Genevieve Pont-Kingdon1, Elaine Lyon. 1. ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA. pontkig@aruplab.com
Abstract
BACKGROUND: Molecular approaches for the detection of chromosomal abnormalities will allow the development of rapid, cost-effective screening strategies. We present here a molecular alternative for the detection of aneuploidies and, more specifically, trisomy 21. METHODS: We used the quantitative value of melting curve analysis of heterozygous genetic loci to establish a relative allelic count. The two alleles of a given single-nucleotide polymorphism (SNP) were differentiated by thermodynamic stability with a fluorescently labeled hybridization probe and were quantified by relative areas of derivative melting curves detected after fluorescence resonance energy transfer. Heterozygous SNPs provided internal controls for the assay. RESULTS: We selected six SNPs, heterozygous in at least 30% of a random population, to form a panel of informative loci in the majority of a random population. After normalization to a heterozygous control, samples segregated into three categories; nontrisomic samples had mean allele ratios of 0.96-1.09, whereas trisomic samples had mean ratios of 1.84-2.09 or 0.46-0.61, depending on which allele was duplicated. Within-run mean CVs of ratios were 6.5-27%, and between-assay mean CVs were 13-24%. CONCLUSIONS: The use of melting curve analysis of multiple SNPs is an alternative to the use of small tandem repeats for the detection of trisomies. Because of the high density of SNPs, the approach may be specifically useful for very fine mapping of the regions of chromosome 21 that are critical for Down syndrome; it is also applicable to aneuploidies other than trisomy 21 and to specimens that are not amenable to cytogenetic analysis.
BACKGROUND: Molecular approaches for the detection of chromosomal abnormalities will allow the development of rapid, cost-effective screening strategies. We present here a molecular alternative for the detection of aneuploidies and, more specifically, trisomy 21. METHODS: We used the quantitative value of melting curve analysis of heterozygous genetic loci to establish a relative allelic count. The two alleles of a given single-nucleotide polymorphism (SNP) were differentiated by thermodynamic stability with a fluorescently labeled hybridization probe and were quantified by relative areas of derivative melting curves detected after fluorescence resonance energy transfer. Heterozygous SNPs provided internal controls for the assay. RESULTS: We selected six SNPs, heterozygous in at least 30% of a random population, to form a panel of informative loci in the majority of a random population. After normalization to a heterozygous control, samples segregated into three categories; nontrisomic samples had mean allele ratios of 0.96-1.09, whereas trisomic samples had mean ratios of 1.84-2.09 or 0.46-0.61, depending on which allele was duplicated. Within-run mean CVs of ratios were 6.5-27%, and between-assay mean CVs were 13-24%. CONCLUSIONS: The use of melting curve analysis of multiple SNPs is an alternative to the use of small tandem repeats for the detection of trisomies. Because of the high density of SNPs, the approach may be specifically useful for very fine mapping of the regions of chromosome 21 that are critical for Down syndrome; it is also applicable to aneuploidies other than trisomy 21 and to specimens that are not amenable to cytogenetic analysis.
Authors: Subeen Hong; Seung Mi Lee; Sohee Oh; So Yeon Kim; Young Mi Jung; Sun Min Kim; Chan-Wook Park; Jong Kwan Jun; Byoung Jae Kim; Joong Shin Park Journal: Sci Rep Date: 2022-01-07 Impact factor: 4.379