Rapid Diagnosis of Aneuploidy by High-Resolution Melting Analysis of Segmental Duplications

Abstract

BACKGROUND: Several molecular methods, such as quantitative fluorescence PCR and multiplex ligationdependent probe amplification, currently serve as important adjuncts to traditional karyotyping for the diagnosis of aneuploidy; however, the performance or throughput limitations of these methods hinder their use for routine prenatal diagnosis and populationbased postnatal screening. We developed a novel approach, called "high-resolution melting analysis of segmental duplications," to detect common aneuploidies.

Rapid Prenatal Diagnosis of Common Numerical Chromosomal Abnormalities by High-Resolution Melting Analysis of Segmental Duplications

“…Based on our previous work (Guo et al, 2012), we selected eight pairs of segmental duplicates and redesigned primers within them. Using this strategy, dosages of chromosomes 13, 18, and 21, and the sex chromosomes can be dually confirmed by these eight assays.…”

“…However, its cell culturebased protocol is labor intensive and time consuming, which reduces the effectiveness of karyotyping for high-throughput testing. To address these issues, several molecular methods that do not require cell culture have been developed to make prenatal diagnosis simpler and more rapid (Trask, 1991;Mansfield, 1993;Lee et al, 1997;Zimmermann et al, 2002;Pont-Kingdon and Lyon, 2003;Slater et al, 2003;Deutsch et al, 2004;Larrabee et al, 2004;Fan et al, 2009;Guo et al, 2010;Vialard et al, 2011;Yan et al, 2011;Dan et al, 2012;Guo et al, 2012). Most of these methods target trisomies 13, 18, and 21, and sex chromosome aneuploidies, as such aneuploidies are associated with substantial morbidity and mortality and account for the majority of clinically significant chromosomal abnormalities in live births (Driscoll and Gross, 2009).…”

“…Opponents of RAD methods suggest that these assays may miss chromosomal abnormalities other than targeted aneuploidies, and those which result from RAD screening should be followed up with karyotyping (Test and Technology Transfer Committee, 2000;Caine et al, 2005;de Jong et al, 2009). However, proponents argue that chromosomal abnormalities with adverse outcomes missed by RAD are rare, because prenatal chromosomal diagnosis is mainly offered to pregnant women at a high risk who are selected by prenatal screening programs targeting trisomies 21 and 18 (Boormans et al, 2009;Ogilvie et al, 2009;Gekas et al, 2011). However, this debate has not hampered the widespread use of well-recognized RAD methods, and, indeed, many new RAD methods have also emerged in recent years (Vialard et al, 2011;Yan et al, 2011;Dan et al, 2012;Guo et al, 2012). Technically, each RAD method possesses both advantages and limitations.…”

“…In previous studies, we described a novel one-step RAD method called high-resolution melting analysis of segmental duplications (SD-HRM) (Guo et al, 2012). It was shown that SD-HRM could accurately differentiate samples of patients with common aneuploidies from those of unaffected controls, while markedly simplifying assay setup and analysis and reducing time and costs compared with existing RAD methods.…”

Copy Number Assessment by Competitive PCR with Limiting Deoxynucleotide Triphosphates and High-Resolution Melting

“…Competitive PCR typically amplifies both a target and a competitor with the same primer set to retain their quantitative relationship (12 ). Duplex melting with a single primer set that amplifies segmental duplications can quantify trisomies (13 ), and a similar method using homologous sequences can identify microdeletions or microinsertions (14 ). The reference and target PCR products are on different chromosomes with distinct melting temperatures (T m s) so they can be compared.…”