Quantitative Real-Time Polymerase Chain Reaction for the Verification of Genomic Imbalances Detected by Microarray-Based Comparative Genomic Hybridization

Abstract

The American College of Medical Genetics guidelines for microarray analysis for constitutional cytogenetic abnormalities require abnormal or ambiguous results from microarray-based comparative genomic hybridization (aCGH) analysis be confirmed by an alternative method. We employed quantitative real-time polymerase chain reaction (qPCR) technology using SYBR Green I reagents for confirmation of 93 abnormal aCGH results (50 deletions and 43 duplications) and 54 parental samples. A novel qPCR protocol using DNA sequences coding for X-linked lethal diseases in males for designing reference primers was established. Of the 81 sets of test primers used for confirmation of 93 abnormal copy number variants (CNVs) in 80 patients, 71 sets worked after the initial primer design (88%), 9 sets were redesigned once, and 1 set twice because of poor amplification. Fifty-four parental samples were tested using 33 sets of test primers to follow up 34 CNVs in 30 patients. Nineteen CNVs were confirmed as inherited, 13 were negative in both parents, and 2 were inconclusive due to a negative result in a single parent. The qPCR assessment clarified aCGH results in two cases and corrected a fluorescence in situ hybridization result in one case. Our data illustrate that qPCR methodology using SYBR Green I reagents is accurate, highly sensitive, specific, rapid, and cost-effective for verification of chromosomal imbalances detected by aCGH in the clinical setting.

Single exon-resolution targeted chromosomal microarray analysis of known and candidate intellectual disability genes

“…The B21% validation rate we achieved is in keeping with other CMA research studies using whole-genome arrays. 16 Four individuals (patients 931, 331, 600, and 513) exhibited two de novo CNVs each. For each of these four patients, one CNV was clearly pathogenic and the contribution, if any, of the second CNV is unknown.…”

“…16 Because of the research nature of this project, the settings to identify a CNV were liberal in order to detect previously unrecognized ID loci or uncover novel intragenic causative CNVs, and therefore a high false positive rate is expected. In terms of the specificity of our design, because of our Canadian health care structure, not all patients run on our custom array were fortunate to also have a clinical array performed.…”

16p13.11 duplication is a risk factor for a wide spectrum of neuropsychiatric disorders

“…There were no other genomic abnormalities with clinical relevance in these patients. The genomic imbalances on 16p12.3-p13.11 in these patients were confirmed by quantitative real-time PCR technique targeting to the MYH11 and NDE1 genes within 16p13.11 region based on the procedures published before 16 (data not shown). The quantitative PCR method was also carried out for available parental follow-up studies.…”

Cardiac Defects Are Infrequent Findings in Individuals With 8p23.1 Genomic Duplications Containing GATA4

“…The genomic imbalances containing GATA4 identified by aCGH also were verified by quantitative real-time polymerase chain reaction using test primers targeted to GATA4 as reported previously (onlineonly Data Supplement Table I). 18 The quantitative real-time polymerase chain reaction method also was carried out for parental follow-up studies.…”