Simultaneous amplification, detection, and analysis of common mutations in the galactose-1-phosphate uridyl transferase gene

Abstract

Classic galactosemia is an autosomal recessive inherited error of galactose metabolism. It is caused by lack of galactose-1-phosphate uridyl transferase, an enzyme that is required to metabolize galactose-1-phosphate to uridine diphosphate galactose. The build up of galactose-1-phosphate is toxic at high levels and can damage the liver, brain, eyes, and other vital organs. Over 200 mutations have been identified in affected individuals. We describe an assay to identify nine target mutations or variants in the galactose-1-phosphate uridyl transferase gene, namely p.Q188R, p.S135L, p.K285N, p.L195P, p.T138M, p.Y209C, IVS2-2 A>G, p.L218L, and p.N314D. A single long-range PCR is followed by a multiplexed nucleotide extension assay (single nucleotide extension) and capillary electrophoresis to detect simultaneously all nine target mutations/variants. Fifty-four previously characterized samples (47 clinical samples and seven controls) gave a 100% concordance. We also report a nontarget novel mutation, p.L192X, and its profile using single nucleotide extension. This assay can complement the enzyme activity assay and identify familial mutations for testing additional family members.

Figure 1

Schematic of primer locations for the generation of artificial template for IVS 2-2 A>G mutation. A: IVS 2-2 forward primer; B: IVS 2-2 reverse primer; C: artificial oligodeoxyribonucleotides template forward primer; and D: artificial oligodeoxyribonucleotides template reverse primer. “X” indicates the position of the mutation.

Figure 2

Multiplex genotyping of nine common mutations/variants from eight galactosemia samples. A normal wild-type DNA and nine mutations/variants are shown. For each sample, all nine alleles are analyzed and detected as a wild type or a mutant in each size range (bin). Mutations are labeled as “MUT _”. Vertical scales are peak heights measured in relative fluorescent units, and horizontal scales are ranges of oligodeoxyribonucleotides sizes for normal and mutant genotypes detected with each allele. a: Wild-type genotype; b: heterozygous IVS2-2, A>G, artificial template; c: heterozygous p.S135L, C>T; d: heterozygous p.T138M (reverse strand genotyped), G>A mutation; e: compound heterozygous p.Q188R, A>G and p.Y209C, T>C (reverse strand genotyped); f: compound heterozygous p.L195P, T>C and p.N314D, A>G; g: heterozygous p.L218L, G>A (reverse strand genotyped), and p.N314D, A>G; h: heterozygous p.K285N, G>T.

Figure 3

a: Sample with nontargeted mutation at locus p.L218L, showing a C base incorporated instead of the expected A base and a shift in the size. b: DNA sequence analysis showing the variant to be p.L218Q due to c.652delC.