Quantification of PCR Bias Caused by a Single Nucleotide Polymorphism in SMN Gene Dosage Analysis

Abstract

Approximately 94% of patients with spinal muscular atrophy lack both copies of SMN1 exon 7, and most carriers have only one copy of SMN1 exon 7. We described previously the effect of SMN1/SMN2 heteroduplex formation on SMN gene dosage analysis, which is a multiplex quantitative PCR assay to determine the copy numbers of SMN1 and SMN2 using DraI digestion to differentiate SMN2 from SMN1. We describe herein the quantification of PCR bias between SMN1 exon 7 and SMN2 exon 7, which differ by only one nucleotide that is not present in either primer binding site. Using samples from 272 individuals with various SMN genotypes, we found that the amplification efficiency of SMN2 was consistent only approximately 80% that of SMN1. Thus, even a single nucleotide polymorphism, not in primer binding sites, can cause reproducible PCR bias. The precision and accuracy of our SMN gene dosage analysis are high because our assay design and controls take advantage of the consistency of the PCR bias. As additional clinically significant single nucleotide polymorphisms (SNPs) are discovered, assessment of PCR bias, and judicious selection of standards and controls, will be increasingly important for quantitative PCR assays. Spinal muscular atrophy (SMA: type I, MIM no. 253300; type II, MIM no. 253550; type III, MIM no. 253400) is an autosomal recessive disorder associated with loss of motor neurons in the anterior horn of the spinal cord and caused by mutations in the Survival Motor Neuron 1 gene (SMN1; MIM no. 600354) on 5q13. 1 Coding regions of SMN1 and its centromeric homologue, SMN2 (MIM no. 601627), differ in only one base. 2 This C-to-T substitution in SMN2 exon 7 affects the activity of an exonic splice enhancer and alters the splicing pattern of SMN2 mRNA, 3 resulting in a lower level of full-length SMN transcript from SMN2 than from SMN1. 4 -6 SMN2 was shown to be unique to Homo sapiens. 7 Approximately 94% of clinically typical SMA patients lack both copies of SMN1 exon 7. 8 SMN gene dosage analysis, a method to determine the copy number of SMN1, can be used to identify SMA carriers. Exon 7 of SMN1 and SMN2 are co-amplified with genomic and internal standards. The PCR products are then digested with DraI, which cuts only SMN2 exon 7 PCR products, followed by quantification of the PCR products. 9 -11 Other methods for SMN gene dosage analysis have been described. 12-14 A single copy of SMN1 by gene dosage analysis confirms carrier status; this analysis is therefore of clinical importance. A single-copy result also supports the diagnosis of SMN1-related SMA in an affected individual, who may have one deleted allele and one allele with a small intragenic mutation. However, the final diagnosis depends largely on the index of clinical suspicion. 15 This is because the frequency of single-copy carriers in the general population (ϳ2%) approaches the frequency of individuals affected with SMN1-related SMA who have a single-copy test result (ϳ3.6% 12 ). 16 The copy number of SMN2 correlates inversely with disease severity. 9,10,[12]...

Precision and Performance Characteristics of Bisulfite Conversion and Real-Time PCR (MethyLight) for Quantitative DNA Methylation Analysis

“…One should also be aware of PCR bias in quantitative molecular assays, 42 so we further normalized for a potential difference in PCR efficiencies between the gene of interest and COL2A1 by dividing the gene:COL2A1 ratio in a sample of interest by that ratio in SssI-treated DNA (presumably fully methylated). For all three genes tested, we showed good precision in threshold cycle (Ct) values in terms of both bisulfite-to-bisulfite variation (among seven bisulfite-treated aliquots B1 to B7) in the same run and MethyLight-to-MethyLight run-to-run variation (among five repeated MethyLight runs M1 to M5) in the same bisulfite-treated DNA sample.…”

New insights on the evolution of the SMN1 and SMN2 region: simulation and meta-analysis for allele and haplotype frequency calculations

“…SMN gene dosage analysis was developed and validated as described previously. 10,19,20 SMN1 and SMN2 copy numbers were determined by quantification of the PCR products after DraI digestion to differentiate SMN1 from SMN2, and normalizations to genomic standards, internal standards, and two-SMN1-copy controls. All samples were assayed in duplicate.…”

“…Hence, by starting with data on individuals affected with SMA for our calculations, we are potentially introducing an ascertainment bias against the 0-0 haplotype, since we would never expect to see the 0:0 genotype among living individuals. However, the 0-0 haplotype is sufficiently rare that the existence of the 0:0 genotype can be ignored, and the far more common 0:1 genotype can be used, to estimate e. Using the data in Table 3, and assuming Hardy-Weinberg equilibrium, To decrease errors associated with small numbers of subjects, we used equations containing larger numerators (ie, formulae (16) through (19) and (21) through (23)). Likewise, to calculate e, f, and g, we used population data based on large numbers of SMA patients (ie, formulae (7) through (10)), rather than small numbers of carrier individuals (ie, formulae (11) through (15)).…”

Inverse correlation between SMN1 and SMN2 copy numbers: evidence for gene conversion from SMN2 to SMN1

“…The quantitative accuracy of this method was validated as described. 7,11,12 All test samples were analyzed in duplicate.…”