Composite karyotype designations for G-bands and FISH CN for satII/III and CKS1B
probes are provided in Table S1
. All patients showed complex karyotypes by G-banding, and 47 patients showed only whole-arm unbalanced jumping translocations (JT1q) and/or isochromosome 1q. Twenty patients showed both proximal duplications/triplications and/or JT1qs (#s 4, 5, 6, 12, 16, 18, 24, 26, 28, 30, 31, 39, 43, 49, 56, 60, 63, 64, 66, 67). FISH analysis showed one patient (1%) with two copies of CKS1B
, thirty-nine patients (58%) showed 3-4, 16 patients (23%) with 5-6, and 11 (16%) with a CN of 7-9 (#s 3,5,26,28,39,49,58,60,64,65,66) (Table S1
). Six patients (#s 26,28,57,63,64, 66) were of particular interest because they demonstrated both a higher CN and the pattern of two red (sat II/II) signals flanking two green (CKS1B
) signals, indicating an inverted duplication (). Five of these six patients also showed multiple copies of inverted duplications, suggesting that the possible origin of the inverted duplications were BFB cycles. In an effort to further characterize the origin for the focal amplifications of 1q12~23 in these six patients, we expanded the analysis by increasing the number of cells analyzed by FISH from 20 to 200 cells, and utilized additional BAC FISH probes for the proximal 1q, including RP11-550P17 (1q22), RP11-375F2 (1q24.2),RP11-177M16 (1q25.1), RP11-57D16 (1q25.2) ().
Modal chromosome number and copy number for FISH BAC probes in 1q12~23 region.
Strikingly, after expanded analysis, in three of the six patients (#s 66, 64, 28) we identified similar types of unstable intermediates demonstrating ladder-like structures of inverted duplications (-). The ladders showed equal-spaced organization of the region containing copies of probes for CKS1B
bracketed by copies of satII/III on each end. For example, Patient # 66 shows expansion of CN of CKS1B
from four, to eight, to 16, and finally 18 copies in an inverted duplication pattern on the same chromosome arm (). In this patient the initiating breakpoint for the BFB cycles was localized between probes for CKS1B
(1q21) and RP11-550P17 (1q22), apparently resulting from an unbalanced translocation of 1q with chromosome 15. Interestingly, the inverted duplication in this patient consistently showed doublets of CKS1B
, a pattern that suggests the initiation breakpoint for the BFB cycles is in close proximity to CKS1B
. This type of doubling of CN for a gene under selection has been demonstrated previously in rodent cell lines and indicates that the doubling occurs as a result of a very short distance between the initiation breakpoint and the gene under selection (Coquelle et al, 1997
). Moreover, the chromosomes 1 in this case also demonstrated isodicentric intermediates with eight copies of CKS1B
(), and the expected next step of eight copies with the subsequent deletion of 1p (). When this chromosome underwent another cycle it produced unstable isodicentrics with 16 copies of CKS1B
() corresponding to the expected intermediates in the expansion of the amplicon by BFB cycles. The breakpoints between the centromeres of the isodicentrics occurred in or just adjacent to the duplicated pericentromeric regions. In some metaphase chromosomes the sister chromatids show separation except at the fusion of the distal ends of the chromatids involving the satII/III sequences; this is the only point of attachment between the chromatids other than at the centromere ().
Figure 1 Representative metaphase chromosomes demonstrating key structures in the progression of intrachromosomal amplification of 1q12~23. Probes for satII/III (red) and CKS1B (green) are shown on inverted DAPI images of chromosomes depicting G-band-like patterns. (more ...)
Figure 3 (A) Patient # 28 demonstrated pericentromeric decondensation in both chromosomes 1, each with a single copy of CKS1B. The chromosome 1 on the left has a normal 1p, while chromosome1 on right has a visible interstitial deletion of 1p. (B) An inverted duplication (more ...)
Patients # 64 () and # 28 () demonstrated that BFB cycles can occur secondary to an existing inverted duplication on 1q. Chromatid misalignments and unequal sister chromatid exchanges are known to result in direct duplications or, alternatively, a reverse sister chromatid exchange which can produce a large symmetric or asymmetric inverted duplication (Ma et al, 1993
). In these cases the chromosome 1 shows three copies of CKS1B
and two copies of satII/III resulting from the inversion (). As the progression begins, breakage in the distal satII/III signal results in a jumping translocation of the 1q to a non-homologous chromosome () or, alternatively, the loss of the whole 1q as a micronucleus. In Patient # 64 the truncated inverted dup of 1q undergoes SCF at the distal satII/III signal (), resulting in an unstable intermediate isodicentric with two copies of 1p and four copies of CKS1B
(). The FISH confirmation of isodicentric structure of these unstable intermediates was demonstrated by the rehybridization of this same isodicentric chromosome (). This chromosome demonstrated four copies of CKS1B
and three copies of satII/III (on the left), while on the same chromosome (on right) rehybridized with probes for the centromere (alpha sat in red and RP11-153J19 for 1p12 in green) confirms the presence of two centromeric regions and two copies of 1p. Finally, the loss of one of the two copies of the 1p occurs leaving the two copies of the amplicon associated with a single copy of 1p ().
Figure 2 Representative examples of chromosomes 1 in Patient # 64. (A) Chromosome 1 with an inverted duplication of 1q12~23 and whole 1q distal to the duplication. This chromosome 1 shows three copies of CKS1B, two inside the inverted duplication and one in the (more ...)
The third patient (#28) showing the amplicon ladders demonstrated decondensation of 1q12 pericentromeric heterochromatin in both copies of chromosome 1, each showing only a single copy of CKS1B (). A subclone was identified with an inverted duplication in the proximal region of the chromosome 1 which showed the normal 1p (with breakpoints in the inversion between F2 and M16). This inverted duplication resulted in three copies of CKS1B () two of which were bracketed by decondensed regions of satII/III sequences. The distal satII/III sequences demonstrated decondensation (), resulting in breakage in the most distal satII/III sequences () and the formation of a truncated 1q () similar to those seen in Patients #66 and #64 ( and , respectively). The fusion of sister chromatids at the distal satII/III and progression through further cells cycles ultimately produced amplified chromosomes () similar to chromosomes 1 in Patients #66 and #64 ( and , respectively).
The amplicon ladders in Patients # 66, 64 and 28 were transient intermediates which subsequently underwent unbalanced translocations to non-homologous chromosomes or were lost as acentric fragments of amplicons (). Unbalanced translocations between the most distal satII/III sequences and non-homologous chromosomes were documented in two of the patients (#s 66, 64) (). In Patient # 66, a non-homologous chromosome fused with the donor chromosome 1 at the distal pericentromeric region producing an unstable fusion dicentric (). This unstable intermediate chromosome demonstrated the predicted breakage between the centromeres, leaving a shortened amplicon ladder (two copies) on the donor chromosome 1 and a jumping segmental translocation (two copies) on the recipient chromosome (). This same translocation process was seen in Patient #64 as the distal 1q fused with chromosome 21p () creating the unstable fusion dicentric and ultimately breaking between the centromeres translocating an amplicon to 21p (). These unbalanced translocations are the type predicted to occur in both the telomere fusion and CFS models for gene amplification. Further evidence for the breakage-prone nature of satII/III sequences is the presence of acentric amplicon segments found in some cells (not shown). These segments presumably occurred by simultaneous breakage in two different pericentromeric junctions, thus yielding unstable acentric amplicon fragments. These acentric fragments would be subsequently lost as micronuclei with high CNs of CKS1B ().
Figure 4 Secondary aberrations including unbalanced translocations and/or the formation of micronuclei subsequently results in the masking of amplicon ladders. Translocation of intact copies of amplicons to non-homologous chromosomes and loss of amplicons by micronucleus (more ...)
Taken together the findings in this study showed a recurring pattern of 1q12~23 amplification (, , ). Importantly, two types of initiating breaks for the BFB amplification were demonstrated. In the classic model for BFB cycles, a DSB occurs in a region distal to the gene under selection, as in Patient # 66. However, in the other two patients (#s 64 and 28) the initiating DSB occurred in the distal copy of the 1q12 heterochromatin of an existing inverted duplication. Breakage in 1q12 pericentromeric heterochromatin has not been reported in the initiation of BFB cycles. Importantly, all three patients (#s 66, 64, 28) showed that the breakpoints within the pericentromeric heterochromatin ultimately set both the centromeric and telomeric boundaries of the 1q12~23 amplicon. The breakage and fusion occurring within the satII/III sequences accounts for the regular pattern within ladder-like expansions and provides evidence for a mechanism which mediates the progression of the BFB cycles.