Genomic disorders result from DNA rearrangements mediated by non-allelic homologous recombination (NAHR) between large, highly homologous flanking segmental duplications1
. The clinical features of many of the known genomic disorders include mental retardation and developmental delay; and several recent studies of individuals with mental retardation have led to the identification of novel recurrent genomic disorders2
. By whole-genome array CGH screening of 757 patients with mental retardation and/or congenital anomalies, we identified two unrelated individuals with mild to moderate MR, dysmorphic features, and abnormal EEG findings who both have identical de novo
1.5 Mb deletions of 15q13.3. These two deletions share the same distal breakpoint (BP5) with a previously reported 3.95 Mb deletion of 15q133
(, ), with a proximal breakpoint (BP4) within this larger deletion (, ). The shared 1.5 Mb region contains six known genes. Our array CGH screening also detected a single patient with a proximal deletion breakpoint corresponding to breakpoint region 3 (BP3) of the Prader-Willi and Angelman syndrome region and a distal breakpoint at BP4 (Patient 543/06, ). However, the deletion was also detected in the patient’s unaffected father. We therefore interpret this BP3-BP4 deletion as likely representing a benign copy number variation, although we cannot exclude that it may instead be a pathogenic deletion with incomplete penetrance.
Figure 1 High-resolution oligonucleotide array mapping of 15q12-q13.3 rearrangements (chr15:25,700,000–31,400,000). Although there appears to be variation in the exact location of breakpoints, all map to large blocks of segmental duplication at BP3, BP4, (more ...)
Figure 2 Pedigrees and patient photographs of 15q13 deletions. Developmental delay and seizure phenotype is indicated by left- and right-half shaded symbols, respectively. Presence or absence of 15q13 deletions is shown below each symbol in all individuals tested (more ...)
In order to rapidly screen a large collection of affected individuals for deletions in the shared 1.5 Mb interval between BP4 and BP5, we developed two TaqMan quantitative PCR (qPCR) assays targeted to this region and screened 1040 individuals with mental retardation of unknown etiology. This cohort, obtained from the Greenwood Genetic Center (South Carolina), consists of an approximately equal number of individuals of Caucasian and African American descent. qPCR analyses identified four individuals as potentially harboring a deletion of the interval BP4-BP5. Samples were subsequently validated by BAC array comparative genomic hybridization (data not shown) and a custom oligonucleotide array (). Review of pedigrees showed evidence of multiple affected individuals for each case (, Supplementary Figure 1
), and review of the sample collection revealed that, although the series was thought to have only unrelated individuals, two of the patients we identified with deletions are mother (CMS7833) and son (CMS5803).
Review of the phenotypes observed in the nine individuals identified with deletions of 15q13 shows a number of phenotypic features (summarized in and , with full phenotype details in Supplementary Note 1
). The most consistent features among the patients in our series are mild to moderate mental retardation (9/9 cases) and epilepsy and/or abnormal EEG findings, which were noted in seven of the nine individuals. All patients also have mild facial dysmorphism, though these features are variable. Features shared among three or more patients include hypertelorism, upslanting palpebral fissures, prominent philtrum with full everted lips, and short and/or curved fifth finger and short fourth metacarpals. Related to the latter, it is noteworthy that skeletal and/or joint defects of the hand were noted in seven of nine patients (). Testing for the 15q13.3 deletion should therefore be considered in patients with unexplained mental retardation, seizures, and mild dysmorphic features.
Phenotypic features of nine patients with deletions of 15q13
Each breakpoint region corresponds to a complex set of segmental duplications, termed duplication blocks. BP4 is an 818 kb duplication block with three large regions of homology to BP5 (95 kb, 140 kb, and 218 kb with 99.6% identity, http://genome.ucsc.edu/
), each of which lie in an inverted orientation relative to those at BP5 (, Supplementary Table 1
and Supplementary Figure 2
). Consistent with NAHR as the mechanism underlying rearrangements of 15q13.3, our data localized the breakpoints of all five BP4-BP5 deletions and a BP4-BP5 duplication to these large paralogous sequences (, , Supplementary Figure 3
). The BP3 region, which is the common distal breakpoint in Prader-Willi/Angelman syndrome deletions, corresponds to an 843 kb duplication block (hr17/Build 35, chr15:26,053,472–26,896,735). Duplicated sequence within BP3 shows relatively small stretches of homology to BP4 (17 kb with 93% identity) and BP5 (30 kb with 98% identity and 11 kb with 93% identity). BP3 also contains two assembly gaps, and our data from IMR338 and 543/06 suggest that the proximal breakpoint in these deletions occur within the distal gap. Given the likelihood that NAHR underlies rearrangements of 15q13, we suggest that this region contain as-yet uncharacterized segmental duplications paralogous to those at BP4 and BP5 which catalyze recurrent rearrangements of 15q13. Thus, the increased prevalence of rearrangements observed between BP4-BP5 relative to those involving BP3-BP4/BP5 is consistent with the size and homology of duplicons in 15q13. It is interesting that most of the breakpoint regions associated with genomic disorders on chromosome 15 correspond to duplication blocks which harbor copies of the GOLGA
gene family4, 5
. Within the limits of oligonucleotide array comparative genomic hybridization, breakpoint regions appear to overlap with these specific duplicons (Supplementary Figure 4
Figure 3 Duplication architecture of 15q13 breakpoint regions. (a) Paralogy between large (≥10 kb), highly identical (≥95%) segmental duplications (blue bars) is shown between BP3, BP4 and BP5 as pairwise alignments (blue lines). Sequence assembly (more ...)
Figure 4 Duplications of (a) 15q13.1-q13.2 (BP3-BP4), and (b) 15q13.3 (BP4-BP5) identified in 2 of 960 normal control samples using the HumanHap300 Genotyping BeadChip (Illumina, San Diego, CA). Data shows probe position (x-axis) against smoothed LogR intensity (more ...)
Observations in several genomic disorders suggest that microdeletions arise preferentially from chromosomes carrying an inversion of that same region3, 6–9
. Given the opposing orientation of the duplicons at BP4 relative to BP5 in the reference assembly (Supplementary Figure 2
), we hypothesized that 15q13.3 might also be a site of inversion polymorphism which could create a configuration predisposed to recurrent rearrangement. In order to investigate this possibility, we utilized FISH to assay the BP4-BP5 region. Testing of eight HapMap individuals of varied ethnicities showed the presence of a common inversion of this interval, which was present on 7 of the 16 chromosomes assayed (frequency of inversion relative to reference assembly 0.44, 95% CI 0.19–0.68) (, Supplementary Table 2
). In the two cases where we were able to study the parent-of-origin of BP4-BP5 deletions (mother of Patient 69/06 and father of Patient 02961), we found both to be heterozygous for the inversion. Although the high frequency of this inversion in the normal population and the lack of sufficient numbers of parental samples mean that we are currently unable to verify this hypothesis, our observations are consistent with a model in which inversion polymorphism of the BP4-BP5 region results in these flanking duplicons being placed in direct orientation, creating a configuration predisposed to microdeletion/duplication by NAHR. These data strengthen the growing link between the occurrence of large inversion polymorphisms and genomic disorders (reviewed in Sharp et al.
Figure 5 Identification of a common inversion polymorphism in 15q13.3. To detect inversions of the BP4-BP5 microdeletion region, we performed FISH mapping using fosmid probes located proximal and distal to BP5. The separation of these probes in the reference assembly (more ...)
In order to assess the frequency of copy number changes between BP3, BP4, and BP5 in the general population, we screened 960 control individuals at this locus (n=960 unrelated Caucasians genotyped with a high density SNP Genotyping BeadChip11
for variable efficacy of statin drug response on cardiovascular disease [R. Krauss and D. Nickerson, personal communication]). We identified two individuals with duplications of 15q13: one individual with a duplication within the interval BP3-BP4, and a second with a duplication between BP4 and BP5 (). Within the limits of resolution of these data, the BP4-BP5 duplication appears to be reciprocal to the deletions shown in , providing further evidence that NAHR likely underlies rearrangements of 15q13. However, the BP3-BP4 duplication is smaller than the deletion identified in patient 543/06, with breakpoints located within unique sequence (i.e. not within the segmental duplications). We did not detect any large deletions of 15q13 in this control set. While some smaller regions of copy number polymorphism are present within 15q13 (mostly corresponding to the segmental duplication blocks at BP3, BP4, and BP5), deletions or duplications comparable to those that we describe have not been found in previous studies of a further 2002 control individuals12–19
. Together, these results suggest that deletions of BP4-BP5 are pathogenic (6/2082 probands with MR vs. 0/2962 controls, p=0.005, Fisher’s Exact test). In contrast, our limited data suggest that duplication events of BP3-BP4 and BP4-BP5 are likely either (i) benign copy number variants, or (ii) are frequently associated with milder phenotypic abnormalities, although additional studies are necessary to confirm this interpretation. Parental origin studies in the two de novo
cases showed paternal origin of the deletion in Patient 02961 and maternal origin in Patient 69/06. In addition, no imprinted genes have been identified to date within BP3-BP5 region (http://www.geneimprint.com/site/genes-by-species
), suggesting that imprinting is unlikely to significantly affect the phenotype of these patients (Supplementary Figure 5
Interestingly, seven of nine deletion carriers presented with seizures or abnormal EEG findings (). One of the genes within the critical region is CHRNA7
(cholinergic receptor, neuronal nicotinic, alpha polypeptide 7), a synaptic ion channel protein that mediates neuronal signal transmission. Linkage studies have suggested CHRNA7
as a susceptibility factor for both juvenile myoclonic epilepsy20
and benign epilepsy of childhood with centrotemporal spikes21
. Further, it has been reported that mice with a knockout of CHRNA7
show a hypersynchronous neocortical EEG phenotype22
. As a result, CHRNA7
represents an excellent candidate gene, haplo-insufficiency for which may underlie the epilepsy/seizure phenotype seen in the patients we describe. As copy-number variation of a region including CHRNA7
has also been observed in the general population13–16
, the possible involvement of this gene as a genetic risk factor for epilepsy warrants further investigation.
Although we present the first description of recurrent BP4-BP5 microdeletions, there are single reports of apparently similar deletions at BP3-BP4-BP5 (http://decipher.sanger.ac.uk/
). Several previous studies have also described a variety of different structural rearrangements involving 15q13, suggesting that this is a highly unstable genomic region. BAC array CGH studies of isodicentric(15q) chromosomes, marker(15) chromosomes, atypical deletions associated with Angelman syndrome, and deletions of 15q14 have suggested that the breakpoints of these rearrangements often map to the segmental duplication blocks at BP3, BP4, and BP523–26
. We therefore investigated a number of other rearrangements of chromosome 15 using high resolution oligonucleotide array CGH. Consistent with previous studies25
, data obtained in two unrelated inv dup(15) carriers were similar. Results showed that both inv dup(15) chromosomes are complex, being composed of two copies of the region 15cen-BP4 and a single copy of region BP4-BP5. This was subsequently confirmed by FISH (data not shown). Results in a patient with an Angelman syndrome class II deletion and in a second case with a mar(15) showed that in both cases the distal breakpoints mapped to the large segmental duplication block at BP3 (Supplementary Figure 6
Our screen of 2082 patients with idiopathic mental retardation identified six unrelated individuals with 15q13.3 deletions, suggesting that this microdeletion accounts for ~0.3% of mental retardation of unknown etiology. The 1040 individuals screened by qPCR are a subset of a larger population of patients who receive services through the South Carolina Department of Disabilities and Special Needs27
. We compared the frequency of the 15q13.3 deletion to that of other known microdeletions resulting in mental retardation in the larger cohort from which our population was obtained (10,997 patients of both European and African ancestry from South Carolina). Within the larger cohort, Prader-Willi syndrome was diagnosed in 0.22%, Angelman syndrome in 0.34%, and Williams syndrome in 0.31%27
. In comparison, we identified 15q13.3 deletions in 0.29% of the subgroup (3 unrelated probands of 1040 tested) and 0.29% of the European populations tested by array CGH (3 unrelated probands of 1042 tested), suggesting the frequency of this 15q13.3 microdeletion syndrome may be comparable to that of the disorders listed above (estimated frequency in MR patients 0.29%; 95% CI 0.06–0.52%). Two pedigrees were of African American descent with the remainder of Caucasian origin, indicating this syndrome is found in patients of different ethnic backgrounds. Given that this microdeletion is well below the resolution of conventional cytogenetics—it could only be detected using techniques such as array CGH—we anticipate that the increasing resolution of these studies will lead to the future identification of additional microdeletion syndromes. Assuming a prevalence of moderate mental retardation in the general population of ~0.8%28
, we estimate an approximate population incidence for this 15q13.3 microdeletion of 1/40,000. Given this relatively high frequency and the multiethnic distribution, we recommend testing for this disorder in individuals with features similar to the patients presented here.