Chromosomal Breakpoint Regions Research Articles

MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR and ddPCR.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL), B-cell chronic lymphocytic leukemia (CLL), and multiple myeloma and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. Real-time quantitative PCR (RQ-PCR) targeting circulating lymphoma cells is still the gold standard for MRD detection in indolent B-NHL and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, CLL, and FL with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy (IGH) and light chain genes. Complete and incomplete IGH and additionally IG kappa light chain rearrangements can be used as targets for MRD. Next-generation sequencing (NGS) of IG-rearrangements (IG-NGS) as new sequencing-based technology can overcome the limitation of PCR-based approaches and has a potential for higher sensitivity. Chromosomal translocations like the t(14;18)(q32;q21) translocation associated with IGH::BCL2 fusion in FL and t(11;14)(q13;q32) translocation in MCL leading to the IGH::CCND1 fusion can be used as MRD target in selected lymphoma subtypes. In patients with CLL, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10-4 is achieved.MRD diagnostics targeting the IG loci is complex and requires extensive knowledge and experience because the junctional regions of each clonal rearranged gene have to be identified before the patient-specific PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation within the rearranged IGH gene occurring during B-cell development of germinal center and post-germinal center B-cell lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. PCR-based methods using allele-specific primers can reach a high sensitivity of up to 10-5. This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IG gene rearrangements and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results. This is available by the EuroMRD network ( https://euromrd.org ), a subgroup of ESHLO ( https://eslho.org ).

Analysis for common chromosomal breakpoint regions among 586 carriers of reciprocal translocations from Henan Province

To determine the frequency, common chromosomal karyotypes and breakpoints, and involved regions among carriers of reciprocal translocations from Henan Province, and to explore the influence of common breakpoint regions on pregnancy and fetal development. For 586 carriers of reciprocal translocations, the above features were retrospectively analyzed. The 586 reciprocal translocations were identified among 62 477 subjects, which yielded a frequency of 0.94%. Among these, 572 (0.92%) had abnormal fertility, and 14 (0.02%) had a history of abnormal fetal development. Statistical analysis showed that chromosomes 1, 4, 7 and 11 were most frequently involved, with t(11;22)(q25;q13) being the most common type of translocation. In total 437 breakpoint regions were identified, with 11q23, 22q13 and 1p36 being most frequently involved, which resulted in infertility, abortion, embryo death, congenital malformation, development delay, mental retardation or a normal phenotype. Above results indicated a 0.92% carrier rate for reciprocal chromosomal translocations in Henan. The location of breakpoint regions may affect the pregnancy and/or fetal development. Discovery of such regions may enable more accurate genetic, reproductive and developmental counseling for carriers, and provide reference for delineation of function and pathogenetic mechanism of the relevant genes.

The Genome of Blue-Capped Cordon-Bleu Uncovers Hidden Diversity of LTR Retrotransposons in Zebra Finch.

Avian genomes have perplexed researchers by being conservative in both size and rearrangements, while simultaneously holding the blueprints for a massive species radiation during the last 65 million years (My). Transposable elements (TEs) in bird genomes are relatively scarce but have been implicated as important hotspots for chromosomal inversions. In zebra finch (Taeniopygia guttata), long terminal repeat (LTR) retrotransposons have proliferated and are positively associated with chromosomal breakpoint regions. Here, we present the genome, karyotype and transposons of blue-capped cordon-bleu (Uraeginthus cyanocephalus), an African songbird that diverged from zebra finch at the root of estrildid finches 10 million years ago (Mya). This constitutes the third linked-read sequenced genome assembly and fourth in-depth curated TE library of any bird. Exploration of TE diversity on this brief evolutionary timescale constitutes a considerable increase in resolution for avian TE biology and allowed us to uncover 4.5 Mb more LTR retrotransposons in the zebra finch genome. In blue-capped cordon-bleu, we likewise observed a recent LTR accumulation indicating that this is a shared feature of Estrildidae. Curiously, we discovered 25 new endogenous retrovirus-like LTR retrotransposon families of which at least 21 are present in zebra finch but were previously undiscovered. This highlights the importance of studying close relatives of model organisms.

MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL) and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. MRD diagnostics performed by real-time quantitative PCR (RQ-PCR) is still the gold standard and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy chain gene (IGHV). Chromosomal translocations like the t(14;18) translocation in FL and t(11;14) translocation in MCL can be used as MRD target in selected lymphoma subtypes. In patients with B-cell chronic lymphocytic leukemia, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10-4 shall be achieved.MRD diagnostics targeting the IGHV gene is complex and requires extensive knowledge and experience because the junctional regions of each lymphoma have to be identified before the patient-specific RQ-PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation (SHM) within the rearranged IG heavy variable (IGHV) gene occurring as during B-cell development of germinal center and post-germinal center lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. RQ-PCR-based methods can reach a good sensitivity (≤10-4). This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IGHV gene rearrangement and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results.

Analysis of t(9;17)(q33.2;q25.3) chromosomal breakpoint regions and genetic association reveals novel candidate genes for bipolar disorder.

Breakpoints of chromosomal abnormalities facilitate identification of novel candidate genes for psychiatric disorders. Genome-wide significant evidence supports the linkage between chromosome 17q25.3 and bipolar disorder (BD). Co-segregation of translocation t(9;17)(q33.2;q25.3) with psychiatric disorders has been reported. We aimed to narrow down these chromosomal breakpoint regions and to investigate the associations between single nucleotide polymorphisms within these regions and BD as well as schizophrenia (SZ) in large genome-wide association study samples. We cross-linked Danish psychiatric and cytogenetic case registers to identify an individual with both t(9;17)(q33.2;q25.3) and BD. Fluorescent in situ hybridization was employed to map the chromosomal breakpoint regions of this proband. We accessed the Psychiatric Genomics Consortium BD (n=16,731) and SZ (n=21,856) data. Genetic associations between these disorders and single nucleotide polymorphisms within these breakpoint regions were analysed by BioQ, FORGE, and RegulomeDB programmes. Four protein-coding genes [coding for (endonuclease V (ENDOV), neuronal pentraxin I (NPTX1), ring finger protein 213 (RNF213), and regulatory-associated protein of mammalian target of rapamycin (mTOR) (RPTOR)] were found to be located within the 17q25.3 breakpoint region. NPTX1 was significantly associated with BD (p=0.004), while ENDOV was significantly associated with SZ (p=0.0075) after Bonferroni correction. Prior linkage evidence and our findings suggest NPTX1 as a novel candidate gene for BD.

Detection of Chromosomal Breakpoints in Patients with Developmental Delay and Speech Disorders

Delineating candidate genes at the chromosomal breakpoint regions in the apparently balanced chromosome rearrangements (ABCR) has been shown to be more effective with the emergence of next-generation sequencing (NGS) technologies. We employed a large-insert (7–11 kb) paired-end tag sequencing technology (DNA-PET) to systematically analyze genome of four patients harbouring cytogenetically defined ABCR with neurodevelopmental symptoms, including developmental delay (DD) and speech disorders. We characterized structural variants (SVs) specific to each individual, including those matching the chromosomal breakpoints. Refinement of these regions by Sanger sequencing resulted in the identification of five disrupted genes in three individuals: guanine nucleotide binding protein, q polypeptide (GNAQ), RNA-binding protein, fox-1 homolog (RBFOX3), unc-5 homolog D (C.elegans) (UNC5D), transmembrane protein 47 (TMEM47), and X-linked inhibitor of apoptosis (XIAP). Among them, XIAP is the causative gene for the immunodeficiency phenotype seen in the patient. The remaining genes displayed specific expression in the fetal brain and have known biologically relevant functions in brain development, suggesting putative candidate genes for neurodevelopmental phenotypes. This study demonstrates the application of NGS technologies in mapping individual gene disruptions in ABCR as a resource for deciphering candidate genes in human neurodevelopmental disorders (NDDs).

Intrachromosomal rearrangements in avian genome evolution: evidence for regions prone to breakpoints

It is generally believed that the organization of avian genomes remains highly conserved in evolution as chromosome number is constant and comparative chromosome painting demonstrated there to be very few interchromosomal rearrangements. The recent sequencing of the zebra finch (Taeniopygia guttata) genome allowed an assessment of the number of intrachromosomal rearrangements between it and the chicken (Gallus gallus) genome, revealing a surprisingly high number of intrachromosomal rearrangements. With the publication of the turkey (Meleagris gallopavo) genome it has become possible to describe intrachromosomal rearrangements between these three important avian species, gain insight into the direction of evolutionary change and assess whether breakpoint regions are reused in birds. To this end, we aligned entire chromosomes between chicken, turkey and zebra finch, identifying syntenic blocks of at least 250 kb. Potential optimal pathways of rearrangements between each of the three genomes were determined, as was a potential Galliform ancestral organization. From this, our data suggest that around one-third of chromosomal breakpoint regions may recur during avian evolution, with 10% of breakpoints apparently recurring in different lineages. This agrees with our previous hypothesis that mechanisms of genome evolution are driven by hotspots of non-allelic homologous recombination.

Atypical mRNA fusions in PML‐RARA positive, RARA‐PML negative acute promyelocytic leukemia

Reciprocal RARA-PML transcripts are not detected in approximately 25% of patients with PML-RARA positive acute promyelocytic leukemia (APL), but the reasons for this are poorly understood. We studied 21 PML-RARA positive/RARA-PML negative cases by bubble PCR and multiplex long template PCR to identify the genomic breakpoints. Additional RT-PCR analysis was performed based on the DNA findings. Three cases were found to have complex rearrangements involving a third locus: the first had a PML-CDC6-RARA forward DNA fusion and expressed a chimeric PML-CDC6-RARA mRNA in addition to a PML-RARA. The other two had HERC1-PML and NT_009714.17-PML genomic fusion sequences at their respective reciprocal breakpoints. Six patients were falsely classified as RARA-PML negative due to deletions on chromosome 15 and/or 17, or alternative splicing leading to atypical RARA-PML fusion transcripts, which were not identified by conventional RT-PCR assays. This study demonstrates that the frequency of RARA-PML expression has been underestimated and highlights remarkable complexity at chromosomal breakpoint regions in APL even in cases with an apparently simple balanced t(15;17)(q24;q12).

Molecular characterization of a balanced rearrangement of chromosome 12 in two siblings with Noonan syndrome

The etiology of Noonan syndrome (NS) has been greatly elucidated with the discovery of the disease causative genes PTPN11, KRAS, SOS1, and RAF1, all involved in the RAS/MAPK-signaling cascade. Given that overall mutations are identified in about 70% of patients, identification of other NS associated genes remains a high priority to fully understand the etiopathogenesis of the condition. We report two affected siblings with an apparently balanced rearrangement of chromosome 12 ins(12)(q12p11.2p12.3) which segregates with the Noonan phenotype. The rearrangement was inherited from the phenotypically normal mother who had mosaicism for the derivative chromosome 12. There were no mutations of PTPN11, KRAS, SOS1, or RAF1 genes detected in the probands. Using fluorescence in situ hybridization analysis we identified the three breakpoints involved at 12p12.3, 12p11.2, and 12q12. By microarray analysis, there were no gains or losses near the breakpoints. Neither, the PTPN11 or KRAS region on chromosome 12 was involved in the rearrangement. We hypothesize that other NS candidate gene(s) may be located in the breakpoint regions of chromosome 12 causing the Noonan phenotype in both of these children.

The role of Alu repeat clusters as mediators of recurrent chromosomal aberrations in tumors.

There is increasing evidence for the involvement of repetitive DNA sequences as facilitators of some of the recurrent chromosomal rearrangements observed in human tumors. The high densities of repetitive DNA, such as Alu elements, at some chromosomal translocation breakpoint regions has led to the suggestion that these sequences could provide hot spots for homologous recombination, and could mediate the translocation process and elevate the likelihood of other types of chromosomal rearrangements taking place. The Alu core sequence itself has been suggested to promote DNA strand exchange and genomic rearrangement, and it has striking sequence similarity to chi (which has been shown to stimulate recBCD-mediated recombination in Escherichia coli). Alu repeats have been shown to be involved in the generation of many constitutional gene mutations in meiotic cells, attributed to unequal homologous recombination and consequent deletions and/or duplication events. It has recently been demonstrated that similar deletion events can take place in neoplasia because several types of leukemia-associated chromosomal rearrangements frequently have submicroscopic deletions immediately adjacent to the translocation breakpoint regions. Significantly, these types of deletions appear to be more likely to take place when the regions subject to rearrangement contain a high density of Alu repeats. With the completion of the Human Genome Project, it will soon be possible to create more comprehensive maps of the distribution and densities of repetitive sequences, such as Alu, throughout the genome. Such maps will offer unique insights into the relative distribution of cancer translocation breakpoints and the localization of clusters of repetitive DNA.

Clonality analysis of hematopoietic cell lineages in acute myeloid leukemia and translocation (8;21): only myeloid cells are part of the malignant clone.

Bone marrow from six patients with acute myeloid leukemia (AML) and t(8;21) (q22;q22) or a variant t(8;13;21) was studied by simultaneous analysis of cell morphology and karyotype. Combination of May-Grünwald-Giemsa (MGG) and fluorescence in situ hybridization (FISH) using probes specific for the breakpoint regions of chromosome 8 and 21 allowed us to establish the extent of cell-lineage involvement of the translocation. The translocation was found in all myeloid blasts and in high percentages of the more mature neutrophilic cells. In one patient we could demonstrate the translocation in the eosinophils as well. Erythroblasts and lymphocytes did not show the t(8;21) abnormality. These results indicate that the t(8;21) in AML is restricted to the myeloid (granulocytic) lineage.