Molecular genetic studies of complex phenotypes

Abstract

The approach to molecular genetic studies of complex phenotypes evolved considerably during the recent years. The candidate gene approach, which is restricted to an analysis of a few single-nucleotide polymorphisms (SNPs) in a modest number of cases and controls, has been supplanted by the unbiased approach of genome-wide association studies (GWAS), wherein a large number of tagger SNPs are typed in many individuals. GWAS, which are designed on the common disease-common variant hypothesis (CD-CV), identified several SNPs and loci for complex phenotypes. However, the alleles identified through GWAS are typically not causative but rather in linkage disequilibrium (LD) with the true causal variants. The common alleles, which may not capture the uncommon and rare variants, account only for a fraction of heritability of the complex traits. Hence, the focus is being shifted to rare variants-common disease (RV-CD) hypothesis, surmising that rare variants exert large effect sizes on the phenotype. In conjunctional with this conceptual shift, technologic advances in DNA sequencing techniques have dramatically enhanced whole genome or whole exome sequencing capacity. The sequencing approach affords identification of not only the rare but also the common variants. The approach-whether used in complementation with GWAS or as a stand-alone approach-could define the genetic architecture of the complex phenotypes. Robust phenotyping and large-scale sequencing studies are essential to extract the information content of the vast number of DNA sequence variants (DSVs) in the genome. To garner meaningful clinical information and link the genotype to a phenotype, the identification and characterization of a large number of causal fields beyond the information content of DNA sequence variants would be necessary. This review provides an update on the current progress and limitations in identifying DSVs that are associated with phenotypic effects.

Figure 1. Gradients of disease prevalence and the effect sizes of the causative alleles

Prevalence of disease, number of determinant DNA sequence variants (DSVs) and their effect sizes are shown. Single gene disorders are caused by rare variants with large effect sizes. In addition to the main causal variant, which typically exhibits a Mendelian pattern of inheritance, several other non-Mendelian variants contribute to expression of the phenotype. On the opposite end of the spectrum are the common complex traits, which are caused, in part, by the cumulative effects of a very large number of DSVs, each imparting a modest effect size. In oligogenetic phenotypes, several alleles with moderate size effects and a large number of alleles with small effect sizes contribute to the phenotype.

Figure 2. Determinants of a complex phenotype

Cardiac hypertrophy is used as an example of a complex phenotype and various other potential determinants of phenotypic expression of cardiac hypertrophy, including genetics, genomics and external environmental factors are shown (not meant to indicate scale of effect sizes).

Figure 3. Non-homogenous target capture

A segment of LMNA gene showing locations of exons (blue boxes), capture regions (green boxes; exons plus 150bp flanking sequences), capture probes (purple-blue boxes) and depth of coverage of sequence reads from one (light gray) and 2 rounds of capture (dark grey). The depth of coverage is reflected by the shades of grey.

Figure 4. An example of sequence output of a Next Generation Sequencing (NGS) platform

Multiple copies of each DNA fragment is sequenced in parallel and analyzed. Four heterozygous nucleotides are identified in the sequenced fragment. The Figure illustrates the significance of an adequate coverage of each nucleotide for robust allele calling as well the potential for mis-calling because of inadequate coverage at each nucleotide position.

Figure 5. Approach to sub-genomic capture and sequencing

The diagram illustrates steps involved in target capture and sequencing. The steps include fragmentation of genomic DNA through sonication, adapter ligation, design of capture probes, and quality control to ensure specificity of hybridization, efficiency and uniformity of the capture. The analysis includes BLAST screening against repetitive DNAs and for specificity. Commonly several base long priming sites are added to the ends of all probes, allowing amplification of the probe set following synthesis. Probe pools are amplified using primers to the flanking, common priming sites. Several hundred-fold molar excesses of each probe sequence are used in hybridization. Typically, after one round of capture and enrichment, targets are enriched several thousand-fold. The enriched targets are then loaded onto a NGS platform for parallel sequencing of DNA strands.