GENETICS AND GENOMICS OF CHRONIC INFLAMMATORY DISORDERS, III: BRONCHIAL ASTHMA

GENETICS AND GENOMICS OF CHRONIC INFLAMMATORY DISORDERS, III: BRONCHIAL ASTHMA

INTRODUCTION

Bronchial asthma is characterized by an abnormal mucosa of the airways, inflammation, and symptoms of wheezing and shortness of breath. Asthma affects more than one child in 10 in the developed world, and there are 300 million cases worldwide.1 Although some effective therapies for mild asthma exist, severe asthma remains difficult to treat. The societal cost of the disease is substantial in Westernized societies,2,3 and it is likely to become overwhelmingly important in the developing world as asthma rises in prevalence there.

Asthma should be considered a syndrome rather than a disease. Childhood- onset disease has a strong association with atopy (diagnosed by positive skin tests, or elevations of allergen-specific and total serum immunoglobulin E [IgE] concentrations) and is more common in males. Adult-onset disease typically comes on after 40 years of age, is more common in women, is not associated with atopy beyond the general population prevalence of the condition, is at times associated with cigarette smoking, and is often resistant to therapy.

Occupational asthma is attributable to, or is made worse by, environmental exposures in the workplace and may be present in 10–15% of cases of adult disease.4 It serves as an exemplar for dose–response relationships in allergen- induced disease (for example, in bakers) and disease induced by small molecules (such as isocyanate paint additives). Other clinical variants of asthma may include severe disease, therapy-resistant asthma, brittle asthma, and asthma that results in fixed airway diseases.

Asthma   runs   strongly   in   families   and   has   a   heritability   (variance attributable to genetics) of up to 60%.5 Genetic studies therefore offer a structured means of understanding the causes of asthma as well as identifying targets for treating the syndrome. As with other common complex diseases, large-scale genetic studies have led to considerable advances in the understanding of asthma. In general, robust genetic effects have been identified that carry substantial population-attributable risks. Several genes act in pathways that communicate the presence of epithelial damage to the adaptive immune system, providing a new focus for effective therapies. Genetic findings have also led to a reassessment of the primacy of atopy in both asthma and eczema (atopic dermatitis), suggesting that atopy is secondary to epithelial or other events in both diseases.

Despite these advances, only a small component of the overall genetic contribution to asthma has been identified. This missing heritability may be due to multiple small effects (polygenes) or rare, highly penetrant mutations, or epigenetic modifications of gene function.

Asthma was a rare disorder in the nineteenth century, and a sharp increase in the prevalence of asthma has been observed in many countries during the last decades of the twentieth century. Phase I of the International Study of Asthma and Allergies in Childhood (ISAAC I) found that the prevalence of symptoms of asthma in children differed as much as 20-fold between study centers around the world.1 It has been established with clarity that a rich microbial environment in childhood confers significant protection from the development of asthma, hay fever, and atopy,68 particularly in the context of farming environments across Europe.8 Recent studies in Poland indicate that the prevalence of asthma and atopy are profoundly altered by a rapid movement of the population from traditional rural to urban dwelling following Poland’s entry into the European Community.9

These findings indicate the presence of strong environmental factors underlying disease susceptibility, and suggest that asthma may be prevented by discovery of these factors. The search for the exact microbial modifiers is becoming progressively refined.10,11

This review will describe in detail the progress made by genetic studies of asthma and will outline what are likely to be the next frontiers in genetic and epigenetic investigations. I will conjecture about the implications of specific loci for therapeutic intervention and interpret the overall findings in the context of investigations of the airway microbiome.

GENETICS

GENOME-WIDE ASSOCIATION STUDIES

Large-Scale Studies

Adequately powered genome-wide association studies (GWAS), in which hundreds of thousands of genetic markers are genotyped in  thousands of cases and controls, have been the method of choice for identifying complex disease genes.12 Several of these have now been completed for asthma, with gratifying and consistent results.

A first-generation GWAS of approximately 1000 children with asthma and 1000 controls showed in 2007 that markers on chromosome 17q21 were strongly associated with childhood-onset asthma.13,14 The GWAS was extended by measuring global gene expression in Epstein-Barr virus– transformed lymphoblastoid cell lines (LCL) from asthmatic children and their siblings.15 This showed that the asthma-associated SNPs were also strongly associated (P = 10–23) with transcript abundance of ORMDL3.13 Subsequently, we and others have shown that the locus also regulates expression of the neighboring gene, GSDMB.16,17

A multidisciplinary study to identify the genetic and environmental causes of asthma in the European community (GABRIEL collaboration) was a large international initiative that completed a second-generation GWAS of 10,000 asthmatics and 16,000 controls, including those with adult-onset asthma as well as childhood disease.18 Associations were found on chromosome 2 within IL1RL1/IL18R1; on chromosome 6 within HLA-DQ; on  chromosome 9 flanking IL33; on chromosome 15 within SMAD3, and on chromosome 22 within IL2RB. Association within the chromosome 17q21 ORMDL3/GSDMB locus was confined to childhood-onset disease, and the HLA-DQ locus was the only significant hit in the adult-onset group. Markers at two additional genes, SLC22A5 and RORA, were identified just below genome-wide significance in the whole group, and markers within TSLP were seen at a similar level of significance in the patients with the most severe disease. Associations within IL1RL1 and IL33 had been identified previously, through the intermediate phenotype of elevated eosinophil counts,19 and TSLP variants  had  also  been  previously  associated  with  include  SNPs within PDE4D20 and DENND1B.21

The study of populations with diverse ancestry and environmental exposures adds depth to genetic studies. A GWAS of pediatric asthma in the Japanese population showed pediatric asthma to be associated primarily to the major histocompatibility complex (MHC), with association to HLA-DP alleles DPA1*0201 and DPB1*0901, which are in strong linkage- disequilibrium with each other were strongly associated with pediatric asthma.22 This study found no effects from the ORMDL3 locus, although association to this locus had been confirmed in other Japanese subjects (Hirota PMID 18155279). A study of asthma in Japanese adults showed the strongest associations to the MHC, between the complement genes and the HLA-DRA locus, and confirmed association to the TSLP locus.23  These results suggest a differing balance of etiologies to asthma in Japan and Europe. The HLA-DP association may indicate the presence of an antigen driving the process, and it may be relevant that HLA-DP associations to aspirin-induced24 and isocyanate asthma25 have been observed previously, both involving small molecules.

A well-powered meta-analysis of GWAS in ethnically diverse North American populations from the EVE consortium (meta-analysis of genome- wide association studies of asthma in ethnically diverse North American populations) confirmed the previously reported loci near ORMDL3, IL1RL1, TSLP, and IL33, and showed that these loci were associated with asthma risk in Hispanic and African-American groups. The authors also identified association to PYHIN1, with the association being specific to individuals of African descent.26 This result has yet to be confirmed.

More recently, an Australian study that combined new data with published results showed new associations to the interleukin-6 receptor (IL6R) gene and markers on chromosome 11q13.5 near the leucine-rich repeat containing 32 gene (LRRC32, also known as GARP). The LRRC32 locus was significantly associated with atopic status among asthmatics.27 It had first been found to be a susceptibility locus for atopic dermatitis28 and was shown in the ALSPAC population to be a novel susceptibility factor for atopic asthma and hay fever.29

The asthma genetics community is currently consolidating all  the data from these international GWAS, so the number of asthmatics included in a single meta-analysis will rise from 10,000 to 25,000. The experience with other diseases30 indicates that this exercise may double the number  of verified asthma-susceptibility loci. It is likely, however, that the strongest and most consistent genetic effects on asthma have already been identified.

The summary from all of these studies is that ORMDL3 single biggest risk factor for childhood asthma; that childhood-onset asthma consistently shows the strongest genetic effects18; that IL33, IL33R/IL18R, and TSLP communicate epithelial damage to the adaptive immune system; that SMAD3 and 1L2RB downregulate inflammation; and that the MHC has diverse effects that may center on the presentation of specific foreign antigens.

Heterogeneity and Atopy

These studies point to a heterogeneity underlying asthma that has long been suspected by clinicians. The ORMDL3 locus is confined to childhood-onset disease and is associated with severity31 but not with atopy. The MHC is the strongest risk factor for adult-onset disease and shows different allelic relationships to disease in different populations and age groups. The LRRC32 locus may have its strongest effects in atopic asthma in conjunction with atopic dermatitis.

GWAS studies for total serum IgE levels have been successfully carried out, identifying effects around FCER1A, STAT6, and IL13.18,28,32,33 With the exception of the IL13 locus, it is notable that the genes that regulate IgE production have a minimal effect on asthma susceptibility, and vice versa.18 Whilst these results do not negate the importance of atopic mechanisms in asthma, they do indicate that atopy is secondary to asthma, rather than its driving force. Similarly, disorders in the epidermal barrier proteins SPINK534 and FLG35 lead to strong atopic manifestations that must be secondary to inflammatory events that follow barrier failure.

Overlap of GWAS Hits with Inflammatory Bowel  Disease

Several of the robust asthma associations are also found in studies of inflammatory bowel disease (IBD).36 Clinically, IBD is well recognized to be accompanied by low-grade airway inflammation. Loci that contribute to both diseases include SMAD3, ORMDL3, HLA-DR, and DENNDB1.36 The overlap suggests that both diseases may result from the disordered interactions between bacteria and mucosal surfaces. The functions of these genes and  the potential role of the airway microbiome are described below. Notably, genes that identify defective processing of intracellular bacteria in Crohn’s disease (such as NOD2, IRGM, and ATG16L1) are not implicated in asthma. It is also of interest that genetic evidence has demonstrated the importance of the barrier function to the development of ulcerative colitis, although the genes involved (HNF4A, LAMB1, CDH1, and GNA12) are different from those that mediate the barrier function in asthma and atopic dermatitis.36

FUNCTIONS OF ASTHMA-ASSOCIATED GENES

ORMDL3 and GSDMB

The association signals on human chromosome 17 with asthma are maximal within an island of linkage- disequilibrium that contains ORMDL3 and GSDMB. Asthma-associated SNPs were strongly associated with transcript abundance of ORMDL3 and more weakly with the neighboring GSDMB, so it is feasible that the locus contributes to asthma through the differential regulation of both genes. A single promoter upstream of ORMDL3 is a promising target region to search for functional SNPs that affect this regulation.

The locus covers an area of approximately 200 kb. ORMDL3 and GSDMB reside in one island of linkage disequilibrium that contains all the maximally associated SNPs. However, statistically independent associations are detectable telomerically near the GSDMA and PSDM3 genes,13 which may make additional contributions to asthma susceptibility.

ORMDL3 protein is found in the membranes of the endoplasmic reticulum (ER). ER stress is linked to cellular responses to inflammation.37 Enforced X- box-binding protein 1 (XBP1S) is a transcriptional activator that is a key element in the development of the ER. ORMDL3 has been found to be upregulated in XBP-1(S) transuded NIH-3T3 fibroblasts.38 It has been shown that changes in ORM gene expression cause dysregulation of sphingolipid metabolism.39 Sphingolipids are amphiphatic molecules formally derived from sphingosine. Sphingosine phosphorylation leads to sphingosine-1- phosphate (S1P) and acylation to ceramide. Sphingolipids mediate cell survival, proliferation, apoptosis, differentiation, and cell-cycle arrest.40 Clinical observation shows that sphingosines and ceramide are increased in asthmatic airways.41

It has also been suggested that ORMDL3 modifies SERCA in the ER resulting in an unfolded-protein response and inducing inflammation.42 A recent study showed mononuclear cells significantly increased  IL-17 secretion in 17q21 risk allele–carrier children.43 All these results show that ORMDL3 may influence multiple pathways in the ER that mediate inflammation during asthma.

The GSDM family of genes have been best studied in mice. They are expressed predominantly in the gastrointestinal (GI) tract and in the skin44 in a highly tissue-specific manner.45 In humans, GSDMA and GSDMB are expressed in the gastrointestinal and bronchial epithelium (http://www.proteinatlas.org/). Members of the gene family may have a role in the regulation of apoptosis.46

IL33, IL18R1, and IL1RL1

IL33, IL18, and IL1 belong to the IL1 family of cytokines that alter host responses to inflammatory and infectious challenges. They exert their functions through a family of receptors that belong to the Toll-like receptor- IL-1 receptor (TLR-IL-1R) superfamily. An important feature of IL1 receptor signaling is the activation of transcription factor NF-κB and mitogen- activated protein (MAP) kinases p38, JNK, and ERK1/2.47

IL33 was originally identified as a nuclear factor in vascular endothelial cells,48 and was subsequently detected in airway epithelial cells.49,50 The activities of IL33 as a nuclear factor remain unclear,51 but it may repress gene expression of pro-inflammatory factors. In contrast to inducible cytokines, IL33 is constitutively expressed and is thought to function as an endogenous danger signal or alarmin to alert the immune system after endothelial or epithelial cell damage during trauma or infection.52 IL33 is formed as a prodomain containing polypeptide, which after activation with caspase-1, is realized as mature IL33. A mouse gene knockout has shown Il33 works as a crucial amplifier of innate immunity.53 IL-33 expression is induced  by a range of environmental and endogenous triggers, suggesting an essential role during infection, inflammation, and tissue damage.54

IL33 activates a heterodimeric receptor complex containing IL1RL1 (ST2) and IL-1 receptor accessory protein (IL1RAP), leading to activation of NF- κB and MAP kinases and drives production of Th2-associated cytokines such as IL4, IL5, and IL13.49

The IL18R1 gene, along with four other members of the interleukin 1 receptor family (IL1R2, IL1R1, ILRL2 [IL-1Rrp2], and IL1RL1 [T1/ST2]), form a cluster on chromosome 2q. IL18R1 and IL1RL1 flank each other with the same orientation of translation. They are within the same island of linkage disequilibrium, and it has not yet been possible to assign the genetic effects at this locus to one gene or the other. It is possible that both genes may be co- regulated.

As stated above, IL1RLI encodes the receptor of IL33. IL18 is closely related to IL3349 and synergizes with IL12 to induce the production of interferon gamma and to promote TH1 responses.55 These loci therefore identify a pathway for the communication of epithelial damage to  the adaptive immune system and a potential switch point for choosing between TH1 or TH2 responses.

Thymic stromal lymphopoietin (TSLP)

Despite the initial identification of TSLP in the culture supernatant of a thymic stromal cell line, this cytokine is expressed mainly by epithelial cells at barrier surfaces (skin, gut and lung).56,57 The cell populations with the highest known co-expression of the TSLP receptor (TSLPR) and its associated subunit IL-7Rα are myeloid dendritic cells (DCs).56 Treatment of human DCs with TSLP induces improved survival, upregulation of major histocompatibility complex class II and co-stimulatory molecules, and the production of a variety of chemokines.56 It promotes TH2 cytokine– associated inflammation by directly promoting the effector functions of CD4+ TH2 cells, basophils, and other granulocyte populations while simultaneously limiting the expression of DC-derived proinflammatory cytokines and promoting regulatory T-cell responses in peripheral tissues.57

SMAD3

SMAD3 encodes SMAD (“mothers against  decapentaplegic homolog”) family member 3 and came to attention for its role in modifying tumour growth57,58,59 through the transforming growth factor-beta (TGFB) pathway.60  SMAD3 is concentrated in the nuclei of bronchial epithelial cells and macrophages (http://www.proteinatlas.org/) and functions as a transcriptional modulator activated by TGFB. TGFB family members exert fundamental effects on the maintenance of immune function in the lung,61 and TGFB signaling pathways are activated after allergen challenge in mild asthma.62 A mouse knockout of Smad3 showed accelerated wound healing and an impaired local inflammatory response,63 even though mice lacking Smad3 may exhibit increased baseline levels of proinflammatory cytokines in their lungs.64 Smad3 signaling is required for myogenic differentiation of myoblasts,65 perhaps pointing to a role in airway smooth muscle hypertrophy.

IL2RB

IL12RB encodes the beta receptor of IL2. IL2 is secreted by antigen-activated T cells. It controls the survival and proliferation of regulatory T cells66 and plays a crucial role in the maintenance of natural immunological self- tolerance.67 The IL2 receptor is composed of α (CD25), β (CD122), and γ chains.66 The β chain (IL2RB) is a signal transduction element that is also present in the IL15 receptor. It belongs to the type I cytokine receptor family and is devoid of intrinsic kinase activity.68 The receptor modulates T cell– mediated immune responses through endocytosis, whereby ectodomain shedding of IL2Rβ generates an intracellular fragment with biological functions.69 In a murine model of asthma, local blockade of Il2rb restored an immunosuppressive cytokine milieu that ameliorated lung inflammation.70

IL4 and IL13

IL4 and IL13 are adjacent to each other on chromosome 5 and downstream of RAD50. The locus is exceptional in showing strong association to IgE in addition to doctor-diagnosed asthma. The 3′ end of RAD50 contains several conserved non-coding sequences and enhancer elements that act as a locus- control region for IL4 and IL13.71 RAD50 encodes a gene that mediates DNA double-stranded break repair, and it is most likely that the effects at this locus are mediated through IL4 and IL4 expression.

The Major Histocompatibility Complex

The MHC has long been a focus of interest in understanding the genetics of asthma, and it is one of few candidate regions that have survived the stringent criteria imposed by GWAS. As described above, most MHC associations to asthma and related phenotypes have emerged around the HLA class II genes, with apparently independent effects for adult-onset asthma in Europeans,18 asthma in Japanese adults,23 asthma in Japanese children,22 and total serum IgE levels in Europeans.18 Previously, HLA-DQ and HLA-DR alleles have been associated with IgE responses to individual allergens,7274 and TNF alleles in the same region have been related to the presence of asthma and its response to therapy.75 Intriguingly, polymorphisms within the MHC are strongly related to the levels of gene expression of many genes, including the classical HLA antigens.15

The HLA class II proteins classically restrict immune responses to particular external antigens, and it is possible that the GWAS findings are pointing to different types of antigen, perhaps bacterial (see below) as well as typical allergens. A concerted collaborative international effort is needed to disentangle these observations.

PHARMACOGENETICS AND ASTHMA

Pharmacogenetic studies may define variants that modify an individual patient’s response to therapy. Small or moderate beneficial effects  will require studies that are as large as current GWAS for disease susceptibility. GWAS of therapeutic response have to date been most successful in showing side effects of drugs to result from variation in their metabolism.76 A recent study of the response of asthmatics to glucocorticoid therapy has shown that patients who were homozygous for an allele mapped to glucocorticoid- induced transcript 1 gene (GLCCI1) had an improvement in respiratory function that was only about one-third of that seen in similarly treated subjects who were homozygous for the wild-type allele (3.2 ± 1.6% vs. 9.4 ± 1.1%). The same subjects’ risk of a poor response was significantly higher, with genotype accounting for about 6.6% of overall inhaled glucocorticoid response variability.77 These associations were supported by functional studies that showed the risk allele to be associated with decrements in GLCCI1 expression. These intriguing results are worthy of replication.

EPIGENETICS

Epigenetic effects, mediated through mechanisms other than sequence variation, are another possible cause of familial clustering. The patterns of gene expression that determine cellular type and function become stably restricted during development, partly through methylation of CpG sequences and gene silencing.78 Islands of CpG sequences are positioned at the 5′ UTRs of many human genes.79 About one-fifth of islands are variably methylated, and in one-third of these, methylation status correlates with transcript abundance.80 Abnormalities of DNA methylation are well recognized in single-gene disorders and in cancer,81 and it is postulated that epigenetic changes in methylation may be of importance to common human diseases81 such as asthma and chronic obstructive pulmonary disease (COPD).

Whilst it is very likely that genome-wide studies of methylation status at various loci may identify new genes and pathways that mediate airway diseases, it is important to recognize that age,80,82,83 sex,80,82 genetic polymorphism,84,85 and environmental factors83,85 have all been strongly associated with altered methylation at selected loci. The relative contribution of these factors to methylation at loci genome-wide is not known, and it is not certain to what extent true epigenetic inheritance with transmission between generations takes place. These factors will have to be taken into account if methylation changes at individual loci are to help us understand complex diseases.

THE MICROBIOME

Asthma is due to complex interactions between genes and the environment. Whilst the genetic studies described above have shown the  central importance of the respiratory epithelium, a worthwhile understanding of the causes of asthma needs to reconcile consistent epidemiological indications of the importance of the microbiome to the disease. These include the protection afforded by a rich microbial environment in early life,10,86 observations that the bronchial tree contains characteristic flora that are disturbed by the presence of pathogens such as Haemophilus infuenzae in asthma,11,87 birth cohort studies showing that the presence of the same pathogens in throat swabs predicts the later development of asthma,88  and recognition that  these bacteria have consistently been associated with exacerbations of asthma.89 Ninety  percent  of  the  cells  in  the  human  body  are      microorganisms including   bacteria,   parasites,   and archaea.90   These   microorganisms  are commensal on body surfaces exposed to the external environment, including the gut, respiratory tract, and skin. Although most bacteria are not cultivable with standard methods,91 the membership of complex microbial communities can be quantified and classified by DNA sequencing of the conserved bacterial 16S rRNA gene.92,93 Bacteria are classified by these sequences into operational taxonomic units (OTUs). OTUs approximate closely, but not completely, to taxonomy derived from classical techniques, but sequences of other regions may be necessary for precise discrimination at the species level. A small study from our group compared the airway microbiota at three levels in adult patients with asthma, the related condition of COPD, and controls.11 Bronchial lavage from asthmatic children and controls was also investigated.11 The bronchial tree was not sterile, and contained a mean of 2,000 bacterial genomes per square centimeter surface sampled. Pathogenic proteobacteria, particularly Haemophilus spp., were much more frequent in bronchi of adult asthmatics or patients with COPD than in controls. Highly significant increases in proteobacteria were also seen in asthmatic children.

A recent study used a microarray technique to test for relationships between the composition of the airway bacterial microbiota and clinical features of asthma, establishing that bacterial burden and diversity were higher among asthmatic patients compared to controls, although not identifying the organisms responsible for this effect.87

Of potential importance is the finding that organisms commonly found in healthy airways (such as Bacteroidetes, particularly Prevotella spp.) were significantly reduced in asthmatic airways.11 As described above, genetic studies indicate an overlap in mechanisms underlying asthma and IBD. It is increasingly recognized that a normal bacterial flora is essential in maintaining a healthy bowel mucosa,94 and similar mechanisms are likely to be important in the airways.9597 Murine studies have shown that sterility of the airways and intestinal tract results in markedly enhanced inflammatory responses to a variety of stimuli.96,98

The study of the role of the microbiome of asthma is in its infancy. Even at this early stage, however, there are many questions that suggest a structured set of experiments that will elucidate how the microbiome operates in  health and disease.

CLINICAL IMPLICATIONS

GENETIC RISK

The primary motivation for genetic investigations of asthma has been to develop a systematic understanding of otherwise impenetrable disease processes. The success of GWAS for complex diseases and the rapidly declining costs of sequencing individual human genomes have led to a high level of public interest in using DNA to provide an estimated individual risk of disease, with the expectation that high-risk individuals may benefit from preventive and therapeutic interventions.99 It is appropriate that the value of individual genetic screening has been questioned on the grounds of interpretation of statistical risk estimates,99 the quality control of genotyping, and the need to establish the validity of predictive tests from appropriately designed trials.100

The outstanding finding from asthma GWAS has been the GSDMB- ORMDL3 locus on chromosome 17q21.13,18 In the initial GWAS,13 the odds ratio (OR) for the most strongly associated SNP in cases of childhood asthma recruited through hospital clinics and compared to controls was 1.84 per additional risk allele. The per-allele OR at this locus for childhood-onset asthma was 1.32 in the GABRIEL consortium GWAS, which contained a high proportion of asthmatics from epidemiological surveys,18 and was 2.02 in severe therapy-resistant asthmatics attending tertiary referral clinics.101 These substantial differences in ORs reflect the different ascertainment criteria and types of asthma for each study, and show that genetic risk is highest in children with early-onset disease that is resistant to therapy, and that environmental factors are important modifiers of this risk.

The size of these effects can be put into perspective by considering well- known epidemiological effects on asthma. The OR for the protective effect of farming on the prevalence of childhood asthma in the PARSIFAL study (Prevention of allergy risk factors for sensitization in children related to farming and anthroposophic lifestyle) was 0.62 (inverse 1.61) and was 0.86 (1.16) in the GABRIEL advanced survey.10 At school age, the typical ORs associated with either parent’s smoking were 1.24 for wheezing in the last year and 1.21 for asthma.102 Although these figures are derived from other populations, genetic risks seem comparable to those conferred by known environmental factors.

Genetic Risks in Populations

The population-attributable risk (PAR; also known as the population- attributable risk fraction, PARF) is widely used to estimate the burden of a risk factor in population surveys. Applied to genetic studies, the PARF estimates the fraction of cases that would not occur if no one in  the population carried the risk allele.99 Usage of the PARF to model how genetic variants at the six GABRIEL GWAS significant loci impacted jointly on the burden of childhood disease in the GABRIEL populations18 indicated that 38% of childhood-onset asthma was attributable to SNP  combinations at these loci, and in an independent population-based replication sample, the same SNPs accounted for 49% of asthma risk from birth to middle age.18 These findings indicated that the genetic variants at these loci and their presumed functional outcomes had a significant impact on the burden of asthma in the community. A large PARF does not imply that the measured genotypes can provide clinically useful risk predictions,99 but it does suggest that therapies directed at the functional consequences of the genes identified by the GWAS are likely to be of benefit to many asthmatics.

Genetic Risk in Individuals

The GABRIEL GWAS is the largest set of data analyzed to date for the ability of genetic variants to determine the risk of an individual developing asthma.18 We assessed individual risk for the seven SNPs associated with childhood asthma in a classification analysis based on a logistical regression model. Taking as a cut-point the risk score exceeded by 25% of the non- asthmatic population (specificity, 75%), the sensitivity of classifying asthma was just 35% (false-negative rate, 65%). Assuming the prevalence of asthma in the general population is between 5% and 10%, the positive predictive value of a genetic risk score above this arbitrary cut-point would be in the range from 6.9% to 13.5%. The clear conclusion from this is that  the common genetic polymorphisms measured or imputed by GWAS are not useful  in  predicting  which  children  or  infants  are  at  risk  of   developing asthma.

Familial Risk and Heritability

A third component of genetic risk relates to families. The increase in risk in the siblings of affected individuals is known as the familial relative risk (FRR). A related measurement is that of heritability, which is defined as the variance in a complex trait due to inherited factors. The heritability of childhood asthma has been estimated to be as high as 60%, but the  variance in asthma prevalence accounted for by the loci in the GABRIEL large-scale GWAS was only 4%. This is consistent with other complex human traits such as height,103 Crohn’s disease,104 and obesity,105 and the search for the “missing heritability” is a major current focus for geneticists who study complex diseases.106

Applying heritability estimates from rigorously ascertained family studies to general population samples with their attendant environmental variation is inherently unreliable. Additionally, GWAS are directed at SNPs with minor allele frequencies greater than 5% or 10%, because the power to detect genetic associations is only high when a disease and its underlying susceptibility alleles are both common,107 and because parametric statistical tests of association are unreliable when allele counts are sparse. However, the clustering of asthma within families may be due to private mutations that are rare in the general population. The search for missing heritability, and for the ability to predict individual risks of disease, should perhaps therefore be concentrated on rare mutations with high penetrance.

THERAPEUTIC TARGETS

The increasing scale and level of international cooperation have provided several clear directions for therapeutic interventions in asthma. It is not safe to assume that GWAS will identify targets that can be accessed either by small molecules (drugs) or biologics (antibodies and proteins). Of the top GWAS hits for asthma, it is perhaps ORMDL3 that offers the most promise. ORM proteins act as a brake on sphingolipid metabolism with increased dihydrospingosine and ceramide levels observed in knockdown cells.39 Sphingolipids have a wide range of actions in modifying inflammatory processes,  providing  a  potential  mechanism  and  a  therapeutic  target for modulating airway inflammation.108

The IL33/IL18 ST2/IL18R axis may be crucial in determining the type of adaptive immune response to airway damage and should be the focus of therapeutic endeavors. Despite much effort, however, the exact roles of IL33 and its receptor in inflammation have been difficult to define with precision. IL33 knockouts have been difficult to develop, the nuclear binding sites for IL33 are not known, there has not yet been a clear demonstration of free IL33 in inflamed airways, and it is not yet a definite target for therapy. Based on the results of murine knockouts, IL18 and its receptor IL18R may be more tractable therapeutic targets, but the field requires the development of an appropriate model system for IL18/IL18R interactions.

TSLP has a well-defined biology, and it represents a good target for biologic therapies. It may be anticipated that an anti-TSLP antibody would have its most profound effects on dendritic cells and granulocytes.

The IL2RB and SMAD3 proteins may provide a means of downregulating inflammation and promoting healing. SMAD3 is, however, concentrated in the nucleus, and nothing is yet known of its nuclear binding sites and partners. IL2RB is expressed at the cell surface, and murine knockdown studies indicate that biologics directed against it may have therapeutic benefits.

MANIPULATING THE MICROBIOME

The finding of a disordered microbiome in asthma inevitably leads to direct manipulation of the airway bacterial community. Clinical investigators have supported these findings with direct interventions with long courses of antibiotics.109 A conclusion from these studies is that chronic bacterial infections are relevant in a subgroup of preschool children with persistent wheezing, and such children benefit significantly from antibiotic therapy.109 Persistent bacterial bronchitis (PBB) is a syndrome of childhood wheezing with a chronic productive cough that is often diagnosed as asthma.110 It may be relevant that Haemophilus influenzae and Streptococcus pneumoniae are the most commonly isolated organisms, and that long courses of antibiotics are very effective in treating the disease.110

Many patients with asthma receive antibiotics at different times without noticeable changes in the course of their disease. Rational antibiotic therapy would probably necessitate direct sampling of the airway mucosa with brushings or lavage, together with assessment of the key elements of the local inflammatory response. Antibiotics have profound effects on microbial communities, and restoration of a normal bacterial flora may be a necessary part of the therapeutic approach. In this context, it is remarkable that replacement of a normal bacterial community in the bowel has been very effective in patients with IBD,111,112 and curative in patients with Clostridium difficile necrotizing enteroclotitis.113,114

CONCLUSIONS AND FUTURE PERSPECTIVES

GWAS of asthma have provided a rich harvest of knowledge about the mechanisms of asthma. Next steps in genetics will include a comprehensive meta-analysis of all GWAS data internationally, currently being undertaken by the applied genetics core (TAGC). This exercise may double the number of loci proven to be influencing asthma.

The contribution of rare mutations to asthma is not known, and full genomic sequencing of multiple diseased individuals is the gold standard to be applied to this problem. Whilst the community waits for sequencing costs to fall to acceptable levels before undertaking full sequencing, whole-exome sequencing with enrichment of exonic sequences by hybridization before ultra-deep sequencing115 has proven to be extremely effective in the identification of rare mutations in Mendelian diseases,116,117 and merits application to complex disorders such as asthma. Another interim solution may come from the Illumina exome chip, which is derived from assembled information on 12,000 sequenced genomes and exomes and catalogues, for each variant that potentially affects protein structure, the total number of times it was seen, and the total number of datasets that included the variant. Non-synonymous variants seen at least three times across two datasets are to be included on the chip.

It is unlikely that many important genetic effects will remain undiscovered after completion of global meta-analysis and identification of any important rare mutations. The source of the missing asthma heritability is most likely to be polygenic aggregation on multiple small effects. The large number of loci identified through candidate gene studies118 that do not show up in GWAS may point to this eventuality.

Molecular genetic studies identify unexpected pathways and potential targets on the basis of sequence variation in DNA. The essential components of many disease-related pathways may, however, not be subject to genetic variation. Epigenetic variation may be another means to identify  novel disease pathways and therapeutic targets.

Whatever the outcome of future genetic studies, the biggest advances in understanding and treating asthma will come from full functional analyses of the genes already identified, from reagents that manipulate their actions, and from disentangling their relationship to the microbial partners on the other side of the respiratory mucosa.

REFERENCES

1.   ISAAC. Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema: ISAAC. The International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee. Lancet 1998;351:1225–1232.

2.  Smith DH, Malone DC, Lawson KA, Okamoto LJ, Battista C, Saunders WB. A national estimate of the economic costs of asthma. Am J Respir Crit Care Med 1997;156:787–793.

3.  Asthma Agenda. London: National Asthma Campaign; 1998.

4.  Vandenplas O, Toren K, Blanc PD. Health and socioeconomic impact of work-related asthma. Eur Respir J 2003;22:689–697.

5.  Duffy DL, Martin NG, Battistutta D, Hopper JL, Mathews JD. Genetics of asthma and hay fever in Australian twins. Am Rev Respir Dis 1990;142:1351–1358.

6.  Strachan DP. Hay fever, hygiene, and household size. Brit Med J 1989;299:1259–1260.

7.   von Mutius E, Fritzsch C, Weiland SK, Roll G, Magnussen H. Prevalence of asthma and allergic disorders among children in united Germany: a descriptive comparison. Brit Med J 1992;305:1395–1399.

8.   Braun-Fahrlander C, Gassner M, Grize L, et al. Prevalence of hay fever and allergic sensitization in farmer’s children and their peers living in the same rural community. SCARPOL team: Swiss Study on Childhood Allergy and Respiratory Symptoms with Respect to Air Pollution. Clin Exp Allergy 1999;29:28–34.

9.   Sozanska B, Macneill SJ, Kajderowicz-Kowalik M, et al. Atopy and asthma in rural Poland: a paradigm for the emergence of childhood respiratory allergies in Europe. Allergy 2007;62:394– 400.

10.  Ege MJ, Mayer M, Normand AC, et al. Exposure to environmental microorganisms and childhood asthma. N Engl J Med 2011;364:701–709.

11.  Hilty M, Burke C, Pedro H, et al. Disordered microbial communities in asthmatic airways. PLoS One 2010;5:e8578.

12.  Manolio TA. Genomewide association studies and assessment of the risk of disease. N Engl J Med 2010;363:166–176.

13.   Moffatt MF, Kabesch M, Liang L, et al. Genetic variants regulating ORMDL3  expression contribute to the risk of childhood asthma. Nature 2007;448:470–473.

14.  Bouzigon E, Corda E, Aschard H, et al. Effect of 17q21 variants and smoking exposure in early- onset asthma. N Engl J Med 2008;359:1985–1994.

15.    Dixon AL, Liang L, Moffatt MF, et al. A genome-wide association study of global gene expression. Nat Genet  2007;39:1202–1207.

16.   Cookson W, Liang L, Abecasis G, Moffatt M, Lathrop M. Mapping complex disease traits with global gene expression. Nat Rev Genet 2009;10:184–194.

17.   Verlaan DJ, Berlivet S, Hunninghake GM, et al. Allele-specific chromatin remodeling in the ZPBP2/GSDMB/ORMDL3 locus associated with the risk of asthma and autoimmune disease. Am J Hum Genet 2009;85:377–393.

18.   Moffatt MF, Gut IG, Demenais F, et al. A large-scale, consortium-based genomewide association study of asthma. N Engl J Med 2010;363:1211–1221.

19.  Gudbjartsson DF, Bjornsdottir US, Halapi E, et al. Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction. Nat Genet 2009;41:342–347.

20.   Himes BE, Hunninghake GM, Baurley JW, et al. Genome-wide association analysis identifies PDE4D as an asthma-susceptibility gene. Am J Hum Genet 2009;84:581–593.

21.   Sleiman PM, Flory J, Imielinski M, et al. Variants of DENND1B associated with asthma  in children. N Engl J Med 2010;362:36–44.

22.   Noguchi E, Sakamoto H, Hirota T, et al. Genome-wide association study identifies HLA-DP as a susceptibility gene for pediatric asthma in Asian populations. PLoS Genet 2011;7:e1002170.

23.   Hirota T, Takahashi A, Kubo M, et al. Genome-wide association study identifies three new susceptibility loci for adult asthma in the Japanese population. Nat Genet 2011;43:893–896.

24.  Young RP, Dekker JW, Wordsworth BP, Cookson WOCM. HLA-DR and HLA-DP genotypes and immunoglobulin E responses to common major allergens. Clin Exp Allergy 1994;24:431–439.

25.   Choi JH, Lee KW, Kim CW, et al. The HLA DRB1*1501-DQB1*0602-DPB1*0501 haplotype is a risk factor for toluene diisocyanate-induced occupational asthma. Int Arch Allergy Imm 2009;150:156–163.

26.   Torgerson DG, Ampleford EJ, Chiu GY, et al. Meta-analysis of genome-wide association studies of asthma in ethnically diverse North American populations. Nat Genet 2011;43:887–892.

27.   Ferreira MA, Matheson MC, Duffy DL, et al. Identification of IL6R and chromosome 11q13.5 as risk loci for asthma. Lancet 2011;378:1006–1014.

28.  Esparza-Gordillo J, Weidinger S, Folster-Holst R, et al. A common variant on chromosome 11q13 is associated with atopic dermatitis. Nat Genet 2009;41:596–601.

29.   Marenholz I, Bauerfeind A, Esparza-Gordillo J, et al. The eczema risk variant on chromosome 11q13 (rs7927894) in the population-based ALSPAC cohort: a novel susceptibility factor  for asthma and hay fever. Hum Mol Genet 2011;20:2443–2449.

30.  Anderson CA, Boucher G, Lees CW, et al. Meta-analysis identifies 29 additional ulcerative colitis risk loci, increasing the number of confirmed associations to 47. Nat Genet 2011;43:246–252.

31.   Cookson W, Moffatt M, Strachan DP. Genetic risks and childhood-onset asthma. J Allergy Clin Immunol 2011;128:266–270; quiz 71–72.

32.  Weidinger S, Gieger C, Rodriguez E, et al. Genome-wide scan on total serum IgE levels identifies FCER1A as novel susceptibility locus. PLoS Genet 2008;4:e1000166.

33.   Granada M, Wilk JB, Tuzova M, et al. A genome-wide association study of plasma total IgE concentrations in the Framingham Heart Study. J Allergy Clin Immunol 2011;129(3):840– 845.e21. doi:10.1016/j.jaci.2011.09.029.

34.   Walley AJ, Chavanas S, Moffatt MF, et al. Gene polymorphism in Netherton and common atopic disease. Nat Genet 2001;29:175–178.

35.   Palmer CN, Irvine AD, Terron-Kwiatkowski A, et al. Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nat Genet 2006;38:441–446.

36.   Lees CW, Barrett JC, Parkes M, Satsangi J. New IBD genetics: common pathways with other diseases. Gut 2011;60:1739–1753.

37.   Zhang K, Kaufman RJ. From endoplasmic-reticulum stress to the inflammatory response. Nature 2008;454:455–462.

38.   Sriburi R, Bommiasamy H, Buldak GL, et al. Coordinate regulation of phospholipid biosynthesis and secretory pathway gene expression in XBP-1(S)-induced endoplasmic reticulum biogenesis. J Biol Chem 2007;282:7024–7034.

39.   Breslow DK, Collins SR, Bodenmiller B, et al. Orm family proteins mediate sphingolipid homeostasis. Nature;463:1048–1053.

40.  Uhlig S, Gulbins E. Sphingolipids in the lungs. Am J Respir Crit Care Med 2008;178:1100–1114.

41.  Ammit AJ, Hastie AT, Edsall LC, et al. Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma. FASEB J 2001;15:1212–1214.

42.   Cantero-Recasens G, Fandos C, Rubio-Moscardo F, Valverde MA, Vicente R. The asthma- associated ORMDL3 gene product regulates endoplasmic reticulum-mediated calcium signaling and cellular stress. Hum Mol Genet;19:111–121.

43.  Lluis A, Schedel M, Liu J, et al. Asthma-associated polymorphisms in 17q21 influence cord blood ORMDL3 and GSDMA gene expression and IL-17 secretion. J Allergy Clin Immunol 2011;127:1587–1594 e6.

44.   Saeki N, Kuwahara Y, Sasaki H, Satoh H, Shiroishi T. Gasdermin (Gsdm) localizing to mouse Chromosome 11 is predominantly expressed in upper gastrointestinal tract but significantly suppressed in human gastric cancer cells. Mamm Genome 2000;11:718–724.

45.   Tamura M, Tanaka S, Fujii T, et al. Members of a novel gene family, GSDM, are expressed exclusively in the epithelium of the skin and gastrointestinal tract in a highly  tissue-specific manner. Genomics 2007;89:618–629.

46.  Saeki N, Usui T, Aoyagi K, et al. Distinctive expression and function of four GSDM family genes (GSDMA-D) in normal and malignant upper gastrointestinal epithelium. Gene Chromosome Canc 2009;48:261–271.

47.   Dunne A, O’Neill LA. The interleukin-1 receptor/Toll-like receptor superfamily:  signal transduction during inflammation and host defense. Science’s STKE [electronic resource] 2003;171:2003, pre3.

48.   Baekkevold ES, Roussigne M, Yamanaka T, et al. Molecular characterization of  NF-HEV,  a nuclear factor preferentially expressed in human high endothelial venules. Am J Pathol 2003;163:69–79.

49.   Schmitz J, Owyang A, Oldham E, et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 2005;23:479–490.

50.   Carriere V, Roussel L, Ortega N, et al. IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo. Proc Natl Acad Sci U S A 2007;104:282–287.

51.  Kurowska-Stolarska M, Hueber A, Stolarski B, McInnes IB. Interleukin-33: a novel mediator with a role in distinct disease pathologies. J Intern Med 2011;269:29–35.

52.   Moussion C, Ortega N, Girard JP. The IL-1-like cytokine IL-33 is constitutively expressed in the nucleus of endothelial cells and epithelial cells in vivo: a novel  “alarmin”?  PLoS One 2008;3:e3331.

53.   Oboki K, Ohno T, Kajiwara N, et al. IL-33 is a crucial amplifier of innate rather than acquired immunity. Proc Natl Acad Sci U S A;107:18581–18586.

54.   Lloyd CM. IL-33 family members and asthma—bridging innate and adaptive immune responses. Curr Opin Immunol 2010;22:800–806.

55.   Fukao T, Matsuda S, Koyasu S. Synergistic effects of IL-4 and IL-18 on IL-12-dependent IFN- gamma production by dendritic cells. J Immunol 2000;164:64–71.

56.   Reche PA, Soumelis V, Gorman DM, et al. Human thymic stromal lymphopoietin preferentially stimulates myeloid cells. J Immunol 2001;167:336–343.

57.   Ziegler SF, Artis D. Sensing the outside world: TSLP regulates barrier immunity. Nat Immunol 2010;11:289–293.

58.   Yang YA, Zhang GM, Feigenbaum L, Zhang YE. Smad3 reduces susceptibility  to hepatocarcinoma by sensitizing hepatocytes to apoptosis through downregulation of Bcl-2. Cancer Cell 2006;9:445–457.

59.   Tian F, DaCosta Byfield S, Parks WT, et al. Reduction in Smad2/3 signaling enhances tumorigenesis but suppresses metastasis of breast cancer cell lines. Cancer Res 2003;63:8284– 8292.

60.  Daly AC, Vizan P, Hill CS. Smad3 protein levels are modulated by Ras activity and during the cell cycle to dictate transforming growth factor-beta responses. J Biol Chem 2010;285:6489–6497.

61.  Lloyd CM, Hawrylowicz CM. Regulatory T cells in asthma. Immunity 2009;31:438–449.

62.   Kariyawasam HH, Pegorier S, Barkans J, et al. Activin and transforming growth factor-beta signaling pathways are activated after allergen challenge in mild asthma. J Allergy Clin Immunol 2009;124:454–462.

63.   Ashcroft GS, Yang X, Glick AB, et al. Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nat Cell Biol 1999;1:260–266.

64.  Anthoni M, Wang G, Leino MS, Lauerma AI, Alenius HT, Wolff HJ. Smad3—signalling and Th2 cytokines in normal mouse airways and in a mouse model of asthma. Int J Biol Sci 2007;3:477– 485.

65.   Ge X, McFarlane C, Vajjala A, et al. Smad3 signaling is required for satellite cell function and myogenic differentiation of myoblasts. Cell Res.2011;21(11):1591–1604. doi:10.1038/cr.2011.72.

66.   Letourneau S, Krieg C, Pantaleo G, Boyman O. IL-2—and CD25-dependent immunoregulatory mechanisms in the homeostasis of T-cell subsets. J Allergy Clin Immunol 2009;123:758–762.

67.   Setoguchi R, Hori S, Takahashi T, Sakaguchi S. Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. JEM 2005;201:723–735.

68.  Gaffen SL. Signaling domains of the interleukin 2 receptor. Cytokine 2001;14:63–77.

69.   Montes de Oca P, Malarde V, Proust R, Dautry-Varsat A, Gesbert F. Ectodomain shedding of interleukin-2 receptor beta and generation of an intracellular functional fragment. J Biol Chem;285:22050–22058.

70.   Doganci A, Karwot R, Maxeiner JH, et al. IL-2 receptor beta-chain signaling controls immunosuppressive CD4+ T cells in the draining lymph nodes and lung during allergic airway inflammation in vivo. J Immunol 2008;181:1917–1926.

71.  Lee GR, Fields PE, Griffin TJ, Flavell RA. Regulation of the Th2 cytokine locus by a locus control region. Immunity 2003;19:145–153.

72.  Marsh DG, Meyers DA, Bias WB. The epidemiology and genetics of atopic allergy. N Engl J Med 1981;305:1551–1559.

73.   Marsh DG, Blumenthal MN, Ishikawa T, et al. HLA and specific immune respoinsiveness to allergens. In: Tsuji K, Aizawa M, Sazazuki T, eds. Proceedings of the Eleventh International Histocompatability Workshop and Conference. Oxford, UK: Oxford University Press; 1992:765– 767.

74.   Moffatt MF, Young A, Schou C, Faux J, et al. Involvement of TCR-a/d and HLA-DR in specific allergy. Allergy Suppl 1995;50:164.

75.   Moffatt MF, Cookson WO. Tumour necrosis factor haplotypes and asthma. Hum Mol Genet 1997;6:551–554.

76.   Link E, Parish S, Armitage J, et al. SLCO1B1 variants and statin-induced myopathy—a genomewide study. N Engl J Med 2008;359:789–799.

77.   Tantisira KG, Lasky-Su J, Harada M, et al. Genomewide association between  GLCCI1  and response to glucocorticoid therapy in asthma. N Engl J Med 2011;365:1173–1183.

78.  Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002;16:6–21.

79.  Bird AP. CpG islands as gene markers in vertebrate nucleus. Trends Genet 1987;3:342–347.

80.   Eckhardt F, Lewin J, Cortese R, et al. DNA methylation profiling of human chromosomes 6, 20 and 22. Nat Genet  2006;38:1378–1385.

81.   Feinberg AP. Phenotypic plasticity and the epigenetics of human disease. Nature 2007;447:433– 440.

82.   Liu J, Morgan M, Hutchison K, Calhoun VD. A study of the influence of sex on genome wide methylation. PLoS One 2010;5: e10028.

83.  Christensen BC, Houseman EA, Marsit CJ, et al. Aging and environmental exposures alter tissue- specific DNA methylation dependent upon CpG island context. PLoS Genet 2009;5:e1000602.

84.   Boks MP, Derks EM, Weisenberger DJ, et al. The relationship of DNA methylation with age, gender and genotype in twins and healthy controls. PLoS One 2009;4:e6767.

85.  Heijmans BT, Kremer D, Tobi EW, Boomsma DI, Slagboom PE. Heritable rather than age-related environmental and stochastic factors dominate variation in DNA methylation of the human IGF2/H19 locus. Hum Mol Genet 2007;16:547–554.

86.  Eder W, Ege MJ, von Mutius E. The asthma epidemic. N Engl J Med 2006;355:2226–2235.

87.  Huang YJ, Nelson CE, Brodie EL, et al. Airway microbiota and bronchial hyperresponsiveness in patients with suboptimally controlled asthma. J Allergy Clin Immunol 2011;127:372–381 e3.

88.  Bisgaard H, Hermansen MN, Buchvald F, et al. Childhood asthma after bacterial colonization of the airway in neonates. N Engl J Med 2007;357:1487–1495.

89.  Kraft M. The role of bacterial infections in asthma. Clin Chest Med 2000;21:301–313.

90.  Savage DC. Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol 1977;31:107–133.

91.  Staley JT, Konopka A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 1985;39:321–146.

92.   Turnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM, Knight R, Gordon JI. The human microbiome project. Nature 2007;449:804–810.

93.   Ahmed S, Macfarlane GT, Fite A, McBain AJ, Gilbert P, Macfarlane S.  Mucosa-associated bacterial diversity in relation to human terminal ileum and colonic biopsy samples. Appl Environ Microbiol 2007;73:7435–7442.

94.  Maslowski KM, Vieira AT, Ng A, et al. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature 2009;461:1282–1286.

95.   Noverr MC, Huffnagle GB. The “microflora hypothesis” of allergic diseases. Clin Exp Allergy 2005;35:1511–1520.

96.   Herbst T, Sichelstiel A, Schar C, Yadava K, Burki K, et al. Dysregulation of allergic airway inflammation in the absence of microbial colonization. Am J Respir Crit Care Med 2011;184: 198–205

97.   Nembrini C, Sichelstiel A, Kisielow J, Kurrer M, Kopf M, Marsland BJ. Bacterial-induced protection against allergic inflammation through a multicomponent immunoregulatory mechanism. Thorax 2011;66:755–763.

98.   Noverr MC, Huffnagle GB. Does the microbiota regulate immune responses outside  the  gut? Trends Microbiol 2004;12:562–568.

99.  Kraft P, Wacholder S, Cornelis MC, et al. Beyond odds ratios—communicating disease risk based on genetic profiles. Nat Rev Genet 2009;10:264–269.

100.  Hunter DJ, Khoury MJ, Drazen JM. Letting the genome out of the bottle—will we get our wish? N Engl J Med 2008;358: 105–107.

101.  Binia A, Khorasani N, Bhavsar PK, et al. Chromosome 17q21 SNP and severe asthma. J Hum Genet 2011;56:97–98.

102.  Cook DG, Strachan DP. Health effects of passive smoking. 3. Parental smoking and prevalence of respiratory symptoms and asthma in school age children. Thorax 1997;52:1081–1094.

103.  Weedon MN, Lango H, Lindgren CM, et al. Genome-wide association analysis identifies 20 loci that influence adult height. Nat Genet 2008;40:575–583.

104.  Barrett JC, Hansoul S, Nicolae DL, et al. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn’s disease. Nat Genet 2008;40:955–962.

105.  Blakemore AI, Froguel P. Investigation of Mendelian forms of obesity holds out the prospect of personalized medicine. Ann N Y Acad Sci 2010;1214:180–189.

106.  Maher B. Personal genomes: The case of the missing heritability. Nature 2008;456:18–21.

107.  Risch NJ. Searching for genetic determinants in the new millennium. Nature 2000;405:847–856.

108.  Nixon GF. Sphingolipids in inflammation: pathological implications and potential therapeutic targets. Br J Pharmacol 2009;158:982–993.

109.  Schwerk N, Brinkmann F, Soudah B, Kabesch M, Hansen G. Wheeze in preschool  age  is associated with pulmonary bacterial infection and resolves after antibiotic therapy. PLoS One 2011;6:e27913.

110.  Donnelly D, Critchlow A, Everard ML. Outcomes in children treated for persistent bacterial bronchitis. Thorax 2007;62:80–84.

111.  Bennet JD, Brinkman M. Treatment of ulcerative colitis by implantation of normal colonic flora. Lancet 1989;1:164.

112.  Borody TJ, Warren EF, Leis S, Surace R, Ashman O. Treatment of ulcerative colitis using fecal bacteriotherapy. J Clin Gastroenterol 2003;37:42–47.

113.  Eiseman B, Silen W, Bascom GS, Kauvar AJ. Fecal enema as an adjunct in the treatment of pseudomembranous enterocolitis. Surgery 1958;44:854–859.

114.  Hamilton MJ, Weingarden AR, Sadowsky MJ, Khoruts A. Standardized Frozen Preparation for Transplantation of Fecal Microbiota for Recurrent Clostridium difficile Infection. Am J Gastroenterol 2012.; 107(5):761–767. doi:10.1038/ajg.2011.482.

115.  Ng SB, Turner EH, Robertson PD, et al. Targeted capture and massively parallel sequencing of 12 human exomes. Nature 2009;461:272–276.

116.  Ng SB, Buckingham KJ, Lee C, et al. Exome sequencing identifies the cause of a mendelian disorder. Nat Genet 2010;42:30–35.

117.  Biesecker LG. Exome sequencing makes medical genomics a reality. Nat Genet 2010;42:13–14.

118.  Vercelli D. Advances in asthma and allergy genetics in 2007. J Allergy Clin Immunol 2008;122:267–271.

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