Analysis of the t(3;8) of hereditary renal cell carcinoma: a palindrome-mediated translocation.
ABSTRACT: It has emerged that palindrome-mediated genomic instability generates DNA-based rearrangements. The presence of palindromic AT-rich repeats (PATRRs) at the translocation breakpoints suggested a palindrome-mediated mechanism in the generation of several recurrent constitutional rearrangements: the t(11;22), t(17;22), and t(8;22). To date, all reported PATRR-mediated translocations include the PATRR on chromosome 22 (PATRR22) as a translocation partner. Here, the constitutional rearrangement, t(3;8)(p14.2;q24.1), segregating with renal cell carcinoma in two families, is examined. The chromosome 8 breakpoint lies in PATRR8 in the first intron of the RNF139 (TRC8) gene, whereas the chromosome 3 breakpoint is located in an AT-rich palindromic sequence in intron 3 of the FHIT gene (PATRR3). Thus, the t(3;8) is the first PATRR-mediated, recurrent, constitutional translocation that does not involve PATRR22. Furthermore, we detect de novo translocations similar to the t(11;22) and t(8;22), involving PATRR3 in normal sperm. The breakpoint on chromosome 3 is in proximity to FRA3B, the most common fragile site in the human genome and a site of frequent deletions in tumor cells. However, the lack of involvement of PATRR3 sequence in numerous FRA3B-related deletions suggests that there are several different DNA sequence-based etiologies responsible for chromosome 3p14.2 genomic rearrangements.
Project description:BACKGROUNDS:The t(8;22)(q24.13;q11.2) has been identified as one of several recurrent constitutional translocations mediated by palindromic AT-rich repeats (PATRRs). Although the breakage on 22q11 utilizes the same PATRR as that of the more prevalent constitutional t(11;22)(q23;q11.2), the breakpoint region on 8q24 has not been elucidated in detail since the analysis of palindromic sequence is technically challenging. RESULTS:In this study, the entire 8q24 breakpoint region has been resolved by next generation sequencing. Eight polymorphic alleles were identified and compared with the junction sequences of previous and two recently identified t(8;22) cases . All of the breakpoints were found to be within the PATRRs on chromosomes 8 and 22 (PATRR8 and PATRR22), but the locations were different among cases at the level of nucleotide resolution. The translocations were always found to arise on symmetric PATRR8 alleles with breakpoints at the center of symmetry. The translocation junction is often accompanied by symmetric deletions at the center of both PATRRs. Rejoining occurs with minimal homology between the translocation partners. Remarkably, comparison of der (8) to der(22) sequences shows identical breakpoint junctions between them, which likely represent products of two independent events on the basis of a classical model. CONCLUSIONS:Our data suggest the hypothesis that interactions between the two PATRRs prior to the translocation event might trigger illegitimate recombination resulting in the recurrent palindrome-mediated translocation.
Project description:We have demonstrated that the breakpoints of the constitutional t(11;22) are located at palindromic AT-rich repeats (PATRRs) on 11q23 and 22q11. As a mechanism for this recurrent translocation, we proposed that the PATRR forms a cruciform structure that induces the genomic instability leading to the rearrangement. A patient with neurofibromatosis type 1 (NF1) had previously been found to have a constitutional t(17;22) disrupting the NF1 gene on 17q11. We have localized the breakpoint on 22q11 within the 22q11-specific low-copy repeat where the breakpoints of the constitutional t(11;22)s reside, implying a similar palindrome-mediated mechanism for generation of the t(17;22). The NF1 gene contains a 195-bp PATRR within intron 31. We have isolated the junction fragments from both the der(17) and the der(22). The breakpoint on 17q11 is close to the center of the PATRR. A published breakpoint of an additional NF1-afflicted patient with a constitutional t(17;22) is also located close to the center of the same PATRR. Our data lend additional support to the hypothesis that PATRR-mediated genomic instability can lead to a variety of translocations.
Project description:Constitutional translocations at the same 22q11.21 low copy repeat B (LCR-B) breakpoint involved in the recurrent t(11;22) are relatively abundant. A novel 46,XY,t(8;22)(q24.13;q11.21) rearrangement was investigated to determine whether the recurrent LCR-B breakpoint is involved. Investigations demonstrated an inversion of the 3Mb region typically deleted in patients with the 22q11.2 deletion syndrome. The 22q11.21 inversion appears to be mediated by low copy repeats, and is presumed to have taken place prior to translocation with 8q24.13. Despite predictions based on inversions observed in other chromosomes harboring low copy repeats, this 22q11.2 inversion has not been observed previously. The current studies utilize novel laser microdissection and MLPA (multiplex ligation-dependent probe amplification) approaches, as adjuncts to FISH, to map the breakpoints of the complex rearrangements of 22q11.21 and 8q24.21. The t(8;22) occurs between the recurrent site on 22q11.21 and an AT-rich site at 8q24.13, making it the fifth different chromosomal locus characterized at the nucleotide level engaged in a translocation with the unstable recurrent breakpoint at 22q11.21. Like the others, this breakpoint occurs at the center of a palindromic sequence. This sequence appears capable of forming a perfect 145 bp stem-loop. Remarkably, this site appears to have been involved in a previously reported t(3;8) occurring between 8q24.13 and FRA3B on 3p14.2. Further, the fragile site-like nature of all of the breakpoint sites involved in translocations with the recurrent site on 22q11.21, suggests a mechanism based on delay of DNA replication in the initiation of these chromosomal rearrangements.
Project description:Palindrome-mediated genomic instability has been associated with chromosomal translocations, including the recurrent t(11;22)(q23;q11). We report a syndrome characterized by extremity anomalies, mild dysmorphia, and intellectual impairment caused by 3:1 meiotic segregation of a previously unrecognized recurrent palindrome-mediated rearrangement, the t(8;22)(q24.13;q11.21). There are at least ten prior reports of this translocation, and nearly identical PATRR8 and PATRR22 breakpoints were validated in several of these published cases. PCR analysis of sperm DNA from healthy males indicates that the t(8;22) arises de novo during gametogenesis in some, but not all, individuals. Furthermore, demonstration that de novo PATRR8-to-PATRR11 translocations occur in sperm suggests that palindrome-mediated translocation is a universal mechanism producing chromosomal rearrangements.
Project description:There is an emerging consensus that secondary structures of DNA have the potential for genomic instability. Palindromic AT-rich repeats (PATRRs) are a characteristic sequence identified at each breakpoint of the recurrent constitutional t(11;22) and t(17;22) translocations in humans, named PATRR22 (approximately 600 bp), PATRR11 (approximately 450 bp) and PATRR17 (approximately 190 bp). The secondary structure-forming propensity in vitro and the instability in vivo have been experimentally evaluated for various PATRRs that differ regarding their size and symmetry. At physiological ionic strength, a cruciform structure is most frequently observed for the symmetric PATRR22, less often for the symmetric PATRR11, but not for the other PATRRs. In wild-type E. coli, only these two PATRRs undergo extensive instability, consistent with the relatively high incidence of the t(11;22) in humans. The resultant deletions are putatively mediated by central cleavage by the structure-specific endonuclease SbcCD, indicating the possibility of a cruciform conformation in vivo. Insertion of a short spacer at the centre of the PATRR22 greatly reduces both its cruciform extrusion in vitro and instability in vivo. Taken together, cruciform extrusion propensity depends on the length and central symmetry of the PATRR, and is likely to determine the instability that leads to recurrent translocations in humans.
Project description:Recently, it has emerged that palindrome-mediated genomic instability contributes to a diverse group of genomic rearrangements including translocations, deletions, and amplifications. One of the best studied examples is the recurrent t(11;22) constitutional translocation in humans that has been well documented to be mediated by palindromic AT-rich repeats (PATRRs) on chromosomes 11q23 and 22q11. De novo examples of the translocation are detected at a high frequency in sperm samples from normal healthy males, but not in lymphoblasts or fibroblasts. Cloned breakpoint sequences preferentially form a cruciform configuration in vitro. Analysis of the junction fragments implicates frequent double-strand-breaks (DSBs) at the center of both palindromic regions, followed by repair through the non-homologous end joining (NHEJ) pathway. We propose that the PATRR adopts a cruciform structure in male meiotic cells, creating genomic instability that leads to the recurrent translocation.
Project description:Translocation is one of the most frequently occurring human chromosomal aberrations. The constitutional t(11;22)(q23;q11), which is the only known recurrent non-Robertsonian translocation, represents a good model for studying translocations in humans. Here we demonstrate polymorphisms of the palindromic sequence at the t(11;22) breakpoint that affect the frequency of de novo translocations in sperm from normal males. A typical allele consists of a perfect palindrome, producing ~10-5 de novo t(11;22) translocations. Alleles with an asymmetric center do not form the t(11;22). Our data show the importance of genome sequence on chromosomal rearrangements, a class of human mutation that is thought to be random.
Project description:Repetitive DNA sequences constitute 30% of the human genome, and are often sites of genomic rearrangement. Recently, it has been found that several constitutional translocations, especially those that involve chromosome 22, take place utilizing palindromic sequences on 22q11 and on the partner chromosome. Analysis of translocation junction fragments shows that the breakpoints of such palindrome-mediated translocations are localized at the center of palindromic AT-rich repeats (PATRRs). The presence of PATRRs at the breakpoints indicates a palindrome-mediated mechanism involved in the generation of these constitutional translocations. Identification of these PATRR-mediated translocations suggests a universal pathway for gross chromosomal rearrangement in the human genome. De novo occurrences of PATRR-mediated translocations can be detected by PCR in normal sperm samples but not somatic cells. Polymorphisms of various PATRRs influence their propensity for adopting a secondary structure, which in turn affects de novo translocation frequency. We propose that the PATRRs form an unstable secondary structure, which leads to double-strand breaks at the center of the PATRR. The double-strand breaks appear to be followed by a non-homologous end-joining repair pathway, ultimately leading to the translocations. This review considers recent findings concerning the mechanism of meiosis-specific, PATRR-mediated translocations.
Project description:The constitutional t(11;22) is the most frequent recurrent non-Robertsonian translocation in humans, the breakpoints of which are located within palindromic AT-rich repeats on 11q23 and 22q11 (PATRR11 and PATRR22). Genetic variation of the PATRR11 was found to affect de novo t(11;22) translocation frequency in sperm derived from normal healthy males, suggesting the hypothesis that polymorphisms of the PATRR22 might also influence the translocation frequency. Although the complicated structure of the PATRR22 locus prevented determining the genotype of the PATRR22 in each individual, genotyping of flanking markers as well as identification of rare variants allowed us to demonstrate an association between the PATRR22 allele type and the translocation frequency. We found that size and symmetry of the PATRR22 affect the de novo translocation frequency, which is lower for the shorter or more asymmetric versions. These data lend support to our hypothesis that the PATRRs form secondary structures in the nucleus that induce genomic instability leading to the recurrent translocation.
Project description: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.