ENT FEATURE Discovery in the genetics of complex disease: Otitis media BY SARRA E JAMIESON titis media (OM), a common suggests that the SNP either directly (i.e. Odds Ratio [OR] ≤1.5) expected to disease of childhood, is increases risk of the disease (i.e. it is a contribute to a complex trait such considered to be a complex causal variant) or that it is in linkage as OM. Otrait with multiple genetic disequilibrium (LD) with the true Regardless of the study design and environmental factors expected to causal variant. chosen, it is essential that replication contribute to a child’s risk of developing The alternative approach has been studies are subsequently performed recurrent acute OM (rAOM; ≥3 episodes to interrogate the whole genome using to reduce the number of false in 6 months or ≥4 episodes in a year) either a genome-wide linkage study positive genetic risk factors reported. or chronic OM with effusion (COME; (GWLS) or a genome-wide association Replication is ideally performed in persistent middle ear fluid for ≥3 study (GWAS) design. In a GWLS an independent case/control sample months). Heritability studies confirm hundreds of microsatellite markers drawn from the same population that the genetic component is likely to or thousands of SNPs spaced evenly uses a comparable phenotype and be substantial, explaining 40-70% of across the genome are genotyped in which is of sufficient size to replicate the variation in OM phenotype [1]. In sets of affected relatives to identify the genetic effect size observed in the order to identify the genes involved regions that are co-inherited due to original analysis. Without replication, in susceptibility to OM several study genetic linkage. The linked genomic results should be interpreted with designs have been employed to regions identified tend to be large (often some caution. date and are described briefly in this megabases in size) and frequently Our understanding of the genetic risk article (Table 1). harbour tens to hundreds of genes, any factors that are important in childhood The most commonly used approach one of which could ultimately be the OM has advanced considerably in has been the candidate gene association disease locus. As such, results from a recent years. The results of numerous study. This study design investigates GWLS have to be followed up by fine- candidate gene studies have been the alleles observed at a small mapping of all genes under the linkage reported across several different number (10-100) of single nucleotide peak, a task usually achieved via an populations and have identified polymorphisms (SNPs) located at association study. positive association at more than 20 or close to specific genes, either in More recently the GWAS approach individual genes (recently reviewed a cohort of individuals with disease has been utilised to identify the genes [2]). This includes several genes (cases) compared to those without involved in susceptibility to OM. In involved in the immune response, disease (controls) or within families a GWAS the frequencies of (usually) such as Toll-like Receptor 4 (TLR4), (usually parent/child trios). The specific several million SNPs are compared members of the Interleukin (IL) family genes investigated are usually selected across the whole genome in hundreds of cytokines (IL1A, IL1B, IL6, IL10), on the basis of plausible biological or thousands of unrelated cases versus Tumour Necrosis Factor alpha (TNF) function or due to their homology with ethnically matched controls. Again, and several members of the Mucin disease genes identified in relevant statistical evidence that specific SNP gene family (MUC2, MUC5AC, MUC5B). murine models. Statistical evidence alleles differ in frequency between However, it is important to note that that specific SNP alleles differ in cases and controls suggests a role for many of these reported candidate gene frequency between cases and controls those SNPs, or ones they are in LD with, associations have been based on small in disease. The GWAS has become a sample sizes and very few have been popular approach for the investigation replicated in independent populations of many complex diseases as previously to date. One notable exception is the unsuspected genetic risk factors can association reported at the F-Box only “The most commonly be identified which may provide novel Protein 11 (FBXO11), a gene initially insight into gene networks / pathways identified in a murine model of OM used approach has important in disease pathogenesis. In [3]. Association has been observed at the context of OM the GWAS approach variants in this gene in four independent been the candidate has only recently been undertaken for (predominantly Caucasian) cohorts gene association several reasons, the key one of which from Australia, the USA and the UK is the availability of large case/control making this one of the strongest genetic study.” cohorts. Such large case/ risk factors for OM reported to date control sample sizes are essential to [4]. Subsequent functional work in the detect the small genetic effect sizes Fbxo11 mouse model has highlighted ent and audiology news | NOVEMBER/DECEMBER 2014 | VOL 23 NO 5 | www.entandaudiologynews.com ENT FEATURE the role of middle ear hypoxia in OM TABLE 1: GLOSSARY OF GENETIC TERMINOLOGY and points to molecules that target hypoxia-mediated pathways as Term Definition potential novel therapies to alleviate Allele One member of a pair at a specific locus or single middle ear hypoxia, accumulation of nucleotide polymorphism. middle ear fluid and the associated conductive hearing loss [5]. Association Statistical test to determine whether a specific allele is Several genome-wide studies associated with disease. Can be done by comparison of investigating OM genetic risk factors allele frequencies between cases and ethnically matched have also been reported. This includes controls or by looking at the rates of allele transmission from parents to affected children. two independent GWLS that have identified several regions of genetic Complex trait A trait that is not attributable to a single gene but is linkage on chromosomes 3p25, 10q22, instead likely to arise through the action of multiple genetic and 10q26, 17q12 and 19q13, all of which environmental risk factors. appear to contain disease susceptibility Exome The portion of the genome encodes proteins. loci [6, 7]. To date one independent replication study has been reported Fine-mapping Method to narrow a region of genetic linkage known to contain a disease susceptibility locus down to a specific which confirmed the genetic linkage gene. reported at chromosome 10q26 only. Subsequent fine mapping of the 10q26 Genetic effect Usually given as an odds ratio, quantifies the risk region suggests a role for the Protein size attributable to a genetic variant in cases compared to Phosphatase 2, Regulatory Subunit B, controls. Delta (PPP2R2D) gene, a modulator Genome-wide GWAS; Analysis of large numbers of single nucleotide of the Transforming Growth Factor ß study association polymorphisms across the genome in large (TGF-ß) pathway [8]. The remaining numbers of affected cases and unaffected controls to linkage regions have yet to be replicated identify disease genes. and the causative disease genes Genome-wide GWLS; Method to map a disease locus to a chromosomal contained within them identified. linkage study region. This method relies on the close proximity More recently the results from the between the unknown disease locus and one or more first GWAS of OM have been reported. polymorphic markers which means they tend to be The first GWAS of OM identified inherited together. associations at the Calpain 14 (CAPN14) Heritability The proportion of observed phenotype variation and N-acetylgalactosaminyltransferase amongst individuals in a population that is attributable to genetic 14 (GALNT14) genes on chromosome differences. 2p23.1, as well as association at two Intergenic A region of DNA located between genes. Such regions members of the Bacterial Permeability can contain functionally important elements. Increasing Protein Family (BPIFA3 and BPIFA1) cluster on chromosome Linkage LD; The non-random association of alleles at two or more 20q11.21 [9]. These gene products play disequilibrium polymorphic markers. In the presence of LD certain a role in the invasion, adherence or combinations of alleles are more likely to occur together recognition of respiratory bacterial than would be seen by chance. pathogens respectively, pointing to Locus The location of a gene or genetic marker on a the potential functional significance chromosome. of these novel genetic risk factors. LOD Score A statistical estimate of linkage that illustrates whether The second GWAS of OM identified two loci (i.e. a disease locus and marker locus) associations at several different genes/ are inherited together more frequently than expected. regions, including at the Kinesin Family Member 7 (KIF7) gene and a SNP within Microsatellite A simple repeating DNA sequence, usually 1-6 base pairs in length, in which the repeating unit differs in number an intergenic region on chromosome between individuals. 2q31.1 that has been shown to regulate the Low-Density Lipoprotein Receptor Next generation NGS; various high-throughput sequencing technologies (LDLR) expressed on ciliated airway sequencing that parallelise the sequencing process allowing epithelial cells [10]. To date, the thousands or millions of sequences to be generated concurrently. association at the 2q31.1 intergenic SNP is the only GWAS association to Polymorphism Differences in DNA sequence between individuals at have been replicated in an independent specific loci that are commonly seen (i.e. >1% frequency) cohort. in the population. Whilst it is known that microbial Single Differences in DNA sequence between individuals at a and environmental factors play an nucleotide single base pair. important role in OM there is also a polymorphism clear role for genetic factors, one that we are only just starting to understand. Variant allele The alternative allele to the wild-type The recent formation of OTIGEN, an (or common) allele.