ORIGINAL RESEARCH ARTICLE ©American College of Medical Genetics and Genomics Exon-level array CGH in a large clinical cohort demonstrates increased sensitivity of diagnostic testing for Mendelian disorders Swaroop Aradhya, PhD1, Rachel Lewis, MS1, Tahrra Bonaga, BS1, Nnenna Nwokekeh, MS1, Amanda Stafford, MS1, Barbara Boggs, PhD1, Kathleen Hruska, PhD1, Nizar Smaoui, MD1, John G. Compton, PhD1, Gabriele Richard, MD1 and Sharon Suchy, PhD1 Purpose: Mendelian disorders are most commonly caused by muta- somal dominant disorders, with a detection rate of 2.9%. For auto- tions identifiable by DNA sequencing. Exonic deletions and duplica- somal recessive disorders, array CGH was usually performed after a tions can go undetected by sequencing, and their frequency in most single mutation was identified by sequencing. Among 138 individu- Mendelian disorders is unknown. als tested for recessive disorders, 10.1% had intragenic deletions. For X-linked disorders, 3.5% of 313 patients carried a deletion or Methods: We designed an array comparative genomic hybridiza- duplication. tion (CGH) test with probes in exonic regions of 589 genes. Targeted testing was performed for 219 genes in 3,018 patients. We demon- Conclusion: Our results demonstrate that exon-level array CGH strate for the first time the utility of exon-level array CGH in a large provides a robust option for intragenic copy number analysis and clinical cohort by testing for 136 autosomal dominant, 53 autosomal should routinely supplement sequence analysis for Mendelian recessive, and 30 X-linked disorders. disorders. Results: Overall, 98 deletions and two duplications were identified Genet Med 2012:14(6):594–603 in 53 genes, corresponding to a detection rate of 3.3%. Approxi- Key words: array CGH; copy number; deletion; duplication; exon; mately 40% of positive findings were deletions of only one or two Mendelian exons. A high frequency of deletions was observed for several auto- INTRODUCTION clinical setting.5,6 We constructed a custom high-density oligo- The majority of mutations in Mendelian disorders are detected nucleotide array with probes targeted to the individual exons of by DNA sequencing. However, in recent years the significance 589 genes associated with known genetic disorders to identify and frequency of pathogenic intragenic deletions or duplications whole and partial gene deletions or duplications. Our results (copy number mutations) have become increasingly evident. from testing 3,018 patients with this array demonstrate that Quantitative methods are now used for molecular diagnosis of exon array CGH complements DNA sequencing and increases a limited number of disorders.1 In a variety of genetic disor- the mutation detection rate in the molecular diagnosis of auto- ders, including Rett syndrome, Smith–Magenis syndrome, and somal and X-linked Mendelian disorders. Prader–Willi syndrome, routine testing includes copy number analysis by multiplex ligation-dependent amplification (MLPA) MATERIALS AND METHODS assay, quantitative PCR (qPCR), or fluorescence in situ hybrid- Array design ization. The same methods have also been used to identify The 3,018 clinical cases tested by exon array CGH were ana- larger chromosomal rearrangements, including those affecting lyzed on one of the two array versions. On the first array version, subtelomeric regions.2,3 Array comparative genomic hybridiza- 1,584 cases were tested; 1,434 cases were tested on the second. tion (CGH) has proven to be a powerful tool for copy number The first version included two or more probes in most exons of analysis but has been used mostly for cytogenetic analysis to 465 targeted genes and three probes in each intron, regardless detect large genomic deletions and duplications that extend of size. Coverage of some small exons included probes in the hundreds of kilobases to megabases.4 Some recent reports have intronic sequence immediately flanking the exon, based on the shown that high-resolution array CGH with probes densely premise that any deletion/duplication of that exon would prob- distributed across individual genes can detect small deletions or ably extend into the intron. Genes with unprocessed pseudo- duplications, but this approach has not been widely applied in a genes were not included in the target list. The second version 1GeneDx, Gaithersburg, Maryland, USA. Correspondence: Swaroop Aradhya ([email protected]) Submitted 21 September 2011; accepted 28 November 2011; advance online publication 1 March 2012. doi:10.1038/gim.2011.65 594 Volume 14 | Number 6 | June 2012 | GENETICS in MEDICINE Exon-level array CGH | ARADHYA et al ORIGINAL RESEARCH ARTICLE of the exon array included seven probes across each exon and online Universal Probe Library assay design center (https:// the flanking 250-bp intronic sequence on either side. The array www.roche-applied-­science.com). Seventy nanograms each was designed to include probe coverage of 589 genes, including of patient sample and control DNA samples were added to all 465 genes from the first array version. Intronic regions other separate reaction mixtures containing a TaqMan probe and than the 250-bp exonic flanks were not covered, and deletions FastStart TaqMan mastermix (Roche) and locus-­specific prim- or duplications in those regions would not be detected. Genes ers. The amplification was carried out at 60 °C annealing tem- with processed pseudogenes (e.g., PTEN) were furnished with perature for 45 cycles on a Stratagene Mx3000P/3005P machine extra probe coverage within the 250-bp exon-flanking intronic (Agilent Technologies). Three normal genomes were tested in sequences. Untranslated regions of the exons were also covered triplicate along with the patient sample. In a valid positive assay, if part of those exons included coding sequences. The average the Ct for the clinical sample showed a difference of at least one gene size and the exon size in this collection of 589 genes were PCR cycle relative to the normal sample, the triplicates devi- 104 kb and 741 bp, respectively. Each array version was vali- ated by <0.30 Ct, and all negative controls showed normal copy dated with 60–80 DNA samples that previously showed either number for the locus tested. The assay used an internal nor- normal copy number or a known MLPA- or qPCR-confirmed malizing target (SOD1 gene in 21q22.11) to ensure that equal deletion or duplication in one of a variety of genes (data not amounts of DNA were used in all tested samples. The difference shown). in Ct ­values between the clinical sample and one normal control Following validation of the exon array, prospective studies sample was expressed as a fold change in the copy number of were performed on blood samples from patients referred to our the target gene. clinical laboratory for deletion/duplication testing for various Mendelian disorders. For autosomal recessive disorders, dele- RESULTS tion/duplication testing was performed if DNA sequencing Clinical testing of 3,018 cases identified only a single mutation. For individuals with an auto- Exon array CGH was used to examine 3,018 patients for dele- somal dominant disorder or for female carriers of an X-linked tions or duplications in 219 genes. More than one gene was disorder (affected or unaffected), deletion/duplication testing analyzed in 307 individuals affected with a genetically heteroge- was either offered as a second test if gene sequencing was nega- neous disorder. Therefore, a total of 4,354 genes were analyzed tive or in combination with the sequencing test when that dis- in the 3,018 individuals. Exon array CGH identified 98 partial order had a known high frequency of deletions. or whole-gene deletions and two duplications, corresponding to a detection rate of 3.3% in the individuals tested (Figure 1). Array hybridization and data analysis qPCR, MLPA, or whole-genome array CGH confirmed copy DNA was extracted from blood samples on the QiaCube number mutations detected by exon array CGH. Table 1 and (Qiagen, Valencia, CA) automated system. Labeling was carried Supplementary Table S5 online lists all the copy number out with the Enzo CGH labeling kit for oligo arrays (Enzo Life mutations identified in our cohort, andFigure 2 shows selected Sciences, Plymouth Meeting, PA). Array CGH was performed examples of partial gene deletions or duplications detected by with 0.5 μg of DNA according to the manufacturer’s protocol exon array CGH. Copy number mutations were identified in (Agilent Technologies, Santa Clara, CA). The data for each 53 of the 219 genes tested. No deletions or duplications were patient were examined only for the specific gene (or gene panel) found in the remaining 166 genes, although most of these were requested. Data were analyzed using the ADM-1 algorithm in evaluated in 10 cases or fewer. Forty percent of the 3,018 cases DNA Analytics/Genomic Workbench software. Reportable 12.0 data were based on log2 ratio deviations >0.25 and including two or more adjacent probes. 10.1% 10.0 Confirmation of array findings es 8.0 Probe deviations including two or more adjacent probes were confirmed by qPCR with custom-designed primers, MLPA or, 6.0 5.2% 5.3% in one case, whole-genome array CGH. MLPA was performed 3.5% 4.0 2.9% 3.3% according to the manufacturer’s protocols (MRC-Holland, ercentage of positiv 2.0 Amsterdam, The Netherlands). The following SALSA MLPA P kits were validated for clinical use: P067 PTCH1, kit P187 0.0 Holoprosencephaly HPE, P225-B1 PTEN, P101 STK11, P219-B1 AD AD except AR XL All All except PTEN PTEN PAX6, P313 CREBBP, P215 EXT, P180 Limb Malformations-2, Cases by category and P015 MECP2. qPCR analysis was performed using a TaqMan assay based on the Human Universal Probe Library Figure 1 Percentage of exon array CGH results classified according to mode of inheritance in 100 positive cases. Results are shown with set from Roche Applied Science (Roche, Indianapolis, IN; and without the PTEN cases included.