Accurate microsatellite typing and inter-study comparison: pitfalls and solutions using interferon-gamma (IFNG) and natural resistance-associated macrophage protein 2 (NRAMP2) genes as examples.
Microsatellite typing is frequently used in disease diagnosis and in genetic association studies. Inter-study consistency and comparability is essential in both applications. In this study, we show that the interlaboratory comparison of microsatellite sizes is often discrepant and misleading. This is a matter of great concern in the recent literature. However, accurate allele designation is easily attainable by the simple procedures we report, which are applicable to all gel-based genotyping methods. These involve: 1) the creation of dedicated standards for a specific microsatellite by PCR-amplifying representative alleles to generate an allelic ladder with comparable electrophoretic characteristics; 2) including both internal and external standards during electrophoresis to facilitate alignment. In addition, we recommend procedures that will improve inter-study comparability of all microsatellite analyses regardless of genotyping method. These involve: 1) cloning and sequencing representative microsatellite alleles to obtain accurate size designation; 2) sharing alleles of known sequence between laboratories to use as standards. We report on the typing of natural resistance-associated macrophage protein (NRAMP2) and interferon-y (IFNG) gene microsatellites as examples, the latter of which is crucial to many pathogenic processes. We describe in detail the varying allele sizes obtained by different methods, which prevent meaningful inter-study comparisons.
Rossouw
Manda
M
Department of Medical Biochemistry, Faculty of Health Sciences, Stellenbosch University, South Africa.
Warren
Robin
R
Hoal
Eileen G
EG
D000483:Alleles; D027682:Cation Transport Proteins / Q000235:genetics*; D019411:Clinical Laboratory Techniques / Q000592:standards*; D003001:Cloning, Molecular; D030561:Databases, Nucleic Acid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005838:Genotype; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics*; D033862:Iron-Binding Proteins / Q000235:genetics*; D018895:Microsatellite Repeats / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D000099236:Solute Carrier Family 11, Member 2
D027682:Cation Transport Proteins; D007371:Interferon-gamma; D033862:Iron-Binding Proteins; D000099236:Solute Carrier Family 11, Member 2