Discovering susceptibility genes for asthma and allergy

Asthma and asthma-related traits are complex diseases with strong genetic and environmental components. Rapid progress in asthma genetics has led to the identification of several candidate genes that are associated with asthma-related traits. Typically the phenotypic impact of each of these genes, including the ones most often replicated in association studies, is mild, but larger effects may occur when multiple variants synergize within a permissive environmental context. Despite the achievements made in asthma genetics formidable challenges remain. The development of novel, powerful tools for gene discovery, and a closer integration of genetics and biology, should help to overcome these challenges.     

Multiple genes for essential-hypertension susceptibility on chromosome 1q

Essential hypertension, defined as elevated levels of blood pressure (BP) without any obvious cause, is a major risk factor for coronary heart disease, stroke, and renal disease. BP levels and susceptibility to development of essential hypertension are partially determined by genetic factors that are poorly understood. Similar to other efforts to understand complex, non-Mendelian phenotypes, genetic dissection of hypertension-related traits employs genomewide linkage analyses of families and association studies of patient cohorts, to uncover rare and common disease alleles, respectively. Family-based mapping studies of elevated BP cover the large intermediate ground for identification of genes with common variants of significant effect. Our genomewide linkage and candidate-gene-based association studies demonstrate that a replicated linkage peak for BP regulation on human chromosome 1q, homologous to mouse and rat quantitative trait loci for BP, contains at least three genes associated with BP levels in multiple samples: ATP1B1, RGS5, and SELE. Individual variants in these three genes account for 2-5-mm Hg differences in mean systolic BP levels, and the cumulative effect reaches 8-10 mm Hg. Because the associated alleles in these genes are relatively common (frequency >5%), these three genes are important contributors to elevated BP in the population at large.     

Identification of candidate genes for dyslexia susceptibility on chromosome 18

Background

Six independent studies have identified linkage to chromosome 18 for developmental dyslexia or general reading ability. Until now, no candidate genes have been identified to explain this linkage. Here, we set out to identify the gene(s) conferring susceptibility by a two stage strategy of linkage and association analysis.

Methodology/Principal Findings

Linkage analysis: 264 UK families and 155 US families each containing at least one child diagnosed with dyslexia were genotyped with a dense set of microsatellite markers on chromosome 18. Association analysis: Using a discovery sample of 187 UK families, nearly 3000 SNPs were genotyped across the chromosome 18 dyslexia susceptibility candidate region. Following association analysis, the top ranking SNPs were then genotyped in the remaining samples. The linkage analysis revealed a broad signal that spans approximately 40 Mb from 18p11.2 to 18q12.2. Following the association analysis and subsequent replication attempts, we observed consistent association with the same SNPs in three genes; melanocortin 5 receptor (MC5R), dymeclin (DYM) and neural precursor cell expressed, developmentally down-regulated 4-like (NEDD4L).

Conclusions

Along with already published biological evidence, MC5R, DYM and NEDD4L make attractive candidates for dyslexia susceptibility genes. However, further replication and functional studies are still required.     

High-resolution linkage mapping for susceptibility genes in human polygenic disease: Insulin-dependent diabetes mellitus and chromosome 11q

Insulin-dependent diabetes mellitus (IDDM) has a complex pattern of genetic inheritance. In addition to genes mapping to the major histocompatibility complex (MHC), several lines of evidence point to the existence of other genetic susceptibility factors. Recent studies of the nonobese diabetic mouse (NOD) model of IDDM have suggested the presence, on mouse chromosome 9, of a susceptibility gene linked to the locus encoding the T-cell antigen, Thy-1. A region on human chromosome 11q is syntenic to this region on mouse chromosome 9. We have used a set of polymorphic DNA markers from chromosome 11q to investigate this region for linkage to a susceptibility gene in 81 multiplex diabetic pedigrees. The data were investigated by maximization of lod scores over genetic models and by multiple-locus affected-sib-pair analysis. We were able to exclude the presence of a susceptibility gene (location scores less than -2) throughout greater than 90% of the chromosome 11q homology region, under the assumption that the susceptibility factor would cause greater than 50% of affected sib pairs to share two alleles identical by descent. Theoretical estimates of the power to map susceptibility genes with a high-resolution map of linked markers in a candidate region were made, using HLA as a model locus. This result illustrates the feasibility that IDDM linkage studies using mapped sets of polymorphic DNA markers have, both for other areas of the genome in IDDM and for other polygenic diseases. The analytic approaches introduced here will be useful for affected-sib-pair studies of other complex phenotypes.     

Evidence for a susceptibility gene for autism on chromosome 2 and for genetic heterogeneity

Although there is considerable evidence for a strong genetic component to idiopathic autism, several genomewide screens for susceptibility genes have been performed with limited concordance of linked loci, reflecting either numerous genes of weak effect and/or sample heterogeneity. Because decreasing sample heterogeneity would increase the power to identify genes, the effect on evidence for linkage of restricting a sample of autism-affected relative pairs to those with delayed onset (at age >36 mo) of phrase speech (PSD, for phrase speech delay) was studied. In the second stage of a two-stage genome screen for susceptibility loci involving 95 families with two or more individuals with autism or related disorders, a maximal multipoint heterogeneity LOD score (HLOD) of 1.96 and a maximal multipoint nonparametric linkage (NPL) score of 2.39 was seen on chromosome 2q. Restricting the analysis to the subset of families (n=49) with two or more individuals having a narrow diagnosis of autism and PSD generated a maximal multipoint HLOD score of 2.99 and an NPL score of 3.32. The increased scores in the restricted sample, together with evidence for heterogeneity in the entire sample, indicate that the restricted sample comprises a population that is more genetically homogeneous, which could therefore increase the likelihood of positional cloning of susceptibility loci.     

Resequencing candidate genes implicates rare variants in asthma susceptibility

Common variation in over 100 genes has been implicated in the risk of developing asthma, but the contribution of rare variants to asthma susceptibility remains largely unexplored. We selected nine genes that showed the strongest signatures of weak purifying selection from among 53 candidate asthma-associated genes, and we sequenced the coding exons and flanking noncoding regions in 450 asthmatic cases and 515 nonasthmatic controls. We observed an overall excess of p values <0.05 (p = 0.02), and rare variants in four genes (AGT, DPP10, IKBKAP, and IL12RB1) contributed to asthma susceptibility among African Americans. Rare variants in IL12RB1 were also associated with asthma susceptibility among European Americans, despite the fact that the majority of rare variants in IL12RB1 were specific to either one of the populations. The combined evidence of association with rare noncoding variants in IL12RB1 remained significant (p = 3.7 × 10(-4)) after correcting for multiple testing. Overall, the contribution of rare variants to asthma susceptibility was predominantly due to noncoding variants in sequences flanking the exons, although nonsynonymous rare variants in DPP10 and in IL12RB1 were associated with asthma in African Americans and European Americans, respectively. This study provides evidence that rare variants contribute to asthma susceptibility. Additional studies are required for testing whether prioritizing genes for resequencing on the basis of signatures of purifying selection is an efficient means of identifying novel rare variants that contribute to complex disease.     

A major susceptibility gene for asthma maps to chromosome 14q24

Asthma is a complex genetic disorder with a heterogeneous phenotype, largely attributed to the interactions among many genes and between these genes and the environment. Numerous loci and candidate genes have been reported to show linkage and association to asthma and atopy. Although some studies reporting these observations are compelling, no gene has been mapped that confers a sufficiently high risk of asthma to meet the stringent criteria for genomewide significance. Using 175 extended Icelandic families that included 596 patients with asthma, we performed a genomewide scan with 976 microsatellite markers. The families were identified by cross-matching a list of patients with asthma from the Department of Allergy/Pulmonary Medicine of the National University Hospital of Iceland with a genealogy database of the entire Icelandic nation. We detected linkage of asthma to chromosome 14q24, with an allele-sharing LOD score of 2.66. After we increased the marker density within the locus to an average of one microsatellite every 0.2 cM, the LOD score rose to 4.00. We designate this locus "asthma locus one" (AS1). Taken together, these results provide evidence of a novel susceptibility gene for asthma on chromosome 14q24.     

Evidence for involvement of a second genetic locus on chromosome 11q in porphyrin metabolism

Chester porphyria is a distinct type of acute porphyria, which shows a biochemical overlap with acute intermittent and variegate porphyrias and has a dual enzyme deficiency of porphobilinogen deaminase (PBGD) and protoporphyrinogen oxidase. Linkage analysis in an extensive family with Chester porphyria was undertaken using multiple polymorphic markers. A maximum two point Lod score of 5.25 at 0.07 recombination (confidence interval 0.01 to 0.14) was observed with D11S351, which has been localised to 11q23.1. Multipoint linkage analysis confirmed the two point results and gave a maximum Lod score of 7.33 at a distance less than 1 cM proximal to D11S351. PBGD also maps to 11q but four recombinants could be identified from ten informative meioses in this family using a PBGD DNA polymorphism. Thus, a separate locus on 11q appears to be the basis of Chester porphyria.     

Multiple seizure susceptibility genes on chromosome 7 in SWXL-4 congenic mouse strains

The SWXL-4 recombinant inbred mouse strain is unusually sensitive to recurrent tonic-clonic seizures upon routine handling and to seizures induced by chemoconvulsants. In a conventional intercross with the ABP/Le strain, we previously mapped a SWXL-4-derived quantitative trait locus called Szf1 (seizure frequency 1) to Chromosome 7. In the present study, we confirm the existence of Szf1 in both an independent cross and a congenic strain. However, derivative congenic recombinant strains show that an interaction between at least two genes on Chromosome 7-each of which has a very small effect on its own-account for Szf1.     

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.     

Congenic mapping and candidate gene analysis for streptozotocin-induced diabetes susceptibility locus on mouse chromosome 11

Streptozotocin (STZ) has been widely used to induce diabetes in rodents. Strain-dependent variation in susceptibility to STZ has been reported; however, the gene(s) responsible for STZ susceptibility has not been identified. Here, we utilized the A/J-11^SM consomic strain and a set of chromosome 11 (Chr. 11) congenic strains developed from A/J-11^SM to identify a candidate STZ-induced diabetes susceptibility gene. The A/J strain exhibited significantly higher susceptibility to STZ-induced diabetes than the A/J-11^SM strain, confirming the existence of a susceptibility locus on Chr. 11. We named this locus Stzds1 (STZ-induced diabetes susceptibility 1). Congenic mapping using the Chr. 11 congenic strains indicated that the Stzds1 locus was located between D11Mit163 (27.72 Mb) and D11Mit51 (36.39 Mb). The Mpg gene, which encodes N-methylpurine DNA glycosylase (MPG), a ubiquitous DNA repair enzyme responsible for the removal of alkylated base lesions in DNA, is located within the Stzds1 region. There is a close relationship between DNA alkylation at an early stage of STZ action and the function of MPG. A Sanger sequence analysis of the Mpg gene revealed five polymorphic sites in the A/J genome. One variant, p.Ala132Ser, was located in a highly conserved region among rodent species and in the minimal region for retained enzyme activity of MPG. It is likely that structural alteration of MPG caused by the p.Ala132Ser mutation elicits increased recognition and excision of alkylated base lesions in DNA by STZ.

     

Variation in conserved non-coding sequences on chromosome 5q and susceptibility to asthma and atopy

Background

Evolutionarily conserved sequences likely have biological function.

Methods

To determine whether variation in conserved sequences in non-coding DNA contributes to risk for human disease, we studied six conserved non-coding elements in the Th2 cytokine cluster on human chromosome 5q31 in a large Hutterite pedigree and in samples of outbred European American and African American asthma cases and controls.

Results

Among six conserved non-coding elements (>100 bp, >70% identity; human-mouse comparison), we identified one single nucleotide polymorphism (SNP) in each of two conserved elements and six SNPs in the flanking regions of three conserved elements. We genotyped our samples for four of these SNPs and an additional three SNPs each in the IL13 and IL4 genes. While there was only modest evidence for association with single SNPs in the Hutterite and European American samples (P < 0.05), there were highly significant associations in European Americans between asthma and haplotypes comprised of SNPs in the IL4 gene (P < 0.001), including a SNP in a conserved non-coding element. Furthermore, variation in the IL13 gene was strongly associated with total IgE (P = 0.00022) and allergic sensitization to mold allergens (P = 0.00076) in the Hutterites, and more modestly associated with sensitization to molds in the European Americans and African Americans (P < 0.01).

Conclusion

These results indicate that there is overall little variation in the conserved non-coding elements on 5q31, but variation in IL4 and IL13, including possibly one SNP in a conserved element, influence asthma and atopic phenotypes in diverse populations.     

Evidence for a new Graves disease susceptibility locus at chromosome 18q21

Graves disease (GD) is a common autoimmune thyroid disorder that is inherited as a complex multigenic trait. By using a single microsatellite marker at each locus, we screened the type 1 diabetes loci IDDM4, IDDM5, IDDM6, IDDM8, and IDDM10 and the fucosyltransferase-2 locus for linkage in sib pairs with GD. This showed a two-point nonparametric linkage (NPL) score of 1.57 (P=.06) at the IDDM6 marker D18S41, but NPL scores were <1.0 at the other five loci. Thus, the investigation of the IDDM6 locus was extended by genotyping 11 microsatellite markers spanning 48 cM across chromosome 18q12-q22 in 81 sib pairs affected with autoimmune thyroid disease (AITD). Multipoint analysis, designating all AITD sib pairs as affected, showed a peak NPL score of 3.46 (P=.0003), at the marker D18S487. Designation of only GD cases as affected (74 sib pairs) showed a peak NPL score of 3.09 (P=.001). Linkage to this region has been demonstrated in type 1 diabetes (IDDM6), rheumatoid arthritis, and systemic lupus erythematosus, which suggests that this locus may have a role in several forms of autoimmunity.     

Birth weight in an African-American population living under moderate ecologic stress

The African-American population of McNary, Arizona, resides at an altitude of 2200 m. The lengthy winters are typically quite cold; the monthly mean temperature from November to April is 1.8 degrees C. Data from 318 singleton full-term births of African-American babies from 1949 to 1972 show a mean weight of 3095 g (s.d. = 427 g). At birth 1.9% of the babies weighed at least 4 kg; 9.7% weighed less than 2.5 kg. These data suggest that altitude may have influenced birth weight in this sample. Significant patterns in birth weight exist for sex, parity, mother's age, and severity of the winter preceding the year of birth. The birth weight of female babies born following warm winters is significantly lighter than those born during years following cold winters. There are relatively fewer high-birth-weight babies, in comparison to other African-American populations. Birth weight is also significantly lighter than three other African-American samples, even though African-American mothers of McNary had full-term professional care. Birth weight of African-American babies born in McNary is consistent with the overwhelming African ancestry of the African-American population of McNary. Sex differences in birth weight of babies born following cold winters can be ascribed to gender-related hereditary or physiologic factors at the level of the fetus. Maternal inactivity during particularly cold winters may be a contributing factor.     

Physical linkage of three CD3 genes on human chromosome 11.

T-cell antigen receptors are associated on T cell surfaces with a complex of proteins called CD3 (formerly T3). Human CD3 consists of at least four proteins, gamma, delta, epsilon and zeta, and all but the latter have been cloned as cDNA. Using standard cloning techniques, together with field inversion gel electrophoresis, we have demonstrated the physical linkage of three CD3 genes. The genes for CD3 gamma and CD3 delta are situated close together, about 1.6 kb apart, organized in a head-to-head orientation. The gene encoding CD3 gamma has been sequenced, and is split into seven exons spread over 9 kb of DNA. Like CD3 delta, CD3 gamma gene has an unusual promoter which lacks a TATA-box and potential Sp1 binding sites. The CD3 gamma-CD3 delta gene pair is within 300 kb of the CD3 epsilon gene, and therefore these genes form a tightly linked cluster in chromosome 11 band q23. The clustering of the CD3 genes may be significant in terms of their simultaneous activation during T-cell development.     

Replication of an African-American GWAS on blood pressure and hypertension in the Korean population

Hypertension is a complex disease that is caused by the interaction of multiple genetic and environmental risk factors, affecting 30% adult in industrialized countries. The identification of genetic factors that impact one’s predisposition to hypertension and its progression is an ongoing challenge. A genome-wide association study of African-Americans, who have one of the highest rates of hypertension in the world, was reported. We replicated the GWAS results in 8842 unrelated Koreans. Fifteen of the 30 reported SNPs were analyzed for their association with blood pressure and hypertension. Linear regression was used to analyze blood pressure as a quantitative trait in 7551 subjects, and a case-control study was performed using 1968 hypertensive cases and 4452 normotensive controls by logistic regression analysis. The quantitative trait study demonstrated a moderate association of 2 SNPs, rs9301196 (p=4.9×10^?3 for diastolic blood pressure) and rs2823756 (p=0.04 for systolic blood pressure), which were also associated with hypertension (p=0.042 and p=6.3×10^?3, respectively). Further, 3 SNPs were associated with hypertension (p=0.042 for rs7902529, p=0.027 for rs10135446, and p=0.01 for rs4613079) but not with blood pressure. Based on the moderate association signals and the low proportion of positive signals, this cross validation between African-Americans and Asians suggests that association studies of blood pressure traits require a larger number of subjects and a more refined design.     

Localization of two genes for Usher syndrome type I to chromosome 11.

The Usher syndromes (USH) are autosomal recessive diseases characterized by congenital sensorineural hearing loss and progressive pigmentary retinopathy. While relatively rare in the general population, collectively they account for approximately 6% of the congenitally deaf population. Usher syndrome type II (USH2) has been mapped to chromosome 1q (W. J. Kimberling, M. D. Weston, C. M?ller, et al., 1990, Genomics 7: 245-249; R. A. Lewis, B. Otterud, D. Stauffer, et al., 1990, Genomics 7: 250-256), and one form of Usher syndrome type I (USH1) has been mapped to chromosome 14q (J. Kaplan, S. Gerber, D. Bonneau, J. Rozet, M. Briord, J. Dufier, A. Munnich, and J. Frezal, 1990. Cytogenet. Cell Genet. 58: 1988). These loci have been excluded as regions of USH genes in our data set, which is composed of 8 French-Acadian USH1 families and 11 British USH1 families. Both of these sets of families show linkage to loci on chromosome 11. Linkage analysis demonstrates locus heterogeneity between these sets of families, with the French-Acadian families showing linkage to D11S419 (Z = 4.20, theta = 0) and the British families showing linkage to D11S527 (Z = 6.03, theta = 0). Genetic heterogeneity of the data set was confirmed using HOMOG and the M test (log likelihood ratio > 10(5)). These results confirm the presence of two distinct USH1 loci on chromosome 11.     

A susceptibility locus for myopia in the normal population is linked to the PAX6 gene region on chromosome 11: a genomewide scan of dizygotic twins

Myopia is a common, complex trait with considerable economic and social impact and, in highly affected individuals, ocular morbidity. We performed a classic twin study of 506 unselected twin pairs and inferred the heritability of refractive error to be 0.89 (95% confidence interval 0.86-0.91). A genomewide scan of 221 dizygotic twin pairs, analyzed by use of optimal Haseman-Elston regression methods implemented by use of generalized linear modeling, showed significant linkage (LOD >3.2) to refractive error at four loci, with a maximum LOD score of 6.1 at 40 cM on chromome 11p13. Evidence of linkage at this locus, as well as at the other linkage peaks at chromosomes 3q26 (LOD 3.7), 8p23 (LOD 4.1), and 4q12 (LOD 3.3), remained the same or became stronger after model fit was checked and outliers were downweighted. Examination of potential candidate genes showed the PAX6 gene directly below the highest peak at the 11p13 locus. PAX6 is fundamental to identity and growth of the eye, but reported mutations usually result in catastrophic congenital phenotypes such as aniridia. Haplotype tagging of 17 single-nucleotide polymorphisms (SNPs), which covered the PAX6 gene and had common minor allele frequencies, identified 5 SNPs that explained 0.999 of the haplotype diversity. Linkage and association analysis of the tagging SNPs showed strong evidence of linkage for all markers with a minimum chi 21 of 7.5 (P=.006) but no association. This suggests that PAX6 may play a role in myopia development, possibly because of genetic variation in an upstream promoter or regulator, although no definite association between PAX6 common variants and myopia was demonstrated in this study.     

Large-scale search of SNPs for type 2 DM susceptibility genes in a Japanese population

The etiology of type 2 diabetes (DM) is polygenic. We investigated here genes and polymorphisms that associate with DM in the Japanese population. Single-nucleotide polymorphisms (SNPs) of 398 derived from 120 candidate genes were examined for association with DM in a population-based case-control study. The study group consisted of 148 cases and 227 controls recruited from Funagata, Japan. No evident subpopulation structure was detected for the tested population. The association tests were conducted with standard allele positivity tables (chi(2) tests) between SNP genotype frequency and case-control status. The independent association of the SNPs from serum triglyceride levels and body mass index was examined by multiple logistic regression analysis. A value of P<0.01 was accepted as statistically significant. Six genes (met proto-oncogene, ATP-binding cassette transporter A1, fatty acid binding protein 2, LDL receptor defect C complementing, aldolase B, and sulfonylurea receptor) were shown to be associated with DM.