Analytical and clinical validity of whole‐genome oligonucleotide array comparative genomic hybridization for pediatric patients with mental retardation and developmental delay

Abstract

We performed a pilot study to establish the analytical and clinical validity of whole genome oligonucleotide array comparative genomic hybridization (oaCGH) using the 44,000 oligonucleotide array from Agilent Technologies. DNA specimens from 10 patients with different chromosomal abnormalities were used as the test group and sex mismatched normal male or female DNA as references. A series of DNA mixtures containing 50%, 33%, and 25% of a known deletion was generated to evaluate the analytical capacity of oaCGH on detecting mosaic pattern. Receiver operating characteristic (ROC) curves were computed to evaluate sensitivity, specificity, and analytical resolution for detecting deletions, duplications, and mosaic patterns. The oaCGH detected the chromosomally recognized deletions, duplications, and additional genomic aberrations. Fluorescent in situ hybridization (FISH) assays using targeted BAC clone probes confirmed oaCGH findings. Failure in detecting marker chromosomes, a polymorphic inversion, and a Robertsonian translocation was also noted. The oaCGH achieved 99% sensitivity and 99% specificity with a resolution of 300-500 Kb. It also demonstrated 85% sensitivity and 95% specificity in detecting 50% mosaicism; however, increased test-to-test variations and reduced sensitivity were noted as the mosaic percentage decreased. Chromosome and oaCGH analyses on 50 pediatric patients with mental retardation (MR) and developmental delay (DD) delineated the genomic content of chromosomal abnormalities in nine cases, pathogenic genomic disorders in three cases and benign genomic variants in six cases. These results affirmed the analytical and clinical validity of oaCGH and prompted a cytogenomic algorithm to integrate oaCGH, chromosome and FISH analyses for genetic diagnosis.

Unique Genomic Structure and Distinct Mitotic Behavior of Ring Chromosome 21 in Two Unrelated Cases

“…For a cell-based analysis, an extended analysis of 100 G-banded metaphase cells or 200-500 directly-prepared interphase cells by FISH will allow the detection of equal or 1 3% or 1-2% of mosaicism with 95% confidence, respectively [Hook, 1977]. As demonstrated in our second case, aCGH could be used to evaluate the level of mosaicism, but a reduced sensitivity for low-level mosaicisms has been noted [Xiang et al, 2008]. For cases with a ring chromosome replacing a normal chromosome, we recommended an integrated cytogenomic approach including (1) routine chromosome analysis of 100 metaphases or a sufficient number for percentages of cells with typical ring, ring variants, and ring loss, (2) FISH using chromosome-specific subtelomeric or targeted probes on direct cell preparations to access terminal deletion, duplication, and ring loss on 200-500 interphase cells, (3) aCGH analysis to characterize the genomic structure and ring stability, and (4) follow-up parental study to determine de novo or familial transmission of the ring chromosome.…”

“…The contiguous deletion and duplication patterns have also been reported in a proportion of ring chromosomes 13, 15, 18, 20, 21, and 22 [Rossi et al, 2008;Conlin et al, 2011] and in cases with subtelomeric abnormalities of 8p, 4q, 5p, and other chromosomes [Xiang et al, 2008;Rossi et al, 2009;Yu et al, 2010]. A chromosomal inverted duplication with a terminal deletion has been induced by segmental duplications such as the 2 olfactory receptor gene clusters flanking a 5-Mb region of 8p23.1 [Yu et al, 2010].…”

“…However, most of the earlier reported cases lacked genomic mapping of the ring chromosome structure which made it difficult to identify candidate genes and infer genotype-phenotype correlations. Recently, genome-wide oligonucleotide array comparative hybridization (aCGH) has been validated for clinical diagnosis and proven to be highly effective in defining the breakpoints and gene content of chromosomal imbalances [Xiang et al, 2008]. The application of genomic analysis defined a simple distal deletion of ring chromosome 21 in 3 cases [Ki et al, 2003;Bertini et al, 2008;Fiorini et al, 2009] and a complete ring chromosome 21 in a fetus [Papoulidis et al, 2010].…”

Copy number detection in discordant monozygotic twins of Congenital Diaphragmatic Hernia (CDH) and Esophageal Atresia (EA) cohorts

“…As described recently, high-resolution (SNP) arrays are suitable for detection of both germ-line and mosaic CNVs. [21][22][23][24][25] Mosaic copy number aberrations are hallmarked by a concomitant change of log2 intensity signal and a shift in b-allele frequency. The detection limit (sensitivity) of the Nexus SNP-FASST algorithm for mosaic CNVs is 20% using a heterozygous imbalance threshold of 0.45.…”

Microarray-Based Genomic DNA Profiling Technologies in Clinical Molecular Diagnostics

“…In general, CNVs can be categorized into 3 major groups: CNVs with established pathogenicity (pCNVs), i.e., deletions/duplications that overlap with re- (14,30,(65)(66)(67)(68)(69)(70), Table 4]. Even when bCNVs and uCNVs are intentionally avoided during array design, a large percentage of the identified imbalances often belong to bCNVs or uCNVs, because a substantial number of novel CNVs are still being discovered (Table 4).…”