Cultured lymphocytes' mitochondrial genome integrity is not altered by cladribine.

Cladribine tablets are a treatment for multiple sclerosis with effects on lymphocytes, yet its mode of action has not been fully established. Here, we analyzed the effects of cladribine on mitochondrial DNA integrity in lymphocytes. We treated cultured human T-cell lines (CCRF-CEM and Jurkat) with varying concentrations of cladribine to mimic the slow cell depletion observed in treated patients. The CCRF-CEM was more susceptible to cladribine than Jurkat cells. In both cells, mitochondrial protein synthesis, mitochondrial DNA copy number, and mitochondrial cytochrome-c oxidase-I mRNA mutagenesis was not affected by cladribine, while caspase-3 cleavage was detected in Jurkat cells at 100 nM concentration. Cladribine treatment at concentrations up to 10 nM in CCRF-CEM and 100 nM in Jurkat cells did not induce significant increase in mitochondrial DNA mutations. Peripheral blood mononuclear cells from eight multiple sclerosis patients and four controls were cultured with or without an effective dose of cladribine (5 nM). However, we did not find any differences in mitochondrial DNA somatic mutations in lymphocyte subpopulations (CD4+, CD8+, and CD19+) between treated versus nontreated cells. The overall mutation rate was similar in patients and controls. When different lymphocyte subpopulations were compared, greater mitochondrial DNA mutation levels were detected in CD8+ (P = 0.014) and CD4+ (P = 0.038) as compared to CD19+ cells, these differences were independent of cladribine treatment. We conclude that T cells have more detectable mitochondrial DNA mutations than B cells, and cladribine has no detectable mutagenic effect on lymphocyte mitochondrial genome nor does it impair mitochondrial function in human T-cell lines.

High Epstein-Barr virus capsid antigen IgG level associates with the carriership of CD8+ T cell somatic mutations in the STAT3 SH2 domain.

High carrier prevalence of STAT3 SH2 domain somatic mutations was recently discovered in CD8+ T cells. We found these low-allele-fraction clones in 26% of donors, without difference between multiple sclerosis (MS) patients and controls. Here we tested whether anti-viral antibodies associate with the carriership of these mutant clones. We compared antibody responses against common viruses in mutation carriers vs. non-carriers. Plasma samples of 152 donors (92 MS patients, 60 controls) were analyzed for antibodies against cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus-6A and parvovirus B19. The mutation carrier status associated with EBV VCA IgG level (p = 0.005) and remained significant after logistic regression (p = 0.036). This association was contributed similarly by MS patients and controls. These results suggest that EBV contributes to the generation or growth of these clones. The pathogenic role of the STAT3 mutant clones in MS is presently unclear, but their detailed characterization warrants further study.

High prevalence of low-allele-fraction somatic mutations in STAT3 in peripheral blood CD8+ cells in multiple sclerosis patients and controls.

Somatic mutations have a central role in cancer, but there are also a few rare autoimmune diseases in which somatic mutations play a major role. We have recently shown that nonsynonymous somatic mutations with low allele fractions are preferentially detectable in CD8+ cells and that the STAT3 gene is a promising target for screening. Here, we analyzed somatic mutations in the STAT3 SH2 domain in peripheral blood CD8+ cells in a set of 94 multiple sclerosis (MS) patients and 99 matched controls. PCR amplicons targeting the exons 20 and 21 of STAT3 were prepared and sequenced using the Illumina MiSeq instrument with 2x300bp reads. We designed a novel variant calling method, optimized for large number of samples, high sequencing depth (>25,000x) and small target genomic area. Overall, we discovered 64 STAT3 somatic mutations in the 193 donors, of which 63 were non-synonymous and 77% have been previously reported in cancer or lymphoproliferative disease. The overall median variant allele fraction was 0.065% (range 0.007-1.2%), without significant difference between MS and controls (p = 0.82). There were 26 (28%) MS patients vs. 24 (24%) controls with mutations (p = 0.62). Two or more mutations were found in 9 MS patients vs. 2 controls (p = 0.03, pcorr = 0.12). Carriership of mutations associated with older age and lower neutrophil counts. These results demonstrate that STAT3 SH2 domain is a hotspot for somatic mutations in CD8+ cells with a prevalence of 26% among the participants. There were no significant differences in the mutation prevalences between MS patients and controls. Further research is needed to elucidate the role of antigenic stimuli in the expansion of the mutant clones. Furthermore, the high discovered prevalence of STAT3 somatic mutations makes it feasible to analyze these mutations directly in tissue-infiltrating CD8+ cells in autoimmune diseases.

CD8+ cell somatic mutations in multiple sclerosis patients and controls-Enrichment of mutations in STAT3 and other genes implicated in hematological malignancies.

Somatic mutations have a central role in cancer but their role in other diseases such as common autoimmune disorders is not clear. Previously we and others have demonstrated that especially CD8+ T cells in blood can harbor persistent somatic mutations in some patients with multiple sclerosis (MS) and rheumatoid arthritis. Here we concentrated on CD8+ cells in more detail and tested (i) how commonly somatic mutations are detectable, (ii) does the overall mutation load differ between MS patients and controls, and (iii) do the somatic mutations accumulate non-randomly in certain genes? We separated peripheral blood CD8+ cells from newly diagnosed relapsing MS patients (n = 21) as well as matched controls (n = 21) and performed next-generation sequencing of the CD8+ cells' DNA, limiting our search to a custom panel of 2524 immunity and cancer related genes, which enabled us to obtain a median sequencing depth of over 2000x. We discovered nonsynonymous somatic mutations in all MS patients' and controls' CD8+ cell DNA samples, with no significant difference in number between the groups (p = 0.60), at a median allelic fraction of 0.5% (range 0.2-8.6%). The mutations showed statistically significant clustering especially to the STAT3 gene, and also enrichment to the SMARCA2, DNMT3A, SOCS1 and PPP3CA genes. Known activating STAT3 mutations were found both in MS patients and controls and overall 1/5 of the mutations were previously described cancer mutations. The detected clustering suggests a selection advantage of the mutated CD8+ clones and calls for further research on possible phenotypic effects.

Diagnostic yield of genetic testing in a heterogeneous cohort of 1376 HCM patients.

BACKGROUND: Genetic testing in hypertrophic cardiomyopathy (HCM) is a published guideline-based recommendation. The diagnostic yield of genetic testing and corresponding HCM-associated genes have been largely documented by single center studies and carefully selected patient cohorts. Our goal was to evaluate the diagnostic yield of genetic testing in a heterogeneous cohort of patients with a clinical suspicion of HCM, referred for genetic testing from multiple centers around the world. METHODS: A retrospective review of patients with a suspected clinical diagnosis of HCM referred for genetic testing at Blueprint Genetics was undertaken. The analysis included syndromic, myopathic and metabolic etiologies. Genetic test results and variant classifications were extracted from the database. Variants classified as pathogenic (P) or likely pathogenic (LP) were considered diagnostic. RESULTS: A total of 1376 samples were analyzed. Three hundred and sixty-nine tests were diagnostic (26.8%); 373 P or LP variants were identified. Only one copy number variant was identified. The majority of diagnostic variants involved genes encoding the sarcomere (85.0%) followed by 4.3% of diagnostic variants identified in the RASopathy genes. Two percent of diagnostic variants were in genes associated with a cardiomyopathy other than HCM or an inherited arrhythmia. Clinical variables that increased the likelihood of identifying a diagnostic variant included: an earlier age at diagnosis (p < 0.0001), a higher maximum wall thickness (MWT) (p < 0.0001), a positive family history (p < 0.0001), the absence of hypertension (p = 0.0002), and the presence of an implantable cardioverter-defibrillator (ICD) (p = 0.0004). CONCLUSION: The diagnostic yield of genetic testing in this heterogeneous cohort of patients with a clinical suspicion of HCM is lower than what has been reported in well-characterized patient cohorts. We report the highest yield of diagnostic variants in the RASopathy genes identified in a laboratory cohort of HCM patients to date. The spectrum of genes implicated in this unselected cohort highlights the importance of pre-and post-test counseling when offering genetic testing to the broad HCM population.

Oligogenic basis of sporadic ALS: The example of SOD1 p.Ala90Val mutation.

OBJECTIVE: To characterize the clinical and neuropathologic features of patients with amyotrophic lateral sclerosis (ALS) with the superoxide dismutase 1 (SOD1) p.Ala90Val mutation, as well as the mutation frequency and the role of oligogenic mechanisms in disease penetrance. METHODS: An index patient with autopsy-proven ALS was discovered to have the SOD1 p.Ala90Val mutation, which was screened in 2 Finnish ALS cohorts (n = 453). Additional contributing variants were analyzed from whole-genome or whole-exome sequencing data. RESULTS: Seven screened patients (1.5%) were found to carry the SOD1 heterozygous mutation. Allele-sharing analysis suggested a common founder haplotype. Common clinical features included limb-onset, long disease course, and sensory symptoms. No TDP43 pathology was observed. All cases were apparently sporadic, and pedigree analysis demonstrated that the mutation has reduced penetrance. Analysis of other contributing genes revealed a unique set of additional variants in each patient. These included previously described rare ANG and SPG11 mutations. One patient was compound heterozygous for SOD1 p.Ala90Val and p.Asp91Ala. CONCLUSIONS: Our data suggest that the penetrance of SOD1 p.Ala90Val is modulated by other genes and indicates highly individual oligogenic basis of apparently sporadic ALS. Additional genetic variants likely contributing to disease penetrance were very heterogeneous, even among Finnish patients carrying the SOD1 founder mutation.

Phosphorylation of Parkin at serine 65 is essential for its activation in vivo.

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Alzheimer risk loci and associated neuropathology in a population-based study (Vantaa 85+).

OBJECTIVE: To test the association of distinct neuropathologic features of Alzheimer disease (AD) with risk loci identified in genome-wide association studies. METHODS: Vantaa 85+ is a population-based study that includes 601 participants aged ≥85 years, of which 256 were neuropathologically examined. We analyzed 29 AD risk loci in addition to APOE ε4, which was studied separately and used as a covariate. Genotyping was performed using a single nucleotide polymorphism (SNP) array (341 variants) and imputation (6,038 variants). Participants with Consortium to Establish a Registry for Alzheimer Disease (CERAD) (neuritic Aß plaques) scores 0 (n = 65) vs score M + F (n = 171) and Braak (neurofibrillary tangle pathology) stages 0-II (n = 74) vs stages IV-VI (n = 119), and with capillary Aß (CapAß, n = 77) vs without (n = 179) were compared. Cerebral amyloid angiopathy (CAA) percentage was analyzed as a continuous variable. RESULTS: Altogether, 24 of the 29 loci were associated (at p < 0.05) with one or more AD-related neuropathologic features in either SNP array or imputation data. Fifteen loci associated with CERAD score, smallest p = 0.0002122, odds ratio (OR) 2.67 (1.58-4.49) at MEF2C locus. Fifteen loci associated with Braak stage, smallest p = 0.004372, OR 0.31 (0.14-0.69) at GAB2 locus. Twenty loci associated with CAA, smallest p = 7.17E-07, ß 14.4 (8.88-20) at CR1 locus. Fifteen loci associated with CapAß smallest p = 0.002594, OR 0.54 (0.37-0.81) at HLA-DRB1 locus. Certain loci associated with specific neuropathologic features. CASS4, CLU, and ZCWPW1 associated only with CAA, while TREM2 and HLA-DRB5 associated only with CapAß. CONCLUSIONS: AD risk loci differ in their association with neuropathologic features, and we show for the first time distinct risk loci for CAA and CapAß.

Common origin of the gelsolin gene variant in 62 Finnish AGel amyloidosis families.

Finnish gelsolin amyloidosis (AGel amyloidosis) is an autosomal dominantly inherited systemic disorder with ophthalmologic, neurologic and dermatologic symptoms. Only the gelsolin (GSN) c.640G>A variant has been found in the Finnish patients thus far. The purpose of this study was to examine whether the Finnish patients have a common ancestor or whether multiple mutation events have occurred at c.640G, which is a known mutational hot spot. A total of 79 Finnish AGel amyloidosis families including 707 patients were first discovered by means of patient interviews, genealogic studies and civil and parish registers. From each family 1-2 index patients were chosen. Blood samples were available from 71 index patients representing 64 families. After quality control, SNP array genotype data were available from 68 patients from 62 nuclear families. All the index patients had the same c.640G>A variant (rs121909715). Genotyping was performed using the Illumina CoreExome SNP array. The homozygosity haplotype method was used to analyse shared haplotypes. Haplotype analysis identified a shared haplotype, common to all studied patients. This shared haplotype included 17 markers and was 361 kb in length (GRCh37 coordinates 9:124003326-124364349) and this level of haplotype sharing was found to occur highly unlikely by chance. This GSN haplotype ranked as the largest shared haplotype in the 68 patients in a genome-wide analysis of haplotype block lengths. These results provide strong evidence that although there is a known mutational hot spot at GSN c.640G, all of the studied 62 Finnish AGel amyloidosis families are genetically linked to a common ancestor.

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

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.

[The pathogenesis of amyotrophic lateral sclerosis and frontal lobe dementia is unraveling: pathology of the nucleus and glutamate sensitivity]. / Amyotrofisen lateraaliskleroosin ja otsalohkodementian patogeneesi avautuu: Tumapatologia ja glutamaattiherkkyys.

The mechanisms of neurodegenerative diseases have begun to become unraveled, thanks to the progress in stem cell research. The repeat expansion in the C90RF72 gene was identified in 2011 as the most common genetic cause of both ALS and frontal lobe dementia. Only over a couple of years the disease mechanisms of this mutation have been revealed and treatment trials have already been conducted in nerve cell cultures differentiated from patients' stem cells. We discuss the role of the repeat expansion in the C90RF72 gene in the epidemiology of the diseases and the resulting disturbances in nerve cell function.

Exome and regulatory element sequencing of neuromyelitis optica patients.

Neuromyelitis optica (NMO) is rare in Finland. To identify rare genetic variants contributing to NMO risk we performed whole exome, HLA and regulatory region sequencing in all ascertained cases during 2005-2013 (n=5) in a Southern Finnish population of 1.6 million. There were no rare variant shared by all patients. Four missense variants were shared by two patients in C3ORF20, PDZD2, C5ORF47 and ZNF606. Another PDZD2 variant was found in a third patient. In the non-coding sequence two predictably functional rare variants were shared by two patients. Our results do not support a homogeneous genetic etiology of NMO in Finland.

Genome-wide association study of neocortical Lewy-related pathology.

OBJECTIVE: Dementia with Lewy bodies is an α-synucleinopathy characterized by neocortical Lewy-related pathology (LRP). We carried out a genome-wide association study (GWAS) on neocortical LRP in a population-based sample of subjects aged 85 or over. METHODS: LRP was analyzed in 304 subjects in the Vantaa 85+ sample from Southern Finland. The GWAS included 41 cases with midbrain, hippocampal, and neocortical LRP and 177 controls without midbrain and hippocampal LRP. The Medical Research Council Cognitive Function and Ageing Study (CFAS) material was used for replication (51 cases and 131 controls). RESULTS: By analyzing 327,010 markers the top signal was obtained at the HLA-DPA1/DPB1 locus (P = 1.29 × 10(-7)); five other loci on chromosomes 15q14, 2p21, 2q31, 18p11, and 5q23 were associated with neocortical LRP at P < 10(-5). Two loci were marked by multiple markers, 2p21 (P = 3.9 × 10(-6), upstream of the SPTBN1 gene), and HLA-DPA1/DPB1; these were tested in the CFAS material. Single marker (P = 0.0035) and haplotype (P = 0.04) associations on 2p21 were replicated in CFAS, whereas HLA-DPA1/DPB1 association was not. Bioinformatic analyses suggest functional effects for the HLA-DPA1/DPB1 markers as well as the 15q14 marker rs8037309. INTERPRETATION: We identified suggestive novel risk factors for neocortical LRP. SPTBN1 is the candidate on 2p21, it encodes beta-spectrin, an α-synuclein binding protein and a component of Lewy bodies. The HLA-DPA1/DPB1 association suggests a role for antigen presentation or alternatively, cis-regulatory effects, one of the regulated neighboring genes identified here (vacuolar protein sorting 52) plays a role in vesicular trafficking and has been shown to interact with α-synuclein in a yeast model.

Anima: modular workflow system for comprehensive image data analysis.

Modern microscopes produce vast amounts of image data, and computational methods are needed to analyze and interpret these data. Furthermore, a single image analysis project may require tens or hundreds of analysis steps starting from data import and pre-processing to segmentation and statistical analysis; and ending with visualization and reporting. To manage such large-scale image data analysis projects, we present here a modular workflow system called Anima. Anima is designed for comprehensive and efficient image data analysis development, and it contains several features that are crucial in high-throughput image data analysis: programing language independence, batch processing, easily customized data processing, interoperability with other software via application programing interfaces, and advanced multivariate statistical analysis. The utility of Anima is shown with two case studies focusing on testing different algorithms developed in different imaging platforms and an automated prediction of alive/dead C. elegans worms by integrating several analysis environments. Anima is a fully open source and available with documentation at www.anduril.org/anima.

C9ORF72 expansion does not affect the phenotype in Nasu-Hakola disease with the DAP12 mutation.

Nasu-Hakola disease (NHD) is a rare autosomal recessive disease that is characterized by cyst-like bone lesions and pathologic fractures combined with an early-onset frontal type of dementia. Mutations in DNAX-activation protein 12 (DAP12) and triggering receptor expressed on myeloid cells 2 (TREM2) are the known genetic causes of NHD. However, the role of both these genes in the neurodegenerative process is still partly unclear, and the input of other modifying factors has been postulated. Frontotemporal lobar degeneration (FTLD) is a neuropathologically and genetically heterogeneous neurodegenerative disease. A hexanucleotide repeat expansion in the chromosome 9-associated open reading frame 72 (C9ORF72) gene is the most common cause of familial FTLD in Finland. Here, we describe a family with 3 siblings with a clinical diagnosis of NHD. All patients had an equivalent age of onset of the behavioral/cognitive symptoms, and brain imaging revealed a similar pattern of brain atrophy and calcification in putamen and caudate nucleus. Case II-3 had the most severe phenotype with epilepsy and a rapid cognitive decline. Genetic analyses were performed in 2 patients (cases II-2 and II-3), and both had a homozygous DAP12 deletion. Because the role of DAP12 and TREM2 in neurodegeneration in NHD is partly unclear, our aim was to evaluate the role of other genetic variations as modifiers. The C9ORF72 expansion was found in case II-2. Exome sequencing did not reveal any other mutations that could be involved in FTLD. Case II-3 had a novel predictably deleterious mutation in the progressive myoclonic epilepsy type 2 (EPM2), which may have influenced his epilepsy as the EPM2 has been implicated in Lafora progressive myoclonic epilepsy. We conclude that the C9ORF72 expansion is probably an incidental finding because it did not have any apparent influence on the phenotype. Exome sequencing identified several rare missense variants and indels. Additional analyses in other NHD patients will be needed to elucidate their clinical relevance.

Use of IgE and IgG4 epitope binding to predict the outcome of oral immunotherapy in cow's milk allergy.

BACKGROUND: Oral immunotherapy (OIT) with cow's milk (CM) has been reported to induce a number of specific antibody responses, but these remain to be fully characterized. Our objective was to explore whether IgE and IgG4 epitope binding profiles could predict the risk of side effects during CM OIT. METHODS: The study population consisted of 32 children (6-17 yr of age) with CM allergy: 26 children who successfully completed OIT and six children who discontinued therapy due to adverse reactions. We investigated sera drawn before and after OIT. We analyzed specific IgE and IgG4 binding to CM protein-derived peptides with a microarray-based immunoassay. Antibody binding affinity was analyzed with a competition assay where CM proteins in solution competed with peptides printed on the microarray. RESULTS: IgE binding to CM peptides decreased and IgG4 binding increased following the OIT in children who attained desensitization. Compared with children who successfully completed OIT, those who discontinued OIT due to adverse reactions developed increased quantities and affinity of epitope-specific IgE antibodies and a broader diversity of IgE and IgG4 binding, but less overlap in IgE and IgG4 binding to CM peptides. CONCLUSIONS: Detailed analysis of IgE and IgG4 binding to CM peptides may help in predicting whether CM OIT will be tolerated successfully. It may thus improve the safety of the therapy.