Chapter 5: Molecular biology and genetics

Molecular biology and genetics

Once the locus of the gene has been determined, one attempts to narrow the region between the flanking markers before proceeding to identify the gene. Today with the sequence of the genome known and many genes having already been mapped to their chromosomal locus, the first approach is to sequence genes in the mapped region as potential candidate genes. If the candidate genes in the region after being sequenced do not contain the responsible mutation, it may be necessary to clone the region and identify novel genes to be sequenced as candidates for the mutation. Once the mutation is identified, one then determines if it is indeed the causative mutation. The minimum requirement is the mutation be found in affected and not in normal family members and be absent in at least 300 normal individuals representative of the population from which the family with the disease was selected (e.g. Caucasian, African, or Chinese).

Overview of phenotyping, genotyping, mapping, and identification of the gene

The overall approach to chromosomal mapping of heredity diseases by linkage analysis and subsequent isolation of the gene may be summarized categorically as follows: (1) collection of data from families having individuals affected by this specific disease through two or three generations; (2) the disease segregates in a Mendelian pattern; (3) clinical assessment to provide an accurate diagnosis of the disease using consistent and objective criteria to separate normal individuals from those affected and those who are indeterminate or unknown; (4) collection of blood samples for extraction of DNA for immediate analysis and subsequent whole-genome amplification should be stored in small aliquots to avoid repeated freezing and thawing; (5) development of a pedigree for analysis of the families; (6) DNA genotyping with a large number of DNA markers of known chromosomal loci that span the human genome; (7) linkage analysis is performed on the genotypes to map the chromosomal locus; (8) development of flanking markers around the region containing the disease locus; (9) isolation and cloning of the region of DNA containing the gene; (10) sequence analysis of the gene to identify the precise mutation causing the disease; (11) demonstration of the causal relationship between the defective gene and the disease by showing segregation of the mutation in affected individuals only and absence in an independent, unrelated normal population.

Modifier genes and phenotypic variability

A common feature of many single-gene disorders, is the presence of significant variability in the phenotypic expression of affected patients. This variability is seen between families and even within affected members of the same family and causative mutation. A significant factor for this genetic background is the presence of genomic DNA polymorphisms or SNPs in genes other than the disease-causing gene. SNPs are located in coding or regulatory regions of genes and can affect the gene expression and function. SNPs imposing functional differences for proteins involved in pathways of cardiac hypertrophy phenotype will alter the end phenotype in single-gene disorders, and thus, are referred to as “modifier genes.” Modifier genes are neither necessary nor sufficient to cause a disease but may influence the severity or risk of the disease. The identity of most modifier genes remains largely unknown. The identification of these modifiers will provide additional substrates for potential therapeutic intervention.

Although phenotypic variability may exist in patients caused by the same gene, due to genetic modifiers, correlation between the causal gene and the degree of disease severity and risk exists. However, due to the low frequency of most mutations consistent correlation to outcome can be made for only a few mutations. Importantly, it is necessary to recognize the limitations of these generalizations. Many confounding variables, such as small number of families with identical mutations, the influence of modifier genes, and coexisting morbidities make strict genotype–phenotype.