Real-Time High Resolution Melting Analysis-PCR for Assessment of Gene Mutations in Human Autosomal Dominant Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common hereditary renal diseases that affect 1 in 400 to 1 in 1000 life birth and cause by mutations in two known genes; PKD1 and PKD2. Although the manifestation of Type1 ADPKD is similar to Type2, it is more severe and patients suffer total renal failure by the fifth decade of life. In finding the mutations occurring in both genes, PCR-based methodology have been used and all require technical expertise, otherwise are time consuming and costly. Therefore, this study aims to develop a much rapid screening procedure that is cost effective but have the same sensitivity to widely used methodology to differentiate ADPKD samples from control. Fourteen primers were selected and used to amplify the target genes by using Real-time High Resolution Melting Analysis PCR (Real-time HRMA PCR) and was compared to PCR-SSCP in terms of rapidity, sensitivity and reliability. PCR-SSCP revealed same number of bands in control and case sample, with different only in the bands’ size. All primers successfully amplified respective genes in Real-time HRMA PCR. Ten primers had two apparent melting curves which enabled control and case samples to be differentiated. The mean melting temperature for control samples of the ten primers was 81.38±1.70oC whereas in case samples the mean was 81.00±1.68oC. The p value was 0.001. The melting curves of control were all shifted to the right having higher melting temperature while case melting curves were shifted to the left due to lower melting temperature. Possible mutations were found to be present in five of these primers due to lone case samples where the melting curve were shifted differently from control and case melting curves and cannot be classified either in control or case group (p<0.001). Three primers were found to be less useful to be used in Real-time HRMA PCR as control and case samples melted in the same manner with same exact temperature, resulting in inconclusive results. While other primers conferred single melting domain, a primer amplifying the PKD1 gene was melted twice and evidence in both samples. The PCR-SSCP needs more time to be completed (mean; 13.93±0.94 hours) before analysis can be done while the Real-time HRMA PCR can be completed in a very short time (mean; 2.40±0.54 hours) including the analysis. Both methodologies are able to discriminate case from control but Real-time HRMA PCR is more rapid (p<0.001). Since Real-time HRMA PCR is more rapid and less procedure is needed, it is proven to be better for gene mutation screening. In conclusion, the Real-time HRMA PCR is not only sensitive, but it can screen for control and ADPKD samples more rapidly and more reliable with the application of commonly used primers in conventional PCR.

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