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Genetics of age-related macular degeneration
Mendelian diseases and complex diseases
The contribution of genetics to human disease has been long recognized. The genetic bases of the diseases may be monogenic or complex in origin. In monogenic Mendelian diseases, a mutation in a single gene transmitted in families predominantly leads to devastating phenotypic outcomes. Therefore, they are termed as simple genetic diseases. , Mendelian diseases, typically rare and very infrequently encountered at physicians’ offices, are exemplified by monogenic macular dystrophies, which include autosomal dominant disorders such as Best disease, North Carolina macular dystrophy, Sorsby macular dystrophy, malattia leventinese/Doyne’ honeycomb dystrophy, pattern dystrophy, late-onset retinal degeneration, and autosomal recessive disorders such as Stargardt disease and mitochondrial diseases.
In addition to monogenic diseases, other diseases develop from a combination of multiple genetic and environmental factors. They are considered as complex genetic diseases. Common complex diseases frequently seen in the real clinical practice, include diabetes and age-related macular degeneration (AMD) ( Fig. 9.1 ).
Heritability of AMD
The importance of heritability of AMD was revealed by twin studies. Meyers et al. reported a cohort study in which 134 twin pairs were recruited from 1986 to 1994. Monozygosity and dizygosity of these twins were confirmed by genetic tests. The criteria for AMD diagnosis included the different size, type, and number of drusen, well-demarcated geographic atrophy (GA), and any neovascular changes, which are consistent with clinical classifications. Based on these criteria, a statistical Fisher’s test was used to determine whether the concordance of AMD differed significantly between monozygotic and dizygotic pairs. The results showed that fundus features and vision loss are strikingly similar in monozygotic twins with AMD but not in dizygotic twins.
In another twin study in 2009, 42 twin pairs with normal visual acuity, matching restricted criteria that exclude clinical AMD were recruited. This cohort comprises age-matched 21 pairs of monozygotic twins and 21 pairs of dizygosity. A series of psychophysical and electroretinographic tests were performed. All color and flicker threshold and cone absolute threshold were significantly higher in the monozygotic pairs than that of the dizygotic pairs. Rod absolute threshold and rod and cone recovery rate were essentially similar for both monozygotic and dizygotic twin pairs. These findings indicated that cone thresholds and flicker thresholds are strongly determined by genetics, whereas, rod thresholds and adaptive abilities may be influenced more by environmental factors. This twin study demonstrated that genetic and environmental factors contribute differently to neuronal processes in the retina, which may influence the disease risk and diseases severity in various stages of AMD.
Molecular genetics of AMD
Methods of molecular genetics of AMD
AMD is a spectrum of macular disease with heterogeneous disease manifestations and varied disease severity. Heritability in complex genetic diseases cannot be defined by the methodology through which identifying Mendelian disease genes has been successfully applied. In 2001, the Human Genome Project was completed. It provided a necessary map of the human genome and the crucial information in which polymorphism data had been collected over the years ( http://www.ncbi.nlm.nih.gov/projects/SNP/index.htm ) and the database of resequencing project ( http://hapmap.ncbi.nlm.nih.gov/ ) was established. From 2000 to 2005, genome-wide association studies (GWAS) were developed to identify genetic variants responsible for disease risk. Typical GWAS obey the concept of using phenotype-first, in which the participants are classified by their clinical manifestation(s), not by genotype-first approach. And then a large collection of DNA samples of well-phenotyped subjects is collected and analyzed. This is a case-control DNA collection. In GWAS for AMD, the people with AMD (cases) and age- and gender-matched people without AMD (controls) are recruited for the comparative study of DNA sequence. Designing large longitudinal cohort studies provides many advantages for GWAS, in which not only disease phenotypes but also quantitative phenotypes, for example stratifying drusen size, type, and number of subjects in the case group, can be analyzed. The longitudinal design of such cohort studies may also provide a gold standard in “case definition” in epidemiology, by which the incidence rate, not prevalence rate, can be determined.
The genomic variation in GWAS is represented by single nucleotide polymorphisms (SNPs). SNP is the basic unit of genetic variation that refers to single base-pair changes in the DNA sequence of an individual’s genome. The method of GWAS is evolving rapidly. The first GWAS was done with only 100,000 SNPs. Two companies, Affymetrix and Illumina, have produced increasingly dense and more optimal arrays containing up to several millions of SNPs. Genotyping with arrays is based on oligonucleotides specific for a small area (approx. 50 base pairs) surrounding the targeted SNPs. Before GWAS analysis, a modified step called “imputation” was performed. This modification is a process of “guessing the genotype” of adjacent SNPs from the actual SNP obtained by the arrays. The basis of “guessing the genotype” is the existence of the database of HapMap reference genotype, which was derived from few hundred samples. Currently, the reference population has increased in sample size and ethnic diversity. This step allows many more SNPs to be analyzed.
GWAS rely on the phenomenon of linkage disequilibrium, wherein SNPs are not inherited individually but instead are in linkage disequilibrium blocks, with many nearby SNPs being highly associated. This enables the selection of one SNP (the tag SNP) that represents up to 50,000 surrounding base pairs. Through the high-throughput technique, for example, HapMap project that is gradually replaced by the National Center for Biotechnology Information (NCBI) Genomes Project ( http://www.1000genomes.org/ ), genotyping sets of 500,000–1,000,000 tag SNPs can cover approximately 80% of all common SNPs in the genome.
As whole genome sequencing technologies are rapidly improving and becoming less expensive, they will replace the current genotyping technologies. Whole genome sequencing approaches can make up the deficiency in the information obtained separately through genotyping arrays and exome sequencing. Through genotyping arrays such as the products made by Affymetrix and Illumina, GWAS largely evaluated common variants but missed rare variants. Exome analysis may miss many GWAS variants because DNA variations outside the exons can affect gene activity and protein production. The use of a whole-genome sequencing approach defines a genetic architecture characterized by GWAS loci that include the relevant gene, and rare variants of large effect.
In molecular genetics, GWAS has revolutionized the technology delineating the extent of genetic variants in different individuals. It allows us to see if any variant is associated with a trait of disease with complex etiology. Based on GWAS, AMD has been characterized as having polygenic and multifactorial inheritance, wherein heritability is determined by the joint action of multiple genes and their interaction with environmental factors ( Fig. 9.2 ).
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