Department of Genomics of Common Disease, School of Public Health, Imperial College London, London, UK.
Clin Chem. 2011 Aug;57(8):1188-95. doi: 10.1373/clinchem.2010.159558. Epub 2011 Jun 15.
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.
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 M(r) 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.
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.
This approach enabled reliable genotyping of heterozygous trisomic samples and the determination of the origin of nondisjunction in Down syndrome family trios.
准确分配嵌合于三体或重复区域的等位基因，是评估单核苷酸多态性 (SNP) 和基因组拷贝数综合效应的必要前提。这种综合分析具有挑战性，因为该 SNP 的杂合子可能是两种基因型之一，即 AAB 或 ABB。然而，已建立的 SNP 基因分型方法可能难以区分这两种杂合三体基因型。我们开发了一种用于分配杂合三体基因型的方法，该方法使用单碱基延伸分析后通过质谱获得的两个等位基因峰的高度比。
通过多重单碱基延伸分析分析 18 个 COL6A2（胶原，VI 型，α 2）SNP，该分析生成不同分子量值的等位基因特异性寡核苷酸，通过 MALDI-TOF 质谱检测。从杂合正常个体的样本中确定等位基因峰高度比的参考数据（均值和标准差）。通过计算每个三体等位基因峰高度比的 z 分数，并考虑 z 分数的符号（+/-），来分配杂合三体基因型。
对于每个分析的 SNP，96.1%（范围 89.9%-100%）的样本中都分配了杂合三体基因型。在第二个分析中重新测试了 97 个基因座中的 95 个（97.5%），获得了相同的基因型。随后，在 132 个唐氏综合征患儿的家系三家中确定了非分离的起源。
该方法能够可靠地对杂合三体样本进行基因分型，并确定唐氏综合征家系三家中非分离的起源。
