Rare cases of aggressive B-cell lymphomas with a morphology similar to Burkitt lymphoma (BL) present with the BL-typical immunophenotype, but lacked MYC translocation (MYC-negative Burkitt-like ...lymphomamnBLL). A proportion of those with an imbalance pattern in chromosome 11q has been designated Burkitt-like lymphoma with 11q aberration in the recent update of the World Health Organization (WHO) classification. Because of the problems in the identification of Burkitt-like lymphoma with 11q aberration, our goal was to retrospectively analyze their frequency in a cohort of “candidate” aggressive lymphomas (cohort 1, n=35) such as mnBLL (n=16), diffuse large B-cell lymphoma with similarities to Burkitt lymphoma (DLBCL-BL; n=3), high-grade B-cell lymphomas, not otherwise specified (NOS) (n=16), as well as in a cohort of MYC-negative diffuse large B-cell lymphoma NOS (cohort 2, n=62). In total, 17/33 cohort 1 cases (52%) harbored the typical 11q aberration pattern, predominantly those that had been classified as mnBLL (12/16, 75%), but also as DLBCL-BL (2/3, 67%) and high-grade B-cell lymphomas, NOS (3/14; 21%). The specimens with this typical 11q aberration pattern were usually negative for the BCL2 protein. Of interest and as a new finding, samples harboring the 11q aberration pattern were often characterized by strikingly coarse apoptotic debris within starry sky macrophages facilitating their recognition. In contrast, only 1 of 62 garden variety DLBCL, NOS was positive for the 11q aberration pattern. In 2 DLBCL-BL, a dual MYC translocation/11q aberration pattern was detected. As a diagnostic algorithm, we, therefore, propose analysis of 11q status in MYC-negative high-grade lymphomas with features of BL, especially showing BCL2 negativity and a conspicuous coarse apoptotic debris in starry sky macrophages.
Genetic predisposition is one of the major risk factors for pediatric cancer, with ~10% of children being carriers of a predisposing germline alteration. It is likely that this is the tip of the ...iceberg and many children are underdiagnosed, as most of the analysis focuses on single or short nucleotide variants, not considering the full spectrum of DNA alterations. Hence, we applied optical genome mapping (OGM) to our cohort of 34 pediatric cancer patients to perform an unbiased germline screening and analyze the frequency of structural variants (SVs) and their impact on cancer predisposition. All children were clinically highly suspicious for germline alterations (concomitant conditions or congenital anomalies, positive family cancer history, particular cancer type, synchronous or metachronous tumors), but whole exome sequencing (WES) had failed to detect pathogenic variants in cancer predisposing genes. OGM detected a median of 49 rare SVs (range 27-149) per patient. By analysis of 18 patient-parent trios, we identified three de novo SVs. Moreover, we discovered a likely pathogenic deletion of exon 3 in the known cancer predisposition gene BRCA2, and identified a duplication in RPA1, which might represent a new cancer predisposition gene. We conclude that optical genome mapping is a suitable tool for detecting potentially predisposing SVs in addition to WES in pediatric cancer patients.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Introduction Firstly described in 1967, hyperdiploidy (HD) is the most frequent genetic abnormality in B-cell- precursor acute lymphoblastic leukemia (BCP-ALL) in children, comprising about 25% of ...all cases. A not yet exactly defined proportion have predisposing pathogenic germline variants in DNA repair pathway genes, chromatin remodeling factors, transcription factors regulating B-cell development (particularly ETV6) or receptor tyrosine kinases pathway genes like RAS/RAF. Among the latter, mutations have mostly been described in PTPN11 and SOS1, but not yet in other components of this central regulatory hub of cellular communication. Germline loss-of-function (LOF) LZTR1 mutations are typically linked to hereditary nerve sheath tumors. However, it remains largely unknown, if other tumor entities are associated with LZTR1 LOF germline mutations, potentially broadening the spectrum of malignancies associated with RASopathies. Aim Our aim is to understand the frequency and the impact of LZTR1 germline variants in HD BCP-ALL of childhood. Material and Methods We analyzed WES data of 283 children with BCP-ALL for the presence of pathogenic variants in the LZTR1 cancer predisposition gene. For a part of the detected variants, we performed further functional analyses to investigate the effect of the alteration on protein function. We applied a Drosophila model that is particularly suited for functional evaluation of Ras pathway activity. Results We identified LZTR1 germline variants in 10/283 (3.5%) patients (Figure 1). Interestingly, in 6 out of 283 (2.1%) children the LZTR1 variants were classified as pathogenic/likely pathogenic (P/LP). The majority of patients (5/6) harboring a P/LP germline LZTR1 variant presented with a HD BCP-ALL. Only two of our patients showed concomitant phenotypic features indicative of an underlying syndromic condition. Patient P1 presented with a HD BCP-ALL at the age of 9 years and mild psychomotor delay. Sequencing revealed a likely pathogenic variant (p.Arg283Trp) that was not yet reported in association with RASopathy syndromes. The second child P8, also diagnosed with a HD BCP-ALL, carried a well-known autosomal dominant mutation described in Noonan syndrome (NS) variant (p.Gly248Arg). The child was characterized by facial dysmorphism, as well as mild developmental delay, although a diagnosis of NS was not established prior to development of the BCP-ALL. The other three patients had no clinical peculiarities other than HD BCP-ALL. We functionally characterized patient derived LZTR1 variants in dLztr1-depleted ISC. Like wild-type hLZTR1 wt, hLZTR1 p.Arg283Trp and hLZTR1 p.Lys761Arg restored control-like ISC lineage production, suggesting that hLZTR1 p.Arg283Trp and hLZTR1 p.Lys761Arg still regulate Ras ubiquitination. In contrast, hLZTR1 p.Tyr535Ter results in even 1.8-fold higher ISC lineage production than dLztr1-RNAi. Beyond ISC production as a readout, we employed a second experimental paradigm directly addressing Ras signalling activity with a translocating modified ERK sensor. In line with Ras activity control of LZTR1, hLZTR1 wt significantly reduced ERK activity, while the putatively LOF variant hLZTR1 p.Tyr535Ter variant significantly increased Ras pathway activation by 1.4-fold over controls. We also noticed that hLZTR1 p.Tyr535Ter induced extensive membrane blebbing and nuclear fragmentation of GFP positive cells likely indicating programmed cell death. Quantification of apoptotic cells revealed an 11.1-fold increase compared to controls, which was not observed for hLZTR1 p.Arg283Trp and hLZTR1 p.Lys761Arg. Most notably, forced expression of hLZTR1 p.Arg283Trp andhLZTR1 p.Tyr535Ter increased mitotic recombination by 4-fold and 3.4-fold, respectively, which was not observed for the hLZTR1 p. Lys761Arg variant. Conclusion In our cohort, approximately 2% of all children with BCP-ALL harboured a P/LP LZTR1 germline variation, not necessarily linked with clinical appearance of Noonan-syndrome-like features, but to the development of a high hyperdiploid karyotype. By applying a Drosophila model, we demonstrated that patient-derived LZTR1 germline variants affect RAS pathway activation, ERK accumulation, cell proliferation, DNA recombination and apoptosis. Figure 1: Depiction of the ten LZTR1 variants detected in an unselected pediatric cohort of 283 BCP-ALL patients
Full text
Available for:
IJS, IMTLJ, KILJ, NLZOH, NUK, SAZU, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Burkitt lymphoma is a mature aggressive B-cell lymphoma derived from germinal center B cells. Its cytogenetic hallmark is the Burkitt translocation t(8;14)(q24;q32) and its variants, which juxtapose ...the MYC oncogene with one of the three immunoglobulin loci. Consequently, MYC is deregulated, resulting in massive perturbation of gene expression. Nevertheless, MYC deregulation alone seems not to be sufficient to drive Burkitt lymphomagenesis. By whole-genome, whole-exome and transcriptome sequencing of four prototypical Burkitt lymphomas with immunoglobulin gene (IG)-MYC translocation, we identified seven recurrently mutated genes. One of these genes, ID3, mapped to a region of focal homozygous loss in Burkitt lymphoma. In an extended cohort, 36 of 53 molecularly defined Burkitt lymphomas (68%) carried potentially damaging mutations of ID3. These were strongly enriched at somatic hypermutation motifs. Only 6 of 47 other B-cell lymphomas with the IG-MYC translocation (13%) carried ID3 mutations. These findings suggest that cooperation between ID3 inactivation and IG-MYC translocation is a hallmark of Burkitt lymphomagenesis.
Full text
Available for:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Introduction: Current studies indicate a contribution of germline predisposition in the development of approximately 8.5% of childhood cancers (Zhang J. et al., N Engl J Med, 2015), although their ...apparent rate is estimated to be much higher. Understanding tumor evolution based on a predisposed cell can open unknown doors for prevention and therapy of childhood cancer e.g., leukemia. Here we present a novel rare (MAF<0.1%) germline POT1 variant (Q199*) predisposing to acute myeloid leukemia (AML). POT1 as part of the telomeric shelterin complex is known to play an important role in DNA damage protection, telomere length maintenance and chromosomal stability (Calvete O. et al., Nat. Commun., 2015). POT1 variants are associated with a broad range of cancer, including myeloid and lymphoid neoplasms in adults (Lim T.L. et al., Leukemia, 2021), but not yet described for myeloid malignancies in childhood.
Methods: Whole exome sequencing (WES) was implemented to identify germline variants. To assess the effect of POT1 p.Q199*, patient's fibroblast and stably transfected HEK293T cells were used as cell models. The variant's functional impact was experimentally tested performing yH2AX and 53BP1 immunofluorescence assays for DNA damage detection, qRT-PCR for telomere length measurement and telomere FISH to assess chromosomal instability.
Results: Utilizing WES to detect variants within shelterin complex genes we analyzed genomic data of an unselected German parent-child cohort of children with cancer (n=60, TRIO-DD), as well as a recently published parent-child pediatric cancer cohort (n=158, TRIO-D) (Wagener R. et al., Eur. J. Hum. Genet, 2021). Here, we identified a novel germline POT1 variant in a boy affected with Myelodysplastic syndrome (MDS) and secondary AML (7q-). This novel germline variant constitutes a stop-gain mutation causing a substitution of the amino acid Glutamine by a stop codon (p.Q199*).
QRT-PCR analysis within the patient's fibroblasts showed a significant (student's t-test p=0.0037) reduction of POT1 mRNA expression to ≈0.5 compared to POT1 wildtype. Western Blot analysis revealed reduced POT1 levels, confirming the loss of one POT1 allele mediated by p.Q199*. Thereupon, POT1 p.Q199* cloning and stable transfection into Hek293T cells was performed to test the variant's cooperative functionality in a controlled environment. Subsequently, POT1 p.Q199* lead to a drastically significant (student's t-test p=<0.001) increase of DNA double strand breaks in transfected Hek293T cells determined by yH2AX and 53BP1 immunofluorescence assays, which is in line with a deregulated DNA damage response and inappropriate repair by non-homologous end joining.
In addition, we detected dysregulation of telomere length maintenance. Here, relative telomere length measurement by means of qRT-PCR indicated significant (student's t-test p=0.019) telomere elongation in POT1 p.Q199* fibroblast cells. Furthermore telomere FISH on metaphase chromosomes was performed to analyse chromosomal stability. In POT1 p.Q199* Hek293T cells we identified a significant (student's t-test p=0.002) increase in telomere fragility compared to POT1 WT cells.
Conclusion: Taken together, we present the functional effects of POT1 p.Q199* leading to a significant increase of DNA damage, telomere length and chromosomal instability. Our results on functional dysregulation strengthen a potential genetic predisposition to childhood AML mediated by germline POT1 variants.
No relevant conflicts of interest to declare.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Motivation
Alternative splicing is a biological process of fundamental importance in most eukaryotes. It plays a pivotal role in cell differentiation and gene regulation and has been ...associated with a number of different diseases. The widespread availability of RNA-Sequencing capacities allows an ever closer investigation of differentially expressed isoforms. However, most tools for differential alternative splicing (DAS) analysis do not take split reads, i.e. the most direct evidence for a splice event, into account. Here, we present DIEGO, a compositional data analysis method able to detect DAS between two sets of RNA-Seq samples based on split reads.
Results
The python tool DIEGO works without isoform annotations and is fast enough to analyze large experiments while being robust and accurate. We provide python and perl parsers for common formats.
Availability and implementation
The software is available at: www.bioinf.uni-leipzig.de/Software/DIEGO.
Supplementary information
Supplementary data are available at Bioinformatics online.
Application of next-generation sequencing may lead to the detection of secondary findings (SF) not related to the initially analyzed disease but to other severe medically actionable diseases. ...However, the analysis of SFs is not yet routinely performed. We mined whole-exome sequencing data of 231 pediatric cancer patients and their parents who had been treated in our center for the presence of SFs. By this approach, we identified in 6 children (2.6%) pathogenic germline variants in 5 of the noncancer-related genes on the American College of Medical Genetics and Genomics (ACMG) SF v3.0 list, of which the majority were related to cardiovascular diseases ( RYR2 , MYBPC3 , KCNQ1 ). Interestingly, only the patient harboring the KCNQ1 variant showed at the time point of the analysis signs of the related Long QT syndrome. Moreover, we report 3 variants of unknown significance which, although not classified as pathogenic, have been reported in the literature to occur in individuals with the respective disease. While the frequency of patients with SFs is low, the impact of such findings on the patients' life is enormous, with regard to the potential prevention of life-threatening diseases. Hence, we are convinced that such actionable SF should be routinely analyzed.
Summary
Extra‐nodal marginal zone B‐cell lymphoma (MZBL) of mucosa‐associated lymphoid tissue is an indolent lymphoma mostly affecting the gastrointestinal tract. The lymphoma initially has ...small‐cell morphology (SC‐MZBL) and often arises in the background of Helicobacter pylori‐induced gastritis. In some cases, a clonal malignant progression to large‐cell morphology (LC‐MZBL) is observed. Here, we studied the DNA methylation profile of 30 gastric MZBLs along their progression. Genome‐wide DNA methylation profiling, identified 7698 significantly differentially methylated loci during gastric MZBL progression (σ/σmax ≥0·4, q ≤ 0·001). LC‐MZBL showed hypermethylation in comparison to SC‐MZBL with an enrichment of regions involved in transcriptional regulation. In conclusion, our present data show that the morphological distinction between SC‐ and LC‐MZBL is reflected by characteristic DNA methylation profiles.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
PDF
