Candidate Gene Studies And Genomic Searches

The major problem facing gene hunters is not the sheer number of possible genes that need to be examined, of which—despite the recent completion of the Human Genome Project—the majority are unidentified, but the relatively small contribution each gene may make to the relevant trait. As a rule, genes will be found more easily if they explain more of the variance in a trait. Therefore, gene finding is relatively easy if only a single gene affects the trait. In these instances, a simple Mendelian segregation of a limited number of pheno-types is observed for all possible genotypes at a specific locus. Many rare diseases or disorders (but also Huntington's disease) are Mendelian in nature. As a general strategy to find such Mendelian genes, many DNA markers of known location, evenly dispersed throughout the entire genome, are measured in individuals from multiple generations. DNA markers can be mutations in a single base pair [single nucleotide polymorphisms (SNPs)] or a variable number of repeats of two or more base pairs (microsatellites) and need not be part of a functional gene: They are just landmarks in the genome. As with genes, all individuals have the same markers (in this sense the term "marker" may be misleading); it is the allelic variant of the marker that may differ between individuals. When a given marker is situated near the gene influencing the trait of interest, allelic variants of the marker and the gene will be more likely to be transmitted together to the next generation than if they are distant or on different chromosomes. This so-called cosegregation, however, is not perfect. Sometimes, the marker and the gene may be separated by recombination events during meiosis. The extent to which marker and gene cosegregate is referred to as ''linkage,'' and (this is a crucial assumption) the chance of linkage increases if the marker and the gene are close physically (although not necessarily linearly—not all chromosomal locations have equal chance for recombination). For each marker, evidence for linkage is derived using statistical procedures that trace the cosegregation of the trait (and thus in many instances the gene) and a specific variant of the DNA marker along familial lineages in extended pedigrees. Simply stated, if two children resemble each other for a certain trait and they both received exactly the same variant of a DNA marker from the same parent, that marker might be close to the gene influencing the trait.

Linkage analysis assigns a probability value (LOD score) to all markers, and a LOD score profile is obtained for each chromosome. Evidence for linkage is said to be present when the maximal LOD score exceeds a predefined threshold, which depends on the size of the genome and the number of genotyped markers. The chromosomal region surrounding a marker with a significantly high LOD score will be selected for fine mapping, which is essentially a repetition of the same procedure but now with all markers concentrated in the area of interest on a single chromosome. If the region containing the putative gene is sufficiently small, the DNA in the entire region is sequenced in full for a few persons or animals. Because genes have a specific structure, this identifies all genes in the region. By comparing all base pairs in these genes in many different persons/animals, the sites of allelic variation, also called polymorphisms, within these genes can be identified (mutational analysis). If the trait is a disease or disorder, comparison of the polymorphisms between patients and controls without the disease will ultimately reveal which allelic variant is responsible for the disease. The entire process from significant LOD scores to the actual allelic variants is usually called ''positional cloning.''

In 1993, using classical linkage in pedigrees, Han Brunner and colleagues discovered a point mutation associated with a behavioral phenotype that includes disturbed regulation of impulsive aggression. This mutation affected a gene that codes for one of the two isozymes [monoamine oxidase A (MAOA)] that are responsible for the breakdown of several neurotrans-mitters, including dopamine, noradrenaline, and serotonin. Since the MAOA gene is located on the X chromosome and the mutation is recessive, only males that possess the mutant allele show behavioral changes. One should nonetheless be extremely cautious in calling this gene an ''aggression gene.'' The phenotype is not limited to impulsive aggressive behavior but also extends to borderline mental retardation, arson, attempted rape, and exhibitionism. Aggression is therefore merely one of the impulsive behaviors by which these individuals differ from ''normal'' people.

Because of its power to localize classical Mendelian genes, linkage analysis has been the workhorse for mapping genes for simple monogenic traits/diseases. Unfortunately, most complex traits (depression, intelligence, and aggression) are polygenic, i.e., they are influenced by many different genes, environmental factors, and their possible interactions. These interactions involve gene-gene interactions (epistasis), geneenvironment interactions, and environment-environment interactions. Also, the same trait may be brought about by different subsets of genes in different individuals (genetic heterogeneity). Traits that are influenced by many genes and environmental factors are usually quantitative traits, and each of the genes that influence them is called a polygene. The locus where such a polygene can be found is called a quantitative trait locus (QTL). The contribution of a single polygene to the population variance in most complex behaviors is likely to be very small. Statistical power for the detection of such a QTL remains a major concern for the simple reason that only one gene (explaining 30% of the variation in fruit size in tomatoes) has been identified using these methods. Among the various solutions to boost power are the use of isolated populations to reduce genetic heterogeneity, the use of more DNA markers, and the use of selected family members (e.g., sibling pairs with either very high or very low values for the trait).

Two general alternative approaches to find genes for complex traits can be distinguished.

A. Candidate Genes

In some cases, there may be good theoretical reasons to focus on a single candidate gene. For instance, because neurotransmission is crucial to virtually every behavior, all known genes for receptors, transporters, or synthesis elements for neurotransmitters are usable as candidate genes. The ideal candidate gene has been shown to be functional: It influences the concentration of the (iso)form of a protein, its functionality or efficiency, or, perhaps most important, its responsiveness to environmental factors triggering the expression of the gene. All candidate gene studies are association studies and are similar in design to classic case-control studies in epidemiology. DNA is collected from all participants and the trait is compared across the various allelic variants of the candidate gene. Also, frequencies of the various allelic variants may be compared in subjects with a particular disease to detect an association between a particular allele and the occurrence of the disease.

The main problem of association studies is false positives that arise due to population stratification. The famous example is the "chopstick" gene. Suppose that the genome of random San Francisco inhabitants was used in a study on the complex trait of "using chopsticks to eat'' without stratification for Chinese or Anglo-Saxon background. There would be many genes associated with chopstick eating simply because frequencies differ between Chinese and Anglo-Saxon populations for a multitude of genes.

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