Primer system for single cell detection of double mutation for Tay-Sachs disease.
OBJECTIVE
Nearly 100% of infantile Tay-Sachs disease is produced by two mutations occurring in the alpha chain of the lysosomal enzyme beta-N-acetylhexosaminidase (HEXA) in the Ashkenazi Jewish population. Although others have described primer systems used to amplify both sites simultaneously, few discuss the allele dropout problems inherent in this test. Our goal was to construct a more robust test enabling stronger signal generation for single cell preimplantation genetic diagnosis and to investigate the occurrence of allele dropout.


METHODS
New nested primers were designed to optimize detection of both major Tay-Sachs mutations. Four hundred fifty-seven single cells, including normal cells and those carrying mutations of either the 4bp insertion exon 11 or splice-site intron 12 defects, were used to screen a new primer system.


RESULTS
Based on PCR amplified product analysis, total efficiency of amplification was 85.3%, (390/457). The allele dropout rate for the 4bp insertion mutation in exon 11 and splice-site mutation in intron 12 was 4.8% and 5.8%, respectively.


CONCLUSIONS
Multiple mutation detection and analysis within the Tay-Sachs disease gene (HEXA) is possible using single cells for clinical preimplantation genetic diagnosis. Alternative PCR primers and conditions offer various methods for developing systems compatible to specific program requirements.
Liu
M C
MC
Department of Obstetrics and Gynecology, College of Medicine, University of Florida, Gainesville 32610, USA.
Drury
K C
KC
Kipersztok
S
S
Zheng
W
W
Williams
R S
RS
D001483:Base Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000138:chemical synthesis; D005347:Fibroblasts / Q000166:cytology; D020180:Heteroduplex Analysis; D054818:Hexosaminidase A; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D019836:Preimplantation Diagnosis / Q000379:methods; D013661:Tay-Sachs Disease / Q000235:genetics*; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics*
D017931:DNA Primers; D054818:Hexosaminidase A; D001619:beta-N-Acetylhexosaminidases