A palindrome-driven complex rearrangement of 22q11.2 and 8q24.1 elucidated using novel technologies

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.     

A palindrome-mediated mechanism distinguishes translocations involving LCR-B of chromosome 22q11.2

Two known recurrent constitutional translocations, t(11;22) and t(17;22), as well as a non-recurrent t(4;22), display derivative chromosomes that have joined to a common site within the low copy repeat B (LCR-B) region of 22q11.2. This breakpoint is located between two AT-rich inverted repeats that form a nearly perfect palindrome. Breakpoints within the 11q23, 17q11 and 4q35 partner chromosomes also fall near the center of palindromic sequences. In the present work the breakpoints of a fourth translocation involving LCR-B, a balanced ependymoma-associated t(1;22), were characterized not only to localize this junction relative to known genes, but also to further understand the mechanism underlying these rearrangements. FISH mapping was used to localize the 22q11.2 breakpoint to LCR-B and the 1p21 breakpoint to single BAC clones. STS mapping narrowed the 1p21.2 breakpoint to a 1990 bp AT-rich region, and junction fragments were amplified by nested PCR. Junction fragment-derived sequence indicates that the 1p21.2 breakpoint splits a 278 nt palindrome capable of forming stem-loop secondary structure. In contrast, the 1p21.2 reference genomic sequence from clones in the database does not exhibit this configuration, suggesting a predisposition for regional genomic instability perhaps etiologic for this rearrangement. Given its similarity to known chromosomal fragile site (FRA) sequences, this polymorphic 1p21.2 sequence may represent one of the FRA1 loci. Comparative analysis of the secondary structure of sequences surrounding translocation breakpoints that involve LCR-B with those not involving this region indicate a unique ability of the former to form stem-loop structures. The relative likelihood of forming these configurations appears to be related to the rate of translocation occurrence. Further analysis suggests that constitutional translocations in general occur between sequences of similar melting temperature and propensity for secondary structure.     

A novel sequence-based approach to localize translocation breakpoints identifies the molecular basis of a t(4;22)

Low copy repeats (LCRs) located in 22q11.2, especially LCR-B, are susceptible to rearrangements associated with several relatively common constitutional disorders. These include DiGeorge syndrome, Velocardiofacial syndrome, Cat-eye syndrome and recurrent translocations of 22q11 including the constitutional t(11;22) and t(17;22). The presence of palindromic AT-rich repeats (PATRRs) within LCR-B of 22q11.2, as well as within the 11q23 and 17q11 regions, has suggested a palindrome-mediated, stem-loop mechanism for the generation of such recurring constitutional 22q11.2 translocations. The mechanism responsible for non-recurrent 22q11.2 rearrangements is presently unknown due to the extensive effort required for breakpoint cloning. Thus, we have developed a novel fluorescence in-situ hybridization and primed in-situ hybridization (PRINS) approach and rapidly localized the breakpoint of a non-recurrent 22q11.2 translocation, a t(4;22). Multiple primer pairs were designed from the sequence of a 200 kb, chromosome 4, breakpoint-spanning BAC to generate PRINS probes. Amplification of adjacent primer pairs, labeled in two colors, allowed us to narrow the 4q35.1 breakpoint to a 6.7 kb clonable region. Application of our improved PRINS protocol facilitated fine-mapping the translocation breakpoints within 4q35.1 and 22q11.2, and permitted rapid cloning and analysis of translocation junction fragments. To confirm the PRINS localization results, PCR mapping of t(4;22) somatic cell hybrid DNA was employed. Analysis of the breakpoints demonstrates the presence of a 554 bp palindromic sequence at the chromosome 4 breakpoint and a 22q11.2 location within the same PATRR as the recurrent t(11;22) and t(17;22). The sequence of this breakpoint further suggests that a stem-loop secondary structure mechanism is responsible for the formation of other, non-recurrent translocations involving LCR-B of 22q11.2.     

Regions of genomic instability on 22q11 and 11q23 as the etiology for the recurrent constitutional t(11;22)

The constitutional t(11;22)(q23;q11) is the only known recurrent, non-Robertsonian translocation. To analyze the genomic structure of the breakpoint, we have cloned the junction fragments from the der(11) and der(22) of a t(11;22) balanced carrier. On chromosome 11 the translocation occurs within a short, palindromic AT-rich region (ATRR). Likewise, the breakpoint on chromosome 22 has been localized within an ATRR that is part of a larger palindrome. Interestingly, the 22q11 breakpoint falls within one of the 'unclonable' gaps in the genomic sequence. Further, a sequenced chromosome 11 BAC clone, spanning the t(11;22) breakpoint in 11q23, is deleted within the palindromic ATRR, suggesting instability of this region in bacterial clones. Several unrelated t(11;22) families demonstrate similar breakpoints on both chromosomes, indicating that their translocations are within the same palindrome. It is likely that the palindromic ATRRs produce unstable DNA structures in 22q11 and 11q23 that are responsible for the recurrent t(11;22) translocation.     

Detailed analysis of 22q11.2 with a high density MLPA probe set

The presence of chromosome-specific low-copy repeats (LCRs) predisposes chromosome 22 to deletions and duplications. The current diagnostic procedure for detecting aberrations at 22q11.2 is chromosomal analysis coupled with fluorescence in situ hybridization (FISH) or PCR-based multiplex ligation dependent probe amplification (MLPA). However, there are copy number variations (CNVs) in 22q11.2 that are only detected by high-resolution platforms such as array comparative genomic hybridization (aCGH). We report on development of a high-definition MLPA (MLPA-HD) 22q11 kit that detects copy number changes at 37 loci on the long arm of chromosome 22. These include the 3-Mb region commonly deleted in DiGeorge/velocardiofacial syndrome (DGS/VCFS), the cat eye syndrome (CES) region, and more distal regions in 22q11 that have recently been shown to be deleted. We have used this MLPA-HD probe set to analyze 363 previously well-characterized samples with a variety of different rearrangements at 22q11 and demonstrate that it can detect copy number alterations with high sensitivity and specificity. In addition to detection of the common recurrent deletions associated with DGS/VCFS, variant and novel chromosome 22 aberrations have been detected. These include duplications within as well as deletions distal to this region. Further, the MLPA-HD detects deletion endpoint differences between patients with the common 3-Mb deletion. The MLPA-HD kit is proposed as a cost effective alternative to the currently available detection methods for individuals with features of the 22q11 aberrations. In patients with the relevant phenotypic characteristics, this MLPA-HD probe set could replace FISH for the clinical diagnosis of 22q11.2 deletions and duplications.     

Tandem configurations of variably duplicated segments of 22q11.2 confirmed by fiber-FISH analysis

22q11.2 duplication syndrome has recently been established as a new syndrome manifesting broad clinical phenotypes including mental retardation. It is reciprocal to DiGeorge (DGS)/velo-cardio-facial syndrome (VCFS), in which the same portion of the chromosome is hemizygously deleted. Deletions and duplications of the 22q11.2 region are facilitated by the low-copy repeats (LCRs) flanking this region. In this study, we aimed to identify the directions of the duplicated segments of 22q11.2 to better understand the mechanism of chromosomal duplication. To achieve this aim, we accumulated samples from four patients with 22q11.2 duplications. One of the patients had an atypically small (741?kb) duplication of 22q11.2. The centromeric end of the breakpoint was on LCR22A, but the telomeric end was between LCR22A and B. Therefore, the duplicated segment did not include T-box 1 gene (TBX1), the gene primarily responsible for the DGS/VCFS. As this duplication was shared by the patient's healthy mother, this appears to be a benign copy-number variation rather than a disease-causing alteration. The other three patients showed 3.0 or 4.0?Mb duplications flanked by LCRs. The directions of the duplicated segments were investigated by fiber-fluorescence in situ hybridization analysis. All samples showed tandem configurations. These results support the hypothesized mechanism of non-allelic homologous recombination with flanking LCRs and add additional evidence that many interstitial duplications are aligned as tandem configurations.     

Chromosome aberrations involving 10q22: report of three overlapping interstitial deletions and a balanced translocation disrupting C10orf11

Interstitial deletions of chromosome band 10q22 are rare. We report on the characterization of three overlapping de novo 10q22 deletions by high-resolution array comparative genomic hybridization in three unrelated patients. Patient 1 had a 7.9 Mb deletion in 10q21.3–q22.2 and suffered from severe feeding problems, facial dysmorphisms and profound mental retardation. Patients 2 and 3 had nearly identical deletions of 3.2 and 3.6 Mb, the proximal breakpoints of which were located at an identical low-copy repeat. Both patients were mentally retarded; patient 3 also suffered from growth retardation and hypotonia. We also report on the results of breakpoint analysis by array painting in a mentally retarded patient with a balanced chromosome translocation 46,XY,t(10;13)(q22;p13)dn. The breakpoint in 10q22 was found to disrupt C10orf11, a brain-expressed gene in the common deleted interval of patients 1–3. This finding suggests that haploinsufficiency of C10orf11 contributes to the cognitive defects in 10q22 deletion patients.     

Translocation (5;10)(q22;q24) in a case of acute lymphoblastic leukemia

The activation of genes important to acute lymphoblastic leukemia (ALL) may be evidenced by somatically acquired chromosomal translocations found recurrently in different patient subgroups. It is for this reason that research efforts have focused on the molecular dissection of recurring chromosomal rearrangements. However, even though a large number of leukemia-causing genes have been identified, the genetic basis of many ALL cases remains unknown. We and others have reasoned that novel translocations found in the leukemic cells of ALL patients may mark the location of more frequent gene rearrangements that are otherwise hidden submicroscopically within normal or complex karyotypes. Towards this end, we here describe the first reported association of a t(5;10)(q22;q24) with adult ALL. Fluorescence in situ hybridization (FISH) and Southern blot hybridization studies have eliminated likely involvement of the candidate genes APC and MCC on chromosome 5, and PAX2, TLX1, and NFKB2 on chromosome 10. Results further suggest that the breakpoint on chromosome 5 lies centromeric of APC and the chromosome 10 breakpoint is centromeric of PAX2. The genomic regions disrupted by this t(5;10)(q22;q24) have not previously been associated with leukemia.     

Chromosomes in Ewing's sarcoma. I. An evaluation of 85 cases of remarkable consistency of t(11;22)(q24;q12)

Since our initial reports on chromosomal studies in eight Ewing's sarcomas (ES), we have carried out similar investigations on 23 additional ES specimens following short-term culture of tumor cells (16 cases), and established in vitro cell lines (three cases) and on xenografted tumors in nude mice (four cases). We demonstrated the presence of the reciprocal t(11;22)(q24;q12) in every case except one that exhibited a complex t(11;22;14)(q24;q12;q11). On the basis of results from these additional 23 cases, we confirm the consistency of the t(11;22)(q24;q12) in ES. Moreover, we reviewed 54 ES cases reported by other investigators; when added to our 31 cases, this brings the total number to 85 unrelated cases of ES available for an evaluation of the frequency of involvement of bands 11q24 and 22q12 in translocations in ES. The standard t(11;22)(q24;q12) proved to be a remarkably consistent event, present in 83% of the cases. Five percent of the cases exhibited complex translocations involving a third chromosome in addition to chromosomes #11 and #22. In 4% of the cases variant translocations involved 22q12 but with a chromosome(s) other than #11. The breakpoint on chromosome 22q12 appears to be the most consistently observed event in 92% of the cases, whereas, the breakpoint at chromosome 11q24 was observed in 88% of the cases.     

An unusual three-way translocation t(21;8;1)(q22;q22;q32) in a case of acute myeloid leukemia (M2)

Variant forms of the classic translocation t(8;21) are uncommon and account approximately 3% of all t(8;21)(q22;q22) in acute myeloid leukemia (AML) patients. These forms involve chromosomes 8, 21, and other chromosomes. Here we report a Tunisian patient with a complex rearrangement t(21;8;1)(q22;q22;q32) revealed by conventional chromosomal study at diagnosis. Fluorescence in situ hybridization study revealed the presence of the AML1-ETO chimeric gene on the derivative chromosome 8. To the best of our knowledge, this is the second case of t(21;8;1) of AML-M2 reported in the literature with the involvement of the same breakpoint at 1q32. This illustrates that this complex translocation is rarely encountered in AML and reinforces the fact that this region may harbour a critical gene candidate that may play an important role in the pathogenesis of AML. More cases are needed to elucidate its clinical features and prognosis.     

AT-rich repeats associated with chromosome 22q11.2 rearrangement disorders shape human genome architecture on Yq12

Low copy repeats (LCRs; segmental duplications) constitute approximately 5% of the sequenced human genome. Nonallelic homologous recombination events between LCRs during meiosis can lead to chromosomal rearrangements responsible for many genomic disorders. The 22q11.2 region is susceptible to recurrent and nonrecurrent deletions, duplications as well as translocations that are mediated by LCRs termed LCR22s. One particular DNA structural element, a palindromic AT-rich repeat (PATRR) present within LCR22-3a, is responsible for translocations. Similar AT-rich repeats are present within the two largest LCR22s, LCR22-2 and LCR22-4. We provide direct sequence evidence that the AT-rich repeats have altered LCR22 organization during primate evolution. The AT-rich repeats are surrounded by a subtype of human satellite I (HSAT I), and an AluSc element, forming a 2.4-kb tripartite structure. Besides 22q11.2, FISH and PCR mapping localized the tripartite repeat within heterochromatic, unsequenced regions of the genome, including the pericentromeric regions of the acrocentric chromosomes and the heterochromatic portion of Yq12 in humans. The repeat is also present on autosomes but not on chromosome Y in other hominoid species, suggesting that it has duplicated on Yq12 after speciation of humans from its common ancestor. This demonstrates that AT-rich repeats have shaped or altered the structure of the genome during evolution.     

Structural aberrations of the long arm of chromosome no. 22: Report of a family with translocation t(11;22) (q25;q11)*

A chromosomal translocation t(11;22) (q25;q11) is described in a family. Four members, in two generations, had the same translocation but showed phenotypic variation. Case reports of chromosome aberrations involving the long arm of chromosome 22 associated with and without chronic myeloid leukemia (CML) are reviewed. It appears that the distal segment of the long arm of chromosome 22 is either translocated or deleted, resulting in congenital anomalies, presumably due to chromosome imbalance. In other instances, a specific breakpoint on 22q results in the origin of Philadelphia chromosome (Ph¹) associated with CML.     

PLP1 duplication at the breakpoint regions of an apparently balanced t(X;22) translocation causes Pelizaeus–Merzbacher disease in a girl

Fonseca ACS, Bonaldi A, Costa SS, Freitas MR, Kok F, Vianna‐Morgante AM. PLP1 duplication at the breakpoint regions of an apparently balanced t(X;22) translocation causes Pelizaeus–Merzbacher disease in a girl. PLP1 (proteolipid protein1 gene) mutations cause Pelizaeus–Merzbacher disease (PMD), characterized by hypomyelination of the central nervous system, and affecting almost exclusively males. We report on a girl with classical PMD who carries an apparently balanced translocation t(X;22)(q22;q13). By applying array‐based comparative genomic hybridization (a‐CGH), we detected duplications at 22q13 and Xq22, encompassing 487–546 kb and 543–611 kb, respectively. The additional copies were mapped by fluorescent in situ hybridization to the breakpoint regions, on the derivative X chromosome (22q13 duplicated segment) and on the derivative 22 chromosome (Xq22 duplicated segment). One of the 14 duplicated X‐chromosome genes was PLP1.The normal X chromosome was the inactive one in the majority of peripheral blood leukocytes, a pattern of inactivation that makes cells functionally balanced for the translocated segments. However, a copy of the PLP1 gene on the derivative chromosome 22, in addition to those on the X and der(X) chromosomes, resulted in two active copies of the gene, irrespective of the X‐inactivation pattern, thus causing PMD. This t(X;22) is the first constitutional human apparently balanced translocation with duplications from both involved chromosomes detected at the breakpoint regions.     

The t(II; 22)(pI5.5; qII.23) in a retroperitoneal rhabdoid tumor also includes a regional deletion distal to CRYBB2 on 22q

Translocations and deletions involving chromosomal band 22q 11 are common genetic aberrations in malignant rhabdoid tumors. Previous molecular analyses of a t(11; 22) in the malignant rhabdoid tumor cell line TM87-16 localized the breakpoint distal to BCR on 22q 11. In the present report, we have further refined the map position of this breakpoint between CRYBB2 and D22S258. Moreover, the D22S258, CRYBA4, D22S300, D22SI, and D22S310 loci, which lie between CRYBB2 and D22S42, were found to be deleted, presumably as a result of the translocation event. The identification of this deletion of at least 2 Mb on the long arm of chromosome 22 should be helpful for mapping the gene(s) in the region involved in the development of malignant rhabdoid tumors as well as providing insights into the mechanisms of chromosomal translocation in human solid tumors.     

Array-based comparative genomic hybridization analysis of recurrent chromosome 15q rearrangements

Genomic rearrangements of chromosome 15q11-q13 cause diverse phenotypes including autism, Prader-Willi syndrome (PWS), and Angelman syndrome (AS). This region is subject to genomic imprinting and characterized by complex combinations of low copy repeat elements. Prader-Willi and Angelman syndrome are caused primarily by 15q11-13 deletions of paternal and maternal origin, respectively. Autism is seen with maternal, but not paternal, interstitial duplications. Isodicentric 15q, most often of maternal origin, is associated with a complex phenotype often including autistic features. Limitations of conventional cytogenetic tests preclude a detailed analysis in most patients with 15q rearrangements. We have developed a microarray for comparative genomic hybridization utilizing 106 genomic clones from chromosome 15q to characterize this region. The array accurately localized all breakpoints associated with gains or losses on 15q. The results confirmed the location of the common breakpoints associated with interstitial deletions and duplications. The majority of idic(15q) chromosomes are comprised of symmetrical arms with four copies of the breakpoint 1 to breakpoint 5 region. Patients with less common breakpoints that are not distinguished by routine cytogenetic methods were more accurately characterized by array analysis. This microarray provides a detailed characterization for chromosomal abnormalities involving 15q11-q14 and is useful for more precise genotype-phenotype correlations for autism, PWS, AS, and idic(15) syndrome.     

Human chromosome 15q11-q14 regions of rearrangements contain clusters of LCR15 duplicons

Six breakpoint regions for rearrangements of human chromosome 15q11-q14 have been described. These rearrangements involve deletions found in approximately 70% of Prader-Willi or Angelman's syndrome patients (PWS, AS), duplications detected in some cases of autism, triplications and inverted duplications. HERC2-containing (HEct domain and RCc1 domain protein 2) segmental duplications or duplicons are present at two of these breakpoints (BP2 and BP3) mainly associated with deletions. We show here that clusters containing several copies of the human chromosome 15 low-copy repeat (LCR15) duplicon are located at each of the six described 15q11-q14 BPs. In addition, our results suggest the existence of breakpoints for large 15q11-q13 deletions in a proximal duplicon-containing clone. The study reveals that HERC2-containing duplicons (estimated on 50-400 kb) and LCR15 duplicons ( approximately 15 kb on 15q11-q14) share the golgin-like protein (GLP) genomic sequence. Through the analysis of a human BAC library and public databases we have identified 36 LCR15 related sequences in the human genome, most (27) mapping to chromosome 15q and being transcribed. LCR15 analysis in non-human primates and age-sequence divergences support a recent origin of this family of segmental duplications through human speciation.     

Minimal phenotype in a girl with trisomy 15q due to t(X;15)(q22.3;q11.2) translocation

Few cases of de novo unbalanced X;autosome translocations associated with a normal or mild dysmorphic phenotype have been described. We report a 3-year-old dizygotic female twin with prenatally ascertained increased nuchal translucency. Prenatal chromosome studies revealed nearly complete trisomy 15 due to a de novo unbalanced translocation t(X;15)(q22;q11.2) confirmed postnatally. A mild phenotype was observed with normal birth measurements, minor facial dysmorphic features (hypertelorism, short broad nose, and a relatively long philtrum), and moderate developmental delay at the age of 3 years in comparison to her male fraternal twin. Replication timing utilizing BrdU and acridine-orange staining showed that the der(X) chromosome was late-replicating with variable spreading of inactivation into the translocated 15q segment. The der(X) was determined to be of paternal origin by analyses of polymorphic markers and CGG-repeat at FMR1. Methylation analysis at the SNRPN locus and analysis of microsatellites on 15q revealed paternal isodisomy with double dosage for all markers and the unmethylated SNRPN gene. The Xq breakpoint was mapped within two overlapping BAC clones RP11-575K24 and RP13-483F6 at Xq22.3 and the 15q breakpoint to 15q11.2, within overlapping clones RP11-509A17 and RP11-382A4 that are all significantly enriched for LINE-1 elements (36.6%, 43.0%, 26.6%, 22.0%, respectively). We speculate that the attenuated phenotype may be due to inactivation spreading into 15q, potentially facilitated by the enrichment of LINE-1 elements at the breakpoints. In silico analysis of breakpoint regions revealed the presence of highly identical low-copy repeats (LCRs) at both breakpoints, potentially involved in generating the translocation.     

Association of the X chromosomal region q11→22 and Klinefelter syndrome

A male patient with typical Klinefelter syndrome features was found to have a 47, XXq-Y chromosome complement. The X chromosome with the deletion was late-replicating. We suggest that the region q11→22 of the extra X chromosome is important for expression of the Klinefelter phenotype.     

Familial t(11;13)(q21;q14) and the duplication 11q, 13q phenotype

Cases of duplication of distal 11q or proximal 13q have been reported independently. A specific translocation resulting in duplication of distal 11q, [der(22)t(11;22)(q23;q11)], has been documented in over 40 cases. We report on a male fetus with chromosomal excess of both distal 11q and proximal 13q resulting from a familial translocation. This case supports the causal association of duplication 11q with neural tube defects. © 1993 Wiley-Liss, Inc.     

Parental and chromosomal origins of microdeletion and duplication syndromes involving 7q11.23, 15q11-q13 and 22q11

Non-allelic homologous recombination between chromosome-specific LCRs is the most common mechanism leading to recurrent microdeletions and duplications. To look for locus-specific differences, we have used microsatellites to determine the parental and chromosomal origins of a large series of patients with de novo deletions of chromosome 7q11.23 (Williams syndrome), 15q11-q13 (Angelman syndrome, Prader-Willi syndrome) and 22q11 (Di George syndrome) and duplications of 15q11-q13. Overall the majority of rearrangements were interchromosomal, so arising from unequal meiotic exchange, and there were approximately equal numbers of maternal and paternal deletions. Duplications and deletions of 15q11-q13 appear to be reciprocal products that arise by the same mechanisms. The proportion arising from interchromosomal exchanges varied among deletions with 22q11 the highest and 15q11-q13 the lowest. However, parental and chromosomal origins were not always independent. For 15q11-q13, maternal deletions tended to be interchromosomal while paternal deletions tended to be intrachromosomal; for 22q11 there was a possible excess of maternal cases among intrachromosomal deletions. Several factors are likely to be involved in the formation of recurrent rearrangements and the relative importance of these appear to be locus-specific.