Analysis of common mutations in the galactose-1-phosphate uridyl transferase gene: new assays to increase the sensitivity and specificity of newborn screening for galactosemia

Abstract

Classical galactosemia is a genetic disease caused by mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. Prospective newborn screening for galactosemia is routine and utilizes the universally collected newborn dried blood specimen on filter paper. Screening for galactosemia is achieved through analysis of total galactose (galactose and galactose-1-phosphate) and/or determining the activity of the GALT enzyme. While this approach is effective, environmental factors and the high frequency of the Duarte D2 mutation (N314D) does lead to false positive results. Using DNA derived from the original newborn dried blood specimen and Light Cycler technology a panel of five assays able to detect the four most frequently encountered classical galactosemia alleles (Q188R, S135L, K285N, and L195P) and the N314D Duarte variant mutation are described. The five assays are performed simultaneously using common conditions. Including DNA preparation, set-up, amplification, and analysis the genotype data for all five loci is obtained in less than 2 hours. The assays are easily interpreted and amenable to high-throughput newborn screening. Mutational analysis is useful to reduce false positive results, differentiate D/G mixed heterozygotes from classical galactosemia, and to clearly identify a very high percentage of those affected by classical galactosemia.

Figure 2.

A–E: In A–C (N314D, Q188R, S135L) specimens that are wild-type, heterozygous, homozygous mutant, and a no DNA control are displayed. In D–E specimens that are wild-type, heterozygous, and a no DNA control are displayed.

Figure 1.

Hybridization probes to detect GALT mutations by fluorescence resonance energy transfer and melting curve analysis. Detection probes are framed while anchor probes are highlighted. All detection probes match the mutant sequence. The mutated nucleotide is shown in bold font on the sense strand and the wild-type nucleotide is shown in parenthesis above the mutated nucleotide. The detection probes for the N314D and Q188R assays are labeled 5′ with LC red 640 and 3′ phosphorylated, while the detection probes for S135L, K285N, and L195P are labeled 3′ with FITC. Anchor probes for the N314D and Q188R assays are labeled 3′ with FITC, while the anchor probes for S135L, K285N, and L195P are labeled 5′ with LC red 640 and 3′ phosphorylated.