Timothy D. Howard

Gene-gene interaction in asthma: IL4RA and IL13 in a Dutch population with asthma.

Asthma is a common respiratory disease that is characterized by variable airways obstruction caused by acute and chronic bronchial inflammation; associated phenotypes include bronchial hyperresponsiveness (BHR), elevated total serum immunoglobulin E (IgE) levels, and skin tests positive to common allergens. Binding of interleukin-13 (IL13) or interleukin-4 (IL4) to the IL4 receptor (IL4R) induces the initial response for Th2 lymphocyte polarization. Both IL13 and IL4 are produced by Th2 cells and are capable of inducing isotype class-switching of B-cells to produce IgE after allergen exposure. These cytokines also share a common receptor component, IL4R alpha. We have investigated five IL4RA single-nucleotide polymorphisms in a population of Dutch families ascertained through a proband with asthma. By considering the probands and their spouses as an unrelated sample, we observed significant associations of atopy and asthma-related phenotypes with several IL4RA polymorphisms, including S478P and total serum IgE levels (P=.0007). A significant gene-gene interaction between S478P in IL4RA and the -1111 promoter variation in IL13, previously shown to be associated with BHR (P=.003), was detected. Individuals with the risk genotype for both genes were at almost five times greater risk for the development of asthma compared to individuals with both non-risk genotypes (P=.0004). These data suggest that variations in IL4RA contribute to elevated total serum IgE levels, and interaction between IL4RA and IL13 markedly increases an individuals susceptibility to asthma.

Common Estrogen Receptor Polymorphism Augments Effects of Hormone Replacement Therapy on E-Selectin but Not C-Reactive Protein

Background—The estrogen receptor-&agr; (ER-&agr;) IVS1-401 polymorphism identifies a group of women (≈20%) who have augmented effects of hormone replacement therapy (HRT) on levels of HDL cholesterol. This study sought to determine if this augmentation extends to HRT regulation of E-selectin and C-reactive protein (CRP) and to explore possible mechanisms by which this polymorphism might influence estrogen action. Methods and Results—Serum levels of soluble E-selectin and CRP were measured at baseline and 1 year in 264 postmenopausal women randomized to treatment with oral conjugated equine estrogen (0.625 mg/d), estrogen plus progestin (medroxyprogesterone acetate 2.5 mg/d), or placebo. Women with the ER-&agr; IVS1-401 C/C genotype receiving HRT had nearly a 2-fold greater reduction in E-selectin compared with C/T or T/T women (P for interaction=0.02). In contrast, there was no augmentation of the HRT-associated increase in CRP among the C/C women compared with C/T or T/T women (P for interaction=0.54). Of luciferase reporter constructs containing sequences spanning the IVS1-401 T/C polymorphism, expression of the construct containing the C allele was enhanced >10-fold, with cotransfection of a constitutively expressed B-myb vector. In contrast, B-myb resulted in only a 2.5-fold increase in expression of the T allele construct. Conclusions—Women with the ER-&agr; IVS1-401 C/C genotype have greater reductions in E-selectin but no further increases in CRP with HRT. The C allele produces a functional binding site for the transcription factor B-myb. The impact of this polymorphism on ER-&agr; transcription and other estrogen-sensitive intermediate and clinical end points has not yet been established.

Genomewide Screen and Identification of Gene-Gene Interactions for Asthma-Susceptibility Loci in Three U.S. Populations: Collaborative Study on the Genetics of Asthma

The genomewide screen to search for asthma-susceptibility loci, in the Collaborative Study on the Genetics of Asthma (CSGA), has been conducted in two stages and includes 266 families (199 nuclear and 67 extended pedigrees) from three U.S. populations: African American, European American, and Hispanic. Evidence for linkage with the asthma phenotype was observed for multiple chromosomal regions, through use of several analytical approaches that facilitated the identification of multiple disease loci. Ethnicity-specific analyses, which allowed for different frequencies of asthma-susceptibility genes in each ethnic population, provided the strongest evidence for linkage at 6p21 in the European American population, at 11q21 in the African American population, and at 1p32 in the Hispanic population. Both the conditional analysis and the affected-sib-pair two-locus analysis provided further evidence for linkage, at 5q31, 8p23, 12q22, and 15q13. Several of these regions have been observed in other genomewide screens and linkage or association studies, for asthma and related phenotypes. These results were used to develop a conceptual model to delineate asthma-susceptibility loci and their genetic interactions, which provides a promising basis for initiation of fine-mapping studies and, ultimately, for gene identification.

Linkage of Genetic Markers on Human Chromosomes 20 and 12 to NIDDM in Caucasian Sib Pairs With a History of Diabetic Nephropathy

The potential contribution of maturity-onset diabetes of the young (MODY) genes to NIDDM susceptibility in African-American and Caucasian NIDDM-affected sibling pairs with a history of adult-onset diabetic nephropathy has been evaluated. Evidence for linkage to NIDDM was found with polymorphic loci that map to the long arms of human chromosomes 20 and 12 in regions containing the MODY1 and MODY3 genes. Nonparametric analysis of chromosome 20 inheritance data collected with the MODYl-linked marker D20S197 provides evidence forlinkage to NIDDM with a P value of 0.005 in Caucasian sib pairs using affected sibpair (ASP) analyses. Nonparametric analysis of chromosome 12 inheritance data collected with the MODY3-linked markers D12S349 and D12S86 provides evidence for linkage to NIDDM with P values of 0.04 and 0.006, respectively, in Caucasian sib pairs using similar analyses. No evidence for linkage of MODY1 and MODY3 markers to NIDDM in African-American sib pairs was observed. In addition, no evidence for linkage to MODY2 (glucokinase-associated MODY) was observed with either study population. Results of multipoint maximum logarithm of odds (LOD) score analysis were consistent with the ASP results. A maximum LOD score of 1.48 was calculated for linkage to MODYl-linked loci and 1.45 to MODY3-linked loci in Caucasian sib pairs. Tabulation of allele sharing in affected sib pairs with D20S197 and D12S349 suggests that affected sibling pairs may inherit susceptibility genes simultaneously from chromosome 20 and chromosome 12. The results suggest that genes contributing to NIDDM in the general Caucasian population are located in the regions containing the MODY1 and MODY3 genes.

Fine Mapping and Positional Candidate Studies Identify HLA-G as an Asthma Susceptibility Gene on Chromosome 6p21

Asthma affects nearly 14 million people worldwide and has been steadily increasing in frequency for the past 50 years. Although environmental factors clearly influence the onset, progression, and severity of this disease, family and twin studies indicate that genetic variation also influences susceptibility. Linkage of asthma and related phenotypes to chromosome 6p21 has been reported in seven genome screens, making it the most replicated region of the genome. However, because many genes with individually small effects are likely to contribute to risk, identification of asthma susceptibility loci has been challenging. In this study, we present evidence from four independent samples in support of HLA-G as a novel asthma and bronchial hyperresponsiveness susceptibility gene in the human leukocyte antigen region on chromosome 6p21, and we speculate that this gene might contribute to risk for other inflammatory diseases that show linkage to this region.

Polymorphisms of the ADAM33 gene are associated with accelerated lung function decline in asthma

Background Asthma is a genetically complex disease characterized by respiratory symptoms, intermittent airway obstruction and airway hyper‐responsiveness due to airway inflammation and remodelling. The ADAM33 gene is associated with asthma and airway hyper‐responsiveness and is postulated as a gene for airway remodelling.

Major Genes Regulating Total Serum Immunoglobulin E Levels in Families with Asthma

Immunoglobulin E (IgE) has a major role in the pathogenesis of allergic disorders and asthma. Previous data from 92 families, each identified through a proband with asthma, showed evidence for two major genes regulating total serum IgE levels. One of these genes mapped to 5q31-33. In the current study, the segregation analysis was extended by the addition of 108 probands and their families, ascertained in the same manner. A mixed recessive model (i.e., major recessive gene and residual genetic effect) was the best-fitting and most-parsimonious one-locus model of the segregation analysis. A mixed two-major-gene model (i.e., two major genes and residual genetic effect) fit the data significantly better than did the mixed recessive one-major-gene model. The second gene modified the effect of the first recessive gene. Individuals with the genotype aaBB (homozygous high-risk allele at the first gene and homozygous low-risk allele at the second locus) had normal IgE levels (mean 23 IU/ml), and only individuals with genotypes aaBb and aabb had high IgE levels (mean 282 IU/ml). A genomewide screening was performed using variance-component analysis. Significant evidence for linkage was found for a novel locus at 7q, with a multipoint LOD score of 3. 36 (P=.00004). A LOD score of 3.65 (P=.00002) was obtained after genotyping additional markers in this region. Evidence for linkage was also found for two previously reported regions, 5q and 12q, with LOD scores of 2.73 (P=.0002) and 2.46 (P=.0004), respectively. These results suggest that several major genes, plus residual genetic effects, regulate total serum IgE levels.

Genome-wide Association Study for Coronary Artery Calcification with Follow-up in Myocardial Infarction

Background— Coronary artery calcification (CAC) detected by computed tomography is a noninvasive measure of coronary atherosclerosis, which underlies most cases of myocardial infarction (MI). We sought to identify common genetic variants associated with CAC and further investigate their associations with MI. Methods and Results— Computed tomography was used to assess quantity of CAC. A meta-analysis of genome-wide association studies for CAC was performed in 9961 men and women from 5 independent community-based cohorts, with replication in 3 additional independent cohorts (n=6032). We examined the top single-nucleotide polymorphisms (SNPs) associated with CAC quantity for association with MI in multiple large genome-wide association studies of MI. Genome-wide significant associations with CAC for SNPs on chromosome 9p21 near CDKN2A and CDKN2B (top SNP: rs1333049; P=7.58×10−19) and 6p24 (top SNP: rs9349379, within the PHACTR1 gene; P=2.65×10−11) replicated for CAC and for MI. Additionally, there is evidence for concordance of SNP associations with both CAC and MI at a number of other loci, including 3q22 (MRAS gene), 13q34 (COL4A1/COL4A2 genes), and 1p13 (SORT1 gene). Conclusions— SNPs in the 9p21 and PHACTR1 gene loci were strongly associated with CAC and MI, and there are suggestive associations with both CAC and MI of SNPs in additional loci. Multiple genetic loci are associated with development of both underlying coronary atherosclerosis and clinical events.

Factor V Leiden, Hormone Replacement Therapy, and Risk of Venous Thromboembolic Events in Women With Coronary Disease

Oral contraceptive use in women with factor V Leiden is associated with increased rates of venous thromboembolic events (VTEs). However, the effects of hormone replacement therapy (HRT) in postmenopausal women with factor V Leiden are not known. A nested case-control study was conducted among women with established coronary disease enrolled in 2 randomized clinical trials of HRT, the Heart and Estrogen/Progestin Replacement Study (HERS) and the Estrogen Replacement and Atherosclerosis (ERA) trial. The Leiden mutation was present in 8 (16.7%) of 48 cases with VTE compared with only 7 (6.3%) of 112 controls (odds ratio [OR]Leiden 3.3, 95% CI 1.1 to 9.8;P =0.03). In women without the factor V Leiden mutation, risk associated with HRT use was significantly increased (ORHRT 3.7, 95% CI 1.4 to 9.4;P <0.01). On the other hand, in women with the factor V Leiden mutation, the estimated risk associated with HRT was increased nearly 6-fold, although the CIs were wide and included unity (ORHRT 5.7, 95% CI 0.6 to 53.9;P =0.13). The OR for women with the Leiden mutation who were also assigned to HRT compared with wild-type women assigned to placebo was 14.1 (95% CI 2.7 to 72.4, P =0.0015). In women with the factor V Leiden mutation who were treated with HRT, the estimated absolute incidence of VTE was 15.4 of 1000 per year compared with 2.0 of 1000 per year in women without the mutation who were taking a placebo (P =0.0015). On the basis of these data, in women with coronary disease, the estimated number needed to screen for factor V Leiden to avoid an HRT-associated VTE during 5 years of treatment is 376. If factor V Leiden genotyping becomes less expensive, it could be cost effective to screen for the presence of the mutation before instituting HRT in women with coronary disease.

Identification of PCDH1 as a novel susceptibility gene for bronchial hyperresponsiveness.

RATIONALE Asthma is a chronic inflammatory airway disease that affects more than 300 million individuals worldwide. Asthma is caused by interaction of genetic and environmental factors. Bronchial hyperresponsiveness (BHR) is a hallmark of asthma and results from increased sensitivity of the airways to physical or chemical stimulants. BHR and asthma are linked to chromosome 5q31-q33. OBJECTIVES To identify a gene for BHR on chromosome 5q31-q33. METHODS In 200 Dutch families with asthma, linkage analysis and fine mapping were performed, and the Protocadherin 1 gene (PCDH1) was identified. PCDH1 was resequenced in 96 subjects from ethnically diverse populations to identify novel sequence variants. Subsequent replication studies were undertaken in seven populations from The Netherlands, the United Kingdom, and the United States, including two general population samples, two family samples, and three case-control samples. PCDH1 mRNA and protein expression was investigated using polymerase chain reaction, Western blotting, and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS In seven out of eight populations (n = 6,168) from The Netherlands, United Kingdom, and United States, PCHD1 gene variants were significantly associated with BHR (P values, 0.005-0.05) This association was present in both families with asthma and general populations. PCDH1 mRNA and protein were expressed in airway epithelial cells and in macrophages. CONCLUSIONS PCDH1 is a novel gene for BHR in adults and children. The identification of PCDH1 as a BHR susceptibility gene may suggest that a structural defect in the integrity of the airway epithelium, the first line of defense against inhaled substances, contributes to the development of BHR.