis has also demonstrated no effect in susceptibility to MM. But, in haplotype analysis, the haplotype of T-G-T was significantly more common than healthy controls (12.6% in MM group vs. 1.7% in control group, odds ratios (ORs) = 8.7, 95% confidence interval (CI) 3.3-22.8, Pc < 0.01). Our results pointed out that comparable allele, genotype, and diplotype frequencies among MM patients and controls in Chinese Jiangsu Han population were found; the frequency of T-G-T haplotype was significantly increased in MM group compared with the control group, which indicated that this haplotype might be associated with the susceptibility to MM.

NALP3-Inflammasome-Related Gene Polymorphisms in Patients with Prehypertension and Coronary Atherosclerosis.

Objectives. Prehypertension is an early stage of hypertension that is characterized by inflammatory factors. Inflammation also plays an essential role in the development of coronary atherosclerosis (CAS). The present study evaluated the NALP3-inflammasome and its related genes, NLRP3, NOD2, and CARD8, using SNP linkage and gene haplotypes in prehypertensive patients. Methods. A total of 576 patients with prehypertension and suspected coronary heart disease (CHD) were enrolled. According to coronary angiography, patients were divided into two groups: arterial stenosis <50% of the diameter (control) and arterial stenosis >50% of the diameter (case). Fifteen polymorphisms in the NOD2, NLRP3, and CARD8 genes were analyzed, and serum levels of C-reactive protein (CRP) were measured. Results. When comparing allele frequencies, none of these 15 SNPs in NOD2, CARD8, and NLPR3 genes showed a significant difference using multiple logistic regression. However, the CTACATAA (p = 0.0064) and CCACATAG (p = 0.0126) haplotypes of the NOD2 gene SNPs were significantly different between cases and controls. Conclusions. Although our study excludes a significant association of selected SNPs in these genes with CHD in prehypertension patients, this work suggests that the CTACATAA and CCACATAG haplotypes were associated with CHD in the NOD2 locus. This work suggests that the CTACATAA and CCACATAG haplotypes were associated with CHD in prehypertension patients in the NOD2 locus.

Killer Cell Immunoglobulin-like Receptor Genotype and Haplotype Investigation of Natural Killer Cells from an Australian Population of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis Patients.

Killer cell immunoglobulin-like receptor (KIR) genes encode for activating and inhibitory surface receptors, which are correlated with the regulation of Natural Killer (NK) cell cytotoxic activity. Reduced NK cell cytotoxic activity has been consistently reported in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) patients, and KIR haplotypes and allelic polymorphism remain to be investigated. The aim of this article was to conduct a pilot study to examine KIR genotypes, haplotypes, and allelic polymorphism in CFS/ME patients and nonfatigued controls (NFCs). Comparison of KIR and allelic polymorphism frequencies revealed no significant differences between 20 CFS/ME patients and 20 NFCs. A lower frequency of the telomeric A/B motif (P < 0.05) was observed in CFS/ME patients compared with NFCs. This pilot study is the first to report the differences in the frequency of KIR on the telomeric A/B motif in CFS/ME patients. Further studies with a larger CFS/ME cohort are required to validate these results.

Variation in the Zinc Finger of PRDM9 is Associated with the Absence of Recombination along Nondisjoined Chromosomes 21 of Maternal Origin.

Variation in the zinc finger-binding domain (ZFBD) of the protein PR Domain-Containing Protein 9 (PRDM9) is associated with altered placement of recombination in the human genome. As both the absence and altered placement of recombination are observed among chromosomes 21 that nondisjoin, we genotyped the PRDM9 ZFBD among mothers of children with Trisomy 21 in efforts to determine if variation within this region is associated with the recombination-related risk for chromosome 21 nondisjunction (NDJ). In our approach, PCR was used to amplify the ZFBD of PRDM9 and products were then subjected to bi-directional Sanger sequencing. DNA sequencing reads were aligned and compared to the sequence of the PRDM9 alleles previously identified. Chi-Square analysis was used to compare allele frequencies between cases (N=235, mothers of children with maternally-derived Trisomy 21) and controls (N=48, fathers of children with maternally-derived Trisomy 21). Results of our analysis showed that the frequency of PRDM9 ZF minor alleles is significantly increased among women displaying NDJ of chromosome 21 and no recombination on 21q (p=0.02). Even more, when compared to those for the PRDM9 major A-allele, these minor alleles displayed fewer predicted binding sites on 21q. These findings suggest that allelic variation in the ZF of PRDM9 may play a role in the risk for chromosome 21 NDJ by leading to reduced recombination on 21q.

Association of single-nucleotide polymorphisms rs2197076 and rs2241883 of FABP1 gene with polycystic ovary syndrome.

The objective of this study was to evaluate the association between single-nucleotide polymorphisms (SNPs) rs2197076 and rs2241883 in fatty acid-binding protein 1 (FABP1) gene and polycystic ovary syndrome (PCOS). The two alleles rs2197076 and rs2241883 in FABP1 gene in 221 PCOS women and 198 normal women were amplified and sequenced. Allele frequency comparison was performed between the PCOS and control groups, and genotype-phenotype correlation analysis was performed using dominant and recessive models to assess the association of FABP1 and the main features of PCOS. Allele frequency analyses showed a strong association of SNPs rs2197076 and rs2241883 of FABP1 gene with PCOS (P < 0.001). The additive, dominant, and recessive genotype model analyses further supported this association even after adjusting for age and body mass index (BMI). The minor allele frequency (MAF) of rs2241883 in obese PCOS women was less than that in obese control women. Further genotype-phenotype correlation analysis showed that SNP rs2197076 had a stronger association with the main features of PCOS than SNP rs2241883. In the association of SNPs in FABP1 gene with PCOS, rs2197076 was more closely associated with its main features than rs2241883 and seemed to play a more important role in the pathogenesis of PCOS.

Polymorphisms Associated with the Arab-Indian Haplotype of Sickle Cell Anemia Are Candidate Fetal Hemoglobin Gene Modulators

Fetal hemoglobin (HbF) inhibits HbS polymerization. Because of this, sufficient HbF in most sickle erythrocytes can lead to a milder disease phenotype. HbF levels differ amongst the β-globin gene (HBB) cluster haplotypes of sickle cell anemia. In the Arab-Indian (AI) haplotype, HbF was about 20% compared with 5-10% in the Bantu, Benin, and Senegal haplotypes. Functional elements linked to the HBB haplotype are likely to regulate the expression of HbF in addition to the effects of trans-acting modulators. To identify cis-acting SNPs in the HBB gene cluster that differentiate the AI haplotype from all others, including the Senegal haplotype-the Senegal haplotype shares some SNPs with the AI haplotype but its carriers have lower HbF-we studied patients with sickle cell anemia who were homozygous for HBB haplotypes by genome-wide SNP association analysis (GWAS; Table). First, we compared the results of GWAS of 42 Saudi AI haplotype homozygotes with GWAS in 71 Saudi Benin haplotype homozygotes. The only variants distinguishing these 2 populations with genome-wide significance (p-values between 9.6E-07 and 2.7E-45) were 223 SNPs in chromosome 11p15 from positions 3.5 to 6.5 mb. This region included the HBB gene cluster, its locus control region (LCR) and the upstream and downstream olfactory receptor gene clusters. The minor allele frequency of SNPs in MYB (chr 6q23), BCL11A (chr 2p16) and KLF1 (chr 19), trans-acting loci that affect expression of the HbF genes, were similar in these 2 cohorts. A novel candidate trans-acting locus was not found, however our power to detect such an association was low. We followed-up these observations by comparing allele frequencies in 303 African American cases homozygous for the haplotypes shown in the Table. Thirteen GWAS-significant SNPs, in addition to rs7482144 and rs10128556, were present in all AI haplotype cases but not in 83 Senegal haplotype chromosomes. The allele frequency of these SNPs was replicated in 62 independent AI haplotype cases. Rs2472530 is in the coding region of OR52A5; rs16912979, rs4910743 and rs4601817 are in the HBB gene cluster LCR; rs16912979 in DNase I hypersensitive site-4 altered motifs for POLR2A, GATA1, and GATA2 binding.The minor allele of rs10837771 causes a missense mutation in OR51B4 an upstream olfactory receptor gene. To see if any of these or other alleles might sometimes be associated with HbF in the Bantu and Benin haplotyes, we selected homozygotes and compound heterozygous for these haplotypes who had unexplained and uncharacteristically high HbF. Thirty-one African Americans, aged ≥5 yrs. who had a HbF of 21% were compared with 350 similar cases who had a mean HbF of 3%. Four additional SNPs on chromosome 11, from positions ranging from 5536415 to 5543705 in the UBQLNL/HBG2, region and present in 45-48% of AI haplotype and 3-13% of other haplotypes, were found at higher frequencies in the high HbF group compared with the low HbF group. These SNPs also altered transcription factor binding motifs. Loci marked by SNPs that distinguish the AI from the Senegal and other HBB haplotypes might contain functionally important polymorphisms and account in part for high HbF in AI haplotype sickle cell anemia, independent of, or in addition to, the effects associated with rs7482144 or rs10128556. They might also be rarely associated with high HbF found in other haplotypes. These observations provide a foundation for mechanistic studies focused on the role of these variants in the expression of their linked HbF genes. Table 1non-codedallelegenomic locationSaudi AI(n=42)Saudi ben.ben(n=71)AA ben.ben(n=264)AA ban.ban(n=31)AA sen.sen(n=8)HbF (%)1711669rs10837771Gexon OR51B410.020.0200rs4601817GLCR10.020.0400rs4910743CLCR10.010.0100rs16912979CLCR00.960.920.111rs10488675Gintron HBE110.01000rs7482144*AHBG210001rs10128556#TIntron HBBP110001rs7935470COR51V110.020.0300rs10837582GOR51V1100.0200rs11036227TOR51V110000rs10734485COR51A1P00.990.9711rs10837461AOR52A110.01000rs2472522GOR52A110.01000rs2472530Gexon OR52A510.01000rs2499948TOR52A510.020.010.020Allele frequencies in haplotypes of sickle cell anemia. * Xmn1 5' HBG 2 restriction site. This SNP, not present on the SNP microarray, was genotyped independently; # LD with rs7482144; AA designates African Americans; ben-Benin; ban-Bantu; sen-Senegal. DisclosuresNo relevant conflicts of interest to declare.

Exome Capture with Heterologous Enrichment in Pig (Sus scrofa).

The discovery of new protein-coding DNA variants related to carcass traits is very important for the Italian pig industry, which requires heavy pigs with higher thickness of subcutaneous fat for Protected Designation of Origin (PDO) productions. Exome capture techniques offer the opportunity to focus on the regions of DNA potentially related to the gene and protein expression. In this research a human commercial target enrichment kit was used to evaluate its performances for pig exome capture and for the identification of DNA variants suitable for comparative analysis. Two pools of 30 pigs each, crosses of Italian Duroc X Large White (DU) and Commercial hybrid X Large White (HY), were used and NGS libraries were prepared with the SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library (Agilent). A total of 140.2 M and 162.5 M of raw reads were generated for DU and HY, respectively. Average coverage of all the exonic regions for Sus scrofa (ENSEMBL Sus_scrofa.Sscrofa10.2.73.gtf) was 89.33X for DU and 97.56X for HY; and 35% of aligned bases uniquely mapped to off-target regions. Comparison of sequencing data with the Sscrofa10.2 reference genome, after applying hard filtering criteria, revealed a total of 232,530 single nucleotide variants (SNVs) of which 20.6% mapped in exonic regions and 49.5% within intronic regions. The comparison of allele frequencies of 213 randomly selected SNVs from exome sequencing and the same SNVs analyzed with a Sequenom MassARRAY® system confirms that this “human-on-pig” approach offers new potentiality for the identification of DNA variants in protein-coding genes.

Abstract 322: Establishment and genomic characterization of enteroid cultures from human colonic adenomas and adenocarcinomas

Abstract Introduction Characterized enteroid cultures of human colon cancer can more precisely model the diversity of colonic neoplasia for the study of cancer initiation, progression and potentially prevention. Using tissue from colon resections and endoscopic biopsies, we have successfully isolated and cultured 14 colorectal adenomas and 2 adenocarcinomas to date. We have maintained these enteroid cultures for up to 2 years and established a working cryorepository. Specific epithelial cell lineage markers and the stem cell marker Lgr5 can be detected throughout the culture period. Methods Enteroid cultures have been initiated and maintained in a serum-free medium containing EGF and pituitary extract. However, approximately half of all neoplasms do not establish in this reduced medium. In contrast, most neoplasms develop and expand in an enriched culture medium containing serum, Wnt, R-spondin, Noggin, and EGF. We have also created a mouse xenograft from an adenoma expanded in the reduced media; this graft was then successfully reintroduced into culture. Using whole exome sequencing, we are investigating how the genetic background of individual patients contributes to 1) variability in the establishment and expansion of enteroid cultures 2) tumor heterogeneity in neoplasms and xenografts, and 3) the stability of genomic signatures in enteroids over time in culture. Results Twenty-two damaging somatic variants identified in a single colon tumor were preserved in enteroid culture after 2 months in reduced medium. Variants included a frameshift mutation in APC and missense mutations in KRAS and TP53. The allele frequency of most variants increased in enteroid culture, suggesting that cells lacking these mutations failed to propagate (including stromal/immune cells), or cells carrying these mutations expanded at a faster rate. Ten mutations not present in the original tumor were acquired over time in enteroid culture. These mutations included a missense variant of TRPS1, a putative prognosticator of colon cancer. The mutations acquired in enteroid culture may reflect genetic instability in the source neoplastic tissue, or the emergence of subpopulations that were below level of detection in the source tissue. Three mutations were present in the tumor and lost in enteroid culture; this presumably reflects the loss of a subset of cells expressing these variants. Conclusion Changes in allele frequencies suggest that neoplasms are heterogeneous, with shifting cell populations that are differentially affected by culture conditions. This heterogeneity can be further interrogated by comparing allele frequencies in the original tumor with those in enteroids established in reduced or enriched media. This platform can provide further understanding of genetic determinants that underlie the risk for colorectal cancer, as well as strategic insights into the enteroid model as a sophisticated system for the study of tumor biology. Citation Format: Michael K. Dame, Shannon D. McClintock, Durga Attili, Becky Simon, Kelly Copley, Stacy Finkbeiner, Christopher Altheim, Jason Spence, Henry Appelman, D Kim Turgeon, Linda C. Samuelson, Dean E. Brenner, James Varani. Establishment and genomic characterization of enteroid cultures from human colonic adenomas and adenocarcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 322. doi:10.1158/1538-7445.AM2015-322

Abstract 2692: High allele frequency of KRAS functional mutations predicts resistance to MEK inhibitors: Evidence from cell lines and human tumor xenograft models

Abstract Introduction KRAS mutant tumors represent a high unmet need for effective therapy. Even though there is no targeted therapy for KRAS, inhibitors of downstream targets like MEK have shown efficacy in preclinical studies. However, clinical trials of MEK inhibitors in KRAS-mutant tumors have not been successful, although there have been some responders. We sought to understand resistance to MEK inhibition in KRAS-mutant tumors and to develop biomarkers for patient stratification. Results Using DNA and RNA sequencing data from cell lines, we found that high variant allele frequency (&amp;gt;50%) of KRAS functional mutations (G12, G13, Q61) was associated with baseline resistance to MEK inhibitors AZD6244 and MEK162 across multiple tumor types. Low allele frequency of KRAS mutation was found to be necessary but not sufficient for sensitivity to MEK inhibitors, suggesting the existence of additional resistance mechanisms. Comparison of copy number and variant allele frequency of KRAS showed that MEK inhibitor-resistant cell lines have either amplified mutant alleles or lost WT alleles. This prediction of high KRAS mutant allele frequency being associated with MEKi resistance was successfully validated in vivo in 19 primary colorectal tumor xenograft models treated with MEK162. High KRAS mutant allele frequency and high KRAS expression were also associated with acquired resistance to BKM120+MEK162 combination treatment in an in vivo model of HeyA8, an ovarian cell line with a KRAS G12D mutation. This prediction was also supported by the observation in a Phase II clinical trial with MEK inhibitor refametinib + gemcitabine in pancreatic cancer [1]. Patients with disease progression had a significantly higher KRAS mutant allele frequency compared to partial responders and stable disease. Conclusion High allele frequency of KRAS functional mutations is associated with resistance to MEK inhibitors. Excluding patients with high KRAS mutant allele frequency has the potential to improve clinical trial outcomes and benefit patients, with the added benefit that it may not require any additional screening or novel biomarkers.

Association of genetic variants with dyslipidemia.

Although genetic variants, which regulate lipid metabolism, have been extensively investigated in Caucasian populations, the genes, which confer susceptibility to dyslipidemia in Japanese individuals, remain to be elucidated. The aim of the present study was to examine a possible association among hypertriglyceridemia, hypo‑high density lipoprotein (HDL)‑cholesterolemia or hyper‑low density lipoprotein (LDL)‑cholesterolemia in Japanese individuals with 29 polymorphisms observed to confer susceptibility for coronary heart disease. This was performed through meta‑analyses of genome‑wide association studies in Caucasian populations. The study population comprised 2,354 individuals with dyslipidemia (hypertriglyceridemia, hypo‑HDL‑cholesterolemia or hyper‑LDL‑cholesterolemia) and 3,106 control individuals. To compensate for multiple comparisons of genotypes, a false discovery rate (FDR) of <0.05 was adopted to determine the statistical significance of the associations. Comparisons of allele frequencies using the χ2 test revealed that rs964184 of zinc finger gene (ZPR1; FDR=2.1x10‑7), rs4845625 of interleukin 6 receptor (IL6R; FDR=0.032), rs46522 of ubiquitin‑conjugating enzyme E2Z gene (UBE2Z; FDR=0.032) and rs17514846 of furin (FDR=0.041) were significantly associated with hypertriglyceridemia. The χ2 test revealed that rs599839 of proline/serine‑rich coiled‑coil 1 (PSRC1; FDR=0.004) and rs2075650 of translocase of outer mitochondrial membrane 40 homolog (TOMM40; FDR=0.004) were significantly associated with hyper‑LDL‑cholesterolemia. Multivariate logistic regression analysis with adjustment for age, gender and body mass index revealed that rs964184 of ZPR1 (P=5.1x10‑7; odds ratio, 1.37; dominant model), rs4845625 of IL6R (P=0.0019, odds ratio, 1.25; dominant model) and rs46522 of UBE2Z (P=0.0039, odds ratio, 1.19; dominant model) were significantly associated with hypertriglyceridemia, and that rs599839 of PSRC1 (P=0.0004, odds ratio, 0.70; dominant model) and rs2075650 of TOMM40 (P=0.0004, odds ratio, 1.43; dominant model) were significantly associated with hyper‑LDL‑cholesterolemia. Therefore, ZPR1, IL6R, and UBE2Z may be susceptibility loci for hypertriglyceridemia, whereas PSRC1 and TOMM40 may be such loci for hyper-LDL-cholesterolemia in Japanese individuals.