Triplications embedded in duplications spanning MECP2
We previously identified six complex rearrangements (triplications embedded within duplications) in a cohort of 30 patients with MECP2
duplication by high-resolution human genome analysis using customized high-density array CGH9
. An additional four subjects with a complex DUP-TRP-DUP pattern were identified by array CGH suggesting that complex rearrangements are a relatively frequently observed outcome of genomic alterations at this locus. We systematically investigated these complex rearrangements to characterize the molecular features of the rearrangement product. In total, we studied nine patients with triplications embedded in duplications; in five cases the MECP2
gene is included within the triplicated segment ().
Individuals carrying complex triplications of Xq28
Both triplication and duplication sizes are unique in each family and ranged from 41 kb to 537 kb and from 444 kb to 5.7 Mb, respectively. The triplicated region includes the entire MECP2 gene in five patients: BAB2797, BAB2801, BAB2805, BAB3053 and BAB3114 (). Oligonucleotide-array CGH revealed that all of the complex rearrangements were inherited from a carrier mother, except for BAB3053 who harbors a translocation to Yq11.22. The breakpoint at Yq11.22 was not precisely mapped due to the paucity of unique sequences on the Y chromosome.
We independently confirmed genomic triplications in each of the nine families by both Multiplex Ligation-Dependent Probe Amplification (MLPA) and Fluorescence in situ
Hybridization (FISH) (Supplementary Fig. 1
and data not shown). The mothers and grandmothers when available for study were tested by both CGH and MLPA and were shown to carry the same complex rearrangement as their sons or grandsons in all but one family (pedigree HOU1217, ), in which aCGH studies revealed that the complex rearrangement was a de novo
event in the mother (BAB3115) (). X-chromosome inactivation (XCI) studies revealed 100% advantageously skewed XCI patterns in all carrier females tested (data not shown); i.e. consistent with preferential inactivation of the X chromosome harboring the complex rearrangement. Family pedigrees are displayed in Supplementary Fig. 2
Triplicated segments are inserted in inverted orientation amid the duplications
Complex rearrangements can be defined by multiple breakpoint junctions or join points that juxtapose discreet genomic segments. The genomic rearrangement complexity was revealed by aCGH; however, aCGH provides neither orientation nor genomic positional information for the complex rearrangement but rather only copy number information. Based on aCGH results that demonstrated distinct transitions at gains of genomic intervals (i.e. duplication versus normal, triplication versus duplication), we initially hypothesized the existence of at least four potential breakpoints per patient; two for transitions to and from duplications (proximal and distal) and two for transitions to and from triplications (proximal and distal) ( and ). However, the simplest hypothesis is that each of the two duplication/triplication breakpoints was joined during rearrangement formation, ultimately producing only two breakpoint junctions, designated breakpoint junction 1, jct1 and breakpoint junction 2, jct2 ().
To test this latter hypothesis, we first sought to obtain breakpoint junctions using both conventional and long-range PCR and by attempting to use primer pairs in all possible orientations; i.e. inwardly-facing, outwardly-facing, forward primer pairs, and reverse primer pairs. These primers were designed at the apparent boundaries, as denoted by transitions signifying a gain of each duplicated or triplicated segment relative to the reference genome as inferred from the aCGH results. In cases of failure to obtain breakpoint junctions using this assay, alternative experimental approaches were attempted. These alternative approaches included inverse PCR (iPCR) or Southern analyses, both of which have the advantage of not relying on any preconceived notion of genome structure for the rearrangement.
Southern blotting was used to analyze the recurrent breakpoint junctions mapping to the inverted repeat pair of low copy repeats (LCRs) K, which is involved in six our of eight independent complex rearrangements in our cohort (). This assay was performed as described previously10
; for males, the expected result was either a 30.7 kb band corresponding to a reference size structural variation haplotype (H1) or an 18.2 kb band corresponding to a polymorphic inversion of the region flanked by the LCR K1 and the LCR K2 that is present in 18% of the population of European-descent10
(H2) (). Females could potentially carry either one allele (the 30.7 kb or 18.2 kb) in the homozygous state or both alleles as heterozygotes (NA15510, ). To our surprise, all male samples carrying dup/trip involving the LCR K1 and the LCR K2 (BAB2772, BAB2796/BAB2980, BAB2797, BAB2801, BAB2805 and BAB3114) yielded the same pattern consisting of two bands, 18.2 kb and 30.7 kb, corresponding to those usually observed with the H2 and H1 inversion haplotype structures, respectively. We surmised that the unexpected presence of both bands in all male patients was a result of rearrangement formation which suggests that all seven samples have a common jct1
structure. In addition, an 18.2 kb band is expected if the centromeric-flanking region (which contains the TKTL1
gene) is duplicated and inverted whilst still flanking the LCR K1 and the LCR K2 on either the reference (H1dup) or the inverted structure (H2dup) on the ancestral chromosome (). Therefore, we hypothesized that the 18.2 kb band corresponds to jct1
and, by inference that the 30.7 kb band corresponds to the ancestral state (H1 structure) in these chromosomes. We confirmed this hypothesis using the haplotype data obtained from SNP arrays; all patients in our cohort carry the SNP haplotype associated with the H1 structure (Supplementary Fig. 5
Southern blot analysis of the region flanked by LCRs K1 and K2 at the Xq28 chromosome
Three important conclusions can be drawn from these experimental observations: 1) the inverted LCRs K1 and K2 likely mediated the rearrangements; 2) the new segment copied (containing the TKTL1 gene) was inserted in an inverted orientation with respect to the original copy; and 3) a second event, likely represented by jct2, must have occurred in order to “reverse” the inversion process. Supporting our experimental observations, a de novo complex rearrangement occurring in association with sporadic disease in family HOU1217 revealed a novel formation of the 18.2 kb band in addition to the 30.7 kb band already present in all family members. As anticipated, BAB2769 has a 30.7 kb band size corresponding to the reference structural H1 haplotype but no 18.2 kb band, consistent with the fact that BAB2769 is the only sample for which the complex rearrangement does not include the LCRs K1 and K2.
Jct1 for patient BAB2769 was obtained by sequencing across the junction using reverse primer pairs positioned at the proximal ends of the duplication and triplication, respectively. Remarkably, the junction consists of two identical 149 bp segments, present as two small inverted repeats (856 bp) located 317.8 kb apart from each other in the haploid reference human genome sequence (). These inverted repeats are 98% identical in sequence. Thus, in all seven cases in which jct1 were identified, an inverted repeat was located at the breakpoint junction.
Rearrangement structure for patients BAB2769, BAB2772, BAB2796/BAB2980, BAB2797 and BAB2805 based on aCGH, Southern blotting and breakpoint sequencing
Jct2 in five out of eight rearrangements (BAB2769, BAB2772, BAB2796/BAB2980, BAB2797 and BAB2805) was obtained by PCR (regular, long-range or inverted PCR). For patients BAB2772, BAB2796/BAB2980, BAB2797 and BAB2805, the breakpoints were obtained using reverse primers at the proximal ends of the duplication and the triplication, respectively. Jct2 in patient BAB2769 was obtained using forward primer pairs designed at the distal ends of the duplication and triplication, respectively. Routine PCR was attempted first followed by sequencing of the PCR products. One junction was obtained by iPCR (BAB2805); three samples (BAB2801, BAB3053 and BAB3114) were refractory to all attempts to amplify a unique breakpoint junction.
Analysis of the breakpoint sequences of jct2
revealed that the triplicated segment is inverted relative to the duplicated segment in all patients (BAB2769, BAB2772, BAB2796/BAB2980, BAB2797 and BAB2805). Microhomologies of 2 to 4 nucleotides were observed in two out of five cases (BAB2772 and BAB2769, ); in two cases, one nucleotide A
or two nucleotides AA
were inserted at the junction (BAB2796/BAB2980 and BAB2797); in one case (BAB2805), the junction was perfectly joined. In all five cases, one of the breakpoints occurred within or adjacent to a repetitive sequence element such as a SINE or a LINE (). A few nucleotide dissimilarities flanking the junctions were observed in two cases (BAB2772: transversion C=>G; BAB2805: deletion of one G, ); we interpret these dissimilarities to be likely population polymorphisms that are not yet deposited in the dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/
). Alternatively, there is evidence that the polymerase(s) involved in break-induced-replication (BIR) are ‘error prone’, with poor processivity2
at initiation followed by lower replication fidelity compared to normal DNA replication11
. The jct2
for subject BAB2769 reveals a break that we interpret as two template-switching events. The first event is represented by a GC
microhomology that connects the distal duplication breakpoint to the distal triplication breakpoint; the second event is represented by a microhomology of CAGC
accompanying a deletion of 23 bp on the distal triplication side ().
Presence of inverted repeats at the breakpoint junctions of genomic triplications observed in the present study and in the literature
In summary, analysis of two breakpoint junctions (jct1
) from each of five unrelated patients with triplications embedded within duplications at the Xq28 chromosome reveals a common structure in that the triplication was inserted in an inverted orientation within the duplication (i.e. DUP-TRP/INV-DUP). FISH experiments in patient BAB2805 reveal a pattern consistent with this DUP-TRP/INV-DUP genomic structure (Supplementary Fig. 6
). Furthermore, in all cases one of the junctions of the rearrangement involved an inverted repeat pair, with the inverted genomic segments either closely approximated (38 kb) or separated by a sizable distance (>300 kb). These shared genomic architectural features are observed at breakpoints of all complex duplication/triplication alterations at the MECP2
locus analyzed herein.
Phenotypic consequences of DUP-TRP/INV-DUP
The complex DUP-TRP/INV-DUP products vary in size for both triplicated and duplicated intervals (). In the case of complex Xq28 genomic rearrangements, the MECP2
gene was either duplicated or triplicated. This distinction provided a unique opportunity to assess the phenotypic consequences of incremental increases in MECP2
gene dosage. The MECP2
gene was entirely mapped within the triplicated genomic interval in five patients with the complex DUP-TRP/INV-DUP rearrangement. Similar to observations in a previous case report8
and observations in patients without precise breakpoint junction mapping13
, the phenotype associated with MECP2
triplication was more severe than that observed for MECP2
The most salient clinical findings are summarized in Supplementary Table 3
(for complete clinical descriptions, please see Supplementary Note
). Note that early respiratory insufficiency with an oxygen or ventilation requirement, early dysphagia and requirement for a feeding tube, hearing loss, and minor cardiac defects are much more commonly observed with MECP2
triplication (100%) compared with MECP2
duplication (0% to 25%), a robust observation even when compared to the collective published data on boys with MECP2
. Moreover, polyhydramnios and intestinal pseudoobstruction were observed clinically only in subjects with triplication. Interestingly, patients BAB2805 and BAB3114 were reported to have Xq28/MECP2
duplications by the diagnostic laboratories that performed their clinical chromosome microarray analysis. We correctly anticipated that the MECP2
gene was triplicated in patients BAB2805 and BAB3114 based on the observed clinical phenotype. Routine clinical diagnostic testing correctly identified Xq28/MECP2
triplication in the remaining three children with MECP2