SF3B1 mutations are recurrent in chronic lymphocytic leukemia (CLL), particularly enriched in clinically aggressive stereotyped subset #2. To investigate their impact, we conducted RNA-sequencing of 18 SF3B1MUT and 17 SF3B1WT subset #2 cases and identified 80 significant alternative splicing events (ASEs). Notable ASEs concerned exon inclusion in the non-canonical BAF (ncBAF) chromatin remodeling complex subunit, BRD9, and splice variants in eight additional ncBAF complex interactors. Long-read RNA-sequencing confirmed the presence of splice variants, and extended analysis of 139 CLL cases corroborated their association with SF3B1 mutations. Overexpression of SF3B1K700E induced exon inclusion in BRD9, resulting in a novel splice isoform with an alternative C-terminus. Protein interactome analysis of the BRD9 splice isoform revealed augmented ncBAF complex interaction, while exhibiting decreased binding of auxiliary proteins, including SPEN, BRCA2, and CHD9. Additionally, integrative multi-omics analysis identified a ncBAF complex-bound gene quartet on chromosome 1 with higher expression levels and more accessible chromatin in SF3B1MUT CLL. Finally, Cancer Dependency Map analysis and BRD9 inhibition displayed BRD9 dependency and sensitivity in cell lines and primary CLL cells. In conclusion, spliceosome dysregulation caused by SF3B1 mutations leads to multiple ASEs and an altered ncBAF complex interactome, highlighting a novel pathobiological mechanism in SF3B1MUT CLL.

• MeSH
• Alternative Splicing MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell * genetics   pathology   metabolism   MeSH
• Phosphoproteins * genetics   metabolism   MeSH
• Humans MeSH
• Mutation * MeSH
• Bromodomain Containing Proteins MeSH
• Chromatin Assembly and Disassembly * MeSH
• RNA Splicing Factors * genetics   metabolism   MeSH
• Spliceosomes * metabolism   genetics   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Most succinate dehydrogenase (SDH)-deficient renal cell carcinomas (RCCs) demonstrate stereotypical morphology characterized by bland eosinophilic cells with frequent intracytoplasmic inclusions. However, variant morphologic features have been increasingly recognized. We therefore sought to investigate the incidence and characteristics of SDH-deficient RCC with variant morphologies. We studied a multi-institutional cohort of 62 new SDH-deficient RCCs from 59 patients. The median age at presentation was 39 years (range 19-80), with a slight male predominance (M:F = 1.6:1). A relevant family history was reported in 9 patients (15%). Multifocal or bilateral tumors were identified radiologically in 5 patients (8%). Typical morphology was present at least focally in 59 tumors (95%). Variant morphologies were seen in 13 (21%) and included high-grade nuclear features and various combinations of papillary, solid, and tubular architecture. Necrosis was present in 13 tumors, 7 of which showed variant morphology. All 62 tumors demonstrated loss of SDHB expression by immunohistochemistry. None showed loss of SDHA expression. Germline SDH mutations were reported in all 18 patients for whom the results of testing were known. Among patients for whom follow-up data was available, metastatic disease was reported in 9 cases, 8 of whom had necrosis and/or variant morphology in their primary tumor. Three patients died of disease. In conclusion, variant morphologies and high-grade nuclear features occur in a subset of SDH-deficient RCCs and are associated with more aggressive behavior. We therefore recommend grading all SDH-deficient RCCs and emphasize the need for a low threshold for performing SDHB immunohistochemistry in any difficult to classify renal tumor, particularly if occurring at a younger age.

• MeSH
• Adult MeSH
• Hyperplasia MeSH
• Immunohistochemistry MeSH
• Carcinoma, Renal Cell * genetics   pathology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Kidney Neoplasms * genetics   pathology   MeSH
• Necrosis MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Succinate Dehydrogenase genetics   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Key processes in the onset and evolution of chronic lymphocytic leukemia (CLL) are thought to include chronic (antigenic) activation of mature B cells through the B cell receptor (BcR), signals from the microenvironment, and acquisition of genetic alterations. Here we describe three families in which two or more siblings were affected by CLL. We investigated whether there are immunogenetic similarities in the leukemia-specific immunoglobulin heavy (IGH) and light (IGL/IGK) chain gene rearrangements of the siblings in each family. Furthermore, we performed array analysis to study if similarities in CLL-associated chromosomal aberrations are present within each family and screened for somatic mutations using paired tumor/normal whole-genome sequencing (WGS). In two families a consistent IGHV gene mutational status (one IGHV-unmutated, one IGHV-mutated) was observed. Intriguingly, the third family with four affected siblings was characterized by usage of the lambda IGLV3-21 gene, with the hallmark R110 mutation of the recently described clinically aggressive IGLV3-21R110 subset. In this family, the CLL-specific rearrangements in two siblings could be assigned to either stereotyped subset #2 or the immunogenetically related subset #169, both of which belong to the broader IGLV3-21R110 subgroup. Consistent patterns of cytogenetic aberrations were encountered in all three families. Furthermore, the CLL clones carried somatic mutations previously associated with IGHV mutational status, cytogenetic aberrations and stereotyped subsets, respectively. From these findings, we conclude that similarities in immunogenetic characteristics in familial CLL, in combination with genetic aberrations acquired, point towards shared underlying mechanisms behind CLL development within each family.

• Publication type
• Journal Article MeSH

Chronic lymphocytic leukemia (CLL) major stereotyped subset 2 (IGHV3-21/IGLV3-21, ∼2.5% of all cases of CLL) is an aggressive disease variant, irrespective of the somatic hypermutation (SHM) status of the clonotypic IGHV gene. Minor stereotyped subset 169 (IGHV3-48/IGLV3-21, ∼0.2% of all cases of CLL) is related to subset 2, as it displays a highly similar variable antigen-binding site. We further explored this relationship through next-generation sequencing and crystallographic analysis of the clonotypic B-cell receptor immunoglobulin. Branching evolution of the predominant clonotype through intraclonal diversification in the context of ongoing SHM was evident in both heavy and light chain genes of both subsets. Molecular similarities between the 2 subsets were highlighted by the finding of shared SHMs within both the heavy and light chain genes in all analyzed cases at either the clonal or subclonal level. Particularly noteworthy in this respect was a ubiquitous SHM at the linker region between the variable and the constant domain of the IGLV3-21 light chains, previously reported as critical for immunoglobulin homotypic interactions underlying cell-autonomous signaling capacity. Notably, crystallographic analysis revealed that the IGLV3-21-bearing CLL subset 169 immunoglobulin retains the same geometry and contact residues for the homotypic intermolecular interaction observed in subset 2, including the SHM at the linker region, and, from a molecular standpoint, belong to a common structural mode of autologous recognition. Collectively, our findings document that stereotyped subsets 2 and 169 are very closely related, displaying shared immunoglobulin features that can be explained only in the context of shared functional selection.

• MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell genetics   MeSH
• Gene Rearrangement MeSH
• Genes, Immunoglobulin Heavy Chain genetics   MeSH
• HEK293 Cells MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Models, Molecular MeSH
• Protein Domains MeSH
• Receptors, Antigen, B-Cell chemistry   genetics   MeSH
• Gene Expression Regulation, Leukemic MeSH
• Somatic Hypermutation, Immunoglobulin MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

• MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell genetics   MeSH
• Gene Frequency MeSH
• Gene Rearrangement MeSH
• Humans MeSH
• Somatic Hypermutation, Immunoglobulin MeSH
• Immunoglobulin Heavy Chains genetics   MeSH
• Immunoglobulin Variable Region genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

CD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling. Here we use single cell RNA sequencing (scRNA-seq) to classify mouse thymocyte selection intermediates by coreceptor gene expression. In the unperturbed thymus, Cd4+Cd8a- selection intermediates appear before Cd4-Cd8a+ selection intermediates, but the timing of these subsets is flexible according to the strength of TCR signals. Our data show that selection intermediates discriminate MHC class prior to the loss of coreceptor expression and suggest a model where signal strength informs the timing of coreceptor gene activity and ultimately CD4/CD8 lineage choice.

• MeSH
• Lymphocyte Activation genetics   MeSH
• Principal Component Analysis MeSH
• Cell Differentiation immunology   MeSH
• Cell Lineage immunology   MeSH
• CD4-Positive T-Lymphocytes cytology   immunology   MeSH
• CD8-Positive T-Lymphocytes cytology   immunology   MeSH
• Cytokines metabolism   MeSH
• DNA-Binding Proteins metabolism   MeSH
• Histocompatibility Antigens metabolism   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Core Binding Factor Alpha 3 Subunit metabolism   MeSH
• Receptors, Antigen, T-Cell metabolism   MeSH
• Gene Expression Regulation MeSH
• Signal Transduction MeSH
• Thymus Gland cytology   immunology   MeSH
• Transcription Factors metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Chronická lymfatická leukémie (CLL) je nejběžnější formou leukemie u dospělé populace v západním světě. Jde o biologicky heterogenní onemocnění s různorodým klinickým dopadem, které je v dnešní době neléčitelné. Překvapivě, až u třetiny pacientů s CLL je možno identifikovat velice podobné B-buněčné receptory, které lze využít k rozdělení případů do podskupin (CLL podskupin). Případy mají v rámci jedné podskupiny nečekaně homogenní klinický průběh i biologické znaky. Majoritní častí naší bioinformatické práce by bylo navrhnout, zaplnit, analyzovat, „vybavit“ a sdílet encyklopedii CLL podskupin. Měla by za cíl komplexním a opakovatelným způsobem provázet vývojem, výzkumem a klinickou aplikací CLL podskupin, stala by se potřebným referenčním bodem v celém oboru. Díky možnosti porovnávání nových CLL případů s touto znalostní databází by encyklopedie našla využití v aplikacích personalizované medicíny, jako je návrh nových biomarkerů nebo výběr konkrétní efektivní léčby, což by mělo přímý dopad na desítky tisíc pacientů.; Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the West, biologically and clinically heterogeneous with varying clinical outcome, and currently incurable. Remarkably, highly similar, or stereotyped, B cell receptors can be identified in one out of three CLL patients – leading to their inclusion in subsets of cases, or CLL subsets, with intriguing homogeneity in clinical presentation and outcome as well as biological features. Our bioinformatics-heavy proposal is to design, populate, analyse, ‘equip’, and share the Encyclopedia of CLL Subsets. It has the ambition to guide research, development, and application of CLL subsets in a linkable and comparable way, becoming a much-needed reference point for the field. And by connecting new CLL cases to an encyclopedia rich with up-to-date knowledge, it will eventually enable personalised biomedical applications, such as the use of novel predictive biomarkers and treatment targets, and the design of effective therapy – with an impact on many tens of thousands of patients.

• MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell diagnosis   classification   MeSH
• Databases, Factual MeSH
• Precision Medicine methods   MeSH
• Humans MeSH
• Biomarkers, Tumor MeSH
• B-Lymphocyte Subsets MeSH
• Prognosis MeSH
• Computational Biology methods   MeSH
• Check Tag
• Humans MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• onkologie
• knihovnictví, informační věda a muzeologie
• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

Bi-allelic loss-of-function variants in genes that encode subunits of the adaptor protein complex 4 (AP-4) lead to prototypical yet poorly understood forms of childhood-onset and complex hereditary spastic paraplegia: SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). Here, we report a detailed cross-sectional analysis of clinical, imaging and molecular data of 156 patients from 101 families. Enrolled patients were of diverse ethnic backgrounds and covered a wide age range (1.0-49.3 years). While the mean age at symptom onset was 0.8 ± 0.6 years [standard deviation (SD), range 0.2-5.0], the mean age at diagnosis was 10.2 ± 8.5 years (SD, range 0.1-46.3). We define a set of core features: early-onset developmental delay with delayed motor milestones and significant speech delay (50% non-verbal); intellectual disability in the moderate to severe range; mild hypotonia in infancy followed by spastic diplegia (mean age: 8.4 ± 5.1 years, SD) and later tetraplegia (mean age: 16.1 ± 9.8 years, SD); postnatal microcephaly (83%); foot deformities (69%); and epilepsy (66%) that is intractable in a subset. At last follow-up, 36% ambulated with assistance (mean age: 8.9 ± 6.4 years, SD) and 54% were wheelchair-dependent (mean age: 13.4 ± 9.8 years, SD). Episodes of stereotypic laughing, possibly consistent with a pseudobulbar affect, were found in 56% of patients. Key features on neuroimaging include a thin corpus callosum (90%), ventriculomegaly (65%) often with colpocephaly, and periventricular white-matter signal abnormalities (68%). Iron deposition and polymicrogyria were found in a subset of patients. AP4B1-associated SPG47 and AP4M1-associated SPG50 accounted for the majority of cases. About two-thirds of patients were born to consanguineous parents, and 82% carried homozygous variants. Over 70 unique variants were present, the majority of which are frameshift or nonsense mutations. To track disease progression across the age spectrum, we defined the relationship between disease severity as measured by several rating scales and disease duration. We found that the presence of epilepsy, which manifested before the age of 3 years in the majority of patients, was associated with worse motor outcomes. Exploring genotype-phenotype correlations, we found that disease severity and major phenotypes were equally distributed among the four subtypes, establishing that SPG47, SPG50, SPG51 and SPG52 share a common phenotype, an 'AP-4 deficiency syndrome'. By delineating the core clinical, imaging, and molecular features of AP-4-associated hereditary spastic paraplegia across the age spectrum our results will facilitate early diagnosis, enable counselling and anticipatory guidance of affected families and help define endpoints for future interventional trials.

• MeSH
• Adaptor Protein Complex 4 genetics   MeSH
• Corpus Callosum diagnostic imaging   MeSH
• Child MeSH
• Adult MeSH
• Cohort Studies MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Magnetic Resonance Imaging methods   trends   MeSH
• Adolescent MeSH
• Young Adult MeSH
• Child, Preschool MeSH
• Cross-Sectional Studies MeSH
• Registries MeSH
• Spastic Paraplegia, Hereditary diagnostic imaging   genetics   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

AIMS: This is a nation-wide survey of chronic lymphocytic leukemia (CLL) patients at six large hematology centers in the Czech Republic. The aim was to identify specific populations, social, and health characteristics of CLL subgroups divided according to the immunogenetic features of their B cell receptors (BCRs) and clonality. PATIENTS AND METHODS: Questionnaires directed to specific health, social, and environmental conditions were collected in a cohort of 573 CLL patients. For these patients, immunoglobulin heavy chain gene rearrangements were also analyzed in order to gain information about their clonality, IGHV mutational status, and the presence of stereotyped BCRs. Data extracted from the questionnaires were analyzed statistically in the context of immunogenetic features of the cohort. RESULTS: There were no statistically significant differences in the data collected in the survey between patients with mutated and patients with unmutated IGHV. However, patients with oligoclonal CLL reported health conditions such as hypercholesterolemia, hypertension, herpes simplex, tumors, and also, separately, CLL in 1st degree relatives, more often than their monoclonal counterparts. In patients with stereotyped BCRs, we found more frequent alcohol consumption and gastric infections in subset #1 cases and frequent cholecystectomies and familial CLL in subset #2 cases. CONCLUSION: To the best of our knowledge, this study is the first to investigate CLL immunogenetic features and clonality in the context of epidemiological data. We reported statistically significant associations suggesting the influence of certain health and social conditions on a number of clonal populations expanding in CLL and also on characteristic BCR features, especially stereotypy.

• MeSH
• Biological Factors * MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell epidemiology   genetics   immunology   MeSH
• Adult MeSH
• Genetic Variation * MeSH
• Human Genetics MeSH
• Genotype MeSH
• Immunogenetics * MeSH
• Cohort Studies MeSH
• Middle Aged MeSH
• Humans MeSH
• Receptors, Antigen, B-Cell genetics   MeSH
• Risk Factors MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Geographicals
• Czech Republic MeSH

Chronic lymphocytic leukemia (CLL) patients with differential somatic hypermutation status of the immunoglobulin heavy variable genes, namely mutated or unmutated, display fundamental clinico-biological differences. Considering this, we assessed prognosis separately within mutated (M-CLL) and unmutated (U-CLL) CLL in 3015 patients, hypothesizing that the relative significance of relevant indicators may differ between these two categories. Within Binet A M-CLL patients, besides TP53 abnormalities, trisomy 12 and stereotyped subset #2 membership were equivalently associated with the shortest time-to-first-treatment and a treatment probability at five and ten years after diagnosis of 40% and 55%, respectively; the remaining cases exhibited 5-year and 10-year treatment probability of 12% and 25%, respectively. Within Binet A U-CLL patients, besides TP53 abnormalities, del(11q) and/or SF3B1 mutations were associated with the shortest time-to-first-treatment (5- and 10-year treatment probability: 78% and 98%, respectively); in the remaining cases, males had a significantly worse prognosis than females. In conclusion, the relative weight of indicators that can accurately risk stratify early-stage CLL patients differs depending on the somatic hypermutation status of the immunoglobulin heavy variable genes of each patient. This finding highlights the fact that compartmentalized approaches based on immunogenetic features are necessary to refine and tailor prognostication in CLL. Copyright © 2019 Ferrata Storti Foundation.

• MeSH
• Time-to-Treatment MeSH
• Chromosome Aberrations MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell * MeSH
• Immunogenetics MeSH
• Kaplan-Meier Estimate MeSH
• Humans MeSH
• Mutation MeSH
• Disease Susceptibility * MeSH
• Biomarkers, Tumor * MeSH
• Prognosis MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Neoplasm Staging MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH