BACKGROUND
The accurate assignment of alleles embedded within trisomic or duplicated regions is an essential prerequisite for assessing the combined effects of single-nucleotide polymorphisms (SNPs) and genomic copy number. Such an integrated analysis is challenging because heterozygotes for such a SNP may be one of 2 genotypes—AAB or ABB. Established methods for SNP genotyping, however, can have difficulty discriminating between the 2 heterozygous trisomic genotypes. We developed a method for assigning heterozygous trisomic genotypes that uses the ratio of the height of the 2 allele peaks obtained by mass spectrometry after a single-base extension assay.
METHODS
Eighteen COL6A2 (collagen, type VI, alpha 2) SNPs were analyzed in euploid and trisomic individuals by means of a multiplexed single-base extension assay that generated allele-specific oligonucleotides of differing Mr values for detection by MALDI-TOF mass spectrometry. Reference data (mean and SD) for the allele peak height ratios were determined from heterozygous euploid samples. The heterozygous trisomic genotypes were assigned by calculating the z score for each trisomic allele peak height ratio and by considering the sign (+/−) of the z score.
RESULTS
Heterozygous trisomic genotypes were assigned in 96.1% (range, 89.9%–100%) of the samples for each SNP analyzed. The genotypes obtained were reproduced in 95 (97.5%) of 97 loci retested in a second assay. Subsequently, the origin of nondisjunction was determined in 108 (82%) of 132 family trios with a Down syndrome child.
CONCLUSIONS
This approach enabled reliable genotyping of heterozygous trisomic samples and the determination of the origin of nondisjunction in Down syndrome family trios.
Qualitative Single Base Extension assay for Identification of Single Nucleotide Polymorphism in Mumps Virus Genome
