A high throughput beta-globin genotyping method by multiplexed melting temperature analysis.

For a population-based newborn screening program, challenges exist in using technological advances to improve the quality and efficiency of the existing screening program and to develop new diagnostic capabilities. A newly developed genotyping method for screening of common mutations within the beta-globin gene is described here. This genotyping system consists of three major components: an automation system for high throughput DNA extraction and PCR setup, a conventional thermal cycler, and a LightTyper instrument for post-PCR melting temperature analysis. Briefly, genomic DNA is extracted from dried blood on a filter paper using methanol and Tris buffer. Genetic fragments of interest are amplified by asymmetric PCR. Fluorescent labeled probes are added during PCR setup, which eliminates the need for any post-PCR sample handling process. Melting temperature analysis is achieved through fluorescent resonance energy transfer (FRET) reaction using the LightTyper instrument. The assay is designed to simultaneously detect three common beta-globin mutations, S(A173T), C(G172A), and E(G232A), and can identify any of the eight possible genotypes in a single reaction: AA, AE, EE, AS, SC, SS, AC, and CC (A represents wild type allele). The method was validated with a large number of samples in both a retrospective and parallel study. Results were compared to those obtained by isoelectric focusing electrophoresis. The accuracy of this genotyping method is greater than 99%.

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.

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.