Sumit Bhattacharyya

Extent and distribution of linkage disequilibrium in three genomic regions

The positional cloning of genes underlying common complex diseases relies on the identification of linkage disequilibrium (LD) between genetic markers and disease. We have examined 127 polymorphisms in three genomic regions in a sample of 575 chromosomes from unrelated individuals of British ancestry. To establish phase, 800 individuals were genotyped in 160 families. The fine structure of LD was found to be highly irregular. Forty-five percent of the variation in disequilibrium measures could be explained by physical distance. Additional factors, such as allele frequency, type of polymorphism, and genomic location, explained <5% of the variation. Nevertheless, disequilibrium was occasionally detectable at 500 kb and was present for over one-half of marker pairs separated by <50 kb. Although these findings are encouraging for the prospects of a genomewide LD map, they suggest caution in interpreting localization due to allelic association.

Positional cloning of a quantitative trait locus on chromosome 13q14 that influences immunoglobulin E levels and asthma

Atopic or immunoglobulin E (IgE)-mediated diseases include the common disorders of asthma, atopic dermatitis and allergic rhinitis. Chromosome 13q14 shows consistent linkage to atopy and the total serum IgE concentration. We previously identified association between total serum IgE levels and a novel 13q14 microsatellite (USAT24G1; ref. 7) and have now localized the underlying quantitative-trait locus (QTL) in a comprehensive single-nucleotide polymorphism (SNP) map. We found replicated association to IgE levels that was attributed to several alleles in a single gene, PHF11. We also found association with these variants to severe clinical asthma. The gene product (PHF11) contains two PHD zinc fingers and probably regulates transcription. Distinctive splice variants were expressed in immune tissues and cells.

Positional cloning of a novel gene influencing asthma from chromosome 2q14

Asthma is a common disease in children and young adults. Four separate reports have linked asthma and related phenotypes to an ill-defined interval between 2q14 and 2q32 (refs. 1–4), and two mouse genome screens have linked bronchial hyper-responsiveness to the region homologous to 2q14 (refs. 5,6). We found and replicated association between asthma and the D2S308 microsatellite, 800 kb distal to the IL1 cluster on 2q14. We sequenced the surrounding region and constructed a comprehensive, high-density, single-nucleotide polymorphism (SNP) linkage disequilibrium (LD) map. SNP association was limited to the initial exons of a solitary gene of 3.6 kb (DPP10), which extends over 1 Mb of genomic DNA. DPP10 encodes a homolog of dipeptidyl peptidases (DPPs) that cleave terminal dipeptides from cytokines and chemokines, and it presents a potential new target for asthma therapy.

Transmission ratio distortion at the INS-IGF2 VNTR.

defined as a statistically significant departure from mendelian transmission. So far, evidence of this in humans has been limited or controversial1–4, and the few established examples involve chromosome rearrangements in lower organisms5. The variable number of tandem repeat (VNTR) polymorphism 596 bp 5 ́ of the insulin gene (INS) regulates expression of both INS and the gene encoding insulinlike growth factor 2 (IGF2). The VNTR can be subdivided into two main allele sizes, class I and class III, in Europeans. Class III alleles are associated with reduced expression of INS and IGF2 in the pancreas and placenta6. As lower expression of VNTR class III-associated INS/IGF2 alleles early in fetal development may reduce the chances of survival in utero, the locus is considered a candidate for exhibiting TRD. Our initial study comprised 381 families with 1,128 children not ascertained by disease status (Table 1). We used the transmission/disequilibrium test7 (TDT) and found evidence for excess transmission of the class I allele (TRD=0.54; P = 0.04). To replicate this finding we analysed existing data for an independent set of 1,072 nuclear families with 1,079 children (Table 1) and obtained an identical TRD of 0.54 (P=0.02). Overall, the TRD of 0.54 was significant at P=0.002 (95% confidence limits=0.51<TRD<0.56). As TRD may be parent-of-origin specific or dependent on the sex of the offspring, we compared TRD in paternal and maternal meioses and analysed transmissions to males and females separately. We found no evidence of a parent-of-origin or a sex-specific effect (data not shown). Transmission ratio distortion at the INS-IGF2 VNTR

Sequence variants in the melatonin-related receptor gene (GPR50) associate with circulating triglyceride and HDL levels

The gene encoding the melatonin-related receptor (GPR50) is highly expressed within hypothalamic nuclei concerned with the control of body weight and metabolism. We screened GPR50 for mutations in an obese cohort and identified an insertion of four amino acid residues (TTGH) at position 501, two common coding polymorphisms (T528A and V602I), and one noncoding polymorphism (C-16X2GPR50T). Single-nucleotide polymorphisms were then typed in 500 English Caucasian subjects, and associations were sought to intermediate obesity phenotypes. Although no association was seen with body mass index, carriers of two copies of the mutant allele at C-16X2GPR50T, Ins501Del, and A1582G had significantly higher fasting circulating triglyceride levels (P < 0.05). In a separate set of 585 subjects, the associations were replicated, with statistically significant effects of similar magnitude and direction. The association of C-16X2GPR50T with fasting triglycerides was highly significant (P < 0.001). In addition, a significant association between C-16X2GPR50T and circulating HDL levels was observed in the combined population, with C-16X2GPR50T carriers having significantly lower circulating HDL-cholesterol levels (1.39 mM) than wild-type subjects (1.47 mM) (P < 0.01). These findings suggest a previously unexpected role for this orphan receptor in the regulation of lipid metabolism that warrants further investigation.

Positive association to IgE levels and a physical map of the 13q14 atopy locus.

Linkage of atopy and associated traits to a locus on chromosome 13q14 has been identified by several studies in diverse populations. We have previously shown the putative atopy gene to be contained within an interval of approximately 5 Mb flanked by D13S328 and D13S1269 and centred on D13S273. We have now extended this work using a top-down approach to physical mapping. A YAC contig was constructed covering the D13S328 and D13S1269 interval. Thirty-one ESTs were mapped to the contig. We constructed a BAC and PAC contig flanking D13S273 by ∼750 kb in either direction. The interval contained 27 of the 31 ESTS from the YAC contig. Seven previously unknown microsatellites were recovered and then typed in two subject panels. A positive association between the total serum Immunoglobulin E concentration and the novel USAT24G1 microsatellite was discovered (Pcorrected<0.005) and replicated in a second panel of families. The discovery of a region of positive association within the BAC/PAC contig will permit identification of the atopy gene from this locus.

Association Between Physical Activity and Blood Pressure Is Modified by Variants in the G-Protein Coupled Receptor 10

Abstract—Hypertension is strongly related to cardiovascular disease and all-cause mortality. Exercise reduces blood pressure but the response varies between individuals. The mechanisms by which physical activity energy expenditure (PAEE) modifies blood pressure are not fully defined but include modulation of sympathetic tone. Novel polymorphisms in the G-protein coupled receptor (GPR10) have been linked with high blood pressure. GPR10 may mediate the relationship between PAEE and blood pressure via central nervous mechanisms. We examined whether two GPR10 polymorphisms (G-62A and C914T) modify the association between PAEE and blood pressure in the MRC Ely study (N=687). When stratified by the C914T genotype, there were between-group differences for body mass index (BMI) (P =0.05), diastolic blood pressure (DBP) (P =0.006), and systolic blood pressure (SBP) (P =0.005). No differences were found between G-62A genotypes. The previously reported inverse relationship between PAEE and blood pressure was not observed in minor allele carriers for either polymorphism (A62 carriers: DBP &bgr;-1.11, P =0.52; SBP &bgr;-1.66, P =0.52. T914 carriers: SBP &bgr;=3.27; P =0.60) but was in common allele homozygotes (G62G: DBP &bgr;-6.18 P =0.00001; SBP &bgr;-8.54 P =0.0001. C914C: SBP &bgr;-7.07; P =0.00001). This corresponded to a significant interaction between PAEE and GPR10 polymorphisms on DBP (G-62A:P =0.006) and SBP (G-62A:P =0.008. C914T:P =0.068). Significant interactions were observed between haplotype (derived from G-62A and C914T), PAEE, and blood pressure (DBP:P =0.08; SBP:P =0.023). The effect of physical activity on blood pressure is highly variable at population level. Knowledge of GPR10 genotype may define those who are least likely to benefit from physical activity. These findings may have relevance in the targeted treatment of hypertensive disease.

A detailed genetic map of the chromosome 7 bronchial hyper-responsiveness locus

Non-specific bronchial hyper-responsiveness to various inhaled stimuli is a characteristic of asthma. We have previously shown linkage of bronchial responsiveness to methacholine (measured as dose-response slope (DRS)) and the peripheral blood eosinophil count (EOS) to chromosome 7. We have now further investigated these linkages by genotyping 49 microsatellite markers across the DRS locus on chromosome 7. The markers were spaced on average 2.6 cM apart and spanned a sex averaged cumulative genetic distance of 129 cM. Multipoint linkage to DRS was bimodal and dipped at the centromere. The two peaks of linkage were close to markers D7S484 (P=0.0003) and D7S669 (P=0.006) respectively. Separate testing for linkage to paternally and maternally derived alleles showed that the linkage near D7S484 was paternally derived (P<0.00001): maternally derived alleles did not exhibit significant linkage. The results indicate that two disparate loci may be influencing asthma from chromosome 7.

A randomized trial of the effects of the no-carrageenan diet on ulcerative colitis disease activity

BACKGROUND: Carrageenan is a very common food additive in Western diets, but predictably causes inflammation in thousands of cell-based and animal experiments. OBJECTIVE: To assess the impact of carrageenan exposure on the interval to relapse in patients with ulcerative colitis in remission. METHODS: A randomized, double-blind, placebo-controlled, multicenter, clinical trial was conducted to assess if patients with ulcerative colitis in remission would have a longer interval to relapse if they followed a diet with no carrageenan. All participants were instructed in the no-carrageenan diet and were randomized to either placebo capsules or carrageenan-containing capsules. The carrageenan in the capsules was less than the average daily carrageenan intake from the diet. Relapse was defined as an increase of two or more points on the Simple Clinical Colitis Activity Index (SCCAI) and intensification of treatment for ulcerative colitis. Participants were followed by telephone calls every two weeks until relapse or one year of participation. The occurrence of relapse and inflammatory biomarkers were compared between the two groups. RESULTS: Twelve patients completed study questionnaires. Three patients who received carrageenan-containing capsules relapsed, and none of the patients who received placebo-containing capsules relapsed (p = 0.046, log-rank test). Laboratory tests showed increases in Interleukin-6 (p = 0.02, paired t-test, two-tailed) and fecal calprotectin (p = 0.06; paired t-test, two-tailed) between the beginning and the end of study participation in the carrageenan-exposed group, but not in the placebo-group. CONCLUSION: Carrageenan intake contributed to earlier relapse in patients with ulcerative colitis in remission. Restriction of dietary carrageenan may benefit patients with ulcerative colitis.