Genetic polymorphisms in lung disease: bandwagon or breakthrough?

Abstract

The study of genetic polymorphisms has touched every aspect of pulmonary and critical care medicine. We review recent progress made using genetic polymorphisms to define pathophysiology, to identify persons at risk for pulmonary disease and to predict treatment response. Several pitfalls are commonly encountered in studying genetic polymorphisms, and this article points out criteria that should be applied to design high-quality genetic polymorphism studies.

Figure 1

(a) Several methods to detect specific nucleotide changes (polymorphisms) exist. One method relies on hybridization of oligonucleotides of known sequences to target DNA. The target DNA is generally obtained using the polymerase chain reaction and specific primers. Allele-specific oligonucleotides are then used to detect single base changes in the DNA samples. Typically, target DNA is immobilized on a solid support and denatured. Labeled (radioactive or fluorescent) oligonucleotides are then allowed to anneal. Complementary sequences bind while noncomplementary sequences do not. Sequences that match the oligonucleotide are detected by fluorescence or when the oligonucleotide is radiolabeled by exposure to X-ray film. (b) Another means of rapid screening for DNA variations relies on detecting conformational changes in secondary structure caused by the nucleotide sequence alteration. The change in structure can be detected in a number of ways including denaturing gradient electrophoresis and denaturing gradient high-performance liquid chromatography. SSCP, single-stranded conformational polymorphism. (c) Base mismatch methods begin with creating heteroduplexes between wild-type or normal DNA and target DNA. Heteroduplexes with mismatches are detected by enzymatic or chemical cleavage, with the cleavage products resolved by electrophoresis. (d) DNA sequencing can also be used to detect polymorphisms but is the most labor intensive. The method involves synthesis of DNA using DNA polymerase. Dideoxynucleotides are included in the synthesis mix to randomly terminate synthesis at each nucleotide in the sequence. Generally, each dideoxy nucleotide is labeled with a flourescent tag. Terminated strands are separated by denaturing gel or capillary electrophoresis and are detected using fluorescence.