Accurate Breakpoint Mapping in Apparently Balanced Translocation Families with Discordant Phenotypes Using Whole Genome Mate-Pair Sequencing.
ABSTRACT: Familial apparently balanced translocations (ABTs) segregating with discordant phenotypes are extremely challenging for interpretation and counseling due to the scarcity of publications and lack of routine techniques for quick investigation. Recently, next generation sequencing has emerged as an efficacious methodology for precise detection of translocation breakpoints. However, studies so far have mainly focused on de novo translocations. The present study focuses specifically on familial cases in order to shed some light to this diagnostic dilemma. Whole-genome mate-pair sequencing (WG-MPS) was applied to map the breakpoints in nine two-way ABT carriers from four families. Translocation breakpoints and patient-specific structural variants were validated by Sanger sequencing and quantitative Real Time PCR, respectively. Identical sequencing patterns and breakpoints were identified in affected and non-affected members carrying the same translocations. PTCD1, ATP5J2-PTCD1, CADPS2, and STPG1 were disrupted by the translocations in three families, rendering them initially as possible disease candidate genes. However, subsequent mutation screening and structural variant analysis did not reveal any pathogenic mutations or unique variants in the affected individuals that could explain the phenotypic differences between carriers of the same translocations. In conclusion, we suggest that NGS-based methods, such as WG-MPS, can be successfully used for detailed mapping of translocation breakpoints, which can also be used in routine clinical investigation of ABT cases. Unlike de novo translocations, no associations were determined here between familial two-way ABTs and the phenotype of the affected members, in which the presence of cryptic imbalances and complex chromosomal rearrangements has been excluded. Future whole-exome or whole-genome sequencing will potentially reveal unidentified mutations in the patients underlying the discordant phenotypes within each family. In addition, larger studies are needed to determine the exact percentage for phenotypic risk in families with ABTs.
Project description:The majority of apparently balanced translocation (ABT) carriers are phenotypically normal. However, several mechanisms were proposed to underlie phenotypes in affected ABT cases. In the current study, whole-genome mate-pair sequencing (WG-MPS) followed by Sanger sequencing was applied to further characterize de novo ABTs in three affected individuals. WG-MPS precisely mapped all ABT breakpoints and revealed three possible underlying molecular mechanisms. Firstly, in a t(X;1) carrier with hearing loss, a highly skewed X-inactivation pattern was observed and the der(X) breakpoint mapped ~87kb upstream an X-linked deafness gene namely POU3F4, thus suggesting an underlying long-range position effect mechanism. Secondly, cryptic complexity and a chromothripsis rearrangement was identified in a t(6;7;8;12) carrier with intellectual disability. Two translocations and a heterozygous deletion disrupted SOX5; a dominant nervous system development gene previously reported in similar patients. Finally, a direct gene disruption mechanism was proposed in a t(4;9) carrier with dysmorphic facial features and speech delay. In this case, the der(9) breakpoint directly disrupted NFIB, a gene involved in lung maturation and development of the pons with important functions in main speech processes. To conclude, in contrast to familial ABT cases with identical rearrangements and discordant phenotypes, where translocations are considered coincidental, translocations seem to be associated with phenotype presentation in affected de novo ABT cases. In addition, this study highlights the importance of investigating both coding and non-coding regions to decipher the underlying pathogenic mechanisms in these patients, and supports the potential introduction of low coverage WG-MPS in the clinical investigation of de novo ABTs.
Project description:Genomic structural variants, including translocations, inversions, insertions, deletions, and duplications, are challenging to be reliably detected by traditional genomic technologies. In particular, balanced translocations and inversions can neither be identified by microarrays since they do not alter chromosome copy numbers, nor by short-read sequencing because of the unmappability of short reads against repetitive genomic regions. The precise localization of breakpoints is vital for exploring genetic causes in patients with balanced translocations or inversions. Long-read sequencing techniques may detect these structural variants in a more direct, efficient, and accurate manner. Here, we performed whole-genome, long-read sequencing using the Oxford Nanopore GridION sequencer to detect breakpoints in six balanced chromosome translocation carriers and one inversion carrier. The results showed that all the breakpoints were consistent with the karyotype results with only ~10× coverage. Polymerase chain reaction (PCR) and Sanger sequencing confirmed 8 out of 14 breakpoints; however, other breakpoint loci were slightly missed since they were either in highly repetitive regions or pericentromeric regions. Some of the breakpoints interrupted normal gene structure, and in other cases, micro-deletions/insertions were found just next to the breakpoints. We also detected haplotypes around the breakpoint regions. Our results suggest that long-read, whole-genome sequencing is an ideal strategy for precisely localizing translocation breakpoints and providing haplotype information, which is essential for medical genetics and preimplantation genetic testing.
Project description:Unbalanced translocations are a relatively common type of copy number variation and a major contributor to neurodevelopmental disorders. We analyzed the breakpoints of 57 unique unbalanced translocations to investigate the mechanisms of how they form. Fifty-one are simple unbalanced translocations between two different chromosome ends, and six rearrangements have more than three breakpoints involving two to five chromosomes. Sequencing 37 breakpoint junctions revealed that simple translocations have between 0 and 4 base pairs (bp) of microhomology (n = 26), short inserted sequences (n = 8), or paralogous repeats (n = 3) at the junctions, indicating that translocations do not arise primarily from nonallelic homologous recombination but instead form most often via nonhomologous end joining or microhomology-mediated break-induced replication. Three simple translocations fuse genes that are predicted to produce in-frame transcripts of SIRPG-WWOX, SMOC2-PROX1, and PIEZO2-MTA1, which may lead to gain of function. Three complex translocations have inversions, insertions, and multiple breakpoint junctions between only two chromosomes. Whole-genome sequencing and fluorescence in situ hybridization analysis of two de novo translocations revealed at least 18 and 33 breakpoints involving five different chromosomes. Breakpoint sequencing of one maternally inherited translocation involving four chromosomes uncovered multiple breakpoints with inversions and insertions. All of these breakpoint junctions had 0-4 bp of microhomology consistent with chromothripsis, and both de novo events occurred on paternal alleles. Together with other studies, these data suggest that germline chromothripsis arises in the paternal genome and may be transmitted maternally. Breakpoint sequencing of our large collection of chromosome rearrangements provides a comprehensive analysis of the molecular mechanisms behind translocation formation.
Project description:Most balanced translocations are thought to result mechanistically from nonhomologous end joining or, in rare cases of recurrent events, by nonallelic homologous recombination. Here, we use low-coverage mate pair whole-genome sequencing to fine map rearrangement breakpoint junctions in both phenotypically normal and affected translocation carriers. In total, 46 junctions from 22 carriers of balanced translocations were characterized. Genes were disrupted in 48% of the breakpoints; recessive genes in four normal carriers and known dominant intellectual disability genes in three affected carriers. Finally, seven candidate disease genes were disrupted in five carriers with neurocognitive disabilities (SVOPL, SUSD1, TOX, NCALD, SLC4A10) and one XX-male carrier with Tourette syndrome (LYPD6, GPC5). Breakpoint junction analyses revealed microhomology and small templated insertions in a substantive fraction of the analyzed translocations (17.4%; n = 4); an observation that was substantiated by reanalysis of 37 previously published translocation junctions. Microhomology associated with templated insertions is a characteristic seen in the breakpoint junctions of rearrangements mediated by error-prone replication-based repair mechanisms. Our data implicate that a mechanism involving template switching might contribute to the formation of at least 15% of the interchromosomal translocation events.
Project description:The most frequent cause of familial clear cell renal cell carcinoma (RCC) is von Hippel-Lindau disease and the VHL tumor suppressor gene (TSG) is inactivated in most sporadic clear cell RCC. Although there is relatively little information on the mechanisms of tumorigenesis of clear cell RCC without VHL inactivation, a subset of familial cases harbors a balanced constitutional chromosome 3 translocation. To date nine different chromosome 3 translocations have been associated with familial or multicentric clear cell RCC; and in three cases chromosome 6 was also involved. To identify candidate genes for renal tumorigenesis we characterized a constitutional translocation, t(3;6)(q22;q16.1) associated with multicentric RCC without evidence of VHL target gene dysregulation. Analysis of breakpoint sequences revealed a 1.3-kb deletion on chromosome 6 within the intron of a 2 exon predicted gene (NT_007299.434). However, RT-PCR analysis failed to detect the expression of this gene in lymphoblast, fibroblast, or kidney tumor cell lines. No known genes were disrupted by the translocation breakpoints but several candidate TSGs (e.g., EPHB1, EPHA7, PPP2R3A RNF184, and STAG1) map within close proximity to the breakpoints.
Project description:Four unrelated families with the same unbalanced translocation der(4)t(4;11)(p16.2;p15.4) were analyzed. Both of the breakpoint regions in 4p16.2 and 11p15.4 were narrowed to large ?359-kb and ?215-kb low-copy repeat (LCR) clusters, respectively, by aCGH and SNP array analyses. DNA sequencing enabled mapping the breakpoints of one translocation to 24 bp within interchromosomal paralogous LCRs of ?130 kb in length and 94.7% DNA sequence identity located in olfactory receptor gene clusters, indicating nonallelic homologous recombination (NAHR) as the mechanism for translocation formation. To investigate the potential involvement of interchromosomal LCRs in recurrent chromosomal translocation formation, we performed computational genome-wide analyses and identified 1143 interchromosomal LCR substrate pairs, >5 kb in size and sharing >94% sequence identity that can potentially mediate chromosomal translocations. Additional evidence for interchromosomal NAHR mediated translocation formation was provided by sequencing the breakpoints of another recurrent translocation, der(8)t(8;12)(p23.1;p13.31). The NAHR sites were mapped within 55 bp in ?7.8-kb paralogous subunits of 95.3% sequence identity located in the ?579-kb (chr 8) and ?287-kb (chr 12) LCR clusters. We demonstrate that NAHR mediates recurrent constitutional translocations t(4;11) and t(8;12) and potentially many other interchromosomal translocations throughout the human genome. Furthermore, we provide a computationally determined genome-wide "recurrent translocation map."
Project description:BACKGROUND: Despite the importance of chromosomal translocations in the initiation and/or progression of cancer, a comprehensive catalog of translocation breakpoints in which these are precisely located on the reference sequence of the human genome is not available at present. DESCRIPTION: We have created a database that describes the genomic location of 1,225 translocation breakpoints in human tumors, corresponding to 247 different genes, using information from publicly available sources. Junction sequences from reciprocal translocations were obtained from 655 different references (either from the literature or from nucleotide databases), and were mapped onto the reference sequence of the human genome using BLAST. All translocation breakpoints were thus referred to precise nucleotide positions (949 breakpoints) or gene fragments (introns or exons, 276 breakpoints) within specific Ensembl transcripts. CONCLUSION: TICdb is a comprehensive collection of finely mapped translocation breakpoints, freely available at http://www.unav.es/genetica/TICdb/. It should facilitate the analysis of sequences encompassing translocation breakpoints and the identification of factors driving translocation events in human tumors.
Project description:Translocations are a common class of chromosomal aberrations and can cause disease by physically disrupting genes or altering their regulatory environment. Some translocations, apparently balanced at the microscopic level, include deletions, duplications, insertions, or inversions at the molecular level. Traditionally, chromosomal rearrangements have been investigated with a conventional banded karyotype followed by arduous positional cloning projects. More recently, molecular cytogenetic approaches using fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH), or whole-genome SNP genotyping together with molecular methods such as inverse PCR and quantitative PCR have allowed more precise evaluation of the breakpoints. These methods suffer, however, from being experimentally intensive and time-consuming and of less than single base pair resolution. Here we describe targeted breakpoint capture followed by next-generation sequencing (TBCS) as a new approach to the general problem of determining the precise structural characterization of translocation breakpoints and related chromosomal aberrations. We tested this approach in three patients with complex chromosomal translocations: The first had craniofacial abnormalities and an apparently balanced t(2;3)(p15;q12) translocation; the second has cleidocranial dysplasia (OMIM 119600) associated with a t(2;6)(q22;p12.3) translocation and a breakpoint in RUNX2 on chromosome 6p; and the third has acampomelic campomelic dysplasia (OMIM 114290) associated with a t(5;17)(q23.2;q24) translocation, with a breakpoint upstream of SOX9 on chromosome 17q. Preliminary studies indicated complex rearrangements in patients 1 and 3 with a total of 10 predicted breakpoints in the three patients. By using TBCS, we quickly and precisely defined eight of the 10 breakpoints.
Project description:Somatically-acquired translocations may serve as important markers for assessing the cause and nature of diseases like cancer. Algorithms to locate translocations may use next-generation sequencing (NGS) platform data. However, paired-end strategies do not accurately predict precise translocation breakpoints, and "split-read" methods may lose sensitivity if a translocation boundary is not captured by many sequenced reads. To address these challenges, we have developed "Bellerophon", a method that uses discordant read pairs to identify potential translocations, and subsequently uses "soft-clipped" reads to predict the location of the precise breakpoints. Furthermore, for each chimeric breakpoint, our method attempts to classify it as a participant in an unbalanced translocation, balanced translocation, or interchromosomal insertion.We compared Bellerophon to four previously published algorithms for detecting structural variation (SV). Using two simulated datasets and two prostate cancer datasets, Bellerophon had overall better performance than the other methods. Furthermore, our method accurately predicted the presence of the interchromosomal insertions placed in our simulated dataset, which is an ability that the other SV prediction programs lack.The combined use of paired reads and soft-clipped reads allows Bellerophon to detect interchromosomal breakpoints with high sensitivity, while also mitigating losses in specificity. This trend is seen across all datasets examined. Because it does not perform assembly on soft-clipped subreads, Bellerophon may be limited in experiments where sequence read lengths are short.The program can be downloaded from http://cbc.case.edu/Bellerophon.
Project description:Chromosomal translocations drive the development of many hematological and some solid cancers. Several factors have been identified to explain the non-random occurrence of translocation breakpoints in the genome. These include chromatin density, gene density and CCCTC-binding factor (CTCF)/cohesin binding site density. However, such factors are at least partially interdependent. Using 13,844 and 1563 karyotypes from human blood and solid cancers, respectively, our multiple regression analysis only identified chromatin density as the primary statistically significant predictor. Specifically, translocation breakpoints preferentially occur in open chromatin. Also, blood and solid tumors show markedly distinct translocation signatures. Strikingly, translocation breakpoints occur significantly more frequently in acrocentric chromosomes than in non-acrocentric chromosomes. Thus, translocations are probably often generated around nucleoli in the inner nucleoplasm, away from the nuclear envelope. Importantly, our findings remain true both in multivariate analyses and after removal of highly recurrent translocations. Finally, we applied pairwise probabilistic co-occurrence modeling. In addition to well-known highly prevalent translocations, such as those resulting in BCR-ABL1 (BCR-ABL) and RUNX1-RUNX1T1 (AML1-ETO) fusion genes, we identified significantly underrepresented translocations with putative fusion genes, which are probably subject to strong negative selection during tumor evolution. Taken together, our findings provide novel insights into the generation and selection of translocations during cancer development.