Publications by authors named "Pekka Ellonen"

54 Publications

Somatic mutations in lymphocytes in patients with immune-mediated aplastic anemia.

Leukemia 2021 Mar 30. Epub 2021 Mar 30.

Hematology Research Unit Helsinki, University of Helsinki and Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland.

The prevalence and functional impact of somatic mutations in nonleukemic T cells is not well characterized, although clonal T-cell expansions are common. In immune-mediated aplastic anemia (AA), cytotoxic T-cell expansions are shown to participate in disease pathogenesis. We investigated the mutation profiles of T cells in AA by a custom panel of 2533 genes. We sequenced CD4+ and CD8+ T cells of 24 AA patients and compared the results to 20 healthy controls and whole-exome sequencing of 37 patients with AA. Somatic variants were common both in patients and healthy controls but enriched to AA patients' CD8+ T cells, which accumulated most mutations on JAK-STAT and MAPK pathways. Mutation burden was associated with CD8+ T-cell clonality, assessed by T-cell receptor beta sequencing. To understand the effect of mutations, we performed single-cell sequencing of AA patients carrying STAT3 or other mutations in CD8+ T cells. STAT3 mutated clone was cytotoxic, clearly distinguishable from other CD8+ T cells, and attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality, and can alter T-cell phenotype, warranting further investigation of their role in the pathogenesis of AA.
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http://dx.doi.org/10.1038/s41375-021-01231-3DOI Listing
March 2021

Author Correction: Clonal hematopoiesis in patients with rheumatoid arthritis.

Blood Cancer J 2021 Feb 17;11(2):36. Epub 2021 Feb 17.

Hematology Research Unit Helsinki, University of Helsinki and Department of Hematology, Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland.

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http://dx.doi.org/10.1038/s41408-021-00427-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890050PMC
February 2021

Examining the effect of mitochondrial DNA variants on blood pressure in two Finnish cohorts.

Sci Rep 2021 Jan 12;11(1):611. Epub 2021 Jan 12.

Department of Clinical Chemistry, Fimlab Laboratories and Finnish Cardiovascular Research Center Tampere, Faculty of Medicine and Health Technology, Tampere University, Arvo Ylpön katu 34, PO Box 100, 33014, Tampere, Finland.

High blood pressure (BP) is a major risk factor for many noncommunicable diseases. The effect of mitochondrial DNA single-nucleotide polymorphisms (mtSNPs) on BP is less known than that of nuclear SNPs. We investigated the mitochondrial genetic determinants of systolic, diastolic, and mean arterial BP. MtSNPs were determined from peripheral blood by sequencing or with genome-wide association study SNP arrays in two independent Finnish cohorts, the Young Finns Study and the Finnish Cardiovascular Study, respectively. In total, over 4200 individuals were included. The effects of individual common mtSNPs, with an additional focus on sex-specificity, and aggregates of rare mtSNPs grouped by mitochondrial genes were evaluated by meta-analysis of linear regression and a sequence kernel association test, respectively. We accounted for the predicted pathogenicity of the rare variants within protein-encoding and the tRNA regions. In the meta-analysis of 87 common mtSNPs, we did not observe significant associations with any of the BP traits. Sex-specific and rare-variant analyses did not pinpoint any significant associations either. Our results are in agreement with several previous studies suggesting that mtDNA variation does not have a significant role in the regulation of BP. Future studies might need to reconsider the mechanisms thought to link mtDNA with hypertension.
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http://dx.doi.org/10.1038/s41598-020-79931-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804469PMC
January 2021

Adult-Onset Anti-Citrullinated Peptide Antibody-Negative Destructive Rheumatoid Arthritis Is Characterized by a Disease-Specific CD8+ T Lymphocyte Signature.

Front Immunol 2020 19;11:578848. Epub 2020 Nov 19.

Hematology Research Unit Helsinki, University of Helsinki, Helsinki, Finland.

Rheumatoid arthritis (RA) is a complex autoimmune disease targeting synovial joints. Traditionally, RA is divided into seropositive (SP) and seronegative (SN) disease forms, the latter consisting of an array of unrelated diseases with joint involvement. Recently, we described a severe form of SN-RA that associates with characteristic joint destruction. Here, we sought biological characteristics to differentiate this rare but aggressive anti-citrullinated peptide antibody-negative destructive RA (CND-RA) from early seropositive (SP-RA) and seronegative rheumatoid arthritis (SN-RA). We also aimed to study cytotoxic CD8+ lymphocytes in autoimmune arthritis. CND-RA, SP-RA and SN-RA were compared to healthy controls to reveal differences in T-cell receptor beta (TCRβ) repertoire, cytokine levels and autoantibody repertoires. Whole-exome sequencing (WES) followed by single-cell RNA-sequencing (sc-RNA-seq) was performed to study somatic mutations in a clonally expanded CD8+ lymphocyte population in an index patient. A unique TCRβ signature was detected in CND-RA patients. In addition, CND-RA patients expressed higher levels of the bone destruction-associated TNFSF14 cytokine. Blood IgG repertoire from CND-RA patients recognized fewer endogenous proteins than SP-RA patients' repertoires. Using WES, we detected a stable mutation profile in the clonally expanded CD8+ T-cell population characterized by cytotoxic gene expression signature discovered by sc-RNA-sequencing. Our results identify CND-RA as an independent RA subset and reveal a CND-RA specific TCR signature in the CD8+ lymphocytes. Improved classification of seronegative RA patients underlines the heterogeneity of RA and also, facilitates development of improved therapeutic options for the treatment resistant patients.
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http://dx.doi.org/10.3389/fimmu.2020.578848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732449PMC
November 2020

Somatic mutations and T-cell clonality in patients with immunodeficiency.

Haematologica 2020 12 1;105(12):2757-2768. Epub 2020 Dec 1.

Hematology Research Unit Helsinki, University of Helsinki, HUS, Helsinki, Finland.

Common variable immunodeficiency and other late-onset immunodeficiencies often co-manifest with autoimmunity and lymphoproliferation. The pathogenesis of most cases is elusive, as only a minor subset harbors known monogenic germline causes. The involvement of both B and T cells is however implicated. To study whether somatic mutations in CD4+ and CD8+ T cells associate with immunodeficiency, we recruited 17 patients and 21 healthy controls. Eight patients had late-onset common variable immunodeficiency and nine patients other immunodeficiency and/or severe autoimmunity. In total, autoimmunity occurred in 94% and lymphoproliferation in 65%. We performed deep sequencing of 2533 immune-associated genes from CD4+ and CD8+ cells. Deep T-cell receptor beta sequencing was used to characterize CD4+ and CD8+ T-cell receptor repertoires. The prevalence of somatic mutations was 65% in all immunodeficiency patients, 75% in common variable immunodeficiency and 48% in controls. Clonal hematopoiesis-associated variants in both CD4+ and CD8+ cells occurred in 24% of immunodeficiency patients. Results demonstrated mutations in known tumor suppressors, oncogenes, and genes that are critical for immune- and proliferative functions, such as STAT5B (two patients), C5AR1 (two patients), KRAS (one patient), and NOD2 (one patient). Additionally, as a marker of T-cell receptor repertoire perturbation, common variable immunodeficiency patients harbored increased frequencies of clones with identical complementarity determining region 3 sequences despite unique nucleotide sequences when compared to controls. In conclusion, somatic mutations in genes implicated for autoimmunity and lymphoproliferation are common in CD4+ and CD8+ cells of patients with immunodeficiency. They may contribute to immune dysregulation in a subset of immunodeficiency patients.
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http://dx.doi.org/10.3324/haematol.2019.220889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716374PMC
December 2020

Identifying haplotypes in recessive inherited retinal dystrophies using whole-genome linked-read sequencing.

Clin Genet 2021 Jan 18;99(1):193-198. Epub 2020 Sep 18.

Department of Ophthalmology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Conventional next-generation sequencing methods, used in most gene panels, cannot separate maternally and paternally derived sequence information of distant variants. In recessive diseases, two or more equally plausible causative variants with unsolved phase information prevent accurate molecular diagnosis. In reality, close relatives might be unavailable for segregation analysis. Here, we utilized whole genome linked-read sequencing to assign variants to haplotypes in two patients with inherited retinal dystrophies. Patient 1 with macular dystrophy had variants c.3442T>C, p.(Cys1148Arg), c.4209G>T, p.(Glu1403Asp), and c.1182C>T, p.(Cys394=) in CRB1, and Patient 2 with nonsyndromic retinitis pigmentosa had c.1328T>A, p.(Val443Asp) and c.3032C>G, p.(Ser1011*) in AHI1. The relatives were not available for genotyping. Using whole genome linked-read sequencing we phased the variants to haplotypes providing genetic background for the retinal dystrophies. In future, when the price of sequencing methods that provides long-read data decreases and their read-depth and accuracy increases, they are probably considered the primary or adjunctive sequencing methods in genetic testing, allowing the immediate collection of phase information and thus obviating the need for the carrier testing and segregation analysis.
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http://dx.doi.org/10.1111/cge.13847DOI Listing
January 2021

Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease.

Nat Commun 2020 05 7;11(1):2246. Epub 2020 May 7.

Hematology Research Unit Helsinki, Helsinki University Hospital Comprehensive Cancer Center, 00290, Helsinki, Finland.

Graft versus host disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report studies of a patient with chronic GvHD (cGvHD) carrying persistent CD4 T cell clonal expansion harboring somatic mTOR, NFKB2, and TLR2 mutations. In the screening cohort (n = 134), we detect the mTOR P2229R kinase domain mutation in two additional cGvHD patients, but not in healthy or HSCT patients without cGvHD. Functional analyses of the mTOR mutation indicate a gain-of-function alteration and activation of both mTORC1 and mTORC2 signaling pathways, leading to increased cell proliferation and decreased apoptosis. Single-cell RNA sequencing and real-time impedance measurements support increased cytotoxicity of mutated CD4 T cells. High throughput drug-sensitivity testing suggests that mutations induce resistance to mTOR inhibitors, but increase sensitivity for HSP90 inhibitors. Our findings imply that somatic mutations may contribute to aberrant T cell proliferations and persistent immune activation in cGvHD, thereby paving the way for targeted therapies.
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http://dx.doi.org/10.1038/s41467-020-16115-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206083PMC
May 2020

IDH1 Expression via the R132H Mutation-Specific Antibody in Adrenocortical Neoplasias-Prognostic Impact in Carcinomas.

J Endocr Soc 2020 Apr 18;4(4):bvaa018. Epub 2020 Feb 18.

Department of Pathology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Context: Mutations to isocitrate dehydrogenase (IDH) appear to play a prognostic or predictive role in several neoplasias. Immunohistochemical staining designed to detect a specific R132H mutation to IDH1 showed expression in the normal adrenal cortex, raising interest to study the potential role of IDH1 in the pathogenesis of adrenocortical tumors.

Objective: The objective of this work is to study the role of IDH1 and its mutations in adrenocortical tumors.

Design And Patients: R132H immunohistological staining was performed on a cohort of 197 adrenocortical tumors. The exon of the gene was sequenced in 16 tumors.

Results: Positive IDH1 R132H immunohistochemical staining correlated with a better prognosis among patients with a malignant adrenocortical tumor. However, IDH1 R132H immunohistochemistry did not distinguish between local and metastasized tumors. We were unable to identify IDH1 mutations among our adrenocortical tumors using a targeted next-generation sequencing panel or via exon sequencing.

Conclusions: Among adrenocortical carcinomas, IDH1 R132H immunopositivity correlated with a better prognosis. Thus, IDH1 R132H immunohistochemical staining could serve as a prognostic or as a potential predictive marker in adrenocortical carcinomas. Further research is needed to identify the possible alterations in IDH1 that could explain our findings, because we identified no known mutations to the gene.
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http://dx.doi.org/10.1210/jendso/bvaa018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069839PMC
April 2020

Mutation Is Associated with STAT3 Activation in CD30 ALK ALCL.

Cancers (Basel) 2020 Mar 16;12(3). Epub 2020 Mar 16.

Hematology Research Unit Helsinki, Department of Hematology, University of Helsinki and Helsinki University Hospital Comprehensive Cancer Center, 00290 Helsinki, Finland.

Peripheral T-cell lymphomas (PTCL) are a heterogeneous, and often aggressive group of non-Hodgkin lymphomas. Recent advances in the molecular and genetic characterization of PTCLs have helped to delineate differences and similarities between the various subtypes, and the JAK/STAT pathway has been found to play an important oncogenic role. Here, we aimed to characterize the JAK/STAT pathway in PTCL subtypes and investigate whether the activation of the pathway correlates with the frequency of gene mutations. Patient samples from AITL ( = 30), ALCL ( = 21) and PTCL-NOS ( = 12) cases were sequenced for , and mutations using amplicon sequencing and stained immunohistochemically for pSTAT3, pMAPK, and pAKT. We discovered mutations in 13% of AITL, 13% of ALK ALCL, 38% of ALK ALCL and 17% of PTCL-NOS cases. However, no mutations were found and mutations were only present in ALK ALCL (15%). Concurrent mutations were found in all subgroups except ALK ALCL where mutations were always seen alone. High pY-STAT3 expression was observed especially in AITL and ALCL samples. When studying JAK-STAT pathway mutations, pY-STAT3 expression was highest in PTCLs harboring either or mutations and CD30 phenotype representing primarily ALK ALCLs. Further investigation is needed to elucidate the molecular mechanisms of JAK-STAT pathway activation in PTCL.
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http://dx.doi.org/10.3390/cancers12030702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140109PMC
March 2020

Integrated drug profiling and CRISPR screening identify essential pathways for CAR T-cell cytotoxicity.

Blood 2020 02;135(9):597-609

Hematology Research Unit Helsinki, Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland.

Chimeric antigen receptor (CAR) T-cell therapy has proven effective in relapsed and refractory B-cell malignancies, but resistance and relapses still occur. Better understanding of mechanisms influencing CAR T-cell cytotoxicity and the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T-cell cytotoxicity using >500 small-molecule drugs and genome-scale CRISPR-Cas9 loss-of-function screens. We identified several tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling transcriptional activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells. CRISPR screens identified death receptor signaling through FADD and TNFRSF10B (TRAIL-R2) as a key mediator of CAR T-cell cytotoxicity and elucidated the RIPK1-dependent mechanism of sensitization by SMAC mimetics. Death receptor expression varied across genetic subtypes of B-cell malignancies, suggesting a link between mechanisms of CAR T-cell cytotoxicity and cancer genetics. These results implicate death receptor signaling as an important mediator of cancer cell sensitivity to CAR T-cell cytotoxicity, with potential for pharmacological targeting to enhance cancer immunotherapy. The screening data provide a resource of immunomodulatory properties of cancer drugs and genetic mechanisms influencing CAR T-cell cytotoxicity.
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http://dx.doi.org/10.1182/blood.2019002121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098811PMC
February 2020

SLC18A3 variants lead to fetal akinesia deformation sequence early in pregnancy.

Am J Med Genet A 2019 07 6;179(7):1362-1365. Epub 2019 May 6.

Department of Clinical Genetics, HUSLAB, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Fetal akinesia deformation sequence (FADS) and lethal multiple pterygium syndrome (LMPS) are clinically overlapping syndromes manifesting with reduced or absent fetal movement, arthrogryposis, and several anomalies during fetal life. The etiology of these syndromes is heterogeneous, and in many cases it remains unknown. In order to determine the genetic etiology of FADS in two fetuses with fetal akinesia, arthrogryposis, edema, and partial cleft palate, we utilized exome sequencing. Our investigations revealed a homozygous nonsense variant [c.1116C>A, p.(Cys372Ter)] in the SLC18A3 gene, which encodes for the vesicular acetylcholine transporter (VAChT) responsible for active transport of acetylcholine in the neuromuscular junction. This is the first description of a nonsense variant in the SLC18A3 gene, as only missense variants and whole gene deletions have been previously identified in patients. The previously detected SLC18A3 defects have been associated with congenital myasthenic syndromes, and therefore our findings extend the clinical spectrum of SLC18A3 defects to severe prenatal phenotypes. Our findings suggest that nonsense variants in SLC18A3 cause a more severe phenotype than missense variants and are in line with previous studies showing a lethal phenotype in VAChT knockout mice. Our results underline the importance of including SLC18A3 sequencing in the differential diagnostics of fetuses with arthrogryposis, FADS, or LMPS of unknown etiology.
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http://dx.doi.org/10.1002/ajmg.a.61186DOI Listing
July 2019

Gender-Specific Associations Between Saliva Microbiota and Body Size.

Front Microbiol 2019 10;10:767. Epub 2019 Apr 10.

Folkhälsan Research Center, Helsinki, Finland.

Objective: The human intestinal microbiota likely play an important role in the development of overweight and obesity. However, the associations between saliva microbiota and body mass index (BMI) have been sparsely studied. The aim of this study was to identify the associations between saliva microbiota and body size in Finnish children.

Methods: The saliva microbiota of 900 Finnish children, aged 11-14 years with measured height and weight, was characterized using 16S rRNA (V3-V4) sequencing.

Results: The core saliva microbiota consisted of 14 genera that were present in more than 95% of the Finnish children. The saliva microbiota profiles were gender-specific with higher alpha-diversity in boys than girls and significant differences between the genders in community composition and abundances. Alpha-diversity differed between normal weight and overweight girls and between normal weight and obese boys. The composition was dissimilar between normal weight and obese girls, but not in boys. The relative abundance profiles differed according to body size. Decrease in commensal saliva bacteria were observed in all the body sizes when compared to normal weight children. Notably, the relative abundance of bacteria related to, , , , and was reduced in obese children.

Conclusion: Saliva microbiota diversity and composition were significantly associated with body size and gender in Finnish children. Body size-specific saliva microbiota profiles open new avenues for studying the potential roles of microbiota in weight development and management.
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http://dx.doi.org/10.3389/fmicb.2019.00767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467948PMC
April 2019

TaME-seq: An efficient sequencing approach for characterisation of HPV genomic variability and chromosomal integration.

Sci Rep 2019 01 24;9(1):524. Epub 2019 Jan 24.

Department of Research, Cancer Registry of Norway, Oslo, Norway.

HPV genomic variability and chromosomal integration are important in the HPV-induced carcinogenic process. To uncover these genomic events in an HPV infection, we have developed an innovative and cost-effective sequencing approach named TaME-seq (tagmentation-assisted multiplex PCR enrichment sequencing). TaME-seq combines tagmentation and multiplex PCR enrichment for simultaneous analysis of HPV variation and chromosomal integration, and it can also be adapted to other viruses. For method validation, cell lines (n = 4), plasmids (n = 3), and HPV16, 18, 31, 33 and 45 positive clinical samples (n = 21) were analysed. Our results showed deep HPV genome-wide sequencing coverage. Chromosomal integration breakpoints and large deletions were identified in HPV positive cell lines and in one clinical sample. HPV genomic variability was observed in all samples allowing identification of low frequency variants. In contrast to other approaches, TaME-seq proved to be highly efficient in HPV target enrichment, leading to reduced sequencing costs. Comprehensive studies on HPV intra-host variability generated during a persistent infection will improve our understanding of viral carcinogenesis. Efficient identification of both HPV variability and integration sites will be important for the study of HPV evolution and adaptability and may be an important tool for use in cervical cancer diagnostics.
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http://dx.doi.org/10.1038/s41598-018-36669-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345795PMC
January 2019

Discovery of mitochondrial DNA variants associated with genome-wide blood cell gene expression: a population-based mtDNA sequencing study.

Hum Mol Genet 2019 04;28(8):1381-1391

Department of Clinical Chemistry, Fimlab Laboratories and Finnish Cardiovascular Research Center Tampere, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.

The effect of mitochondrial DNA (mtDNA) variation on peripheral blood transcriptomics in health and disease is not fully known. Sex-specific mitochondrially controlled gene expression patterns have been shown in Drosophila melanogaster but in humans, evidence is lacking. Functional variation in mtDNA may also have a role in the development of type 2 diabetes and its precursor state, i.e. prediabetes. We examined the associations between mitochondrial single-nucleotide polymorphisms (mtSNPs) and peripheral blood transcriptomics with a focus on sex- and prediabetes-specific effects. The genome-wide blood cell expression data of 19 637 probes, 199 deep-sequenced mtSNPs and nine haplogroups of 955 individuals from a population-based Young Finns Study cohort were used. Significant associations were identified with linear regression and analysis of covariance. The effects of sex and prediabetes on the associations between gene expression and mtSNPs were studied using random-effect meta-analysis. Our analysis identified 53 significant expression probe-mtSNP associations after Bonferroni correction, involving 7 genes and 31 mtSNPs. Eight probe-mtSNP signals remained independent after conditional analysis. In addition, five genes showed differential expression between haplogroups. The meta-analysis did not show any significant differences in linear model effect sizes between males and females but identified the association between the OASL gene and mtSNP C16294T to show prediabetes-specific effects. This study pinpoints new independent mtSNPs associated with peripheral blood transcriptomics and replicates six previously reported associations, providing further evidence of the mitochondrial genetic control of blood cell gene expression. In addition, we present evidence that prediabetes might lead to perturbations in mitochondrial control.
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http://dx.doi.org/10.1093/hmg/ddz011DOI Listing
April 2019

Genetic Control of Myelin Plasticity after Chronic Psychosocial Stress.

eNeuro 2018 Jul-Aug;5(4). Epub 2018 Jul 11.

Molecular and Integrative Biosciences Research Program, University of Helsinki, Helsinki FI-00014, Finland.

Anxiety disorders often manifest in genetically susceptible individuals after psychosocial stress, but the mechanisms underlying these gene-environment interactions are largely unknown. We used the chronic social defeat stress (CSDS) mouse model to study resilience and susceptibility to chronic psychosocial stress. We identified a strong genetic background effect in CSDS-induced social avoidance (SA) using four inbred mouse strains: 69% of C57BL/6NCrl (B6), 23% of BALB/cAnNCrl, 19% of 129S2/SvPasCrl, and 5% of DBA/2NCrl (D2) mice were stress resilient. Furthermore, different inbred mouse strains responded differently to stress, suggesting they use distinct coping strategies. To identify biological pathways affected by CSDS, we used RNA-sequencing (RNA-seq) of three brain regions of two strains, B6 and D2: medial prefrontal cortex (mPFC), ventral hippocampus (vHPC), and bed nucleus of the stria terminalis (BNST). We discovered overrepresentation of oligodendrocyte (OLG)-related genes in the differentially expressed gene population. Because OLGs myelinate axons, we measured myelin thickness and found significant region and strain-specific differences. For example, in resilient D2 mice, mPFC axons had thinner myelin than controls, whereas susceptible B6 mice had thinner myelin than controls in the vHPC. Neither myelin-related gene expression in several other regions nor corpus callosum thickness differed between stressed and control animals. Our unbiased gene expression experiment suggests that myelin plasticity is a substantial response to chronic psychosocial stress, varies across brain regions, and is genetically controlled. Identification of genetic regulators of the myelin response will provide mechanistic insight into the molecular basis of stress-related diseases, such as anxiety disorders, a critical step in developing targeted therapy.
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http://dx.doi.org/10.1523/ENEURO.0166-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071195PMC
March 2019

Clonal hematopoiesis in patients with rheumatoid arthritis.

Blood Cancer J 2018 07 26;8(8):69. Epub 2018 Jul 26.

Hematology Research Unit Helsinki, University of Helsinki and Department of Hematology, Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland.

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http://dx.doi.org/10.1038/s41408-018-0107-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066480PMC
July 2018

Case studies investigating genetic heterogeneity between anatomically distinct bone marrow compartments in acute myeloid leukemia.

Leuk Lymphoma 2018 12 4;59(12):3002-3005. Epub 2018 Apr 4.

a Institute for Molecular Medicine Finland, FIMM, University of Helsinki , Helsinki , Finland.

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http://dx.doi.org/10.1080/10428194.2018.1453067DOI Listing
December 2018

Reproducibility and repeatability of six high-throughput 16S rDNA sequencing protocols for microbiota profiling.

J Microbiol Methods 2018 04 18;147:76-86. Epub 2018 Mar 18.

Folkhälsan Research Center, Helsinki, Finland; Faculty of Medicine, University of Helsinki, Helsinki, Finland; Department of Research, Cancer Registry of Norway, Oslo, Norway. Electronic address:

Culture-independent molecular techniques and advances in next generation sequencing (NGS) technologies make large-scale epidemiological studies on microbiota feasible. A challenge using NGS is to obtain high reproducibility and repeatability, which is mostly attained through robust amplification. We aimed to assess the reproducibility of saliva microbiota by comparing triplicate samples. The microbiota was produced with simplified in-house 16S amplicon assays taking advantage of large number of barcodes. The assays included primers with Truseq (TS-tailed) or Nextera (NX-tailed) adapters and either with dual index or dual index plus a 6-nt internal index. All amplification protocols produced consistent microbial profiles for the same samples. Although, in our study, reproducibility was highest for the TS-tailed method. Five replicates of a single sample, prepared with the TS-tailed 1-step protocol without internal index sequenced on the HiSeq platform provided high alpha-diversity and low standard deviation (mean Shannon and Inverse Simpson diversity was 3.19 ± 0.097 and 13.56 ± 1.634 respectively). Large-scale profiling of microbiota can consistently be produced by all 16S amplicon assays. The TS-tailed-1S dual index protocol is preferred since it provides repeatable profiles on the HiSeq platform and are less labour intensive.
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http://dx.doi.org/10.1016/j.mimet.2018.03.003DOI Listing
April 2018

Breakpoint mapping and haplotype analysis of translocation t(1;12)(q43;q21.1) in two apparently independent families with vascular phenotypes.

Mol Genet Genomic Med 2018 01 23;6(1):56-68. Epub 2017 Nov 23.

Department of Health, National Institute for Health and Welfare, Helsinki, Finland.

Background: The risk of serious congenital anomaly for de novo balanced translocations is estimated to be at least 6%. We identified two apparently independent families with a balanced t(1;12)(q43;q21.1) as an outcome of a "Systematic Survey of Balanced Chromosomal Rearrangements in Finns." In the first family, carriers (n = 6) manifest with learning problems in childhood, and later with unexplained neurological symptoms (chronic headache, balance problems, tremor, fatigue) and cerebral infarctions in their 50s. In the second family, two carriers suffer from tetralogy of Fallot, one from transient ischemic attack and one from migraine. The translocation cosegregates with these vascular phenotypes and neurological symptoms.

Methods And Results: We narrowed down the breakpoint regions using mate pair sequencing. We observed conserved haplotypes around the breakpoints, pointing out that this translocation has arisen only once. The chromosome 1 breakpoint truncates a CHRM3 processed transcript, and is flanked by the 5' end of CHRM3 and the 3' end of RYR2. TRHDE, KCNC2, and ATXN7L3B flank the chromosome 12 breakpoint.

Conclusions: This study demonstrates a balanced t(1;12)(q43;q21.1) with conserved haplotypes on the derived chromosomes. The translocation seems to result in vascular phenotype, with or without neurological symptoms, in at least two families. We suggest that the translocation influences the positional expression of CHRM3, RYR2, TRHDE, KCNC2, and/or ATXN7L3B.
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http://dx.doi.org/10.1002/mgg3.346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823676PMC
January 2018

A novel class of somatic mutations in blood detected preferentially in CD8+ cells.

Clin Immunol 2017 02 5;175:75-81. Epub 2016 Dec 5.

Molecular Neurology, Research Programs Unit, University of Helsinki, Department of Neurology, Helsinki University Hospital, Haartmaninkatu 8, FIN-00290 Helsinki, Finland. Electronic address:

Somatic mutations have a central role in cancer but their role in other diseases such as autoimmune disorders is poorly understood. Earlier work has provided indirect evidence of rare somatic mutations in autoreactive T-lymphocytes in multiple sclerosis (MS) patients but such mutations have not been identified thus far. We analysed somatic mutations in blood in 16 patients with relapsing MS and 4 with other neurological autoimmune disease. To facilitate the detection of somatic mutations CD4+, CD8+, CD19+ and CD4-/CD8-/CD19- cell subpopulations were separated. We performed next-generation DNA sequencing targeting 986 immune-related genes. Somatic mutations were called by comparing the sequence data of each cell subpopulation to other subpopulations of the same patient and validated by amplicon sequencing. We found non-synonymous somatic mutations in 12 (60%) patients (10 MS, 1 myasthenia gravis, 1 narcolepsy). There were 27 mutations, all different and mostly novel (67%). They were discovered at subpopulation-wise allelic fractions of 0.2%-4.6% (median 0.95%). Multiple mutations were found in 8 patients. The mutations were enriched in CD8+ cells (85% of mutations). In follow-up after a median time of 2.3years, 96% of the mutations were still detectable. These results unravel a novel class of persistent somatic mutations, many of which were in genes that may play a role in autoimmunity (ATM, BTK, CD46, CD180, CLIP2, HMMR, IKFZF3, ITGB3, KIR3DL2, MAPK10, CD56/NCAM1, RBM6, RORA, RPA1 and STAT3). Whether some of this class of mutations plays a role in disease is currently unclear, but these results define an interesting hitherto unknown research target for future studies.
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http://dx.doi.org/10.1016/j.clim.2016.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341785PMC
February 2017

Genome-wide DNA methylation in saliva and body size of adolescent girls.

Epigenomics 2016 11 20;8(11):1495-1505. Epub 2016 Oct 20.

Genetic Epidemiology Group, Folkhälsan Research Center, Helsinki, Finland.

Aim: We performed an epigenome-wide association study within the Finnish Health in Teens cohort to identify differential DNA methylation and its association with BMI in adolescents.

Materials & Methods: Differential DNA methylation analyses of 3.1 million CpG sites were performed in saliva samples from 50 lean and 50 heavy adolescent girls by genome-wide targeted bisulfite-sequencing.

Results: We identified 100 CpG sites with p-values < 0.000524, seven regions by 'bumphunting' and five CpG islands that differed significantly between the two groups. The ten CpG sites and regions most strongly associated with BMI substantially overlapped with obesity- and insulin-related genes, including MC2R, IGFBPL1, IP6K1 and IGF2BP1.

Conclusion: Our findings suggest an association between the saliva methylome and BMI in adolescence.
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http://dx.doi.org/10.2217/epi-2016-0045DOI Listing
November 2016

Driver Gene and Novel Mutations in Asbestos-Exposed Lung Adenocarcinoma and Malignant Mesothelioma Detected by Exome Sequencing.

Lung 2016 Feb;194(1):125-35

Background: Asbestos is a carcinogen linked to malignant mesothelioma (MM) and lung cancer. Some gene aberrations related to asbestos exposure are recognized, but many associated mutations remain obscure. We performed exome sequencing to determine the association of previously known mutations (driver gene mutations) with asbestos and to identify novel mutations related to asbestos exposure in lung adenocarcinoma (LAC) and MM.

Methods: Exome sequencing was performed on DNA from 47 tumor tissues of MM (21) and LAC (26) patients, 27 of whom had been asbestos-exposed (18 MM, 9 LAC). In addition, 9 normal lung/blood samples of LAC were sequenced. Novel mutations identified from exome data were validated by amplicon-based deep sequencing. Driver gene mutations in BRAF, EGFR, ERBB2, HRAS, KRAS, MET, NRAS, PIK3CA, STK11, and ephrin receptor genes (EPHA1-8, 10 and EPHB1-4, 6) were studied for both LAC and MM, and in BAP1, CUL1, CDKN2A, and NF2 for MM.

Results: In asbestos-exposed MM patients, previously non-described NF2 frameshift mutation (one) and BAP1 mutations (four) were detected. Exome data mining revealed some genes potentially associated with asbestos exposure, such as MRPL1 and SDK1. BAP1 and COPG1 mutations were seen exclusively in MM. Pathogenic KRAS mutations were common in LAC patients (42 %), both in non-exposed (n = 5) and exposed patients (n = 6). Pathogenic BRAF mutations were found in two LACs.

Conclusion: BAP1 mutations occurred in asbestos-exposed MM. MRPL1, SDK1, SEMA5B, and INPP4A could possibly serve as candidate genes for alterations associated with asbestos exposure. KRAS mutations in LAC were not associated with asbestos exposure.
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http://dx.doi.org/10.1007/s00408-015-9814-7DOI Listing
February 2016

Targeted resequencing of the pericentromere of chromosome 2 linked to constitutional delay of growth and puberty.

PLoS One 2015 1;10(6):e0128524. Epub 2015 Jun 1.

Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland.

Constitutional delay of growth and puberty (CDGP) is the most common cause of pubertal delay. CDGP is defined as the proportion of the normal population who experience pubertal onset at least 2 SD later than the population mean, representing 2.3% of all adolescents. While adolescents with CDGP spontaneously enter puberty, they are at risk for short stature, decreased bone mineral density, and psychosocial problems. Genetic factors contribute heavily to the timing of puberty, but the vast majority of CDGP cases remain biologically unexplained, and there is no definitive test to distinguish CDGP from pathological absence of puberty during adolescence. Recently, we published a study identifying significant linkage between a locus at the pericentromeric region of chromosome 2 (chr 2) and CDGP in Finnish families. To investigate this region for causal variation, we sequenced chr 2 between the genomic coordinates of 79-124 Mb (genome build GRCh37) in the proband and affected parent of the 13 families contributing most to this linkage signal. One gene, DNAH6, harbored 6 protein-altering low-frequency variants (< 6% in the Finnish population) in 10 of the CDGP probands. We sequenced an additional 135 unrelated Finnish CDGP subjects and utilized the unique Sequencing Initiative Suomi (SISu) population reference exome set to show that while 5 of these variants were present in the CDGP set, they were also present in the Finnish population at similar frequencies. Additional variants in the targeted region could not be prioritized for follow-up, possibly due to gaps in sequencing coverage or lack of functional knowledge of non-genic genomic regions. Thus, despite having a well-characterized sample collection from a genetically homogeneous population with a large population-based reference sequence dataset, we were unable to pinpoint variation in the linked region predisposing delayed puberty. This study highlights the difficulties of detecting genetic variants under linkage regions for complex traits and suggests that advancements in annotation of gene function and regulatory regions of the genome will be critical for solving the genetic background of complex phenotypes like CDGP.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128524PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4452275PMC
April 2016

mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling.

Cell Rep 2015 Jun 28;11(10):1614-24. Epub 2015 May 28.

Research Programs Unit, Molecular Neurology, Biomedicum-Helsinki, University of Helsinki, 00290 Helsinki, Finland; Department of Neurology, Helsinki University Central Hospital, 00290 Helsinki, Finland; Neuroscience Center, University of Helsinki, 00290 Helsinki, Finland. Electronic address:

mtDNA mutagenesis in somatic stem cells leads to their dysfunction and to progeria in mouse. The mechanism was proposed to involve modification of reactive oxygen species (ROS)/redox signaling. We studied the effect of mtDNA mutagenesis on reprogramming and stemness of pluripotent stem cells (PSCs) and show that PSCs select against specific mtDNA mutations, mimicking germline and promoting mtDNA integrity despite their glycolytic metabolism. Furthermore, mtDNA mutagenesis is associated with an increase in mitochondrial H2O2, reduced PSC reprogramming efficiency, and self-renewal. Mitochondria-targeted ubiquinone, MitoQ, and N-acetyl-L-cysteine efficiently rescued these defects, indicating that both reprogramming efficiency and stemness are modified by mitochondrial ROS. The redox sensitivity, however, rendered PSCs and especially neural stem cells sensitive to MitoQ toxicity. Our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function.
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http://dx.doi.org/10.1016/j.celrep.2015.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4509707PMC
June 2015

The analysis of clonal diversity and therapy responses using STAT3 mutations as a molecular marker in large granular lymphocytic leukemia.

Haematologica 2015 Jan 3;100(1):91-9. Epub 2014 Oct 3.

Hematology Research Unit, Department of Hematology, University of Helsinki and Helsinki University Central Hospital Cancer Center, Helsinki, Finland

T-cell large granular lymphocytic leukemia and chronic lymphoproliferative disorder of natural killer cells are intriguing entities between benign and malignant lymphoproliferation. The molecular pathogenesis has partly been uncovered by the recent discovery of somatic activating STAT3 and STAT5b mutations. Here we show that 43% (75/174) of patients with T-cell large granular lymphocytic leukemia and 18% (7/39) with chronic lymphoproliferative disorder of natural killer cells harbor STAT3 mutations when analyzed by quantitative deep amplicon sequencing. Surprisingly, 17% of the STAT3-mutated patients carried multiple STAT3 mutations, which were located in different lymphocyte clones. The size of the mutated clone correlated well with the degree of clonal expansion of the T-cell repertoire analyzed by T-cell receptor beta chain deep sequencing. The analysis of sequential samples suggested that current immunosuppressive therapy is not able to reduce the level of the mutated clone in most cases, thus warranting the search for novel targeted therapies. Our findings imply that the clonal landscape of large granular lymphocytic leukemia is more complex than considered before, and a substantial number of patients have multiple lymphocyte subclones harboring different STAT3 mutations, thus mimicking the situation in acute leukemia.
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http://dx.doi.org/10.3324/haematol.2014.113142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281318PMC
January 2015

Germline mutation of RPS20, encoding a ribosomal protein, causes predisposition to hereditary nonpolyposis colorectal carcinoma without DNA mismatch repair deficiency.

Gastroenterology 2014 Sep 15;147(3):595-598.e5. Epub 2014 Jun 15.

Department of Medical Genetics, Haartman Institute, University of Helsinki, Helsinki, Finland.

Little is known about the genetic factors that contribute to familial colorectal cancer type X (FCCX), characterized by hereditary nonpolyposis colorectal carcinoma with no mismatch repair defects. Genetic linkage analysis, exome sequencing, tumor studies, and functional investigations of 4 generations of a FCCX family led to the identification of a truncating germline mutation in RPS20, which encodes a component (S20) of the small ribosomal subunit and is a new colon cancer predisposition gene. The mutation was associated with a defect in pre-ribosomal RNA maturation. Our findings show that mutations in a gene encoding a ribosomal protein can predispose individuals to microsatellite-stable colon cancer. Evaluation of additional FCCX families for mutations in RPS20 and other ribosome-associated genes is warranted.
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http://dx.doi.org/10.1053/j.gastro.2014.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4155505PMC
September 2014

Novel TBL1XR1, EPHA7 and SLFN12 mutations in a Sezary syndrome patient discovered by whole exome sequencing.

Exp Dermatol 2014 May;23(5):366-8

Hematology Research Unit Helsinki, Department of Hematology, University of Helsinki and Helsinki University Central Hospital Cancer Center, Helsinki, Finland.

Sezary syndrome (SS) is an aggressive leukaemic variant of cutaneous T-cell lymphoma. Recurrent chromosomal aberrations have been found in SS, but the whole genetic mutation spectrum is unknown. To better understand the molecular pathogenesis of SS, we performed exome sequencing, copy number variation (CNV) and gene expression analysis of primary SS cells. In our index patient with typical SS, we found novel somatic missense mutations in TBL1XR1, EPHA7 and SLFN12 genes in addition to larger chromosomal changes. The mutations are located in biologically relevant genes affecting apoptosis and T-cell maturation. They may play a role in the pathobiology of the disease, but no recurrent mutations were discovered in nine additional patients with SS studied. Thus, screening of larger patient cohorts is needed to confirm their prevalence and biological significance in SS.
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http://dx.doi.org/10.1111/exd.12405DOI Listing
May 2014

Copy number alterations and neoplasia-specific mutations in MELK, PDCD1LG2, TLN1, and PAX5 at 9p in different neoplasias.

Genes Chromosomes Cancer 2014 Jul 24;53(7):579-88. Epub 2014 Mar 24.

Department of Pathology, Haartman Institute and HUSLAB, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland.

Genetic alterations affecting 9p are commonly present in many cancer types and many cancer-related genes are located in this chromosomal region. We sequenced all of the genes located in a 32Mb region of 9p by targeted next generation sequencing (NGS) in 96 patients with different cancer types, including acute lymphoblastic leukemia, bone malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma, fibrosarcoma, Ewing's sarcoma, and lung carcinoma. Copy number alterations (CNA), and mutations were studied from the NGS data. We detected a deletion at the CDKN2A locus as being the most frequent genetic alteration in all cancer types. In addition to this locus, NGS also identified other small regions of copy number loss and gain. However, different cancer types did not reveal any statistically significant differences with regard to CNA frequency or type. Of the 191 genes within the target region, two novel recurrent mutations were found in the MELK and PDCD1LG2 genes. The most commonly mutated gene in sarcomas was TLN1 (8%) and PAX5 in ALL (9%). Mutations in PAX5, and RUSC2, were seen exclusively in ALL patients and those in KIAA1432, CA9, TLN1, and MELK only in sarcomas (MFH, FS, EFT). Thus using targeted NGS of the 9p region, in addition to commonly deleted CDKN2A locus, we were able to identify a number of small deletions and gains, as well as novel recurrent mutations in different cancer types. © 2014 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/gcc.22168DOI Listing
July 2014

Mutated ephrin receptor genes in non-small cell lung carcinoma and their occurrence with driver mutations-targeted resequencing study on formalin-fixed, paraffin-embedded tumor material of 81 patients.

Genes Chromosomes Cancer 2013 Dec 7;52(12):1141-9. Epub 2013 Oct 7.

Department of Pathology, Haartman Institute, University of Helsinki, Finland.

Non-small cell lung carcinoma (NSCLC) is the most common subtype of lung cancer. The oncogenic potential of receptor tyrosine kinases (RTKs) is widely known and they are potential targets for tailored therapy. Ephrin receptors (Ephs) form the largest group of RTKs. Nevertheless, Ephs are not widely studied in NSCLC so far. The aim of our study was to investigate novel mutations of Eph genes (EPHA1-8, EPHB1-4, EPHB6) and their association with clinically relevant mutations in BRAF, EML4-ALK, EGFR, INSR, KDR, KRAS, MET, PDGFRA, PDGFRB, PIK3, PTEN, RET, and TP53 in NSCLC patients. Targeted resequencing was conducted on 81 formalin-fixed, paraffin-embedded NSCLC tumor specimens. We analyzed missense and nonsense mutations harbored in the coding regions of the selected genes. We found 18 novel mutations of Ephs in 20% (16 of 81) of the patients. Nearly half of these mutations occurred in the protein kinase domain. The mutations were not mutually exclusive with other clinically relevant mutations. Our study shows that Ephs are frequently mutated in NSCLC patients, and occur together with other known mutations relevant to the pathogenicity of NSCLC.
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http://dx.doi.org/10.1002/gcc.22109DOI Listing
December 2013