Tracy L Stockley and Peter N Ray 1 Introduction

Tay-Sachs disease is a severe, neurodegenerative disease fatal in childhood that is caused by deficiency of the enzyme P-hexosaminidase A (Hex A) (1). Tay-Sachs is most common in the Ashkenazi Jewish population, with an incidence of 1/3600 affected individuals and a carrier rate of approx 1 in 30 (1). Owing to the severity of the disease and the high incidence, carrier screening for Tay-Sachs disease has been available to Ashkenazi Jewish individuals since the 1970s, which has greatly reduced the number of affected children in this population. The standard method of carrier testing is by biochemical analysis for reduced Hex A activity in serum (2).

Although biochemical testing for Tay-Sachs is an established population screening method, there are several difficulties with the biochemical screening. One difficulty is the presence of an 'inconclusive' range of Hex A activity, in which it cannot be precisely determined if a person is a carrier or noncarrier of Tay-Sachs (3). Another difficulty is that women who are pregnant or using birth control pills can show an artificially reduced Hex A level when serum is tested, necessitating the use of biochemical testing of leukocytes for these women, which may also produce an inconclusive result (2,3). Inaccurate Hex A levels may also result in some individuals from the presence of pseudodeficiency alleles, which metabolize the natural Hex A substrate GM2 ganglio-side appropriately but do not metabolize the artificial substrate 4-methylumbelliferone (4MUG) used in the biochemical enzyme assay analysis (4,5). Thus, these individuals may be labeled as Tay-Sachs carriers although they do not carry a pathogenic mutation.

Due to these limitations with biochemical testing for Tay-Sachs disease, there is interest in providing molecular analysis of the HEXA gene for Tay-Sachs as a complement to biochemical testing. In the Jewish population, there are three common mutations in the HEXA gene causing Tay-Sachs disease. An insertion mutation in exon 11, +TATC1278, accounts for ~82% of mutations (6), a splice error in intron 12, 1421+1G>C, for an additional 11% of mutations (7,8) and a missense mutation 805G>A (G269S) in exon 7 for 3% of mutations (9,10). Together, these three mutations account for a total of ~95% of the mutations in HEXA causing Tay-Sachs disease in the Jewish population.

From: Methods in Molecular Biology, vol. 217: Neurogenetics: Methods and Protocols Edited by: N. T. Potter © Humana Press Inc., Totowa, NJ

Fig. 1. Theory of the ASA assay to detect mutations causing Tay-Sachs disease. For each mutation site, a normal and mutant primer were designed that differ at the 3' base, and correspond at this base to either the normal or mutant allele sequence. The primers are labeled with different fluorescent dyes (blue dye for the normal primer, black dye for the mutant primer, as indicated by either a circle or square respectively in the figure above), so that the resultant PCR products can be discriminated by dye colors. The assay was designed so that if only the normal sequence is present, amplification from only the normal primer is seen, and if only the mutant sequence is present, amplification from only the mutant primer is seen. If both normal and mutant sequences are present, amplification from both primers occurs.

Fig. 1. Theory of the ASA assay to detect mutations causing Tay-Sachs disease. For each mutation site, a normal and mutant primer were designed that differ at the 3' base, and correspond at this base to either the normal or mutant allele sequence. The primers are labeled with different fluorescent dyes (blue dye for the normal primer, black dye for the mutant primer, as indicated by either a circle or square respectively in the figure above), so that the resultant PCR products can be discriminated by dye colors. The assay was designed so that if only the normal sequence is present, amplification from only the normal primer is seen, and if only the mutant sequence is present, amplification from only the mutant primer is seen. If both normal and mutant sequences are present, amplification from both primers occurs.

Two common nonpathogenic mutations, 739C>T (R247W) and 745C>T (R249W), in exon 7 of HEXA also exist, although at higher frequency in the non-Jewish population (4,5). However, due to the importance of detecting pseudodeficiency alleles and the increasing rate of intermarriage between Jewish and non-Jewish individuals, molecular testing should include testing for these two pseudodeficiency alleles.

Most current molecular diagnostic tests for Tay-Sachs HEXA mutations are laborintensive and costly and thus are not suitable for testing large numbers of individuals as required in a population-screening situation. The current molecular tests commonly rely on restriction-enzyme differences between normal and mutant alleles, with the assay involving polymerase chain reaction (PCR) amplification of relevant regions of the HEXA gene and digestion with appropriate restriction enzymes for each mutation to be detected (11).

We have developed an alternate rapid method to simultaneously detect the three pathogenic and two pseudodeficiency HEXA mutations in one tube. This assay is based on allele specific amplification (ASA), which relies on the fact that primers that have a mismatch at the 3' terminal nucleotide will not function in PCR (12,13). In this test, fluorescent dye labeled primers specific to either normal or mutant DNA sequences are used to determine an individual's genotype for Tay-Sachs HEXA mutations or pseudodeficiency alleles, as shown in Fig. 1. For each mutation site, a normal and mutant primer are designed that differ at the 3' base, and correspond at this base to either the normal or mutant allele sequence. The primers are labeled with different flourescent dyes, so that the resultant PCR products can be discriminated by dye colors. The assay was designed so that if only the normal sequence is present, amplification from only the normal primer is seen, and if only the mutant sequence is present, amplification from only the mutant primer is seen. If both normal and mutant sequences are present, amplification from both primers occurs. In some primers, additional base pair mismatches besides the mismatch at the 3' end were required to achieve specific amplification of normal and mutant alleles.

This chapter will present methods for the application of the ASA method to detect the three common pathogenic mutations and the two pseudodeficiency alleles in the HEXA gene. This method has been adapted to simultaneously detect multiple mutations in other disorders for which molecular analysis is required, with appropriate design of primers for specific allele amplification.

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