AT-rich palindromes mediate the constitutional t(11;22) translocation

Abstract

The constitutional t(11;22) translocation is the only known recurrent non-Robertsonian translocation in humans. Offspring are susceptible to der(22) syndrome, a severe congenital anomaly disorder caused by 3&rcolon;1 meiotic nondisjunction events. We previously localized the t(11;22) translocation breakpoint to a region on 22q11 within a low-copy repeat termed "LCR22" and within an AT-rich repeat on 11q23. The LCR22s are implicated in mediating different rearrangements on 22q11, leading to velocardiofacial syndrome/DiGeorge syndrome and cat-eye syndrome by homologous recombination mechanisms. The LCR22s contain AT-rich repetitive sequences, suggesting that such repeats may mediate the t(11;22) translocation. To determine the molecular basis of the translocation, we cloned and sequenced the t(11;22) breakpoint in the derivative 11 and 22 chromosomes in 13 unrelated carriers, including two de novo cases and der(22) syndrome offspring. We found that, in all cases examined, the reciprocal exchange occurred between similar AT-rich repeats on both chromosomes 11q23 and 22q11. To understand the mechanism, we examined the sequence of the breakpoint intervals in the derivative chromosomes and compared this with the deduced normal chromosomal sequence. A palindromic AT-rich sequence with a near-perfect hairpin could form, by intrastrand base-pairing, on the parental chromosomes. The sequence of the breakpoint junction in both derivatives indicates that the exchange events occurred at the center of symmetry of the palindromes, and this resulted in small, overlapping staggered deletions in this region among the different carriers. On the basis of previous studies performed in diverse organisms, we hypothesize that double-strand breaks may occur in the center of the palindrome, the tip of the putative hairpin, leading to illegitimate recombination events between similar AT-rich sequences on chromosomes 11 and 22, resulting in deletions and loss of the palindrome, which then could stabilize the DNA structure.

Figure 1

Definition of the t(11;22) breakpoint interval by PCR analysis. PCR amplification was performed on the separated normal and derivative chromosomes 11 and 22 in somatic hybrid cell lines from two unrelated carriers, BM114 and GM06229b. Five PCR markers—three from chromosome 11q23, D11S1340, APOC3, and b1030-11, and two from chromosome 22q11, D22S941 and D22S264—were amplified from their respective normal chromosomes. A PCR product of the correct size was detected in normal human genomic DNA, and no PCR product was detected using a hamster DNA template (data not shown) or in the absence of any DNA template (H₂O). A DNA ladder was used as a size standard.

Figure 2

Human genomic Southern blot hybridization analysis. A, Partial restriction map of the 11q23 breakpoint interval highlighting the SacI and HindIII restriction endonuclease sites and the two probes—b1030-4, to detect the der(11) chromosome, and b1030-6, to detect the der(22) chromosome. The AT-rich repeat is boxed. B, Genomic DNA from the BM1 14 lymphoblastoid cell line and resulting hamster-human somatic cell hybrid cell lines were probed with the radiolabeled b1030-4 PCR product. C, DNA was purified from the t(11;22) carriers shown and probed with b1030-4. D, DNA was purified from eight unaffected individuals and the der(11) chromosome from BM114 and probed with b1030-4. E, DNA was isolated from carriers and der(22) offspring and probed with b1030-6.

Figure 3

PCR strategy for cloning the der(11) and der(22) junction fragments. The positions of the outer and nested PCR primers in the sequence of the normal chromosome 22 (gray) and normal chromosome 11 (black) sequence are shown. The nested PCR strategy used to amplify the derivative chromosomes is depicted within the schematic of the derivative chromosomes. To amplify the der(22) junction fragment, outer primers AT(26)F3 and b1030-6R were used for the first-round PCR. The PCR product was diluted 50-fold and was used in a nested PCR reaction with primers AT(26)F1a.2 from chromosome 22 and b1030-6Frev from chromosome 11. For the (der)11 junction fragment, primers b1030-5F and LCR22-1R, were used for the first-round PCR, and primers b1030-9F and AT(26)R-1, were used for the second-round, or nested, PCR.

Figure 4

Alignment of sequence from derivative 11 and 22 chromosomes. The PCR strategy outlined in figure 3 was used to amplify der(11) and der(22) junction fragments, ∼800 bp in size, from 13 unrelated carriers of the (11;22) translocation. Two of the carriers, BM737 and DD0185, represent de novo cases. Sequence from positions 1–533, for the der(11) chromosome (4A), and sequence from positions 1–487, for the der(22) chromosome (4B), in the carrier with the least sequence loss is shown for all individuals and was aligned using the Clustal W program. The der(22) offspring and their carrier patients had identical sequence, in both derivative chromosomes, and the results were not shown, except for GM00084a, because it was an isolated case and the parents were not available. Regions of nucleotide alterations are highlighted (white background) and regions of deletions are denoted by dashes. The bold bar at positions 225–232 in the der(11) chromosome and positions 252–259 in the der(22) chromosome depict the positions of the regions of strand exchange proposed elsewhere (Kurahashi et al. 2000a). The bracketed bold bar at positions 373–395 in the der(11) chromosome and positions 220–240 in the der(22) chromosome depict the intervals hypothesized to be the sites of chromosome breakage and strand exchange in the carriers. The hatched bar at positions 385–389 in the der(11) chromosome and the arrow at position 225 in the der(22) chromosome depict the positions of the balanced t(17;22) breakpoint junction (Kehrer-Sawatski et al. 1997). The stars at position 343, in the der(11) chromosome, and position 159, in the der(22) chromosome, represent sequence polymorphisms.

Figure 4

Alignment of sequence from derivative 11 and 22 chromosomes. The PCR strategy outlined in figure 3 was used to amplify der(11) and der(22) junction fragments, ∼800 bp in size, from 13 unrelated carriers of the (11;22) translocation. Two of the carriers, BM737 and DD0185, represent de novo cases. Sequence from positions 1–533, for the der(11) chromosome (4A), and sequence from positions 1–487, for the der(22) chromosome (4B), in the carrier with the least sequence loss is shown for all individuals and was aligned using the Clustal W program. The der(22) offspring and their carrier patients had identical sequence, in both derivative chromosomes, and the results were not shown, except for GM00084a, because it was an isolated case and the parents were not available. Regions of nucleotide alterations are highlighted (white background) and regions of deletions are denoted by dashes. The bold bar at positions 225–232 in the der(11) chromosome and positions 252–259 in the der(22) chromosome depict the positions of the regions of strand exchange proposed elsewhere (Kurahashi et al. 2000a). The bracketed bold bar at positions 373–395 in the der(11) chromosome and positions 220–240 in the der(22) chromosome depict the intervals hypothesized to be the sites of chromosome breakage and strand exchange in the carriers. The hatched bar at positions 385–389 in the der(11) chromosome and the arrow at position 225 in the der(22) chromosome depict the positions of the balanced t(17;22) breakpoint junction (Kehrer-Sawatski et al. 1997). The stars at position 343, in the der(11) chromosome, and position 159, in the der(22) chromosome, represent sequence polymorphisms.

Figure 5

Schematic representation of the chromosome 17, 11, and 22 intervals. A, The relative orientation of palindromic sequence on the three chromosomes in the vicinity of the chromosome breakpoint junction is depicted by the arrows. The wild-type chromosome 17 has a palindromic sequence of 71 bp (dark green) on either side of a unique, nonpalindromic sequence of 55 bp (light green). The wild-type chromosomes 11 and 22 have a palindrome structure of 230 bp (blue) and ⩾225 base pairs (red), respectively. The full sequence of chromosome 22q11 in the vicinity of these breakpoint junctions is not known, indicating that the palindrome may extend well beyond the 225-bp interval shown. The yellow bar denotes the center of symmetry of the palindrome and the position of the deletions in the carriers (bracketed bold bar, fig. 4). The numbers shown here refer to the beginning and end of the palindromic blocks and not the numbers shown infigure 4. The derivative chromosomes represent the recombinant chromosomes, which are no longer palindromic. B, The palindromic sequences in the normal chromosomes 17, 11, and 22 can theoretically form hairpins or cruciform structures as indicated. The hairpin structures on chromosomes 11 and 22 can extend the entire length of the palindrome, and the loop is assumed because of steric hindrance. In contrast, there is a stem-loop structure hypothesized to form on chromosome 17, and the light green interval depicts the region that lacks base pairing.