Development of simple and effective PCR based assay to detect PCCA mutation (c.425G > A) among Saudi carriers and functional study of the homozygous PCCA mutations

Abstract

The aim of this study is to develop a rapid and effective method to screen for Saudi carriers of one of the most common propionic acidemia mutations (c.425G > A) and to study the functional impact of this mutation. Using allele-specific primers, we have developed a qPCR assay that clearly distinguishes heterozygotes from mutated and wild type homozygotes that overcome the dependence on labor-intensive gene sequencing. We show here that (i) qPCR rapid test has strong accuracy in detecting (c.425G > A) mutation in heterozygotes and homozygotes individuals and that the Ct-value cut-offs were estimated to be and 23.37 ± 0.04 (CV-6 %, 95 %CI-7.25) for homozygote, 25.06 ± 0.02 (CV-3.5 %, 95 %CI-7.85) for heterozygote PCCA c.425G > A mutation and 29.55 ± 0.002 (CV-11 %, 95 %CI-1.41) for PCCA wild type; (ii) the incidence of PA heterozygotes/carriers in Saudi population is about 550/100,000; (iii) skin fibroblast assays show that homozygote c.425G > A mutation induced propionyl-CoA carboxylase activity abrogation, (iv) PA patients showed an increased level of propionyl carnitine C3 in blood and 3-hydroxy propionic acid and methyl citrate in urine. Conclusion: qPCR represent an effective strategy to assess for PCCA mutation carriers in the Saudi population and we believe that will help in preventing homozygosity in the population after been implemented in pre-marriage screening program.

Keywords: (c.425G>A) Mutation; Ct, Cycle Threshold; PA, Propionic Acidemia; PCC, Propionyl-CoA Carboxylase; PCR; PCR, Polymerase Chain Reaction; Propionic acidemia; Propionyl-CoA carboxylase activity.

Fig. 1

qPCR method for the detection of PCCA mutation (c.425G > A). A) The design of alleles specific primers was based on the PCCA sequence showing the mutation position on the DNA, B) Sequence of alleles specific primers and C) Detection of mutated allele c.425A > G of the PCCA gene in heterozygote samples compared to wild type samples by Real-time PCR using LightCycler 480 (Roche Applied Science, Indianapolis, USA as mentioned in the Materials and Methods section. D) Screening of Saudis for PCCA mutation (c.425G > A). Mutated allele c.425A > G in the PCCA gene. The result is an illustration of amplification curves from the qPCR showing clear amplification of Homozygote, heterozygote and wildtype controls and 40 samples per run in duplicate.

Fig. 2

Genomic DNA sequencing using amplified coding exons and corresponding flanking sequences of the PCCA gene. While the normal wild type sequencing result was obtained with the reverse primer used (A), a clear deletion was seen with the forward primer used (B). Both the forward and reverse primers gave a homozygous missense variant as shown in (C and D), upper and lower panel-patient #2 and #3. Compressed electrophoretograms of Patient P1, P2 and P3 to distinguish the deletion in P1 from a single nucleotide variation in P2 and P3.