Because its detection rate for pathogenic genomic imbalance is much higher than that of conventional cytogenetic analysis, a consensus has developed that AGH should be used clinically for the evaluation of patients with ID and other birth defects [31
]. It is clear that AGH using "targeted" arrays that only include probes for genomic regions known to be involved in microdeletion or microduplication syndromes has substantially lower detection rates for CNVs that cause ID than AGH using arrays that provide genome-wide coverage [37
]. Beyond this, however, there is no agreement regarding the kind of array, the distribution of probes across the genome, or the resolution that is most appropriate for clinical use. Although BAC arrays were initially used, most clinical laboratories now prefer oligonucleotide arrays because high-quality platforms that produce consistent results are reliably available from commercial sources. In addition, the use of larger numbers of smaller probes on oligonucleotide arrays permits more precise delineation of the breakpoints of CNVs that are detected, which facilitates genotype-phenotype correlation and clinical interpretation. AGH with a SNP array provides the additional advantage of generating genotypes that can be used to verify family relationships and find uniparental disomy as well as a second method (in addition to hybridization intensity) for identifying genomic imbalance [10
We previously reported that 100 K Affymetrix GeneChip®
AGH is a robust platform for the detection of pathogenic CNVs in patients with ID [10
]. Here we show that the detection rate of CNVs among such patients is higher with 500 K GeneChip®
AGH than with 100 K GeneChip®
AGH. We made about four times as many CNV calls overall with the 500 K platform as with the 100 K platform when using the same method of bioinformatic analysis in 54 trios studied with both technologies (Table ). We also found 18 instances of pathogenic genomic imbalance in 16 of 100 children with ID and normal cytogenetic analysis studied by 500 K GeneChip®
AGH (Table ), compared to 11 instances of pathogenic genomic imbalance in 11 of 100 similarly-ascertained children tested by 100 K GeneChip®
AGH in our previous study. Although the higher detection rate we observed with the 500 K platform may have occurred by chance because we just happened to include a few more patients with such genomic changes in the new cohort than in the 100 K cohort, we also found two additional de novo
CNVs by 500 K GeneChip®
AGH among the 44 children whose 100 K AGH was interpreted as normal in our earlier studies (Figure and Table ).
We detected three apparently de novo
CNVs smaller than 200 kb among the 100 trios tested with 500 K GeneChip®
AGH in the new cohort (a 107 kb deletion of chromosome 14 in Patient 818, a 186 kb deletion of chromosome 21 in Patient 8619, and an 89 kb deletion of chromosome 6 in Patient 216 that was actually a homozygous loss inherited from two heterozygous parents), but none of these CNVs was clearly pathogenic. The overall size distribution of pathogenic CNVs detected by 500 K GeneChip®
AGH among 154 children with idiopathic ID in the present study is similar to that observed by 100 K GeneChip®
AGH among 100 children with idiopathic ID whom we studied previously [10
] (see Additional File 2
: Supplemental Figure S1). The higher detection rate on the 500 K array therefore appears to be related more to better probe coverage in relevant genomic regions and an improved ability to distinguish CNVs from background noise, rather than to a capacity to identify much smaller pathogenic CNVs. This is illustrated in Families 3890 and 4840 (Figure ), in which a 1.6 Mb duplication of 8q23.2q23.3 and a 1.5 Mb deletion of 4p16.3, respectively, are obvious on the 500 K AGH but were not called on the 100 K analysis. In retrospect, the 4p16.3 deletion in Patient 4840 can be seen on the 100 K AGH copy number plot despite the noisy data, but it was not called by either the automated analysis or visual inspection of these plots when the initial study was done. Our failure to detect the de novo
duplication of 8q23.2q23.3 in Patient 3890 was probably caused by the noisy data in the father's study.
Distinguishing benign CNVs from those that cause ID and other birth defects is a critical issue in routine clinical use of AGH. Benign CNVs occur in all people and are a major source of genetic variation in the normal population [21
]. Most apparently benign CNVs over 2 kb in size occur as polymorphisms with minor allele frequencies of at least 5% [21
] and are inherited from a parent [21
Benign and pathogenic CNVs can usually be distinguished in patients with ID and other birth defects by inheritance and genotype-phenotype correlation [5
]. In this study, we identified a mean of about 10 CNVs per subject in the 154 ID trios tested by 500 K AGH. The vast majority of these CNVs were characterized as benign because they were inherited from a normal parent. Genomic imbalance that occurs de novo
in a patient with ID whose parents are normal is more likely to be pathogenic than genomic imbalance that was inherited unchanged from a normal parent. We performed AGH on both parents of every child with ID to determine the inheritance of the CNVs found in the child, but this is sometimes not possible in clinical practice. In such instances it is necessary to infer likely de novo
occurrence by information obtained from populations that have previously been studied [19
]. Great care must be taken to avoid misinterpretation when this is done, especially if the available data were obtained with lower resolution AGH, the phenotypic characteristics of the comparison population are uncertain, reported polymorphic CNVs do not have exactly the same breakpoints as the CNV of interest, or the population frequency of a previously-reported CNV is unknown.
Compelling evidence that a CNV in a person with ID is pathogenic exists if the genomic imbalance is known to cause the patient's phenotype in other individuals, e.g., if a child with del 9q34.3 has features of the 9q subtelomeric deletion syndrome (Patient 523), a child with del 1p36.32p36.33 has features of the 1p36 deletion syndrome (Patient 9133), or a child with del 17q21.31 has features of the syndrome associated with this deletion (Patient 2106). Pathogenicity is also supported when a CNV includes a gene that is known to cause the patient's phenotype when inactivated (if the CNV is a deletion) or over-expressed (if the CNV is a duplication). On the other hand, a CNV is unlikely to be pathogenic if it involves a highly polymorphic region in which genomic loss (or gain, whichever is present in the patient) of the entire involved segment is known to occur in normal people.
If a direct genotype-phenotype correlation of this kind cannot be made in a particular case, certain genetic features of the CNV may provide clues to its pathogenicity. CNVs that are larger and those that involve gene-rich regions are more likely to be pathogenic than CNVs that are smaller and involve only gene-poor regions [5
]. In addition, clinical experience suggests that deletions are more likely to be pathogenic than duplications [47
]. The genetic content of a CNV may also make pathogenicity more or less likely. For example, involvement of a gene that lies within a pathway that is known to contain other dosage-sensitive genes associated with a similar phenotype strengthens the possibility of pathogenicity, while a CNV that does not contain any genes that are expressed in relevant tissues during embryogenesis is unlikely to be pathogenic for ID.
There are, of course, exceptions to each of these "rules". Some benign CNVs arise de novo
], as appears to have occurred in the de novo
deletion of chromosome 14q11.2 we found in Patient 818. The 107 kb region involved is highly polymorphic and contains several T-cell receptor variable region genes. On the other hand, some CNVs that are inherited from a normal parent are pathogenic for ID. Examples include maternal transmission of a UBE3A
deletion to a child with Angelman syndrome [51
], maternal transmission of a MECP2
duplication to a son [52
], and CNVs such as dup 22q11.2 [53
] or del 1q21.2 [55
] that can cause ID but exhibit incomplete penetrance.
Although large (> 250 kb) CNVs are often pathogenic, they may be benign [19
]. Most benign CNVs are small (< 250 kb) [19
], and it seems probable that the smaller a CNV, the more likely it is to be benign. Nevertheless, no clear size distinction exists between benign and pathogenic CNVs. We found pathogenic CNVs as small as the 298 kb deletion of 9q34.3 in Patient 523 in this study, and others have reported even smaller pathogenic CNVs [13
]. Expression patterns, functional annotation and animal models can provide important clues to pathogenicity in some cases, but without knowledge of the phenotypic effects of a copy number alteration in humans, one can rarely, if ever, be certain whether a novel gain or loss of a particular genomic region can produce ID or other birth defects.
In our 500 K AGH study of 154 ID trios, 58 de novo CNVs were called by bioinformatic analysis, and 33 of these CNVs were confirmed and shown to be de novo by an independent method. Because we could assess the phenotypes of our patients in detail and correlate the findings with those obtained by AGH, we were able to determine with confidence whether the genomic imbalance we observed was pathogenic or not in every case studied except three (Tables and ). Such genotype-phenotype correlation is critical to determining the effects of novel CNVs detected by AGH in patients with ID.
A CNV of uncertain clinical significance was encountered in three (1.9%) of the 154 trios analyzed in this series - a 362 kb duplication of 22q11.21 in Patient 9979 (Table ), a 186 kb deletion of 21q22.11 in Patient 8619 (Table ) and a 1.6 Mb duplication of chromosome 8q23.2q23.3 in Patient 3890 (Table ). This rate of CNVs of uncertain clinical significance is similar to that reported in large series of patients with ID and other birth defects studied by AGH with "targeted" chips [31
We were uncertain of the clinical significance of either case of UPD that we detected. Although only a few liveborn children with UPD 16 have been recognized, the reported experience does not suggest that UPD 16 can cause the abnormalities observed in Patient 1658 [60
]. Paternal UPD 11p15 can produce Beckwith-Wiedemann syndrome [62
], but this phenotype is very different from that observed in the affected child in Family 6904. However, as both of these cases involved isodisomy of a portion of the chromosome, we cannot rule out the possibility that the abnormal phenotype was produced by homozygosity for a recessive mutant allele [63
]. Although the detection of UPD in addition to alterations of copy number is a theoretical benefit of using an array that includes probes for SNPs, the clinical utility of genome-wide screening for UPD in patients with idiopathic ID and other birth defects is uncertain.
We detected more pathogenic CNVs with 500 K AGH than with 100 K AGH, but some CNVs that were present among our patients were not detected using the 500 K assay. For example, our 500 K GeneChip®
analysis failed to identify a pathogenic 83 kb deletion of chromosome 16p13.3 (3,862,993 bp to 3,945,522 bp) involving the CREBBP
gene (Patient 5121). This de novo
deletion was found by AGH on the Agilent®
244 K platform and was confirmed by MLPA. The patient is an 8 year-old boy whose clinical features are characteristic of the Rubinstein-Taybi syndrome, which has been associated with deletions and other mutations of CREBBP
in other patients [65
]. The 83 kb genomic region deleted in our patient is poorly represented on the 500 K GeneChip®
arrays, with a total of only 15 SNPs. SNP arrays have uneven genomic coverage, and the addition of non-polymorphic oligonucleotide probes to the design of arrays like the one used in this study has been shown to provide substantially better detection of CNVs [21