Susceptibility Haplotype Research Articles

Genetic Factors Contributing to Systemic Lupus Erythematosus in Tunisian Patients

Systematic lupus erythematosus (SLE) is a multi system autoimmune disease characterized by autoantibodies production, multi-organ damage and complex genetic inheritance. Multiple genetic and environemental factors contribute to the pathogenesis of this disease. Recent genome-wide studies, have added substantially to the number of genes associated with SLE. We performed a case control study using 138 SNPs in 93 Tunisian patients affected with lupus and 162 healthy controls. All SNPs were genotyped in a Sequenom platform. To confirm some associations, associated SNPs were analyzed using logistic regression which allows the test of association with a given SNP by adjusting for the effect of confounding variables. Association was especially reported with rs3733197 (P=0.0026, OR=2.04), rs17266594 (P=0.046, OR=1.56) in BANK1 gene, rs2070197 (P=0.0016, OR=2.31), rs2004640 (P=0.024, OR=1.54), rs10954213 (P=0.035, OR=1.53) in IRF5 gene and rs7574865 (P=0.017, OR=1.77) in STAT4 gene: previously confirmed SLE susceptibility genes. rs1800629 (P=0.00036, OR=2.26), rs4147359 (P=0.026, OR=1.55) and rs11575812 (P=0.037, OR=1.57) of TNF-α, IR2RA and IL2 genes respectively were also associated with SLE. Haplotypic analysis reported 2 susceptibility haplotypes: TGG (P=0.00421, OR=1.87) in BANK1 and TCA (P=0.00177, OR=2.34) in IRF5 genes. Our results show that numerous genes, some with known immune related function predispose to lupus.

Vitamin D receptor gene polymorphisms and haplotypes (Apa I, Bsm I, Fok I, Taq I) in Turkish psoriasis patients

SummaryBackgroundPsoriasis is an inflammatory disease characterized by increased squamous cell proliferation and impaired differentiation. Vitamin D, Calcitriol, and its analogues are successfully used for psoriasis therapy. However, it is unknown why some psoriasis patients are resistant to Vitamin D therapy. Vitamin D mediates its activity by a nuclear receptor. It is suggested that polymorphisms and haplotypes in the VDR gene may explain the differences in response to vitamin D therapy.Material/MethodsIn this study, 102 psoriasis patients and 102 healthy controls were studied for VDR gene polymorphisms. The Fok I, Bsm I, Apa I and Taq I polymorphisms were examined by PCR-RFLP, and 50 subjects received vitamin D therapy to evaluate the association between VDR gene polymorphisms and response to vitamin D therapy. Existence of cutting site is shown by capital letters, and lack was shown by lower case. The haplotypes were analysed by CHAPLIN.ResultsThere was significant difference in allele frequency of T and genotype frequency of Tt between cases and controls (p values 0.038 and 0.04, respectively). The Aa and bb genotypes were significantly higher in early onset than late onset psoriasis (p values 0.008 and 0.04, respectively). The genotypes Ff, ff and TT are significantly different between vitamin D3 therapy responders and non-responders (p values 0.04, 0.0001, 0.009, respectively). To the best of our knowledge, this is the first report showing importance of VDR gene haplotypes in psoriasis, the significance of the Wald and LR (Likelihood Ratio) statistics (p=0,0042) suggest that FfBbAatt is a disease-susceptibility haplotype.ConclusionsHaplotype analysis is a recent and commonly used method in genetic association studies. Our results reveal a previously unidentified susceptibility haplotype and indicate that certain haplotypes are important in the resistance to vitamin D3 therapy and the onset of psoriasis. The haplotypes can give valuable data where genotypes unable to do.

HLA-class II and class I genotypes among Japanese children with Type 1A diabetes and their families

To determine the HLA-DRB1, DQB1, DPB1, A, C, and B genotypes among Japanese children with autoimmune type 1 diabetes. Four hundred and thirty patients who were GADAb and/or IA-2Ab-positive (Type 1A) were recruited from 37 medical centers as part of a nationwide multicenter collaborative study. DNA samples from 83 siblings of the children with Type 1A diabetes and 149 parent-child trios were also analyzed. A case-control study and a transmission disequilibrium test (TDT) were then performed. The susceptible and protective DRB1 and DQB1 alleles and haplotypes were confirmed. DPB1 alleles unique to the Japanese population and those common to multiple ethnic groups were also present. A linkage disequilibrium (LD) analysis showed both susceptible and protective haplotypes. The TDT did not reveal any alleles that were transmitted preferentially from the mother or father to children with Type 1A. Homozygosity for DRB1-09:01-DQB1-03:03 and heterozygosity for DRB1-04:05-DQB1-04:01 and DRB1-08:02-DQB1-03:02 were associated with an extremely high risk of Type 1A. A comparison of children with Type 1A and their parents and siblings suggested a dose effect of susceptible DRB1-DQB1 haplotypes and an effect of protective alleles on immunological pathogenesis. DRB1-09:01 appeared to be strongly associated with an early onset in preschool children with Type 1A diabetes. This study demonstrated the characteristic association of HLA-class II and class I genes with Type 1A diabetes among Japanese children. A TDT did not reveal the genomic imprinting of HLA-class II and class I genes in Type 1A diabetes.

Analysis of the HLA and non‐HLA susceptibility loci in Japanese type 1 diabetes

We previously reported the associations of human leukocyte antigen (HLA) (DRB1 and DQB1), INS, CTLA4, IL2RA, ERBB3 and CLEC16A with Japanese type 1 diabetes (T1D). In this study, we jointly analysed these loci in addition to IFIH1 and IL7R. A maximum of 790 T1D patients and 953 control subjects were analysed. HLA was determined by sequencing-based typing. Seven non-HLA single nucleotide polymorphisms were genotyped using TaqMan assay. HLA DRB1*0405, DRB1*0901 and DRB1*0802-DQB1*0302 haplotypes were positively associated with T1D, while the DRB1*15 haplotypes were negatively associated. Non-HLA single nucleotide polymorphisms, INS, IL2RA, ERBB3, CLEC16A and IL7R were associated with T1D. By a prediction model using the HLA loci alone (HLA model) or the non-HLA loci alone (non-HLA model), it was revealed that the cumulative effect of the non-HLA model was much weaker than that of the HLA model (average increase in odds ratio: 1.17 versus 3.14). Furthermore, the area under the receiver operating characteristic curve of the non-HLA model was also much smaller than that of the HLA model (0.65 versus 0.81, p<10(-11)). Finally, a patient-only analysis revealed the susceptible HLA haplotypes and the risk allele of INS to be negatively associated with slower onset of the disease. In addition, the DRB1*0901 haplotype and the risk alleles of ERBB3, CLEC16A and CTLA4 were positively associated with the co-occurrence of thyroid autoimmunity. Although several non-HLA susceptibility genes in Japanese were confirmed trans-racially and appear to contribute to the heterogeneity of the clinical phenotypes, the cumulative effect on the ability to predict the development of T1D was weak.

Evidence for the involvement of the glucocorticoid receptor gene in bipolar disorder in an isolated northern Swedish population

Dysfunction of the hypothalamus-pituitary-adrenal (HPA) axis is one of the most consistent findings in the pathophysiology of mood disorders. The potential role of genes related to HPA axis function has been investigated extensively in major depression. However, in bipolar disorder (BPD) such studies are scarce. We performed a systematic HapMap-based association study of six genes crucial for HPA axis function in relation to BPD. Haplotype tagging single nucleotide polymorphisms (htSNPs) were selected in order to identify all haplotypes with a frequency of more than 1% in the genes encoding the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), corticotrophin releasing hormone receptor 1 (CRH-R1) and 2 (CRH-R2), CRH binding protein (CRH-BP), and FK binding protein 5 (FKBP5). This resulted in a total selection of 225 SNPs that were genotyped and analyzed in 309 BPD patients and 364 matched control individuals all originating from an isolated northern Swedish population. Consistent evidence for an association with BPD was found for NR3C1, the gene encoding GR. Almost all SNPs in two adjacent haplotype blocks contributed to the positive signal, comprised of significant single marker, sliding window, and haplotype-specific p-values. All these results point to a moderately frequent (10-15%) susceptibility haplotype covering the entire coding region and 3' untranslated region (UTR) of NR3C1. This study contributes to the growing evidence for a role of the glucocorticoid receptor gene (NR3C1) in vulnerability to mood disorders, and BPD in particular, and warrants further in vitro investigation of the at-risk haplotypes with respect to disease etiology. However, this association might be restricted to this specific population, as it is observed in a rather small sample from an isolated population without replication, and data from large meta-analyses for genome-wide association studies in BPD do not show the GR as a very strong candidate.

Functional Promoter Haplotypes of Interleukin-18 Condition Susceptibility to Severe Malarial Anemia and Childhood Mortality

Severe malarial anemia (SMA) is a leading cause of morbidity and mortality in children residing in regions where Plasmodium falciparum transmission is holoendemic. Although largely unexplored in children with SMA, interleukin-18 (IL-18) is important for regulating innate and acquired immunity in inflammatory and infectious diseases. As such, we selected two functional single-nucleotide polymorphisms (SNPs) in the IL-18 promoter (-137G→C [rs187238] and -607C→A [rs1946518]) whose haplotypes encompass significant genetic variation due to the presence of strong linkage disequilibrium among these variants. The relationship between the genotypes/haplotypes, SMA (hemoglobin [Hb], <5.0 g/dl], and longitudinal clinical outcomes were then investigated in Kenyan children (n = 719). Multivariate logistic regression analyses controlling for age, gender, sickle cell trait, glucose-6-phosphate dehydrogenase (G6PD) deficiency, HIV-1, and bacteremia revealed that carriage of the -607AA genotype was associated with protection against SMA (odds ratio [OR] = 0.440 [95% confidence interval {CI} = 0.21 to 0.90], P = 0.031) in children with acute infection. In contrast, carriers of the -137G/-607C (GC) haplotype had increased susceptibility to SMA (OR = 2.050 [95% CI = 1.04 to 4.05], P = 0.039). Measurement of IL-18 gene expression in peripheral blood leukocytes demonstrated that elevated IL-18 transcripts were associated with reduced hemoglobin concentrations (ρ = -0.293, P = 0.010) and that carriers of the "susceptible" GC haplotype had elevated IL-18 transcripts (P = 0.026). Longitudinal investigation of clinical outcomes over a 3-year follow-up period revealed that carriers of the rare CC haplotype (∼1% frequency) had 5.76 times higher mortality than noncarriers (P = 0.001). Results presented here demonstrate that IL-18 promoter haplotypes that condition elevated IL-18 gene products during acute infection are associated with increased risk of SMA. Furthermore, carriage of the rare CC haplotype significantly increases the risk of childhood mortality.

Genetic Architecture of Aluminum Tolerance in Rice (Oryza sativa) Determined through Genome-Wide Association Analysis and QTL Mapping

Aluminum (Al) toxicity is a primary limitation to crop productivity on acid soils, and rice has been demonstrated to be significantly more Al tolerant than other cereal crops. However, the mechanisms of rice Al tolerance are largely unknown, and no genes underlying natural variation have been reported. We screened 383 diverse rice accessions, conducted a genome-wide association (GWA) study, and conducted QTL mapping in two bi-parental populations using three estimates of Al tolerance based on root growth. Subpopulation structure explained 57% of the phenotypic variation, and the mean Al tolerance in Japonica was twice that of Indica. Forty-eight regions associated with Al tolerance were identified by GWA analysis, most of which were subpopulation-specific. Four of these regions co-localized with a priori candidate genes, and two highly significant regions co-localized with previously identified QTLs. Three regions corresponding to induced Al-sensitive rice mutants (ART1, STAR2, Nrat1) were identified through bi-parental QTL mapping or GWA to be involved in natural variation for Al tolerance. Haplotype analysis around the Nrat1 gene identified susceptible and tolerant haplotypes explaining 40% of the Al tolerance variation within the aus subpopulation, and sequence analysis of Nrat1 identified a trio of non-synonymous mutations predictive of Al sensitivity in our diversity panel. GWA analysis discovered more phenotype–genotype associations and provided higher resolution, but QTL mapping identified critical rare and/or subpopulation-specific alleles not detected by GWA analysis. Mapping using Indica/Japonica populations identified QTLs associated with transgressive variation where alleles from a susceptible aus or indica parent enhanced Al tolerance in a tolerant Japonica background. This work supports the hypothesis that selectively introgressing alleles across subpopulations is an efficient approach for trait enhancement in plant breeding programs and demonstrates the fundamental importance of subpopulation in interpreting and manipulating the genetics of complex traits in rice.

Spontaneous Aortic Thrombosis Causing Left Main Coronary Occlusion in a Man With Secondary Polycythemia

OBJECTIVE—In 1997, the American Diabetes Association proposed two subcategories for type 1 diabetes: type 1A or immunomediated diabetes and type 1B or idiopathic diabetes characterized by negative β-cell autoimmunity markers, lack of association with HLA, and fluctuating insulinopenia. The aim of this study was to examine clinical characteristics, β-cell function, HLA typing, and mutations in maturity-onset diabetes of the young (MODY) genes in patients with atypical type 1 diabetes (type 1 diabetes diagnosed at onset, without pancreatic autoantibodies and fluctuating insulinopenia). RESEARCH DESIGN AND METHODS—Eight patients with atypical type 1 diabetes (all men, 30.7 ± 7.6 years) and 16 newly diagnosed age- and sex-matched patients with type 1A diabetes were studied retrospectively. Islet cell, GAD, tyrosine phosphatase and insulin antibodies, and basal and stimulated plasma C-peptide were measured at onset and after 1 year. HLA-DRB1-DQA1-DQB1 typing and screening for mutations in the HNF-1α and HNF-4α genes were performed from genomic DNA. RESULTS—Atypical patients displayed significantly higher BMI and better β-cell function at onset and after 12 months. Three patients carried protective or neutral type 1 diabetes haplotypes, five patients displayed heterozygosity for susceptible and protective haplotypes, and seven patients showed Aspβ57. We found a nondescribed variant Pro436Ser in exon 10 of the HNF-4α gene in one atypical patient without susceptible haplotypes. CONCLUSIONS—In our population, there are atypical forms of young adult-onset ketosis-prone diabetes initially diagnosed as type 1 diabetes, differing from type 1 diabetes in the absence of β-cell autoimmunity, persistent β-cell function capacity, fluctuating insulin requirements and ketosis-prone episodes, as well as clinical features of type 2 diabetes. Only one subgroup could be strictly classified as having type 1B diabetes. Additional information is still needed to improve our understanding of the mechanisms that finally lead to the disease.

HLA associations with multiple sclerosis in Greece

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the central nervous system originated by a complex interplay of environmental and genetic factors. The association of MS with the human leukocyte antigen (HLA) class II alleles was investigated in MS patients in northwest Greece, in the geographical region of Epirus. Our aim was to estimate the prevalence of the HLA-DRB1*1501, HLA-DQB1*0602 and HLA-DQA1*0102 alleles, consisting the most common susceptibility haplotype in North European and North American Caucasians. We studied 126 MS patients and 93 age and sex matched healthy controls. HLA typing was performed by a polymerase chain reaction (PCR) amplification with sequence-specific primers (PCR-SSP) method. We found that HLA-DRB1*1501, HLA-DQB1*0602 and HLA-DQA1*0102 alleles were significantly more frequent among patients (34% versus 11%, p=0.00015; 69% versus 51%, p=0.01; 76% versus 55%, p=0.002, respectively). HLA-DRB1*1501, HLA-DQB1*0602, HLA-DQA1*0102 haplotype was significantly more common among patients (p=0.00067). HLA-DRB1*1501 and HLA-DQB1*0602 alleles were more frequently detected in patients with initial symptoms from the brainstem or the cerebellum (p=0.024). No significant correlation was observed among these alleles with sex, disease clinical course, or age at onset. This is the first study to investigate genetic susceptibility to MS in Greece. Our results are in line with previous reports in North European and North American patients.

Postnatal regression of hypertrophic cardiomyopathy in infants of diabetic mothers: A crosstalk between Hox genes and epidermal growth factor (EGF) gene polymorphism?

mothers: A crosstalk between Hox genes and epidermal growth factor (EGF) gene polymorphism? Raffaella Mormile ⁎, Giorgio Vittori , Mario De Michele , Umberto Squarcia , Federico Quaini e a Division of Pediatric and Neonatology, Moscati Hospital, Aversa, Italy b Division of Gynecology, San Carlo di Nancy Hospital, Rome, Italy c Division of Cardiology, Moscati Hospital, Aversa, Italy d Pediatric Cardiology, University of Parma, Italy e Department of Internal Medicine and Biomedical Sciences, Section of Internal Medicine, University of Parma, Italy

Preferential induction of protective T cell responses to Theiler’s virus in resistant (C57BL/6 x SJL)F1 mice (67.14)

Abstract Infection of the CNS with Theiler's murine encephalomyelitis virus (TMEV) induces an immune-mediated demyelinating disease in susceptible mouse strains such as SJL/J (H-2(s)), but not in strains such as C57BL/6 (H-2(b)). In addition, it has been shown that (SJL/J x C57BL/6)F1 mice, which carry both resistant and susceptible MHC haplotypes (H-2(b/s)), are resistant to both viral persistence and TMEV-induced demyelinating disease. In this study, we further analyzed the immune responses underlying resistance of F1 mice. Our study shows that the resistance of F1 mice is associated with a higher level of the initial virus-specific H-2(b)-restricted CD8(+) T cell responses vs. the H-2(s)-restricted CD8(+) T cell responses. In contrast, pathogenic Th17 responses to viral epitopes are lower in F1 mice compared to that in susceptible SJL mice. Dominant effects of resistant genes expressed in antigen-presenting cells of F1 mice on regulating viral replication and inducing protective T cell responses appear to play a crucial role in disease resistance. Although the F1 mice are resistant to disease, the level of viral RNA in the CNS was intermediate between those of SJL/J and C57BL/6 mice, indicating the presence of a threshold of viral expression for pathogenesis.

Autoimmune polyglandular syndrome type 2 shows the same HLA class II pattern as type 1 diabetes†

Autoimmune polyglandular syndrome (APS) type 2 is defined by the manifestation of at least two autoimmune endocrine diseases. Only few data exist on genetic associations of APS type 2. In this controlled study, 98 patients with APS type 2, 96 patients with type 1 diabetes (T1D), and 92 patients with autoimmune thyroid disease, both as a single autoimmune endocrinopathy, were tested for association with alleles of the human leukocyte antigen (HLA) class II loci DRB1, DQA1, and DQB1. Patients with APS type 2 had significantly more often the alleles DRB1*03 (P(c) < 0.0001), DRB1*04 (P(c) < 0.000005), DQA1*03 (P(c) < 0.0001), and DQB1*02 (P(c) < 0.05), when compared with controls. Less frequent in APS were DRB1*15 (P(c) < 0.05), DQA1*01 (P(c) < 0.0005), and DQB1*05 (P(c) < 0.005). With regard to frequency and linkage of these alleles, the susceptible haplotypes DRB1*0301-DQA1*0501-DQB1*0201 and DRB1*0401/04-DQA1*0301-DQB1*0302 were deduced. Protective haplotypes in this study were DRB1*1501-DQA1*0102-DQB1*0602 and DRB1*0101-DQA1*0101-DQB1*0501. Comparing APS patients with vs without AD, no significant differences regarding HLA class II alleles were noted in our collective. Patients with T1D as a singular disease had the same susceptible and protective HLA alleles and haplotypes. The prevalence of DRB1*03 and DRB1*04 in APS patients was not because of the presence of diabetes, as the APS type 2 patients without diabetes had the same allele distribution. In conclusion, these data suggest a common immunogenetic pathomechanism for T1D and APS type 2, which might be different from the immunogenetic pathomechanism of other autoimmune endocrine disease.

Mapping a New Spontaneous Preterm Birth Susceptibility Gene, IGF1R, Using Linkage, Haplotype Sharing, and Association Analysis

Preterm birth is the major cause of neonatal death and serious morbidity. Most preterm births are due to spontaneous onset of labor without a known cause or effective prevention. Both maternal and fetal genomes influence the predisposition to spontaneous preterm birth (SPTB), but the susceptibility loci remain to be defined. We utilized a combination of unique population structures, family-based linkage analysis, and subsequent case-control association to identify a susceptibility haplotype for SPTB. Clinically well-characterized SPTB families from northern Finland, a subisolate founded by a relatively small founder population that has subsequently experienced a number of bottlenecks, were selected for the initial discovery sample. Genome-wide linkage analysis using a high-density single-nucleotide polymorphism (SNP) array in seven large northern Finnish non-consanginous families identified a locus on 15q26.3 (HLOD 4.68). This region contains the IGF1R gene, which encodes the type 1 insulin-like growth factor receptor IGF-1R. Haplotype segregation analysis revealed that a 55 kb 12-SNP core segment within the IGF1R gene was shared identical-by-state (IBS) in five families. A follow-up case-control study in an independent sample representing the more general Finnish population showed an association of a 6-SNP IGF1R haplotype with SPTB in the fetuses, providing further evidence for IGF1R as a SPTB predisposition gene (frequency in cases versus controls 0.11 versus 0.05, P = 0.001, odds ratio 2.3). This study demonstrates the identification of a predisposing, low-frequency haplotype in a multifactorial trait using a well-characterized population and a combination of family and case-control designs. Our findings support the identification of the novel susceptibility gene IGF1R for predisposition by the fetal genome to being born preterm.

Psoriasis-Associated Genetic Polymorphism in North Indian Population in the CCHCR1 Gene and in a Genomic Segment Flanking the HLA-C Region

Psoriasis is a common, chronic, recurrent, inflammatory, hyper proliferative disorder of the skin, which has a relatively high prevalence in the general population (0.6–4.8%). Linkage and association analyses in various populations have revealed a major locus for psoriasis susceptibility, PSORS1, at 6p21.3. Association of the disease with human leukocyte antigen (HLA) Cw6, corneodesmosin (CDSN) and the coiled-coil alpha-helical rod protein-1 (CCHCR1) has also been reported. Though the PSORS1 locus accounts for 30–50% of familial psoriasis in various global population groups, yet no studies have been published from the North Indian population. Some of the SNPs in HLA-C and CCHCR1 genes have been reported as markers for disease susceptibility. Therefore in the present study, DNA samples from psoriasis patients from North India were genotyped for polymorphisms in CCHCR1 and HLA-C genes. The allele frequencies were calculated for patients and controls, and were compared for odds ratio and confidence interval values. SNPn.7*22222 (rs12208888), SNPn.7*22333 (rs12216025), SNPn.9*24118 (rs10456057), CCHCR1_386 (rs130065), CCHCR1_404 (rs130076) and CCHCR1_1364 (rs130071) were found to be significant in psoriasis patients. Linkage disequilibrium analysis revealed two haplotypes (rs12208888,rs2844608,rs12216025,rs10456057,rs130065,rs130066,rs130068,rs130269,andrs12208888,rs2844608,rs12216025,rs130076,rs130066,rs130068,rs130269,rs130071) as highly susceptible haplotypes for psoriasis in the cohort studied. Preliminary analysis of the data also suggests the possibilities of ethnic group specific disease related polymorphisms, pending validation in future studies.

Psoriasis-associated genetic polymorphism in North Indian population in the CCHCR1 gene and in a genomic segment flanking the HLA-C region.

Psoriasis is a common, chronic, recurrent, inflammatory, hyper proliferative disorder of the skin, which has a relatively high prevalence in the general population (0.6–4.8%). Linkage and association analyses in various populations have revealed a major locus for psoriasis susceptibility, PSORS1, at 6p21.3. Association of the disease with human leukocyte antigen (HLA) Cw6, corneodesmosin (CDSN) and the coiled-coil alpha-helical rod protein-1 (CCHCR1) has also been reported. Though the PSORS1 locus accounts for 30–50% of familial psoriasis in various global population groups, yet no studies have been published from the North Indian population. Some of the SNPs in HLA-C and CCHCR1 genes have been reported as markers for disease susceptibility. Therefore in the present study, DNA samples from psoriasis patients from North India were genotyped for polymorphisms in CCHCR1 and HLA-C genes. The allele frequencies were calculated for patients and controls, and were compared for odds ratio and confidence interval values. SNPn.7*22222 (rs12208888), SNPn.7*22333 (rs12216025), SNPn.9*24118 (rs10456057), CCHCR1_386 (rs130065), CCHCR1_404 (rs130076) and CCHCR1_1364 (rs130071) were found to be significant in psoriasis patients. Linkage disequilibrium analysis revealed two haplotypes (rs12208888, rs2844608, rs12216025, rs10456057, rs130065, rs130066, rs130068, rs130269, and rs12208888, rs2844608, rs12216025, rs130076, rs130066, rs130068, rs130269, rs130071) as highly susceptible haplotypes for psoriasis in the cohort studied. Preliminary analysis of the data also suggests the possibilities of ethnic group specific disease related polymorphisms, pending validation in future studies.

Lr34 multi‐pathogen resistance ABC transporter: molecular analysis of homoeologous and orthologous genes in hexaploid wheat and other grass species

The Triticum aestivum (bread wheat) disease resistance gene Lr34 confers durable, race non-specific protection against three fungal pathogens, and has been a highly relevant gene for wheat breeding since the green revolution. Lr34, located on chromosome 7D, encodes an ATP-binding cassette (ABC) transporter. Both wheat cultivars with and without Lr34-based resistance encode a putatively functional protein that differ by only two amino acid polymorphisms. In this study, we focused on the identification and characterization of homoeologous and orthologous Lr34 genes in hexaploid wheat and other grasses. In hexaploid wheat we found an expressed and putatively functional Lr34 homoeolog located on chromosome 4A, designated Lr34-B. Another homoeologous Lr34 copy, located on chromosome 7A, was disrupted by the insertion of repetitive elements. Protein sequences of LR34-B and LR34 were 97% identical. Orthologous Lr34 genes were detected in the genomes of Oryza sativa (rice) and Sorghum bicolor (sorghum). Zea mays (maize), Brachypodium distachyon and Hordeum vulgare (barley) lacked Lr34 orthologs, indicating independent deletion of this particular ABC transporter. Lr34 was part of a gene-rich island on the wheat D genome. We found gene colinearity on the homoeologous A and B genomes of hexaploid wheat, but little microcolinearity in other grasses. The homoeologous LR34-B protein and the orthologs from rice and sorghum have the susceptible haplotype for the two critical polymorphisms distinguishing the LR34 proteins from susceptible and resistant wheat cultivars. We conclude that the particular Lr34-haplotype found in resistant wheat cultivars is unique. It probably resulted from functional gene diversification that occurred after the polyploidization event that was at the origin of cultivated bread wheat.

Preferential Induction of Protective T Cell Responses to Theiler's Virus in Resistant (C57BL/6 × SJL)F1 Mice

Infection of the central nervous system (CNS) with Theiler's murine encephalomyelitis virus (TMEV) induces an immune-mediated demyelinating disease in susceptible mouse strains such as SJL/J (H-2(s)) but not in strains such as C57BL/6 (H-2(b)). In addition, it has been shown that (C57BL/6 × SJL/J)F1 mice (F1 mice), which carry both resistant and susceptible MHC haplotypes (H-2(b/s)), are resistant to both viral persistence and TMEV-induced demyelinating disease. In this study, we further analyzed the immune responses underlying the resistance of F1 mice. Our study shows that the resistance of F1 mice is associated with a higher level of the initial virus-specific H-2(b)-restricted CD8(+) T cell responses than of the H-2(s)-restricted CD8(+) T cell responses. In contrast, pathogenic Th17 responses to viral epitopes are lower in F1 mice than in susceptible SJL/J mice. Dominant effects of resistant genes expressed in antigen-presenting cells of F1 mice on regulation of viral replication and induction of protective T cell responses appear to play a crucial role in disease resistance. Although the F1 mice are resistant to disease, the level of viral RNA in the CNS was intermediate between those of SJL/J and C57BL/6 mice, indicating the presence of a threshold of viral expression for pathogenesis.

The JAK2 46/1 Haplotype Analysis In Essential Thrombocythaemia and Polycythaemia Rubra Vera Reveals That CC Genotype Is Associated with a Higher JAK2V617F and c-MPL W515 Allele Burden

AbstractAbstract 1977First 2 authors contributed equally. Background:Genomic-wide association studies have identified the germline 46/1 haplotype as a predisposing allele associated with JAK2V617F positive myeloproliferative neoplasms (MPN). The present study analysed data on 856 JAK2V617F positive patients, 326 of which had complete clinical data. Aims:To evaluate the JAK2 46/1 haplotype frequencies, JAK2V617F allele burden, c-MPL 515 mutation and risk of transformation. Methods:Genomic DNA from whole peripheral blood or bone marrow patient samples was analysed as follows: JAK2V617F allele burden by Q-PCR, JAK2 exon 12 mutations by Q-PCR and PCR fragment analysis, MPL W515 L and K mutations by allele specific PCR. The 46/1 JAK2 mutation susceptibility haplotype (46/1) tagging SNP rs12343867 (susceptibility allele C) were analysed by pyrosequencing. Results:The allele frequency for the 46/1 tag SNP rs1234867 in the 856 patients was calculated for the total JAK2V617F cohort (0.48) and the clinical entities ET (0.34) and PRV (0.44) confirming that the 46/1 haplotype is greatly over represented in JAK2V617F MPD patients as compared to published the control population (Wellcome Trust Case Control Consortium (WTCCC) (0.24).The Analysis of the 856 patients demonstrated that JAK2V617F and c-MPL W515L/K mutations co-existed in 16 patients(1.9%), the incidence of c-MPL W515L being twice as common as the c-MPL W515K mutations. There was no correlation between these mutations and age or 46/1 haplotype status. The JAK2V617F allele burden (AB) was lower in the c-MPL mutant patients, the average JAK2AB 31%. 3 out 4 c-MPL patients for which clinical information was available had a diagnosis of ET. No JAK2 exon 12 mutations were found in any of the 859 JAK2V617F positive samples suggesting that co-existing JAK2 exon 14 and exon 12 mutations are extremely rare.The genotypic data in ET patients showed: C/C 12%, C/T 44%, T/T 44% and their respective JAK2V617 allele burden (AB) were 46%, 32%, 29%. The genotype data in PRV patients: C/C 18%, C/T 53%, T/T 28.6% and their respective AB were 47%, 31% and 39%. The median AB was 32% (n=121) for ET and 37% (n=103) for PRV.Within a cohort of 255 patients (ET=138, PRV=117) 4% of ET and 6% of PRV patients transformed to acute myeloid leukaemia or myelofibrosis with no predominant haplotype association. In the ET patients, the median AB was 35%, there was no significant difference in the JAK2 V617F AB between those who transformed or not (p=0.45). Interestingly, on the whole ET group C/C genotype patients were more likely to have an allele burden >50% (p=0.058). In the PRV patients, the median AB was 48%. Again, the C/C genotype, PRV patients were more likely to have an AB>50% (p=0.06), although not reaching statistical significance. Conclusions:The 46/1 haplotype in both clinical entities ET and PRV demonstrated a higher allele burden in the C/C genotype in comparison to the other genotypes. No predominant haplotype predicted the risk of transformation to a more aggressive disease such as MF or AML. The analysis also showed that c-MPL W515K/L mutations can co-exist with JAK2V617F. The c-MPL W515K/L mutations did not exhibit a positive correlation with a preferential 46/1, but was associated with a lower allele burden. No co-existing exon 12 and exon 14 mutations were found, suggesting the rarity of this occurrence. Disclosures:No relevant conflicts of interest to declare.

Integrated Genetic and Epigenetic Analysis Identifies Haplotype-Specific Methylation in the FTO Type 2 Diabetes and Obesity Susceptibility Locus

Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D), focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip) and absolute methylation values were estimated using a Bayesian algorithm (BATMAN). Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10−4, permutation p = 1.0×10−3). Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10−7). Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM), encapsulates a Highly Conserved Non-Coding Element (HCNE) that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA) SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within disease-associated loci, thus providing a novel route to aid unravelling common complex diseases.

The PTPN22 Locus and Rheumatoid Arthritis: No Evidence for an Effect on Risk Independent of Arg620Trp

ObjectivesThe Trp620 allotype of PTPN22 confers susceptibility to rheumatoid arthritis (RA) and certain other classical autoimmune diseases. There has been a report of other variants within the PTPN22 locus that alter risk of RA; protective haplotype ‘5’, haplotype group ‘6–10’ and susceptibility haplotype ‘4’, suggesting the possibility of other PTPN22 variants involved in the pathogenesis of RA independent of R620W (rs2476601). Our aim was to further investigate this possibility.MethodsA total of 4,460 RA cases and 4,481 controls, all European, were analysed. Single nucleotide polymorphisms rs3789607, rs12144309, rs3811021 and rs12566340 were genotyped over New Zealand (NZ) and UK samples. Publically-available Wellcome Trust Case Control Consortium (WTCCC) genotype data were used.ResultsThe protective effect of haplotype 5 was confirmed (rs3789607; (OR = 0.91, P = 0.016), and a second protective effect (possibly of haplotype 6) was observed (rs12144309; OR = 0.90, P = 0.021). The previously reported susceptibility effect of haplotype 4 was not replicated; instead a protective effect was observed (rs3811021; OR = 0.85, P = 1.4×10−5). Haplotypes defined by rs3789607, rs12144309 and rs3811021 coalesced with the major allele of rs12566340 within the adjacent BFK (B-cell lymphoma 2 (BCL2) family kin) gene. We, therefore, tested rs12566340 for association with RA conditional on rs2476601; there was no evidence for an independent effect at rs12566340 (P = 0.76). Similarly, there was no evidence for an independent effect at rs12566340 in type 1 diabetes (P = 0.85).ConclusionsWe have no evidence for a common variant additional to rs2476601 within the PTPN22 locus that influences the risk of RA. Arg620Trp is almost certainly the single common causal variant.