Olfactory Receptor Gene Clusters Research Articles

Exploring quantitative traits-associated copy number deletions through reanalysis of UK10K consortium whole genome sequencing cohorts

ObjectivesWe performed comprehensive association analyses of common high-confidence gnomAD-reported copy number deletions (CNDs) with 60 quantitative traits from UK10K consortium WGS data.MethodsThe study made use of data generated by the UK10K Consortium. UK10K consortium WGS data consist of TwinsUK (n = 1754, middle-aged females) and ALSPAC (n = 1867, birth to adolescence) cohorts. UK10K consortium called 18,739 CNDs (hg19) with GenomeSTRiP software. After filtering out variants with minor allele frequency < 0.05 or HWE P < 1.0 × 10− 6, 1222 (TwinsUK) and 1211 (ALSPAC) CNDs remained for association analyses with 60 normalized quantitative traits.ResultsWe identified 23 genome-wide significant associations at 13 loci, among which 2 associations reached experiment-wide significance. We found that two common deletions in chromosome 4, located between WDR1 and ZNF518B (23.3 kb, dbVar ID:nssv15888957, 4:10211262–10,234,569 and 9.8 kb, dbVar ID:nssv15888975, 4:10392422–10,402,191), were associated with uric acid levels (P = 5.23 × 10− 11 and 2.29 × 10− 8, respectively). We also discovered a novel deletion spanning chromosome 18 (823 bp, dbVar ID: nssv15841628, 8:74347187–74,348,010) associated with low HDL cholesterol levels (P = 4.15 × 10− 7). Additionally, we observed two red blood cell traits-associated loci with genome-wide significance, a 13.2 kb deletion in 7q22.1 (nssv15922542) and a 3.7 kb deletion in 12q24.12 (nssv15813226), both of which were located in regions previously reported to be associated with red blood cell traits. Two deletions in 11q11 (nssv15803200 and nssv15802240), where clusters of multiple olfactory receptor genes exist, and a deletion (nssv15929560) upstream to DOCK5 were associated with childhood obesity. Finally, when defining Trait-Associated copy number Deletions (TADs) as CNDs with phenotype associations at sub-threshold significance (P < 10− 3), we identified 157 (97.5%) out of 161 TADs in non-coding regions, with a mean size of 4 kb (range: 209 − 47,942 bp).ConclusionWe conducted a reanalysis of the UK10K Whole Genome Sequencing cohort, which led to the identification of multiple high confidence copy number deletions associated with quantitative traits. These deletions have standard dbVar IDs and replicate previous findings, as well as reveal novel loci that require further replication studies.

Mapping transgene insertion sites reveals the \u03b1-Cre transgene expression in both developing retina and olfactory neurons

The Tg(Pax6-cre,GFP)2Pgr (α-Cre) mouse is a commonly used Cre line thought to be retinal-specific. Using targeted locus amplification (TLA), we mapped the insertion site of the transgene, and defined primers useful to deduce zygosity. Further analyses revealed four tandem copies of the transgene. The insertion site mapped to clusters of vomeronasal and olfactory receptor genes. Using R26R and Ai14 Cre reporter mice, we confirmed retinal Cre activity, but also detected expression in Gα0+ olfactory neurons. Most α-Cre+ olfactory neurons do not express Pax6, implicating the influence of neighboring regulatory elements. RT-PCR and buried food pellet test did not detect any effects of the transgene on flanking genes in the nasal mucosa and retina. Together, these data precisely map α-Cre, show that it does not affect surrounding loci, but reveal previously unanticipated transgene expression in olfactory neurons. The α-Cre mouse can be a valuable tool in both retinal and olfactory research.

Generation of induced pluripotent stem cell GZHMCi005-A from amniotic fluid-derived cells with duplication of chromosome 8p.

Cases in which the duplication of chromosome 8p (dup 8p) is observed are characterized by facial dysmorphism, agenesis/hypoplasia of the corpus callosum, heart defects and severe mental retardation. The frequency of dup 8p cases is higher compared to other chromosomes because of the Non-allelic homologous recombination (NAHR) between two segmental duplication regions (SDs) containing olfactory receptor gene clusters, REPD (repeat-distal) and REPP (repeat-proximal), located in chromosome 8p23.1. Here we generated a human iPSC line from a patient's amniotic fluid cells with a 18Mb duplication in 8p23.3p22, which will serve as useful tools for studying dup 8p syndrome.

LHX2- and LDB1-mediated trans interactions regulate olfactory receptor choice.

SummaryThe genome is partitioned into topologically associated domains (TADs) and genomic compartments of shared chromatin valance. This architecture is constrained by the DNA polymer, which precludes genic interactions between chromosomes. Here, we report a dramatic divergence from this pattern of nuclear organization that occurs in mouse olfactory sensory neurons (OSNs). In situ HiC on FAC-sorted OSNs and their progenitors shows that olfactory receptor (OR) gene clusters from 18 chromosomes make specific and robust interchromosomal contacts that increase with differentiation. These contacts are orchestrated by intergenic OR enhancers, the Greek Islands, which first contribute to the formation of OR compartments and then form a multi-chromosomal super-enhancer that associates with the single active OR. Greek Island-bound transcription factor Lhx2 and adaptor protein Ldb1 regulate the assembly and maintenance of OR compartments, Greek Island hubs, and OR transcription, providing mechanistic insight and functional support for the role of trans interactions in gene expression.

Speciation in sympatry with ongoing secondary gene flow and a potential olfactory trigger in a radiation of Cameroon cichlids.

The process of sympatric speciation in nature remains a fundamental unsolved problem. Cameroon crater lake cichlid radiations were long regarded as one of the most compelling examples; however, recent work showed that their origins were more complex than a single colonization event followed by isolation. Here, we performed a detailed investigation of the speciation history of a radiation of Coptodon cichlids from Lake Ejagham, Cameroon, using whole-genome sequencing data. The existence of the Lake Ejagham Coptodon radiation is remarkable as this 0.5km2 lake offers limited scope for divergence across a shallow depth gradient, disruptive selection is currently weak, and the species are sexually monochromatic. We infer that Lake Ejagham was colonized by Coptodon cichlids soon after its formation 9,000years ago, yet speciation occurred only in the last 1,000-2,000years. We show that secondary gene flow from a nearby riverine species has been ongoing, into ancestral as well as extant lineages, and we identify and date river-to-lake admixture blocks. One block contains a cluster of olfactory receptor genes that introgressed near the time of the first speciation event and coincides with a higher overall rate of admixture. Olfactory signalling is a key component of mate choice and species recognition in cichlids. A functional role for this introgression event is consistent with previous findings that sexual isolation appears much stronger than ecological isolation in Ejagham Coptodon. We conclude that speciation in this radiation took place in sympatry, yet may have benefited from ongoing riverine gene flow.

Cooperative interactions enable singular olfactory receptor expression in mouse olfactory neurons.

The monogenic and monoallelic expression of only one out of >1000 mouse olfactory receptor (ORs) genes requires the formation of large heterochromatic chromatin domains that sequester the OR gene clusters. Within these domains, intergenic transcriptional enhancers evade heterochromatic silencing and converge into interchromosomal hubs that assemble over the transcriptionally active OR. The significance of this nuclear organization in OR choice remains elusive. Here, we show that transcription factors Lhx2 and Ebf specify OR enhancers by binding in a functionally cooperative fashion to stereotypically spaced motifs that defy heterochromatin. Specific displacement of Lhx2 and Ebf from OR enhancers resulted in pervasive, long-range, and trans downregulation of OR transcription, whereas pre-assembly of a multi-enhancer hub increased the frequency of OR choice in cis. Our data provide genetic support for the requirement and sufficiency of interchromosomal interactions in singular OR choice and generate general regulatory principles for stochastic, mutually exclusive gene expression programs.

The chromosome 11 olfactory receptor gene cluster is a candidate reproductive partner-specific compatibility locus

The chromosome 11 olfactory receptor gene cluster is a candidate reproductive partner-specific compatibility locus

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.

The role of a single olfactory receptor in odorant perception: relating receptor space to perceptual space

Although odorant identity is encoded by the activation patterns of olfactory receptors, we do not know how alterations in receptor activation affect the perceptual properties of an odorant. Human beings display considerable variation in odor perception, very little of which has been concretely linked to molecular causes. Here, we take advantage of genetic variations that affect human odorant receptor function to study the resulting alterations in olfactory perception. Based on previous studies demonstrating that amino acid alterations in OR7D4 affect perception of the odorant androstenone, we hypothesized that subjects with a nonfunctional odorant receptor allele will display varied perception of the receptor’s ligand, specifically a reduction in intensity and an increase in valence, in comparison to subjects with functional alleles. To address this issue, we asked 321 human subjects to rate the intensity and valence of 68 odors. We then developed a protocol to sequence each subject’s OR subgenome with high coverage. In a genotype/phenotype association analysis, we identified 27 polymorphisms in 8 distinct gene clusters that are associated with alterations in odorant perception more strongly than the association of the OR7D4 R88W variant with androstenone intensity in the same data set. Additionally, 35 polymorphisms fall above the 5% false discovery rate cutoff calculated by permutation analysis. These polymorphisms are found in 11 distinct olfactory receptor gene clusters, but extensive linkage disequilibrium precludes the identification of a single causal receptor. To address this problem, we generated clones of all major receptor variants found in a cluster on chromosome 11 containing 8 odorant receptor genes. These variants were subsequently tested in a high-throughput luciferase assay optimized for the study of olfactory receptor activation. We found that an OR10G4 allele associated with a significant decrease in the perceived intensity of guaiacol, A9V/M134V/V195E/R235G/K265Q, displayed a greatly reduced ability to respond to this odorant in vitro, suggesting a mechanistic basis for the genotype/phenotype association and linking perceptual variation to alterations in receptor function. This work suggests that despite the combinatorial nature of the olfactory code, alterations in a single odorant receptor can have a significant effect on odor perception and is a first step toward the development of a model relating receptor activation and odor perception.

Common variation contributes to the genetic architecture of social communication traits.

BackgroundSocial communication difficulties represent an autistic trait that is highly heritable and persistent during the course of development. However, little is known about the underlying genetic architecture of this phenotype.MethodsWe performed a genome-wide association study on parent-reported social communication problems using items of the children’s communication checklist (age 10 to 11 years) studying single and/or joint marker effects. Analyses were conducted in a large UK population-based birth cohort (Avon Longitudinal Study of Parents and their Children, ALSPAC, N = 5,584) and followed-up within a sample of children with comparable measures from Western Australia (RAINE, N = 1364).ResultsTwo of our seven independent top signals (P-discovery <1.0E-05) were replicated (0.009 <P-replication ≤0.02) within RAINE and suggested evidence for association at 6p22.1 (rs9257616, meta-P = 2.5E-07) and 14q22.1 (rs2352908, meta-P = 1.1E-06). The signal at 6p22.1 was identified within the olfactory receptor gene cluster within the broader major histocompatibility complex (MHC) region. The strongest candidate locus within this genomic area was TRIM27. This gene encodes an ubiquitin E3 ligase, which is an interaction partner of methyl-CpG-binding domain (MBD) proteins, such as MBD3 and MBD4, and rare protein-coding mutations within MBD3 and MBD4 have been linked to autism. The signal at 14q22.1 was found within a gene-poor region.Single-variant findings were complemented by estimations of the narrow-sense heritability in ALSPAC suggesting that approximately a fifth of the phenotypic variance in social communication traits is accounted for by joint additive effects of genotyped single nucleotide polymorphisms throughout the genome (h2(SE) = 0.18(0.066), P = 0.0027).ConclusionOverall, our study provides both joint and single-SNP-based evidence for the contribution of common polymorphisms to variation in social communication phenotypes.

Genetic modulation of HbF in Brazilians with HbSC disease and sickle cell anemia

Sickle cell anemia (homozygosity for HbS) and HbSC disease (compound heterozygosity for HbS and HbC) are the most common genotypes of sickle cell disease (SCD). Fetal hemoglobin (HbF), the major genetic modulator of SCD, interferes with deoxyHbS polymerization [1]. Patients with HbSC disease have about half the number of complications as do HbS homozygotes [1], however, their mean HbF levels were 2.4% compared with about 6% in patients with sickle cell anemia [2]. Single nucleotide polymorphisms (SNPs) in quantitative trait loci cis and trans to the β-globin gene cluster are associated with HbF. We analyzed the association of SNPs in BCL11A, (chromosome 2p), in the HBS1L-MYB intergenic region (HMIP) (chromosome 6q), and in the 5′ olfactory receptor (OR) gene cluster of chromosome 11p with HbF in Brazilian patients with SCD. 622 patients were studied. They were not treated with hydroxyurea and were aged ≥6 years. Hemoglobin was analyzed by HPLC. SNPs rs766432 and rs6732518 in BCL11A; rs35959442 and rs11759553 in HMIP; and rs4910755 and 7483122 located in OR51B5-OR51B6 were genotyped by ABI TaqMan Assays. The βS and βC globin haplotypes were investigated with PCR and restriction fragment length polymorphism techniques. Mean HbF was compared according to genotype using the additive genetic model adjusting for age, gender and HbS homozygotes versus HbSC. A dominant genetic model adjusting for age, gender and SCD genotype was used to test the association SNPs with HbF. We tested to see if there was an association between HbF and SNPs in the OR gene cluster region after adjusting for age, gender, SCD genotype and the HBB gene cluster haplotype (Central African Republic [CAR] or Bantu versus absence of a CAR haplotype). To determine if there was a synergistic effect between the SNPs or if the effect exerted on HbF was independent, we developed multivariable linear regression models adjusting for age, gender and SCD disease status and ran stepwise linear regression analyses where we added the most significant SNPs from BCL11A, HMIP one-at-a-time. Data analysis was performed using R version 2.14.1 and the cubic root transformation of HbF [3]. 392 patients were HbS homozygotes (HbF 7.4±4.4%) and 230 had HbSC disease (HbF 2.5±2.3%). The association between HbF and SNPs in the BCL11A, HMIP and the OR gene cluster for all patients combined and for HbS homozygotes and HbSC patients separately are shown in Table I a-c (on-line supplementary material). In the combined group, there is a statistically significant association between HbF levels and both BCL11A SNPs and with HMIP SNP rs11759553 after adjusting for age, gender and the SCD genotype. As the number of minor alleles increase, so does HbF level; in HbS homozygotes a similar result was found; in HbSC disease both SNPs in BCL11A and HMIP were associated with HbF. The association between HbF levels and SNPs in the OR genes, after adjusting fo r HBB haplotype was not significant (Table II, on-line supplementary material). In a stepwise linear regression analysis adding the SNPs to a multivariable linear regression model as described, after adjusting for age, gender and SCD genotype rs35959442 is still significantly associated with HbF indicating that its effect on HbF is independent of rs766432 (Table III, on-line supplementary material). We dichotomized the patients according to the HBB gene cluster haplotype. Brazilians with SCD have a higher prevalence of the CAR haplotype compared with African Americans. This haplotype is associated with lower levels of HbF than other haplotypes [4][5]. Sickle cell anemia patients were heterozygous or homozygous for the CAR haplotype chromosome or lacked a CAR haplotype chromosome; HbSC disease patients had or did not have a CAR haplotype chromosome. Most patients with a CAR haplotype had the minor allele for rs4910755 and rs7483122, both of which are located in the 5′ OR gene cluster of chromosome 11p and could be in linkage disequilibrium with SNPs characterizing this haplotype. HbSC disease patients usually have low HbF levels. Nevertheless, it is a milder condition than sickle cell anemia even though patients are more susceptible to clinical events related to high bloodviscosity. Although HbF in HbSC disease is lower than in HbS homozygotes, BCL11A and HMIP modulate HbF in both genotypes.

Identification of Regions Associated with Variation in Sensitivity to Food-Related Odors in the Human Genome

Humans vary in their ability to smell numerous odors [1-3], including those associated with food [4-6]. Odor sensitivity is heritable [7-11], with examples linking genetic variation for sensitivity to specific odors typically located near olfactory receptor (OR) genes [12-16]. However, with thousands of aromas and few deorphaned ORs [17, 18], there has been little progress toward linking variation at OR loci to odor sensitivity [19, 20]. We hypothesized that OR genes contain the variation that explains much of the differences in sensitivity for odors, paralleling the genetics of taste [21, 22], which affect the flavor experience of foods [23-25]. We employed a genome-wide association approach for ten food-related odors and identified genetic associations to sensitivity for 2-heptanone (p = 5.1 × 10(-8)), isobutyraldehyde (p = 6.4 × 10(-10)), β-damascenone (p = 1.6 × 10(-7)), and β-ionone (p = 1.4 × 10(-31)). Each locus is located in/near distinct clusters of OR genes. These findings increase the number of olfactory sensitivity loci to nine and demonstrate the importance of OR-associated variation in sensory acuity for food-related odors. Analysis of genotype frequencies across human populations implies that variation in sensitivity for these odors is widespread. Furthermore, each participant possessed one of many possible combinations of sensitivities for these odors, supporting the notion that everyone experiences their own unique "flavor world."

Smell and Autoimmunity: A Comprehensive Review

The sense of smell is an ancient sensory modality vital for sampling and perceiving the chemical composition of surrounding environments. Olfaction involves a pathway of biochemical and electrophysiological processes, which allows the conversion of molecular information into sensations. Disturbances in the olfactory function have been investigated mainly in neurological/neurodegenerative disorders such as Alzheimer's and Parkinson's diseases; impaired sense of smell has been associated with depressed mood. Only recently, smell capability was tested in other diseases, particularly autoimmune diseases. Shoenfeld and colleagues opened this chapter showing that patients affected with systemic lupus erythematosus (SLE) have disturbances in their olfactory functions and revealed its association with neuropsychiatric manifestations of the disease. This evidence was confirmed in experimental models and replicated in other SLE populations. The connection between autoimmunity and the sense of smell was lately emphasized by studies on patients with Sjögren's syndrome and in patients with other autoimmune/immune-mediated diseases, such as polydermatomyositis, recurrent spontaneous abortion, and hereditary angioedema. Genetic susceptibility and hormonal and environmental factors may play a role in these conditions. Olfactory receptor gene clusters are located in proximity to key locus of susceptibility for autoimmune diseases such as the major histocompatibility complex, suggesting not only a physic linkage, but a functional association. Nonetheless, gender- and hormone-mediated effects are fundamental in the development of autoimmune diseases. The different connections between smell and autoimmunity, genes and hormones may suggest that this is another tessera of a mosaic which is waiting the answer of Oedipus.

A genetic variant near olfactory receptor genes influences cilantro preference

The leaves of the Coriandrum sativum plant, known as cilantro or coriander, are widely used in many cuisines around the world. However, far from being a benign culinary herb, cilantro can be polarizing—many people love it while others claim that it tastes or smells foul, often like soap or dirt. This soapy or pungent aroma is largely attributed to several aldehydes present in cilantro. Cilantro preference is suspected to have a genetic component, yet to date nothing is known about specific mechanisms. Here, we present the results of a genome-wide association study among 14,604 participants of European ancestry who reported whether cilantro tasted soapy, with replication in a distinct set of 11,851 participants who declared whether they liked cilantro. We find a single-nucleotide polymorphism (SNP) significantly associated with soapy-taste detection that is confirmed in the cilantro preference group. This SNP, rs72921001 (p = 6.4 × 10−9, odds ratio 0.81 per A allele), lies within a cluster of olfactory receptor genes on chromosome 11. Among these olfactory receptor genes is OR6A2, which has a high binding specificity for several of the aldehydes that give cilantro its characteristic odor. We also estimate the heritability of cilantro soapy-taste detection in our cohort, showing that the heritability tagged by common SNPs is low, about 0.087. These results confirm that there is a genetic component to cilantro taste perception and suggest that cilantro dislike may stem from genetic variants in olfactory receptors. We propose that one of a cluster of olfactory receptor genes, perhaps OR6A2, may be the olfactory receptor that contributes to the detection of a soapy smell from cilantro in European populations.

Clinical and Genetic Variability of Red Blood Cell Hemolysis in Sickle Cell Anemia

Abstract 1077Intravascular hemolysis is an important pathological mechanism underlying some complications of sickle cell disease and other hemolytic anemias. Hemolysis contributes to endothelial dysfunction, pulmonary and systemic vasculopathy, and platelet and hemostatic activation via nitric oxide catabolism by plasma hemoglobin and arginine catabolism by red blood cell arginase. Little is known about the molecular mechanisms of hemolysis and how the propensity of erythrocytes to hemolyze is modulated. Hemoglobin F concentration and the presence of ∝ thalassemia affect the level of hemolysis but it is likely that other genes and their products are also important. We hypothesize that genetic variation, much of which is outside the β-globin gene-like cluster, underlies the susceptibility of erythrocytes to hemolyze in response to diverse disease stressors.We first characterized hemolysis by creating a principal component analysis (PCA) of age-adjusted values for LDH, AST, reticulocyte count and total bilirubin, but not hemoglobin concentration, to develop a hemolytic component that reflects shared variability among markers. The development of such a component helps to resolve the problem of dealing with correlated predictors in multivariate analyses and confounding variables such as site, and it permits for adjustment for the degree of anemia. To validate the PCA, we measured the plasma hemoglobin levels and red cell microparticle levels in the first and fourth quartile of PCA intensity of hemolysis in 118 HbS-only patients without detectable HbA, from the Walk-PHASST cohort We observed a highly significant increase in plasma hemoglobin (p<0.0001) and red cell microparticles (p=0.0004) based on PCA quartile. Despite the small sample size of this validating cohort we reproduced significant associations between high hemolytic rate and the subphentypes of low arterial oxygen saturation, high pulse pressure, leg ulcers, TRV, high NT-proBNP levels, and low 6-minute walk test distance. More patients with ∝ thalassemia and more females were present in the lower hemolytic index quartile (p=0.006).The hemolytic index and its individual components were then used as phenotypes in genome-wide association studies (GWAS) in the CSSCD (Cooperative Study of Sickle Cell Disease) and walk-PHaSST cohorts to discover novel genes that might be associated with hemolysis. As further validation of our approach using PCA stratification, patients in the quartile with the lowest hemolytic index from the CSSCD also had a much higher prevalence of ∝ thalassemia than patients within the highest quartile of hemolytic index (p=2.2E-16). We first examined 1117 cases from the CSSCD and found 303 SNPs, 265 with a MAF >0.05, that reached a threshold of significance of p<5E-4. For replication, we examined these SNPs in the Walk-PHASST cohort. Eight SNPs replicated with the same effects in a GWAS in 449 subjects from Walk-PHAAST and p-value<0.01. Of the 8 SNPs that replicated, 4 SNPs were in olfactory receptor (OR) genes on chromosome (chr) 11p; OR51L2 (rs7948471, rs7938426. rs1391617), and OR51L1 (rs2445284). Several of these SNPs were also associated with HbF in previous GWAS analyses. Polymorphisms in the OR gene cluster upstream of HBG might modulate HbF levels by altering chromatin structure within the HBB globin gene-like cluster. One SNP in an intron of NPRL3 (rs7203566) on chr16p is ∼34 kb upstream from a SNP causing ∝ thalassemia. In CSSCD cases there was an association of SNPs in NPRL3 with reticulocytes (p=5.1E-0006) and LDH (p=0.0003). In silico analysis did not predict any function for this SNP.Genetic studies to discover new biologic modifiers of hemolysis will help to identify critical molecular determinants of hemolysis for functional studies, to develop new disease severity biomarkers, and to suggest candidate therapies for some common human diseases with intravascular hemolysis. We anticipate that our studies will identify genetic variants enriched in the African-American population primarily determined by the evolved human response to endemic malaria infection. These studies are expected to broadly impact many human diseases and blood banking by providing genomic markers of susceptibility to hemolytic anemia, red cell storage stability and transfusion risk, and insights into novel strategies to reduce anemia and to enhance red blood cell storage and post-transfusion erythrocyte recovery. Disclosures:Gladwin:Patents filed related to treating hemolysis.: Patents & Royalties.

Association between olfactory receptor genes, eating behavior traits and adiposity: Results from the Quebec Family Study

Obesity is a major health problem that can be influenced by eating behaviors. Evidence suggests that the sensory properties of food influence eating behaviors and lead to overeating and overweight. A previous genome-wide linkage scan for eating behavior traits assessed with the Three-Factor Eating Questionnaire (cognitive dietary restraint, disinhibition and hunger) performed in the Quebec Family Study (QFS) revealed a quantitative trait locus for disinhibition on chromosome 19p13. This region encodes a cluster of seven olfactory receptor (OR) genes, including OR7D4, previously associated with odor perceptions. Direct sequencing of the OR7D4 gene revealed 16 sequence variants. Nine OR7D4 sequence variants with minor allele frequency (MAF)>1% as well as 100 SNPs spanning the cluster of OR genes on 19p13 were tested for association with age- and sex-adjusted eating behaviors as well as adiposity traits in 890 subjects. One OR7D4 sequence variant (rs2878329 G>A) showed evidence of association with reduced levels of adiposity (p=0.03), cognitive dietary restraint (p=0.05) and susceptibility to hunger (p=0.008). None of the OR7D4 SNPs was associated with disinhibition, but a SNP (rs2240927) in another OR gene (OR7E24) showed evidence of association (p=0.03). Another SNP in the OR7G3 gene (rs10414255) was also found to be associated with adiposity and eating behaviors. These results are the first to suggest that variations in human olfactory receptor genes can influence eating behaviors and adiposity. The associations reported in the present study should be interpreted with caution considering the number of tests performed and considered as potential new hypotheses about the effects OR polymorphisms on eating behaviors and obesity that need to be further explored in other populations.

Cardiac Defects Are Infrequent Findings in Individuals With 8p23.1 Genomic Duplications Containing GATA4

The GATA4 gene is critical to regulating myocardial differentiation and function. Haploinsufficiency of GATA4 is strongly associated with congenital heart defects (CHD). However, it is inconclusive whether duplicated GATA4 causes CHD. We evaluated 1645 consecutive pediatric patients with various developmental disorders by high-resolution microarray-based comparative genomic hybridization and found 8 probands and 2 relatives with pathogenic genomic imbalances containing GATA4. Four probands contain an ≈4.0-Mb interstitial duplication of 8p23.1 flanked by the 2 olfactory receptor gene clusters REPD and REPP, representing 0.24% (4/1645) of the patients analyzed. None of the 4 patients has CHD or any other heart diseases and 1 mother who transmitted the duplication to her child has a history of aortic stenosis. Two patients who carry multiple genomic abnormalities, including a duplication containing GATA4, have complex CHD. Only 1 of the 3 individuals carrying genomic deletion containing GATA4 has atrial septal and ventricular septal defects. Cardiac defects are infrequent findings in individuals with 8p23.1 genomic duplications containing GATA4. A 0.24% detection rate of this duplication in this study is significantly higher than previously estimated. Observation in 2 patients with multiple genomic abnormalities and complex CHD is consistent with a 2-hit model that emphasizes accumulative effects of >1 insult to the genome, leading to a visible or more severe clinical manifestation. Haploinsufficient GATA4 may show variable expressivity with a wide spectrum of clinical findings, including CHD.

HLA-DPB1-COL11A2 and three additional xMHC loci are independently associated with RA in a UK cohort

The aim of this study was to investigate the complex association pattern of the extended major histocompatibility complex (xMHC) region with rheumatoid arthritis (RA) susceptibility to identify effects independent of HLA-DRB1. A total of 1804 RA cases and 1474 controls were included. High-resolution HLA-DRB1 typing was performed. Subjects were genotyped for 1546 single-nucleotide polymorphisms (SNPs) using Affymetrix GeneChip 500 K (Santa Clara, CA, USA) as part of the Wellcome Trust Case Control Consortium Study. Statistical analysis was carried out using PLINK. To avoid confounding by RA-associated HLA-DRB1 alleles, we analyzed xMHC SNPs using a data set with pairwise matching of cases and controls on DRB1 genotypes. A total of 594 case-control pairs with identical DRB1 genotypes were identified. After this adjustment, 104 SNPs remained significantly associated with RA (P<0.05), suggesting that additional RA loci independent of HLA-DRB1 can be found in the xMHC region. Of these, four loci showed the strongest associations with RA (P<0.005): ZNF391, the olfactory receptor (OR) gene cluster, C6orf26-RDBP and HLA-DPB1-COL11A2. An additional locus mapping to the BTN (butyrophilin) cluster showed independent association with RA in anti-cyclic citrullinated peptide-positive patients exclusively. We have validated the previously described independent association of the HLA-DPB1-COL11A2 locus with RA. In addition, association with three novel independent RA loci in the xMHC region (ZNF391, OR2H1 and C6orf26-RDBP) has been detected.

WordCluster: detecting clusters of DNA words and genomic elements

BackgroundMany k-mers (or DNA words) and genomic elements are known to be spatially clustered in the genome. Well established examples are the genes, TFBSs, CpG dinucleotides, microRNA genes and ultra-conserved non-coding regions. Currently, no algorithm exists to find these clusters in a statistically comprehensible way. The detection of clustering often relies on densities and sliding-window approaches or arbitrarily chosen distance thresholds.ResultsWe introduce here an algorithm to detect clusters of DNA words (k-mers), or any other genomic element, based on the distance between consecutive copies and an assigned statistical significance. We implemented the method into a web server connected to a MySQL backend, which also determines the co-localization with gene annotations. We demonstrate the usefulness of this approach by detecting the clusters of CAG/CTG (cytosine contexts that can be methylated in undifferentiated cells), showing that the degree of methylation vary drastically between inside and outside of the clusters. As another example, we used WordCluster to search for statistically significant clusters of olfactory receptor (OR) genes in the human genome.ConclusionsWordCluster seems to predict biological meaningful clusters of DNA words (k-mers) and genomic entities. The implementation of the method into a web server is available at http://bioinfo2.ugr.es/wordCluster/wordCluster.php including additional features like the detection of co-localization with gene regions or the annotation enrichment tool for functional analysis of overlapped genes.

Observation and prediction of recurrent human translocations mediated by NAHR between nonhomologous chromosomes.

Four unrelated families with the same unbalanced translocation der(4)t(4;11)(p16.2;p15.4) were analyzed. Both of the breakpoint regions in 4p16.2 and 11p15.4 were narrowed to large ∼359-kb and ∼215-kb low-copy repeat (LCR) clusters, respectively, by aCGH and SNP array analyses. DNA sequencing enabled mapping the breakpoints of one translocation to 24 bp within interchromosomal paralogous LCRs of ∼130 kb in length and 94.7% DNA sequence identity located in olfactory receptor gene clusters, indicating nonallelic homologous recombination (NAHR) as the mechanism for translocation formation. To investigate the potential involvement of interchromosomal LCRs in recurrent chromosomal translocation formation, we performed computational genome-wide analyses and identified 1143 interchromosomal LCR substrate pairs, >5 kb in size and sharing >94% sequence identity that can potentially mediate chromosomal translocations. Additional evidence for interchromosomal NAHR mediated translocation formation was provided by sequencing the breakpoints of another recurrent translocation, der(8)t(8;12)(p23.1;p13.31). The NAHR sites were mapped within 55 bp in ∼7.8-kb paralogous subunits of 95.3% sequence identity located in the ∼579-kb (chr 8) and ∼287-kb (chr 12) LCR clusters. We demonstrate that NAHR mediates recurrent constitutional translocations t(4;11) and t(8;12) and potentially many other interchromosomal translocations throughout the human genome. Furthermore, we provide a computationally determined genome-wide "recurrent translocation map."