Rapid, long-range molecular haplotyping of thiopurine S-methyltransferase (TPMT) *3A, *3B, and *3C.
BACKGROUND
Haplotyping is an important technique in molecular diagnostics because haplotypes are often more predictive for individual phenotypes than are the underlying single-nucleotide polymorphisms (SNPs). Until recently, methods for haplotyping SNPs separated by kilobase distances were laborious and not applicable to high-throughput screening. In the case of thiopurine S-methyltransferase (TPMT*), differentiating among TPMT*3A, *3B, and *3C alleles is sometimes necessary for predictive genotyping.


METHODS
The genomic region including the two SNPs that define TPMT*3A, *3B, and *3C alleles was amplified by long-range PCR. The resulting PCR product was circularized by ligation and haplotyped by allele-specific amplification PCR followed by product identification with hybridization probes.


RESULTS
Critical points were the long-range PCR conditions, including choice of buffer and primers, optimization of the ligation reaction, and selection of primers that allowed for strict allele-specific amplification in the second-round PCR. Different underlying TPMT haplotypes could then be differentiated. Results from the haplotyping method were in full agreement with those from our standard real-time PCR method: TPMT*1/*3A (n = 20); TPMT*1/*3C (n = 4); TPMT*1/*1 (n = 6); and TPMT*3A/*3A (n = 6). One TPMT*1/*3A sample failed to amplify, and no whole blood was available for repeat DNA isolation.


CONCLUSIONS
This method for rapid-cycle real-time, allele-specific amplification PCR-assisted long-range haplotyping has general application for the haplotyping of distant SNPs. The procedure is simpler and more rapid than previous methods. With respect to TPMT, haplotyping has the potential to discriminate the genotypes TPMT*1/*3A (intermediate metabolizer) and TPMT*3B/*3C (poor metabolizer).
von Ahsen
Nicolas
N
Department of Clinical Chemistry, George-August University, Göttingen, Germany. nahsen@gwdg.de
Armstrong
Victor W
VW
Oellerich
Michael
M
D004247:DNA / Q000737:chemistry / Q000235:genetics; D005838:Genotype; D006239:Haplotypes / Q000235:genetics; D006801:Humans; D008780:Methyltransferases / Q000235:genetics*; D010597:Pharmacogenetics / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012680:Sensitivity and Specificity
D004247:DNA; D008780:Methyltransferases; C022745:thiopurine methyltransferase