Primer System for Single Cell Detection of Double Mutation for Tay-Sachs Disease

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Abstract
Purpose: Nearly 100% of infantile Tay-Sachs disease isproduced by two mutations occurring in the alpha chain ofthe lysosomal enzyme beta-N-acetylhexosaminidase (HEXA)in the Ashkenazi Jewish population. Although others havedescribed primer systems used to amplify both sitessimultaneously, few discuss the allele dropout problems inherent inthis test. Our goal was to construct a more robust testenabling stronger signal generation for single cellpreimplantation genetic diagnosis and to investigate theoccurrence of allele dropout. Methods: New nested primers were designed to optimizedetection of both major Tay-Sachs mutations. Four hundredfifty-seven single cells, including normal cells and thosecarrying mutations of either the 4bp insertion exon 11 orsplice-site intron 12 defects, were used to screen a newprimer system. Results: Based on PCR amplified product analysis, totalefficiency of amplification was 85.3%, (390/457). The alleledropout rate for the 4bp insertion mutation in exon 11 andsplice-site mutation in intron 12 was 4.8% and 5.8%,respectively. Conclusons: Multiple mutation detection and analysiswithin the Tay-Sachs disease gene (HEXA) is possible usingsingle cells for clinical preimplantation genetic diagnosis.Alternative PCR primers and conditions offer variousmethods for developing systems compatible to specificprogram requirements.

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