Accuracy of polymerase chain reaction-restriction fragment length polymorphism for RET rs2435357 genotyping as Hirschsprung risk.

BACKGROUND:
Recently, the common RET rs2435357 variant has been shown to be strongly related to Hirschsprung disease (HSCR) in the Indonesian population. This association study was conducted in developed areas using high-throughput TaqMan polymerase chain reaction (PCR) assay. Although the TaqMan method is less time-consuming, it requires a special more expensive PCR machine and a highly skilled analyst. In this study, we analyzed the usefulness of the PCR-restriction fragment length polymorphism (RFLP) method for genotyping RET rs2435357 polymorphism in Indonesian HSCR patients given the limitation of resource allocation for more expensive technologies.

MATERIALS AND METHODS:
We compared our previous genotyping results of RET rs2435357 in 53 HSCR patients and 86 controls using the TaqMan PCR assay with the PCR-RFLP technique. Furthermore, we included an additional 40 HSCR patients and 50 controls and subsequently genotyped all subjects using the PCR-RFLP method.

RESULTS:
Compared with our previous genotyping data of RET rs2435357 using the TaqMan PCR assay, the PCR-RFLP method indicated 100% concordant results. The overall accuracy of the PCR-RFLP for RET rs2435357 genotyping was 100%. In addition, case-control analysis demonstrated that RET rs2435357 is significantly correlated with HSCR (P = 2.2 × 10(-13)) with an odds ratio of 5.1 (95% confidence interval = 3.2-8.1). The transmission disequilibrium test revealed that risk allele (T) at rs2435357 is significantly overtransmitted to probands at a transmission rate (τ) of 0.87 (P = 1.5 × 10(-6)).

CONCLUSIONS:
The PCR-RFLP method is reliable and affordable for genotyping of RET rs2435357 polymorphism in developing countries. Our results strengthen the proof that the RET rs2435357 variant is a genetic risk for HSCR in Indonesia.

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