A. Craig Chinault

Homologous recombination of a flanking repeat gene cluster is a mechanism for a common contiguous gene deletion syndrome

Smith–Magenis syndrome (SMS), caused by del(17)p11.2, represents one of the most frequently observed human microdeletion syndromes. We have identified three copies of a low–copy–number repeat (SMS–REPs) located within and flanking the SMS common deletion region and show that SMS–REP represents a repeated gene cluster. We have isolated a corresponding cDNA clone that identifies a novel junction fragment from 29 unrelated SMS patients and a different–sized junction fragment from a patient with dup(17)p11.2. Our results suggest that homologous recombination of a flanking repeat gene cluster is a mechanism for this common microdeletion syndrome.

Development and validation of a CGH microarray for clinical cytogenetic diagnosis

Purpose: We developed a microarray for clinical diagnosis of chromosomal disorders using large insert genomic DNA clones as targets for comparative genomic hybridization (CGH).Methods: The array contains 362 FISH-verified clones that span genomic regions implicated in over 40 known human genomic disorders and representative subtelomeric clones for each of the 41 clinically relevant human chromosome telomeres. Three or four clones from almost all deletion or duplication genomic regions and three or more clones for each subtelomeric region were included. We tested chromosome microarray analysis (CMA) in a masked fashion by examining genomic DNA from 25 patients who were previously ascertained in a genetic clinic and studied by conventional cytogenetics. A novel software package implemented in the R statistical programming language was developed for normalization, visualization, and inference.Results: The CMA results were entirely consistent with previous cytogenetic and FISH findings. For clone by clone analysis, the sensitivity was estimated to be 96.7% and the specificity was 99.1%. Major advantages of this selected human genome array include the following: interrogation of clinically relevant genomic regions, the ability to test for a wide range of duplication and deletion syndromes in a single analysis, the ability to detect duplications that would likely be undetected by metaphase FISH, and ease of confirmation of suspected genomic changes by conventional FISH testing currently available in the cytogenetics laboratory.Conclusion: The array is an attractive alternative to telomere FISH and locus-specific FISH, but it does not include uniform coverage across the arms of each chromosome and is not intended to substitute for a standard karyotype. Limitations of CMA include the inability to detect both balanced chromosome changes and low levels of mosaicism.

Clinical implementation of chromosomal microarray analysis: summary of 2513 postnatal cases.

Background Array Comparative Genomic Hybridization (a-CGH) is a powerful molecular cytogenetic tool to detect genomic imbalances and study disease mechanism and pathogenesis. We report our experience with the clinical implementation of this high resolution human genome analysis, referred to as Chromosomal Microarray Analysis (CMA). Methods and Findings CMA was performed clinically on 2513 postnatal samples from patients referred with a variety of clinical phenotypes. The initial 775 samples were studied using CMA array version 4 and the remaining 1738 samples were analyzed with CMA version 5 containing expanded genomic coverage. Overall, CMA identified clinically relevant genomic imbalances in 8.5% of patients: 7.6% using V4 and 8.9% using V5. Among 117 cases referred for additional investigation of a known cytogenetically detectable rearrangement, CMA identified the majority (92.5%) of the genomic imbalances. Importantly, abnormal CMA findings were observed in 5.2% of patients (98/1872) with normal karyotypes/FISH results, and V5, with expanded genomic coverage, enabled a higher detection rate in this category than V4. For cases without cytogenetic results available, 8.0% (42/524) abnormal CMA results were detected; again, V5 demonstrated an increased ability to detect abnormality. Improved diagnostic potential of CMA is illustrated by 90 cases identified with 51 cryptic microdeletions and 39 predicted apparent reciprocal microduplications in 13 specific chromosomal regions associated with 11 known genomic disorders. In addition, CMA identified copy number variations (CNVs) of uncertain significance in 262 probands; however, parental studies usually facilitated clinical interpretation. Of these, 217 were interpreted as familial variants and 11 were determined to be de novo; the remaining 34 await parental studies to resolve the clinical significance. Conclusions This large set of clinical results demonstrates the significantly improved sensitivity of CMA for the detection of clinically relevant genomic imbalances and highlights the need for comprehensive genetic counseling to facilitate accurate clinical correlation and interpretation.

Prenatal diagnosis of chromosomal abnormalities using array-based comparative genomic hybridization

Purpose: This study was designed to evaluate the feasibility of using a targeted array-CGH strategy for prenatal diagnosis of genomic imbalances in a clinical setting of current pregnancies.Methods: Women undergoing prenatal diagnosis were counseled and offered array-CGH (BCM V4.0) in addition to routine chromosome analysis. Array-CGH was performed with DNA directly from amniotic fluid cells with whole genome amplification, on chorionic villus samples with amplification as necessary, and on cultured cells without amplification.Results: Ninety-eight pregnancies (56 amniotic fluid and 42 CVS specimens) were studied with complete concordance between karyotype and array results, including 5 positive cases with chromosomal abnormalities. There was complete concordance of array results for direct and cultured cell analysis in 57 cases tested by both methods. In 12 cases, the array detected copy number variation requiring testing of parental samples for optimal interpretation. Array-CGH results were available in an average of 6 and 16 days for direct and cultured cells, respectively. Patient acceptance of array-CGH testing was 74%.Conclusion: This study demonstrates the feasibility of using array-CGH for prenatal diagnosis, including reliance on direct analysis without culturing cells. Use of array-CGH should increase the detection of abnormalities relative to the risk, and is an option for an enhanced level of screening for chromosomal abnormalities in high risk pregnancies.

22q11.2 Distal Deletion: A Recurrent Genomic Disorder Distinct from DiGeorge Syndrome and Velocardiofacial Syndrome

Microdeletions within chromosome 22q11.2 cause a variable phenotype, including DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS). About 97% of patients with DGS/VCFS have either a common recurrent approximately 3 Mb deletion or a smaller, less common, approximately 1.5 Mb nested deletion. Both deletions apparently occur as a result of homologous recombination between nonallelic flanking low-copy repeat (LCR) sequences located in 22q11.2. Interestingly, although eight different LCRs are located in proximal 22q, only a few cases of atypical deletions utilizing alternative LCRs have been described. Using array-based comparative genomic hybridization (CGH) analysis, we have detected six unrelated cases of deletions that are within 22q11.2 and are located distal to the approximately 3 Mb common deletion region. Further analyses revealed that the rearrangements had clustered breakpoints and either a approximately 1.4 Mb or approximately 2.1 Mb recurrent deletion flanked proximally by LCR22-4 and distally by either LCR22-5 or LCR22-6, respectively. Parental fluorescence in situ hybridization (FISH) analyses revealed that none of the available parents (11 out of 12 were available) had the deletion, indicating de novo events. All patients presented with characteristic facial dysmorphic features. A history of prematurity, prenatal and postnatal growth delay, developmental delay, and mild skeletal abnormalities was prevalent among the patients. Two patients were found to have a cardiovascular malformation, one had truncus arteriosus, and another had a bicuspid aortic valve. A single patient had a cleft palate. We conclude that distal deletions of chromosome 22q11.2 between LCR22-4 and LCR22-6, although they share some characteristic features with DGS/VCFS, represent a novel genomic disorder distinct genomically and clinically from the well-known DGS/VCF deletion syndromes.

Microarray-based CGH detects chromosomal mosaicism not revealed by conventional cytogenetics†

Somatic chromosomal mosaicism is a well‐established cause for birth defects, mental retardation, and, in some instances, specific genetic syndromes. We have developed a clinically validated, targeted BAC clone array as a platform for comparative genomic hybridization (aCGH) to enable detection of a wide range of pathologic copy number changes in DNA. It is designed to provide high sensitivity to detect well‐characterized submicroscopic micro‐deletion and duplication disorders while at the same time minimizing detection of variation of uncertain clinical significance. In the course of studying 2,585 samples submitted to our clinical laboratory, chromosomal mosaicism was detected in 12 patient samples; 10 of these cases were reported to have had a normal blood chromosome analysis. This enhanced ability of aCGH to detect mosaicism missed by routine chromosome analysis may be due to some combination of testing multiple cell lineages and/or failure of cytogenetically abnormal T lymphocytes to respond to mitogens. This suggests that aCGH may detect somatic chromosomal mosaicism that would be missed by conventional cytogenetics.

Microduplications of 22q11.2 are frequently inherited and are associated with variable phenotypes.

Purpose: Genomic rearrangements of chromosome 22q11.2, including the microdeletion associated with DiGeorge/velocardiofacial syndrome, are mediated by nonallelic homologous recombination between region-specific low-copy repeats. To date, only a small number of patients with 22q11.2 microduplication have been identified.Methods: We report the identification by array-comparative genomic hybridization of 14 individuals from eight families who harbor microduplications within the 22q11.2 region.Results: We have now observed a variety of microduplications, including the typical common ∼3-Mb microduplication, ∼1.5-Mb nested duplication, and smaller microduplications within and distal to the DiGeorge/velocardiofacial syndrome region, consistent with nonallelic homologous recombination using distinct low-copy repeats in the 22q11.2 DiGeorge/velocardiofacial syndrome region. These microduplications likely represent the predicted reciprocal rearrangements to the microdeletions characterized in the 22q11.2 region. The phenotypes seen in these individuals are generally mild and highly variable; familial transmission is frequently observed.Conclusions: These findings highlight the unbiased ability of array-comparative genomic hybridization to identify genomic imbalances and further define the molecular etiology and clinical phenotypes seen in microduplication 22q11.2 syndrome. Our findings also further support that the 22q11.2 region is highly dynamic with frequent rearrangements using alternative low-copy repeats as recombination substrates.

Genomic Imbalances in Neonates With Birth Defects: High Detection Rates by Using Chromosomal Microarray Analysis

OBJECTIVES. Our aim was to determine the frequency of genomic imbalances in neonates with birth defects by using targeted array-based comparative genomic hybridization, also known as chromosomal microarray analysis. METHODS. Between March 2006 and September 2007, 638 neonates with various birth defects were referred for chromosomal microarray analysis. Three consecutive chromosomal microarray analysis versions were used: bacterial artificial chromosome–based versions V5 and V6 and bacterial artificial chromosome emulated oligonucleotide–based version V6 Oligo. Each version had targeted but increasingly extensive genomic coverage and interrogated >150 disease loci with enhanced coverage in genomic rearrangement–prone pericentromeric and subtelomeric regions. RESULTS. Overall, 109 (17.1%) patients were identified with clinically significant abnormalities with detection rates of 13.7%, 16.6%, and 19.9% on V5, V6, and V6 Oligo, respectively. The majority of these abnormalities would not be defined by using karyotype analysis. The clinically significant detection rates by use of chromosomal microarray analysis for various clinical indications were 66.7% for “possible chromosomal abnormality” ± “others” (other clinical indications), 33.3% for ambiguous genitalia ± others, 27.1% for dysmorphic features + multiple congenital anomalies ± others, 24.6% for dysmorphic features ± others, 21.8% for congenital heart disease ± others, 17.9% for multiple congenital anomalies ± others, and 9.5% for the patients referred for others that were different from the groups defined. In all, 16 (2.5%) patients had chromosomal aneuploidies, and 81 (12.7%) patients had segmental aneusomies including common microdeletion or microduplication syndromes and other genomic disorders. Chromosomal mosaicism was found in 12 (1.9%) neonates. CONCLUSIONS. Chromosomal microarray analysis is a valuable clinical diagnostic tool that allows precise and rapid identification of genomic imbalances and mosaic abnormalities as the cause of birth defects in neonates. Chromosomal microarray analysis allows for timely molecular diagnoses and detects many more clinically relevant genomic abnormalities than conventional cytogenetic studies, enabling more informed decision-making and management and appropriate assessment of recurrence risk.

Identification of chromosome abnormalities in subtelomeric regions by microarray analysis: a study of 5,380 cases.

Subtelomeric imbalances are a significant cause of congenital disorders. Screening for these abnormalities has traditionally utilized GTG‐banding analysis, fluorescence in situ hybridization (FISH) assays, and multiplex ligation‐dependent probe amplification. Microarray‐based comparative genomic hybridization (array‐CGH) is a relatively new technology that can identify microscopic and submicroscopic chromosomal imbalances. It has been proposed that an array with extended coverage at subtelomeric regions could characterize subtelomeric aberrations more efficiently in a single experiment. The targeted arrays for chromosome microarray analysis (CMA), developed by Baylor College of Medicine, have on average 12 BAC/PAC clones covering 10 Mb of each of the 41 subtelomeric regions. We screened 5,380 consecutive clinical patients using CMA. The most common reasons for referral included developmental delay (DD), and/or mental retardation (MR), dysmorphic features (DF), multiple congenital anomalies (MCA), seizure disorders (SD), and autistic, or other behavioral abnormalities. We found pathogenic rearrangements at subtelomeric regions in 236 patients (4.4%). Among these patients, 103 had a deletion, 58 had a duplication, 44 had an unbalanced translocation, and 31 had a complex rearrangement. The detection rates varied among patients with a normal karyotype analysis (2.98%), with an abnormal karyotype analysis (43.4%), and with an unavailable or no karyotype analysis (3.16%). Six patients out of 278 with a prior normal subtelomere‐FISH analysis showed an abnormality including an interstitial deletion, two terminal deletions, two interstitial duplications, and a terminal duplication. In conclusion, genomic imbalances at subtelomeric regions contribute significantly to congenital disorders. Targeted array‐CGH with extended coverage (up to 10 Mb) of subtelomeric regions will enhance the detection of subtelomeric imbalances, especially for submicroscopic imbalances.

Bacterial artificial chromosome-emulation oligonucleotide arrays for targeted clinical array-comparative genomic hybridization analyses

Purpose: The goal of this work was to test the ability of oligonucleotide-based arrays to reproduce the results of focused bacterial artificial chromosome (BAC)-based arrays used clinically in comparative genomic hybridization experiments to detect constitutional copy number changes in genomic DNA.Methods: Custom oligonucleotide (oligo) arrays were designed using the Agilent Technologies platform to give high-resolution coverage of regions within the genome sequence coordinates of BAC/P1 artificial chromosome (PAC) clones that had already been validated for use in previous versions of clone arrays used in clinical practice. Standard array-comparative genomic hybridization experiments, including a simultaneous blind analysis of a set of clinical samples, were conducted on both array platforms to identify copy number differences between patient samples and normal reference controls.Results: Initial experiments successfully demonstrated the capacity of oligo arrays to emulate BAC data without the need for dye-reversal comparisons. Empirical data and computational analyses of oligo response and distribution from a pilot array were used to design an optimized array of 44,000 oligos (44K). This custom 44K oligo array consists of probes localized to the genomic positions of >1400 fluorescence in situ hybridization-verified BAC/PAC clones covering more than 140 regions implicated in genetic diseases, as well as all clinically relevant subtelomeric and pericentromeric regions.Conclusions: Our data demonstrate that oligo-based arrays offer a valid alternative for focused BAC arrays. Furthermore, they have significant advantages, including better design flexibility, avoidance of repetitive sequences, manufacturing processes amenable to good manufacturing practice standards in the future, increased robustness because of an enhanced dynamic range (signal to background), and increased resolution that allows for detection of smaller regions of change.