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Letters to the Editor |
1 Arabian Diagnostics Laboratory, Research Centre,2 Department Of Medical Genetics, and,3 National Laboratory for Newborn Screening, King Faisal Specialist Hospital, and Research Center, Riyadh, Saudi Arabia
aAddress correspondence to this author at: Arabian Diagnostics Laboratory, MBC 03, KFSHRC, PO Box 3354, Riyadh 11211, Saudi Arabia. Fax 966-1205-5171; e-mail oalsmadi{at}kfshrc.edu.sa.
To the Editor:
Argininosuccinic aciduria (ASAuria; OMIM 207900) is an autosomal recessive inborn error of the urea cycle caused by deficiency of the enzyme argininosuccinate lyase (ASL; EC 4.3.2.1) (1). A novel nonsense variant, ASL:p.Q354STOP, is the most common variant underlying ASAuria in the Saudi population and accounts for 
50% of all cases (2). Currently, diagnosis for ASAuria is based on electrospray tandem mass spectrometry (ESI-MS/MS) of dried blood spots (DBS) (3); however, although it is rapid and specific, this method is not suitable for prenatal diagnosis and carrier detection.
Another autosomal recessive inborn error of metabolism is 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) lyase deficiency (HMGCLD; OMIM 246450). HMG-CoA plays a key role in ketone body formation. Biochemical diagnosis of HMGCLD is made by MS/MS using DBS and is confirmed by organic acid analysis (4). HMGCLD is relatively common in the Saudi population (5), in which a novel variant, c.122G>A, leading to substitution of glutamine for arginine at position 41 (HMGCL:p.R41Q), has been described (6).
We developed and validated independent real-time PCR assays, using LCGreen I for ASL:p.Q354STOP and for HMGCL:p.R41Q. With these assays, we genotyped anonymous DBS from Saudi patients diagnosed with ASAuria and HMGCLD. DNA was prepared from DBS by whole-genome amplification as described (2). Genomic DNA controls for ASL:p.Q354STOP and HMGCL:p.R41Q were validated by direct sequencing in both directions. LCGreen assays were performed in LightCycler (Roche) capillaries and required 60 min to complete (detailed methodology is described in the Data Supplement that accompanies the online version of this letter at http:www.clinchem.org/content/vol52/issue7). Melting curve data were acquired in continuous mode after completion of PCR. Melting curve derivatives (–dF/dT) were calculated automatically by LightCycler software and plotted against fluorescence. Both purified genomic DNA and whole-genome amplification products generated from DBS were suitable for these assays and produced identical genotypes.
Melting curve analysis of relatively short amplicons in the presence of LCGreen I was used to genotype ASL:p.Q354STOP and HMGCL:p.R41Q. The PCR products for both assays were 60 bp in length, with the variants present in the gap between the 3' ends of the forward and reverse primers. With the exception of the mutation sites, intervening sequences were not variant in the Saudi population, as determined by the sequencing of 100 apparently healthy individuals. The derivative melting curves for all genotypes are shown in Fig. 1
. For ASL:c.1060C
T, the mean (SD) melting temperatures for the CC wild-type and TT variants were 84.4 (0.1) °C and 83.4 (0.1) °C, respectively. Results are based on a single run containing 13 and 15 CC and TT genotype replicates, respectively. The heterozygous (CT) genotype produced dual peaks (Fig. 1
). For HMGCL:c.122G>A genotyping, the mean (SD) melting temperatures for the GG wild-type and AA variants were 78.9 (0.09) °C and 78.2 (0.08) °C, respectively. Results are based on a single run containing 12 and 16 GG and AA genotype replicates, respectively. The heterozygous (GA) genotype produced dual peaks (Fig. 1
). For heterozygotes from both assays, we observed a small decrease of melting temperatures in the dual peaks relative to their equivalent homozygous peaks, reflecting heteroduplex formation.
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LCGreen I, unlike SYBR Green, can be included in the PCR reaction at concentrations that saturate newly synthesized PCR product without inhibiting amplification; it thus enables detection of sequence variants (7). Thermodynamically, A-T hybrids melt at significantly lower temperatures than do G-C hybrids. Fortunately, ASL:p.Q354STOP and HMGCL:p.R41Q contain C/T and G/A variants, respectively, which enhanced the successful development of these assays. The sizes and sequences of the PCR amplicons can also affect melting curve analysis. On this basis, we designed primers to generate short amplicons (60 bp) to maximize the thermal discrimination window and to improve genotyping accuracy (8)(9).
The products of the developed assays were fully concordant with the outcomes of sequencing. Development of real-time assays for detecting these variants provides a reliable tool for molecular diagnosis of ASAuria and HMGCLD, but more importantly, a rapid and robust method for prenatal or carrier detection. These assays are readily applicable for small-scale analysis, inductive screening, and if necessary, for regional population studies. Caution must be exercised to limit false negatives by minimizing the gap between assay primers. Although specific base changes may be associated with melting signatures, confirmation requires sequencing of PCR products. We reduced the likelihood of false positives arising in this manner by ruling out the presence of sequence variants in the region being interrogated within the population under study. Nevertheless, in unknown populations, we recommend conducting sequencing in a limited number of individuals to verify the basis for melting curve profiles of the variants being screened.
References
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