High-resolution human genome analysis by array comparative genomic hybridization (aCGH) has revolutionized our ability to identify both benign copy-number variation (CNV) [Conrad et al., 2010b
; Iafrate et al., 2004
; Redon et al., 2006
; Sebat et al., 2004
] as well as pathogenic copy-number changes associated with genomic disorders [Lupski, 1998
]. The pathogenic mechanism for these disorders, which involve genomic losses or gains of various sizes, is often dosage sensitivity of one or more of the genes within the rearranged genomic interval, but gene interruption, gene fusions, and position effects are increasingly recognized mechanisms mediating downstream effects of CNVs [Lupski and Stankiewicz, 2005
]. Array CGH has enabled the detection of submicroscopic CNV (i.e.
, microdeletions and microduplications). To date, dozens of disorders have been ascribed to this type of genomic aberration [Mefford and Eichler, 2009
; Stankiewicz and Lupski, 2010
]. Recurrent microdeletions and microduplications occur via nonallelic homologous recombination (NAHR), with the “fixed” size of the reciprocal rearrangements reflecting the genomic positions of flanking, directly oriented repeat sequences utilized as homologous recombination substrates [Stankiewicz and Lupski, 2002
]. In contrast, nonrecurrent rearrangements vary in size from genomic alterations involving megabases of DNA, to single-gene duplication/triplication, to CNV of single exons [Zhang et al., 2009a
]. Such nonrecurrent CNV occur by nonhomologous end joining (NHEJ) or by the recently described replication-based mechanisms of fork stalling and template switching/microhomology-mediated break induced replication (FoSTeS/MMBIR) [Hastings et al., 2009a
; Lee et al., 2007
Deletion or addition of one or more exons in a gene can have varied molecular and phenotypic consequences. A shift in reading frame can result in a premature termination codon, typically followed by nonsense-mediated decay (NMD) to create a loss of function allele [Maquat 1995
]. Escape from NMD is possible, which may cause disease by gain of function [Ben-Shachar et al., 2009
; Inoue et al., 2004
]. Rarely, premature stop codons may also promote exon skipping (nonsense-associated altered splicing; NAS), which has the potential to restore the reading frame [Dietz et al., 1993
; J. Wang et al., 2002
]. An in-frame loss or gain may result in an altered [Yatsenko et al., 2003
] or fused [Lifton et al., 1992
; Miyahara et al., 1992
] protein product with reduced or novel function. Thus, although haploinsufficiency may result from exonic CNV [Zhang et al., 2009b
], novel hypomorphic, antimorphic, and even neomorphic mutant alleles may be generated.
Exon-targeted aCGH (i.e.
, aCGH using an array with probes concentrated disproportionately in exons) can have either genome-wide or focused coverage. Genome-wide exonic arrays have been used to measure mRNA expression [Kapur et al., 2007
], which unlike traditional 3′ expression arrays allows alternative splicing to be assessed [Clark et al., 2007
; Gardina et al., 2006
; Thorsen et al., 2008
; Yeo et al., 2007
]. This technique has enabled the discovery of tissue- and tumor-specific splice variants. Similar studies have been performed using locus-specific expression exon arrays [Labeit et al., 2006
In addition to assessing gene expression, exon arrays have been used to assess genomic content. Bailey et al. 
studied nine healthy HapMap individuals using an array with exonic coverage for 2,790 genes. This study uncovered substantial CNV, disproportionately localized to regions containing segmental duplications. Although a catalog of benign intragenic CNV has been found by this and other studies [Conrad et al., 2010b
], array-based detection of clinically relevant intragenic CNV remains in its infancy.
Hegde et al. 
, del Gaudio et al. 
, and Bovolenta et al. 
each designed a genomic microarray spanning the length of the dystrophin (DMD
) gene. Although these single-locus arrays were not strictly exon targeted, the density and distribution of probes were sufficient to detect exonic (and intronic) CNV within the DMD
locus in patients suspected of having mutations in this gene. Wong et al. 
also detected exonic CNV, using an array with dense coverage of 130 nuclear genes implicated in mitochondrial and metabolic disorders. This demonstrated the utility of a single nonexon-targeted array to detect intragenic CNV in multiple related genes.
Dhami et al. 
constructed an exon-specific array with coverage for 162 exons of five genes implicated in unrelated conditions (COL4A5
, and PMP22
). Similarly, Saillour et al. 
performed aCGH to assess copy-number variation among 158 exons in eight disease genes (CFTR
, and six sarcoglycan genes), as did Staaf et al. 
for the exons of six cancer-related genes (BRCA1
, and CDKN2A
). Tayeh et al. 
constructed a targeted array with exonic (and intronic, with slightly diminished resolution) coverage of 71 disease genes, predominantly implicated in lysosomal storage and metabolic disorders. Significantly, this array was used in a clinical diagnostic setting in cases where gene sequencing failed to detect a mutation or mutations sufficient to explain a patient’s disease. The aforementioned studies provided proof-of-concept that a targeted exon array could be used to diagnose disparate disorders caused by intragenic copy-number changes. Yet, as the patients assessed in these studies were a selected population of previously-diagnosed (either clinically or molecularly) individuals, an array-based methodology to detect clinically relevant exonic copy-number changes genome-wide in unscreened or undiagnosed individuals has not yet been described.
As part of our continuing effort to clinically implement high resolution human genome analysis [Cheung et al., 2005
; Lu et al., 2007
; Ou et al., 2008
; Shao et al., 2008
], we sought to indentify CNV of smaller sizes (i.e.
, kilobasepairs in length, containing only one or a few exons) in functionally relevant regions of the human genome. To do this, we designed and developed a whole-genome microarray with coverage of approximately 24,000 exons in over 1,700 clinically relevant and candidate disease genes. This approach enables detection of intragenic copy-number changes in patients with varied clinical presentations that would otherwise be missed by traditional aCGH and would not be detected by gene-specific diagnostic DNA sequencing.