of mutations at any one of these loci caused the disease, but once the first bbs gene, bbs6, was cloned, mutational and haplotype analysis revealed that some mutations did not conform to the expected monogenic, recessive transmission. katsanis, et al. were the first to demonstrate the phenomenon of 'triallelic' inheritance, a form of digenic inheritance, of bbs, which initially seemed to be transmitted in a recessive manner. with genotyping of 163 bbs pedigrees, they demonstrated that manifestation of the disease likely requires the presence of three mutant alleles, including those in bbs2, bbs6, and possibly in other loci and they identified a digenic mechanism of inheritance in what was previously considered to be a simple, mendelian disorder. genetic linkage analysis and association studies are widely used methods of identifying modifier loci. the advent of new genetic markers and automated genotyping methods have led to great diversity in applications of these studies. common methodologies used to identify human disease modifier loci include linkage analysis in families, candidate association studies, and whole-genome association studies. in an example of a familial linkage study, genotyping and linkage analysis conducted in a population of 197 sibpairs and their parents identified a single modifier gene responsible for meconium ileus, an intestinal obstruction phenotype detected in a subset of cf patients. their study provides an example of the utility and feasibility of linkage analysis in identifying modifier loci, by simple genotyping and linkage analysis, especially where phenotypes under study are binary, and a subset of candidate markers can be tested. associations studies, genome-wide or biologically-informed candidate approaches, can also reveal modifier loci. to identify genes responsible for disease, association studies commonly compare case and control populations with and without phenotypic indications of the disease of interest respectively. to identify modifier loci of diseases of interest, for which causative major loci are commonly established, researchers consider a population composed only of individuals affected by the disease. distribution of marker genotype is compared in patients with and without 'modified' phenotypes of interest to detect markers in linkage disequilibrium with potential modifier loci. association studies for detecting markers linked to modifier loci require large populations and dense maps of genomic markers, especially to detect markers of subtle effect on phenotypes for which quantification or dichotomization may be challenging. since these types of association studies are constrained to populations of patients affected by a disease, a primary challenge is collecting a sizable population that will lend the study sufficient statistical power. while, genome-wide approaches to association studies are systematic and comprehensive, candidate approaches spare the researcher some of the rigor of statistical analyses, for instance reducing the need for multiple testing corrections, but require informed selection of candidates. milet et al. conducted a candidate association study for modifiers of genetic hemochromatosis (gh), to explain its variable penetrance. where most cases of gh are caused by mutations in the hfe gene, they focus on genes in two candidate pathways, those involved in non-hfe gh and those involved in expression of the iron storage hormone hepcidin, and find evidence that several common snps in these candidates modify serum ferritin levels. the identified modifier loci have established roles in iron metabolism and, with very simple interpretation, their results suggest future avenues for functional study, a corollary of their informed, candidate approach. corvol et al. conducted a whole-genome association study to identify modifiers of cf lung disease, identifying five associated loci. their methods exemplify the large sample size required to conduct a statistically powerful analysis capable of identifying loci of relatively small effects. to obtain a large population, of 6,365 patients, they combine data on new and previously reported subjects and use linear mixed models that allow for the inclusion of affected siblings. the genetic modifiers of huntington's disease (gem-hd) consortium conducted a similar genome-wide association study, the first to demonstrate modifier loci modulating age of onset phenotype in huntington's disease (hd). their analyses were conducted on a population of 1,089 individuals affected by hd, genetic samples for which were collected over a period of almost 30 years. genotype-phenotype correlations, and linkage and association analyses in humans can effectively identify modifier genes with statistical support, but do not establish functional or causative effects of modifier genes. while genetic backgrounds cannot be experimentally manipulated in human populations, transgenic expression of modifier genes in animal models has been effectively used to show that variation in specific loci can cause phenotypic variation. for example, ikeda et al. established that moth1 is a modifier gene of the tub gene, mutations in which cause obesity, retinal degeneration and hearing loss in tubby mice. in the wild-type form, the protein mtap1a, encoded by neuronally-expressed moth1, is protective against hearing loss. ikeda et al. showed, by transgenic expression in mice, that sequence polymorphisms in mtap1a are crucial in causing the hearing-loss phenotype associated with tub gene mutation. unexplained heritability unexplained variation in human disease phenotype is a complex issue, and with established methodologies of identifying and characterizing modifier genes, it is tempting to pursue them as explanation, with the potential to lead to a better understanding of the biological mechanisms of disease, such as by characterized interactions between gene products. but identifying new modifier loci is challenging, and becomes more so, as those variants most easily detectable by current methods are increasingly identified and characterized. still, for a majority human genetic diseases, less than 20% of heritability is explained by known variants. some argue that unexplained heritability may be due to many rare variants, which typical association studies are not designed to detect, or structural genetic variation. though the challenging identification of new genetic variants may not be the most efficient approach to developing mechanistically-informed drugs, it may lead to the development of more safe, individualized, and effective interventional strategies, risk-assessments and prognoses. zuk et al. argue that biomedical research should focus on interacting molecular mechanisms of genetic variants already discovered. they argue that genetic interactions are common. widely used estimates of narrow-sense heritability, the ratio of additive genetic variance (calculated from measured effects of known variants) to total phenotypic variance (inferred from population data) assume additive effects in the heritability effects of variants. for most human traits, this fraction remains below 0.2. they argue that the assumption that heritability effects are additive overestimates heritability attributable to all genetic variation underlying disease, and thus underestimates the fraction of heritability attributable to already-discovered ones. thus, they argue, research should focus on the molecular bases of already-discovered variants, as estimates of discovered sources of heritability are flawed, and there is insufficient evidence that undiscovered genetic modifiers exist. furthermore, "the proportion of phenotypic variance explained by a variant in the human population is a notoriously poor predictor of the importance of the gene for biology or medicine," and effective therapeutics may target products of genes that explain very little of the clinically-observed variation in phenotype.