UTR SNP Research Articles

Identification and analysis of a SMAD3 cis-acting eQTL operating in primary osteoarthritis and in the aneurysms and osteoarthritis syndrome

SummaryObjectiveThe TGF-β pathway plays a central role in joint development with polymorphism in TGF-β pathway genes implicated in osteoarthritis susceptibility. One association is to rs12901499, within intron 1 of SMAD3. Since rs12901499 is not in linkage disequilibrium with a non-synonymous polymorphism, it is likely the association is operating by influencing expression of SMAD3.DesignUsing tissues from the joints of primary osteoarthritis patients who had undergone joint replacement we measured the overall expression of SMAD3 by quantitative real-time PCR. We also measured allelic expression of SMAD3 using these tissues and vascular smooth muscle cells from patients with aneurysms and osteoarthritis syndrome, a rare condition featuring early-onset osteoarthritis. We tested the functional effect of SNPs in vitro using luciferase assays and assessed association with osteoarthritis using a large osteoarthritis case–control dataset.ResultsWe observed that genotype at rs12901499 did not correlate with overall SMAD3 expression or allelic expression. However, genotype at a 3′UTR SNP, rs8031440, did correlate with SMAD3 expression in cartilage (P = 0.005) which was supported by allelic expression data showing that the G allele correlated with decreased SMAD3 expression in joint tissues and vascular smooth muscle cells. This G allele was underrepresented in osteoarthritis cases vs controls (P = 0.027, odds ratio = 0.921). rs8031440 is in perfect linkage disequilibrium with five other SMAD3 3′UTR SNPs and our luciferase analysis identified rs3743342 and rs12595334 as being functional.ConclusionSMAD3 is subject to cis-acting regulatory polymorphism in the tissues of relevance to both primary osteoarthritis and the aneurysms-osteoarthritis syndrome.

Association of four insulin resistance genes with type 2 diabetes mellitus and hypertension in the Chinese Han population

Insulin resistance plays an important role in the development of type 2 diabetes mellitus (T2DM) and hypertension. The purpose of the present study was to evaluate the association between four insulin resistance genes (ADIPOQ, LEPR, RETN, and TRIB3) and both T2DM and hypertension. A total of 768 Han Chinese subjects were recruited into this study, including 188 cases who had T2DM alone, 223 cases who had hypertension alone, 181 cases with both T2DM and hypertension, and 176 control subjects with neither T2DM nor hypertension. Twenty-three tag SNPs in four insulin resistance genes were genotyped and analyzed for association with T2DM and hypertension. One intron SNP (rs13306519) in LEPR and one 3′UTR SNP (rs1063537) in ADIPOQ demonstrated a significant association with T2DM (P = 0.024 and 0.014 respectively). Another intron SNP (rs12037879) in LEPR and a promoter region SNP (rs266729) in ADIPOQ were significantly associated with hypertension (P = 0.041 and 0.042, respectively). These associations survived the permutation test (P = 0.023, 0.018, 0.026, and 0.035, respectively). These associations were still found to be significant in the additive model after adjusting for potential confounding factors including age, sex, BMI, HDL, LDL, total cholesterol, and triglyceride levels (P = 0.024, 0.016, 0.04, and 0.043, respectively). No other gene variants were found to be significantly associated with T2DM or hypertension (P > 0.05). None of the studied gene variants were found to be significantly associated with T2DM+ hypertension (P > 0.05). A significant interaction was observed between two SNPs rs13306519 in LEPR and rs266729 in ADIPOQ for T2DM (P_int = 0.012, OR_int = 2.67) and hypertension (P_int = 0.0041, OR_int = 2.23). These findings suggest that variants in ADIPOQ and LEPR are risk factors for T2DM and hypertension in the Chinese population and that variants in RETN and TRIB3 are not major risk factors for these diseases.

Associations between variants of FADS genes and omega-3 and omega-6 milk fatty acids of Canadian Holstein cows

BackgroundFatty acid desaturase 1 (FADS1) and 2 (FADS2) genes code respectively for the enzymes delta-5 and delta-6 desaturases which are rate limiting enzymes in the synthesis of polyunsaturated omega-3 and omega-6 fatty acids (FAs). Omega-3 and-6 FAs as well as conjugated linoleic acid (CLA) are present in bovine milk and have demonstrated positive health effects in humans. Studies in humans have shown significant relationships between genetic variants in FADS1 and 2 genes with plasma and tissue concentrations of omega-3 and-6 FAs. The aim of this study was to evaluate the extent of sequence variations within these two genes in Canadian Holstein cows as well as the association between sequence variants and health promoting FAs in milk.ResultsThirty three SNPs were detected within the studied regions of genes including a synonymous mutation (FADS1-07, rs42187261, 306Tyr > Tyr) in exon 8 of FADS1, a non-synonymous mutation (FADS2-14, rs211580559, 294Ala > Val) within FADS2 exon 7, a splice site SNP (FADS2-05, rs211263660), a 3′UTR SNP (FADS2-23, rs109772589), and another 3′UTR SNP with an effect on a microRNA binding site within FADS2 gene (FADS2-19, rs210169303). Association analyses showed significant relations between three out of seven tested SNPs and several FAs. Significant associations (FDR P < 0.05) were recorded between FADS2-23 (rs109772589) and two omega-6 FAs (dihomogamma linolenic acid [C20:3n6] and arachidonic acid [C20:4n6]), FADS1-07 (rs42187261) and one omega-3 FA (eicosapentaenoic acid, C20:5n3) and tricosanoic acid (C23:0), and one intronic SNP, FADS1-01 (rs136261927) and C20:3n6.ConclusionOur study has demonstrated positive associations between three SNPs within FADS1 and FADS2 genes (a SNP within the 3’UTR, a synonymous SNP and an intronic SNP), with three milk PUFAs of Canadian Holstein cows thus suggesting possible involvement of synonymous and non-coding region variants in FA synthesis. These SNPs may serve as potential genetic markers in breeding programs to increase milk FAs that are of benefit to human health.

Association of HSPA1B SNP rs6457452 with Alopecia Areata in the Korean Population

The heat shock 70 kDa protein 1B (HSPA1B), which has been well-studied among the famous heat shock proteins HSPA1A/B/L, is related to autoimmune diseases, including Alopecia Areata (AA). In this study, the association of a 5’-untranslated region (5’UTR) SNP rs6457452 and a promoter SNP rs2763979 (‐1140C > T) of HSPA1B with AA was investigated in 236 controls and 228 AA patients. Statistical analyses using the multiple logistic models were done, according to the onset and the clinical features of AA, including the age of onset, family history, type of AA lesion, nail involvement and body hair involvement. The results showed that rs6457452 was associated with the onset of AA (p < 0.002). In the analysis of clinical features of AA, rs6457452 was weakly related to the age of onset (p ≤ 0.04) and that rs2763979 was only weakly related to the type of AA lesion (p = 0.041). In conclusion, we suggest that the 5’UTR SNP rs6457452 of HSPA1B may be associated with the onset of AA and the T allele of rs6457452 may confer the reduced susceptibility to AA in the Korean population.

Molecular Characterization Of a Patient With Thrombocytopenia-Absent Radii (TAR) Syndrome and Diffuse Langerhans Cell Histiocytosis (LCH): Novel Genetic Findings

TAR is a rare bone marrow failure syndrome comprised of thrombocytopenia and a spectrum of bony abnormalities, the most common being bilateral radial aplasia in the presence of thumbs. Recent research has implicated alterations in the RBM8A gene in the pathogenesis of this disorder. Diffuse LCH is a neoplasm of mature Langerhans cells, a subset of dendritic cells, that has now been demonstrated to be clonal in nature. The co-occurrence of these two rare disorders has only been reported twice (Ingram, BMT 2006; Guastadisegni EJMG 2012). We report the third case of a patient with TAR and LCH and present the molecular and genetic characterization of this patient. A 52 year woman with TAR presented with abdominal mass, splenomegaly and left axillary adenopathy. CT scan showed a right pelvic mass measuring 11.5 x 8.6 cm, retroperitoneal lymphadenopathy, enlarged spleen and patchy areas of radiolucency throughout the spine, pelvis and left femoral head. Excisional biopsy of the left axillary lymph node revealed complete effacement of the normal lymph node architecture by an atypical proliferation of Langerhans cell histiocytes. Bone marrow involvement was documented as well. On immunohistochemistry, these cells were positive for CD1a (membrane staining) and S-100 (nuclear and cytoplasmic staining). As part of the diagnostic evaluation, CGH microarray (Signature Genomics) was performed. A diagnosis of diffuse LCH was made. She was treated with vinblastine and prednisone for a year and remains in complete remission from her disease 14 months later. Genomic DNA was extracted from peripheral blood (normal mononuclear cells) and paraffin-embedded tumor tissue, with written informed consent following the approval of the Institutional Review Board of Thomas Jefferson University. The CGH result on the patient's uninvolved tissue as well as the tumor sample showed a hemizygous deletion of 1q21.1 encompassing the RBM8A gene, consistent with recent TAR literature (Albers, Nat Genet 2012). Sequence analysis of the other RBM8A allele revealed minor allele A at SNP rs139428292 at position chr1:145,507,646 (hg19) in the 5'UTR of RBM8A and nt G at chr1:145,507,777 in the first intron (int1 +44). The coding sequence was normal. Minor allele A at the 5'UTR SNP has been associated with TAR syndrome, along with minor allele C at intronic SNP (rs 201779890, int1 +32) which was major allele G in our patient. However, nt G at intron 1 +44 is a novel variant that has not been reported before in dbSNP137, HapMap or 1000 Genomes (UCSC Genome Browser, August 1, 2013). The CGH result on the patient's tumor sample showed a 1.82 Kb deletion within the MAF gene. We identified in normal and tumor DNA one intact MAF gene and one deleted of all of the coding sequences of MAF isoform 1 (alternatively, complete deletion of exon 1 of MAF isoform 2). In addition, a copy loss in the 14q32.2 region was found in the tumor sample but not in the uninvolved tissue. This region contains the BCL11b gene. Finally, we identified a tumor-specific missense mutation (GTG to GAG) resulting in a change in amino acid from valine to glutamine at codon 600. This is the BRAF V600E mutation previously described in LCH and other malignancies.In summary, we report the third ever reported case of the co-occurrence of two rare disorders, TAR and LCH, in a single patient and identify novel molecular changes. We have found the patient to have the recently reported RBM8A genetic alterations of TAR (microdeletion of 1q21 for one allele, minor 5'UTR SNP on the other allele), plus a novel RBM8A intronic SNP. We found a somatic MAF deletion in our patient, possibly a predisposing factor towards LCH. In addition, BRAF V600E mutation is a tumor-specific mutation in our patient. Whether BRAF inhibitors will be of therapeutic benefit in primary or relapsed LCH is currently unknown. Future research is needed to elucidate the function, if any, of the novel RBM8A SNP, the mechanism(s) by which the identified SNPs lead to reduced Y14 levels, and the potential contribution of Y14 deficiency in tumorigenesis.The authors wish to acknowledge the invaluable role of Dr. Elena Gitelson in the care of this patient. Disclosures:No relevant conflicts of interest to declare.

A functional polymorphism of PON1 interferes with microRNA binding to increase the risk of ischemic stroke and carotid atherosclerosis

ObjectiveSingle nucleotide polymorphisms (SNPs) located at microRNA (miRNA) binding sites (miR-SNPs) can affect the expression of genes. This study aimed to identify the miR-SNPs associated with atherosclerosis and stroke. MethodsPatients with ischemic stroke (n = 657) and stroke- and myocardial infarction-free volunteers (n = 1571) were enrolled. The carotid intima-media thickness (IMT) was measured in the control participants. Seventy-nine stroke susceptibility genes were initially selected and 13 genes were predicted to have miR-SNPs at their 3′ untranslated regions (3′UTR). The miRNA arrays were used to further identify potential miR-SNPs. The miR-SNP rs3735590 at the paraoxonase 1 (PON1) gene was finally selected and its associations with stroke and carotid IMT were evaluated. The 3′UTR reporter and SNP functional assays were then performed to validate the results. ResultsCompared with CC genotype, patients with CT or TT genotype at rs3735590 had lower risk of ischemic stroke (OR = 0.72, p = 0.036; OR = 0.83, p = 0.077, respectively). Among the healthy participants, the CT or TT genotype was associated with thinner IMT in the internal carotid arteries in comparison with CC genotype (β = −0.76, p = 0.003; β = −0.022, p = 0.452, respectively). Our findings suggested that the minor allele T had a protective effect on atherosclerosis. Results from 3′UTR reporter assays showed that PON1 is a direct target gene of miR-616. In plasmid constructs carrying the risk allele C at rs3735590, miR-616 inhibited the genetic expression of PON1. However, substitution of C by T at rs3735590 reduced the miR-616 binding affinity, leading to overexpression of the PON1 gene. ConclusionOur study is the first to show that the miR-SNP at PON1 could affect genetic expression and is associated with an elevated risk for ischemic stroke and subclinical atherosclerosis.

Nucleotide variation in IL‐10 and IL‐12 and their receptors and cervical and vulvar cancer risk: A hybrid case–parent triad and case–control study

Given the important role of cell mediated immunity in viral clearance and control of premalignant lesions, we hypothesize that variation in the IL-12/IL-10 cytokine and cytokine receptor genes may influence cervical and vulvar cancer risk. We evaluated 76 tagSNPs from seven candidate genes (IL-10, IL-12A, IL-12B, IL-10RA, IL-10RB, IL-12RB1, and IL12RB2) in case-parent sets (n=43 cervical squamous cell carcinoma (SCC), n=96 cervical adenocarcinoma, n=53 vulvar SCC), additional cases (n=356 cervical SCC, n=406 cervical adenocarcinoma, and n=473 vulvar SCC) and population based controls (1,111). We calculated log-additive odds ratios (ORs) and 95% confidence intervals (CIs) for the association between tagSNP and cancer risk using a pseudo-likelihood based method which combined genotype information on cases, parents, and population controls. After correction for multiple comparisons, we identified several statistically significant SNP associations. Cervical SCC risk was associated with the minor alleles of the IL10RA rs9610 3' UTR SNP (OR=1.76, 95% CI=1.15-2.68) and two synonymous IL12RB2 SNPs (rs4297265, OR=0.46, 95% CI=0.26-0.82; rs2229546, OR=0.43, 95% CI=0.21-0.87). Cervical adenocarcinoma risk was associated with the minor alleles of the IL10RA rs4252314 intronic SNP (OR=2.23, 95% CI=1.26-3.96) and IL12RB1 rs11575934 non-synonymous SNP (OR=1.51, 95% CI=1.12-2.05). Finally, the minor allele of the IL12B rs3181224 3' UTR SNP was associated with a reduced risk of vulvar SCC (OR=0.30, 95% CI=0.12-0.74). These results raise the possibility that a shift in the balance of the immune response due to genetic variants in key cytokine genes could influence the development of cervical and vulvar cancer.

Allele specific expression of the transthyretin gene in swedish patients with hereditary transthyretin amyloidosis (ATTR V30M) is similar between the two alleles.

BackgroundHereditary transthyretin (TTR) amyloidosis (ATTR) is an autosomal dominant disease characterized by extracellular deposits of amyloid fibrils composed of misfolded TTR. The differences in penetrance and age at onset are vast, both between and within populations, with a generally late onset for Swedish carriers. In a recent study the entire TTR gene including the 3′ UTR in Swedish, French and Japanese ATTR patients was sequenced. The study disclosed a SNP in the V30M TTR 3′ UTR of the Swedish ATTR population that was not present in either the French or the Japanese populations (rs62093482-C>T). This SNP could create a new binding site for miRNA, which would increase degradation of the mutated TTR’s mRNA thus decrease variant TTR formation and thereby delay the onset of the disease. The aim of the present study was to disclose differences in allele specific TTR expression among Swedish V30M patients, and to see if selected miRNA had any effect upon the expression.Methodology/Principal FindingsAllele-specific expression was measured on nine liver biopsies from Swedish ATTR patients using SNaPshot Multiplex assay. Luciferase activity was measured on cell lines transfected with constructs containing the TTR 3′ UTR. Allele-specific expression measured on liver biopsies from Swedish ATTR patients showed no difference in expression between the two alleles. Neither was there any difference in expression between cell lines co-transfected with two constructs with or without the TTR 3′ UTR SNP regardless of added miRNA.Conclusions/SignificanceThe SNP found in the 3′ UTR of the TTR gene has no effect on degrading the variant allele’s expression and thus has no impact on the diminished penetrance of the trait in the Swedish population. However, the 3′ UTR SNP is unique for patients descending from the Swedish founder, and this SNP could be utilized to identify ATTR patients of Swedish descent.

Low prevalence of connexin-40 gene variants in atrial tissues and blood from atrial fibrillation subjects.

BackgroundThe atrial gap junction protein connexin-40 (Cx40) has been implicated to play an important role in atrial conduction and development of atrial fibrillation (AF). However, the frequency of Cx40 mutations in AF populations and their impact on Cx40 expression remains unclear. In this study, we sought to identify polymorphisms in the Cx40 gene GJA5, investigate the potential functional role of these polymorphisms, and determine their allelic frequencies. The prevalence of nonsynonymous Cx40 mutations in blood and atrial tissue was also compared to mutation frequencies reported in prior studies.MethodsWe conducted direct sequencing of the GJA5 coding and 3′ UTR regions in blood samples from 91 lone AF subjects and 67 atrial tissue-derived samples from a lone cohort, a mixed AF cohort, and several transplant donors. Reporter gene transfection and tissue allelic expression imbalance assays were used to assess the effects of a common insertion/deletion polymorphism on Cx40 mRNA stability and expression.ResultsWe identified one novel synonymous SNP in blood-derived DNA from a lone AF subject. In atrial tissue-derived DNA from lone and mixed AF subjects, we observed one novel nonsynonymous SNP, one rare previously reported synonymous SNP, and one novel 3′ UTR SNP. A previously noted 25 bp insertion/deletion polymorphism in the 3′ UTR was found to be common (minor allele frequency = 0.45) but had no effect on Cx40 mRNA stability and expression. The observed prevalence of nonsynonymous Cx40 mutations in atrial tissues derived from lone AF subjects differed significantly (p = 0.03) from a prior atrial tissue study reporting a high mutation frequency in a group of highly selected young lone AF subjects.ConclusionsOur results suggest that Cx40 coding SNPs are uncommon in AF populations, although rare mutations in this gene may certainly lead to AF pathogenesis. Furthermore, a common insertion/deletion polymorphism in the Cx40 3′ UTR does not appear to play a role in modulating Cx40 mRNA levels.

A 5′UTR SNP of GHRHR locus is associated with body weight and average daily gain in Chinese cattle

Growth hormone-releasing hormone receptor (GHRHR) has important functions in the regulation of the growth hormone axis and the development and proliferation of pituitary somatotropes. Moreover, some mutations in mouse GHRHR can induce the dwarfism. The objective of this paper is to reveal the association of GHRHR with growth traits in three Chinese cattle breeds, including Nanyang cattle (NY, 220), Qinchuan cattle (QC, 114), and Jiaxian cattle (JX, 142). A novel single nucleotide polymorphism (NM_181020:c.102C>T) in 5'UTR of GHRHR was identified using PCR-SSCP and DNA sequencing. The frequency of NM_181020:c.102C allele ranged from 0.926 to 0.956. We found that the locus was significantly associated with NY cattle's body weight (BW) of 6 months, with average daily gain (ADG) of 0-6 months, and as well as with ADG of 6-12 months (p < 0.05). The data suggested that the polymorphism (NM_181020:c.102C>T) of the GHRHR could be a molecular marker candidate for breeding of NY cattle in favor of BW.

A MicroRNA-7 Binding Site Polymorphism in HOXB5 Leads to Differential Gene Expression in Bladder Cancer

PurposeTo investigate the biological function of HOXB5 in human bladder cancer and explore whether the HOXB5 3′-UTR SNP (1010A/G), which is located within the microRNA-7 binding site, was correlated with clinical features of bladder cancer.MethodsExpression of HOXB5 in 35 human bladder cancer tissues and 8 cell lines were examined using real-time PCR and immunohistochemistry. Next, we explored the biological function of HOXB5 in vitro using cell proliferation, migration and colony formation assays. Using bioinformatics, a SNP (1010A/G) was found located within the microRNA-7 binding site in the 3′-UTR of HOXB5. Real-time PCR was used to test HOXB5 expression affected by different alleles. Finally, multivariate logistic regression analysis was used to determine the relationship between SNP (1010A/G) frequency and clinical features in 391 cases.ResultsHOXB5 was frequently over-expressed both in bladder cancer tissues and cell lines. Inhibition of HOXB5 suppressed the oncogenic function of cancer cells. Next, we demonstrated that a SNP (1010A/G), located within the microRNA-7 binding site in the 3′-UTR of HOXB5, could affect HOXB5 expression in bladder cancer mainly by differential binding activity of microRNA-7 and SNP-related mRNA stability. Finally, we also showed the frequency of 1010G genotype was higher in cancer group compared to normal controls and correlated with the risk of high grade and high stage.ConclusionHOXB5 is overexpressed in bladder cancer. A miRNA-binding SNP (1010A/G) located within 3′-UTR of HOXB5 is associated with gene expression and may be a promising prognostic factor for bladder cancer.

A novel ARC gene polymorphism is associated with reduced risk of Alzheimer’s disease

Alzheimer's disease (AD) is the most common neurodegenerative disease, and is clinically characterized by cognitive disturbances and the accumulation of the amyloid β (Aβ) peptides in plaques in the brain. Recent studies have shown the links between AD and the immediate-early gene Arc (activity-regulated cytoskeleton-associated protein), involved in synaptic plasticity and memory consolidation. For example, AD mouse models show a decreased expression of Arc mRNA in the brain. In additional, acute Aβ application to brain slices leads to a widespread ARC protein diffusion, unlike the normal defined localization to synapses. In this study, we investigated genetic variation in human ARC and the risk of developing AD. To this end, we genotyped 713 subjects diagnosed with AD and 841 controls without dementia. ARC was sequenced in a group of healthy individuals, and seven previously known SNPs and three novel SNPs were identified. Two of the newly found SNPs were intronic and one, +2852(G/A), was located in the 3'UTR. Three tag SNPs were selected, including the novel SNP +2852(G/A), to relate to risk of AD, Mini Mental State Examination (MMSE) scores and cerebrospinal fluid (CSF) biomarker levels of total tau (T-tau), hyperphosphorylated tau181 (P-tau(181)) and Aβ(1-42). The AA genotype of the newly found 3'-UTR SNP +2852(A/G), was associated with a decreased risk of AD (p (c)=0.005; OR=0.74; 95% CI: 0.61-0.89). No associations of single SNPs or haplotypes with MMSE score or CSF biomarkers were found. Here we report a novel ARC SNP associated with a reduced risk of developing AD. To our knowledge, this is the first study associating a gene variant of ARC with any disease. The location of the SNP within the 3'UTR indicates that dendritic targeting of ARC mRNA could be involved in the molecular mechanisms underlying this protective function. However, further investigation of the importance of this SNP for ARC function, ARC processing and the pathology of AD is needed.

Abstract 1661: Fine-mapping of GWAS variants in BMP4, GREM1, CDH1, and RHPN2 in African American colorectal cancer patients

Abstract Background: Genome-wide association studies (GWAS) of colorectal cancer (CRC) have identified risk variants in candidate genes involved in TGF-ß signaling including BMP4, GREM1, CDH1, RHPN2 and SMAD7. We previously replicated the association with a 3′-UTR SNP in GREM1 (rs10318) in African Americans but did not find evidence of associations in the other genes. This finding could be explained by different genomic structure in African Americans. Therefore, we sought to fine-map these genes to identify CRC risk variants in African Americans who are at highest risk of CRC in the US. Methods: DNA from cases and controls was obtained from two US institutions. In total, we included DNA from 795 AA cases and 985 AA controls. TagSNPs with minor allele frequencies &amp;gt;5% and pairwise r2 &amp;gt;0.8 were identified in five genes using YRI Hapmap data. A total of 81 SNPs were genotyped in cases and controls using Sequenom MassARRAY. We calculated odds ratios and 95% confidence intervals using logistic regression controlling for ancestry, age, gender and multiple testing in PLINK. Results: Association analysis was completed for four genes: BMP4, GREM1, CDH1 and RHPN2. The top tagSNP associations for each gene are shown in the Table. None of the SNPs were significant after permutation testing. In GREM1, the strongest SNP associations were found at the 3′ end of the gene. In BMP4, we identified two SNPs (rs1957852 and rs7146040) that are in LD with rs4444235 previously identified in a European GWAS. In CDH1, rs12931189 is located in the same LD block as the strongest association signal (rs7199991) in a recent fine-mapping study in Europeans. Finally, we have no evidence for associations in RHPN2 in African Americans. Conclusion: These results provide preliminary evidence of associations in African American colorectal cancer patients in BMP4, GREM1 and CDH1 identified by GWAS in Europeans. Ongoing work is testing associations in SMAD7 and further fine-mapping by imputation and resequencing in these regions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1661. doi:1538-7445.AM2012-1661

An evaluation of the impact of MAPT, SNCA and APOE on the burden of Alzheimer's and Lewy body pathology

PurposeThe study investigates the effects of genetic factors on the pathology of Alzheimer's disease (AD) and Lewy body (LB) diseases, including Parkinson's disease and dementia with Lewy bodies.MethodsA multicentre autopsy...

Comparative Mapping and 3'UTR SNP Detection of ANGPTL4 Gene in Beef Cattle

Comparative Mapping and 3'UTR SNP Detection of ANGPTL4 Gene in Beef Cattle

Association of ORAI1 Haplotypes with the Risk of HLA-B27 Positive Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a chronic inflammation of the sacroiliac joints, spine and peripheral joints. The aetiology of ankylosing spondylitis is still unclear. Previous studies have indicated that genetics factors such as human leukocyte antigen HLA-B27 associates to AS susceptibility. We carried out a case-control study to determine whether the genetic polymorphisms of ORAI1 gene, a major component of store-operated calcium channels that involved the regulation of immune system, is a susceptibility factor to AS in a Taiwanese population. We enrolled 361 AS patients fulfilled the modified New York criteria and 379 controls from community. Five tagging single nucleotides polymorphisms (tSNPs) at ORAI1 were selected from the data of Han Chinese population in HapMap project. Clinical statuses of AS were assessed by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), and Bath Ankylosing Spondylitis Global Index (BAS-G). Our results indicated that subjects carrying the minor allele homozygote (CC) of the promoter SNP rs12313273 or TT homozygote of the SNP rs7135617 had an increased risk of HLA-B27 positive AS. The minor allele C of 3′UTR SNP rs712853 exerted a protective effect to HLA-B27 positive AS. Furthermore, the rs12313273/rs7135617 pairwise allele analysis found that C-G (OR 1.69, 95% CI 1.27, 2.25; p = 0.0003) and T-T (OR 1.75, 95% CI 1.36, 2.27; p<0.0001) haplotypes had a significantly association with the risk of HLA-B27-positive AS in comparison with the T-G carriers. This is the first study that indicate haplotypes of ORAI1 (rs12313273 and rs7135617) are associated with the risk of HLA-B27 positive AS.

The first missense mutation of NHS gene in a Tunisian family with clinical features of NHS syndrome including cardiac anomaly

Nance-Horan Syndrome (NHS) or X-linked cataract-dental syndrome is a disease of unknown gene action mechanism, characterized by congenital cataract, dental anomalies, dysmorphic features and, in some cases, mental retardation. We performed linkage analysis in a Tunisian family with NHS in which affected males and obligate carrier female share a common haplotype in the Xp22.32-p11.21 region that contains the NHS gene. Direct sequencing of NHS coding exons and flanking intronic sequences allowed us to identify the first missense mutation (P551S) and a reported SNP-polymorphism (L1319F) in exon 6, a reported UTR-SNP (c.7422 C>T) and a novel one (c.8239 T>A) in exon 8. Both variations P551S and c.8239 T>A segregate with NHS phenotype in this family. Although truncations, frame-shift and copy number variants have been reported in this gene, no missense mutations have been found to segregate previously. This is the first report of a missense NHS mutation causing NHS phenotype (including cardiac defects). We hypothesize also that the non-reported UTR-SNP of the exon 8 (3'-UTR) is specific to the Tunisian population.

UGT1A1 sequence variants and bilirubin levels in early postnatal life: a quantitative approach

BackgroundFundamental to definitively identifying neonates at risk of developing significant hyperbilirubinemia is a better understanding of the genetic factors associated with early bilirubin rise. Previous genetic studies have focused on the UGT1A1 gene, associating common variation in the coding or promoter regions with qualitative assessments of bilirubin (i.e. significantly elevated or not). These studies have had conflicting results and limited success. We chose to approach the problem by focusing on the quantitative (absolute) change in bilirubin levels early in post-natal life. We apply this approach to the UGT1A1 gene - exploring the contribution of both rare and common variants to early bilirubin changes.MethodsWe sequenced the exons, PBREM, 5'-, and 3'- regions of the UGT1A1 gene in 80 otherwise healthy term neonates who had repeat bilirubin levels measured within the first five days of life.ResultsThree novel coding variants were observed, but there was no clear relationship between rare coding variants and bilirubin rise. Adjusted linear regression models fit to evaluate the relationship between changing bilirubin levels and common UGT1A1variants found that among 39 neonates whose bilirubin was resampled within 33 hours, individuals homozygous for the mutant allele of a 3'UTR SNP had significantly smaller changes in bilirubin (P = 0.003) than individuals carrying the wild-type allele.ConclusionsCollectively, rare UGT1A1 coding variants do not appear to play a prominent role in determining early bilirubin levels; however common variants in the 3' UTR of UGT1A1 may modulate the early bilirubin rise. A quantitative approach to evaluating early bilirubin kinetics provides a more robust framework in which to better understand the genetics of neonatal hyperbilirubinemia.

PS1-10: Response to Metformin and Genetic Variants of Organic Cation and Multidrug and Toxin Extrusion Transporters

Background and Aims: Metformin is the most widely-used anti-diabetic drug in the United States. However, medication management fails to achieve therapeutic goals in ~20% of patients. This study attempted to identify genetic polymorphisms associated with metformin treatment failure. We studied the associations between polymorphisms in two liver and kidney organic cation transporter genes (OCT1 & OCT2) and two kidney multidrug and toxin extrusion transporter genes, (MATE1 & MATE2K) and response to metformin monotherapy. Methods: At Kaiser Permanente Southeast and Marshfield Clinic Research Foundation we identified 304 type 2 diabetes (T2DM) patients on metformin monotherapy who: were enrolled for >=6 months prior to initiation; remained on metformin >= 90 days following initiation; and had a HgbA1c measured prior to first metformin prescription and then after initiation. From this group we selected 171 patients who had not received metformin or other anti-diabetics previously, and had baseline HbA1c levels >7.0% prior to metformin initiation. We genotyped 10 SNPs, and assessed response by measuring absolute and relative change in HbA1c after after metformin initiation. Results: Three SNPs were identified that were significantly associated with reduced response to metformin, after adjustment for baseline HbA1c, dose, and ethnicity. In a dominant model, heterozygosity or homozygosity for minor A allele in the MATE1 intronic SNP, rs2289669, resulted in a smaller absolute change in HbA1c (mean of 0.45% less reduction in absolute change in HbA1c; 95% CI 0.17 to 0.74%) compared to individuals homozygous for major G allele. In a recessive model, homozygosity for minor A allele in MATE2K 5′-UTR SNP, rs12943590, resulted in smaller absolute change in HbA1c (mean of 0.43% less reduction in absolute change in HbA1c, 95% CI 0.053 to 0.86%). The reduced function SNPs in OCT1 & OCT2 were not associated with metformin response, except for rs34059508 (G465R). This SNP showed a positive (but not statistically significant) association with smaller absolute change in HbA1c (mean of 0.49%; 95% CI −0.06354 to 1.045%). Conclusions: These three variants of metformin transporter genes may be important for personalizing treatment for patients with T2DM. A replication study in a larger population is currently being planned.

A Functional Variation In the NKG2D Gene Regulates NKG2D Receptor Expression and Is Associated with Better Transplant Outcomes After Fully-HLA-Matched Unrelated Bone Marrow Transplantation

AbstractAbstract 221A functional variation in the NKG2D gene regulates NKG2D receptor expression and is associated with better transplant outcomes after fully-HLA-matched unrelated bone marrow transplantationNKG2D, an activating receptor expressed by NK cells and some T cells, plays important roles in both immunity to microbial infections and cancer immune surveillance. A single nucleotide polymorphism (SNP rs1049174) in the 3′-untranslated region (3′UTR) of the NKG2D gene determines two genotypes, HNK1 genotype with high NK cytotoxicity, and the LNK1 genotype with low NK cytotoxicity. Individuals with the HNK1 genotype have reduced risk of cancer (Cancer Res 2006; 66(1):63-70). In the present study, we analyzed the impact of SNP rs1049174 on transplant outcomes after HLA-matched unrelated bone marrow transplantation. The NKG2D genotypes were retrospectively analyzed in a cohort of 360 pairs of patients and their unrelated donors transplanted through the Japan Marrow Donor Program. The donor HNK1 genotype was associated with lower transplant related mortality (TRM) in patients with standard risk (hazard ratio [HR], 0.58; 95% confidence interval [CI], 0.35–0.97; P=0.04) (Fig.1). The donor HNK1 genotype was also associated with a trend toward better OS (HR, 0.69; 95% CI, 0.46–1.04; P=0.08). The host NKG2D genotypes did not significantly influence the transplant outcomes. NK cells from HNK1 donors expressed higher levels of NKG2D on the cell surface, and also expressed higher levels of NKG2D mRNA. Luciferase reporter gene constructs transfected into NK cells showed that the LNK1 construct more effectively attenuated gene expression compared with the HNK1 construct (60% vs. 35%). Bioinformatics tools predicted an interaction between the rs1049174 SNP motif with the micro-RNA-1245 (miR1245). Next, in an mRNA/miRNA binding assay, the LNK allele showed higher affinity to the miR1245 than the HNK1 allele. Moreover transfection of NK cells possessing the LNK1 allele with a miR1245 mimic resulted in 25 fold decrease in mRNA NKG2D level versus only 2 fold decrease in mRNA NKG2D expression in HNK1 allele derived NK cells, thus suggesting that the 3′UTR SNP may regulate the expression of NKG2D through its binding to the putative repressor miR1245. These findings provide evidence that the NKG2D 3′UTR polymorphism is functional, and that the NK cells from donors with the HNK1 genotype exert higher cytotoxicity through their increased expression of NKG2D, thereby resulting in an improved outcome for bone marrow transplantation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.