In situ detection of PR3-ANCA+ B cells and alterations in the variable region of immunoglobulin genes support a role of inflamed tissue in the emergence of auto-reactivity in granulomatosis with polyangiitis.

Circulating anti-neutrophilic cytoplasmic autoantibodies targeting proteinase 3 (PR3-ANCA) are a diagnostic and pathogenic hallmark of granulomatosis with polyangiitis (GPA). It is, however, incompletely understood if inflamed tissue supports presence or emergence of PR3-ANCA+ B cells. In search of such cells in inflamed tissue of GPA, immunofluorescence staining for IgG and a common PR3-ANCA idiotype (5/7 Id) was undertaken. Few 5/7 Id+/IgG+ B cells were detected in respiratory and kidney tissue of GPA. To gain more insight into surrogate markers possibly indicative of an anti-PR3-response, a meta-analysis comprising IGVH and IGVL genes derived from respiratory tract tissue of GPA (231 clones) was performed. Next generation sequencing-based IGHV genes derived from peripheral blood of healthy donors (244.353 clones) and previously published IGLV genes (148 clones) served as controls. Additionally, Ig genes of three murine and five known human monoclonal anti-PR3 antibodies were analyzed. Primary and probably secondary rearrangements led to altered VDJ usage and an extended complementarity determining region 3 (CDR3) of IGHV clones from GPA tissue. Selection against amino acid exchanges was prominent in the framework region of IGHV clones from GPA tissue. The comparison of V(D)J rearrangements and deduced amino acid sequences of the CDR3 yielded no identities and few similarities between clones derived from respiratory tissue of GPA and anti-PR3 antibodies, arguing against a presence of B cells that carry PR3-ANCA-prone Ig genes among the clones. In line with the scarcity of 5/7 Id+ B lymphocytes in GPA tissue, the results suggest that with respect to a local anti-PR3 response, methods detecting rare clones are required.

Genomewide association study identifies GALC as susceptibility gene for mucous membrane pemphigoid.

Mucous membrane pemphigoid (MMP) is a rare, chronic and often aggressive subepidermal autoimmune blistering disease potentially affecting several mucous membranes with blisters and secondary erosions and scars. The pathogenesis of MMP is poorly understood, and the contribution of genetic predispositions, other than HLA class II allele variants to MMP, is unknown. The objective of this study is to identify susceptibility genes for MMP in a British cohort of MMP patients. A GWAS was conducted in a British cohort of 106 MMP patients. Publicly available genotypes of 2900 blood donors of the UK Blood Service and of 6740 individuals of the 1958 British Birth Cohort served as control. Subsequently, putative susceptibility genes were independently replicated in a German cohort of 42 MMP patients. The GWAS found 38 SNPs in 28 haploblocks with an odds ratio >2 reaching genomewide significance (P < 5.7 × 10-7 ). Replication confirmed an association of MMP with SNPs in rs17203398 (OR: 3.9), located intronically in the ß-galactocerebrosidase gene (GALC) on chromosome 14 and with recessive polymorphisms in rs9936045 (OR: 3.1) in the intergenic region between CASC16 and CHD9 on chromosome 16. The risk of developing MMP is partially genetically determined. SNPs in GALC enhance the risk for MMP, indicating that ß-galactocerebrosidase may be involved in the pathogenesis of MMP. Likewise, impacts of polymorphisms in the intergenic region between CASC16 and CHD9 on the activity of neighbouring genes may facilitate the emergence of MMP. The putative role of both polymorphisms requires functional studies in the future.

Dissecting genetics of cutaneous miRNA in a mouse model of an autoimmune blistering disease.

BACKGROUND: MicroRNAs (miRNAs) are small endogenous non-coding RNAs that control genes at post-transcriptional level. They are essential for development and tissue differentiation, and such altered miRNA expression patterns are linked to the pathogenesis of inflammation and cancer. There is evidence that miRNA expression is genetically controlled similar to the transcription of protein-coding genes and previous studies identified quantitative trait loci (QTL) for miRNA expression in the liver. So far, little attention has been paid to miRNA expression in the skin. Moreover, epistatic control of miRNA expression remains unknown. In this study, we characterize genetic regulation of cutaneous miRNA and their correlation with skin inflammation using a previously established murine autoimmune-prone advanced intercross line. RESULTS: We identified in silico 42 eQTL controlling the expression of 38 cutaneous miRNAs and furthermore found two chromosomal hot-spots on chromosomes 2 and 8 that control the expression of multiple miRNAs. Moreover, for 8 miRNAs an interacting effect from pairs of SNPs was observed. Combining the constraints on genes from the statistical interaction of their loci and further using curated protein interaction networks, the number of candidate genes for association of miRNAs was reduced to a set of several genes. A cluster analysis identified miR-379 and miR-223 to be associated with EBA severity/onset, where miR-379 was observed to be associated to loci on chromosome 6. CONCLUSION: The murine advanced intercross line allowed us to identify the genetic loci regulating multiple miRNA in skin. The recurrence of trans-eQTL and epistasis suggest that cutaneous miRNAs are regulated by yet an unexplored complex gene networks. Further, using co-expression analysis of miRNA expression levels we showed that multiple miRNA contribute to multiple pathways that might be involved in pathogenesis of autoimmune skin blistering disease. Specifically, we provide evidence that miRNA such as miR-223 and miR-379 may play critical role in disease progression and severity.

Genetically driven predisposition leads to an unusually genomic unstable renal cell carcinoma.

Renal cell carcinoma originates from the lining of the proximal convoluted renal tubule and represents the most common type of kidney cancer. Risk factors and comorbidities might be associated to renal cell carcinoma, while a small fraction of 2-3% emerges from patients with predisposing cancer syndromes, typically associated to hereditary mutations in VHL, folliculin, fumarate hydratase or MET genes. Here, we report a case of renal cell carcinoma in patient with concurrent germline mutations in BRCA1 and RAD51 genes. This case displays an unusual high mutational burden and chromosomal aberrations compared to the typical profile of renal cell carcinoma. Mutational analysis on whole genome sequencing revealed an enrichment of the MMR2 mutational signature, which is indicative of impaired DNA repair capacity. Overall, the tumor displayed a profile of unusual high genomic instability which suggests a possible origin from germline predisposing mutations in the DNA repair genes BRCA1 and RAD51. While BRCA1 and RAD51 germline mutations are well-characterised in breast and ovarian cancer, their role in renal cell carcinoma is still largely unexplored. The genomic instability detected in this case of renal cell carcinoma, along with the presence of unusual mutations, might offer support to clinicians for the development of patient-tailored therapies.

Genomic and transcriptomic profiling of hepatocellular carcinoma reveals a rare molecular subtype.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, occurring predominantly in patients with underlying chronic liver disease and cirrhosis. Here, we describe a case of a 62-year-old man that was admitted to our hospital and diagnosed with HCC where the cancer has already metastasized to the retroperitoneum and peritoneum. In order to better characterize the HCC, both the cancerous liver tissue and the adjacent normal liver tissue of the patient were collected and subjected to a genomic, transcriptomic and proteomic analysis. Our patient carries a highly mutated HCC, which is characterized by both somatic mutation in the following genes ALK, CDK6, TP53, PGR. In addition, we observe several molecular alterations that are associated with potential therapy resistance, for example the expression of the organic-anion-transporting polypeptide (OATP) family members B1 and B3, that mediate the transport of the anticancer drugs, has been found decreased. Overall, our molecular profiling potentially classify the patient with poor prognosis and possibly displaying resistance to pharmacological therapy.

Molecular profiling of a bladder cancer with very high tumour mutational burden.

The increasing incidence of urothelial bladder cancer is a notable global concern, as evidenced by the epidemiological data in terms of frequency, distribution, as well as mortality rates. Although numerous molecular alterations have been linked to the occurrence and progression of bladder cancer, currently there is a limited knowledge on the molecular signature able of accurately predicting clinical outcomes. In this report, we present a case of a pT3b high-grade infiltrating urothelial carcinoma with areas of squamous differentiation characterized by very high tumor mutational burden (TMB), with up-regulations of immune checkpoints. The high TMB, along with elevated expressions of PD-L1, PD-L2, and PD1, underscores the rationale for developing a personalized immunotherapy focused on the use of immune-checkpoint inhibitors. Additionally, molecular analysis revealed somatic mutations in several other cancer-related genes, including TP53, TP63 and NOTCH3. Mutations of TP53 and TP63 genes provide mechanistic insights on the molecular mechanisms underlying disease development and progression. Notably, the above-mentioned mutations and the elevated hypoxia score make the targeting of p53 and/or hypoxia related pathways a plausible personalized medicine option for this bladder cancer, particularly in combination with immunotherapy. Our data suggest a requirement for molecular profiling in bladder cancer to possibly select appropriate immune-checkpoint therapy.

A primary luminal/HER2 negative breast cancer patient with mismatch repair deficiency.

Here, we present the case of a 47-year-old woman diagnosed with luminal B breast cancer subtype and provide an in-depth analysis of her gene mutations, chromosomal alterations, mRNA and protein expression changes. We found a point mutation in the FGFR2 gene, which is potentially hyper-activating the receptor function, along with over-expression of its ligand FGF20 due to genomic amplification. The patient also harbors somatic and germline mutations in some mismatch repair (MMR) genes, with a strong MMR mutational signature. The patient displays high microsatellite instability (MSI) and tumor mutational burden (TMB) status and increased levels of CTLA-4 and PD-1 expression. Altogether, these data strongly implicate that aberrant FGFR signaling, and defective MMR system might be involved in the development of this breast tumor. In addition, high MSI and TMB in the context of CTLA-4 and PD-L1 positivity, suggest the potential benefit of immune checkpoint inhibitors. Accurate characterization of molecular subtypes, based on gene mutational and expression profiling analyses, will be certainly helpful for individualized treatment and targeted therapy of breast cancer patients, especially for those subtypes with adverse outcome.

A BRCA2 germline mutation and high expression of immune checkpoints in a TNBC patient.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of mammary carcinoma. Here, we describe a case of an 81-year-old female diagnosed with ductal triple negative breast cancer with a germline pathogenic variant in BReast CAncer gene2 (BRCA2). Genetic testing also revealed the presence of four somatic mutations in the ephrin type-A receptor 3 (EphA3), TP53, BRCA1-associated protein (BAP1), and MYB genes. The BRCA2, TP53, and BAP1 gene mutations are highly predictive of a defective homologous recombination repair system and subsequent chromosomal instability in this patient. Coherently, the patient displayed a strong homologous recombination deficiency signature and high tumor mutational burden status, which are generally associated with increased probability of immune neoantigens formation and presentation, and with tumor immunogenicity. Analysis of immune checkpoint revealed high expression of programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), programmed death 1 (PD1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA 4), suggesting that the patient might likely benefit from immunotherapies. Altogether, these findings support an unveiled link between BRCA2 inactivation, HR deficiency and increased expression of immune checkpoints in TNBC. This clinical case highlights the importance of screening TNBC patients for genetic mutations and TMB biomarkers in order to predict the potential efficacy of immunotherapy.

Analysis of DCM associated protein alterations of human right and left ventricles.

Dilated cardiomyopathy (DCM) is characterized by ventricular chamber enlargement and impaired myocardial function. Endomyocardial biopsies (EMB) enable immunohistochemical and molecular characterization of this disease. However, knowledge about specific molecular patterns and their relation to cardiac function in both ventricles is rare. Therefore, we performed a mass spectrometric analysis of 28 paired EMBs of left (LV) and right ventricles (RV) of patients with DCM or suspected myocarditis allowing quantitative profiling of 743 proteins. We analysed associations between protein abundance of LV and RV as well as the echocardiographic parameters LVEF, TAPSE, LVEDDI, and RVEDDI by linear regression models. Overall, more LV than RV proteins were associated with LV parameters or with RVEDDI. Most LV and RV proteins increasing in level with impairing of LVEF were annotated to structural components of cardiac tissue. Additionally, a high proportion of LV proteins with metabolic functions decreased in level with decreasing LVEF. Results were validated with LV heart sections of a genetic murine heart failure model. The study shows, that remodelling and systolic dysfunction in DCM is mirrored by distinct alterations in protein composition of both ventricles. Loss of LV systolic function is reflected predominantly by alterations in proteins assigned to metabolic functions in the LV whereas structural remodelling was more obvious in the RV. Alterations related to intermediate filaments were seen in both ventricles and highlight such proteins as early indicators of LV loss of function. SIGNIFICANCE: The present study report protein sets in the RV and the LV being associated with ventricular function and remodelling in DCM. Protein abundances in the LV and the RV emphasize and expand current knowledge on pathophysiological changes in heart failure and DCM. While RV and LV EMBs do not differ concerning diagnostic assessment of inflammatory status and virus persistence, additional information reflecting disease severity associated protein alterations can be gained by EMB protein profiling. RV and LV protein data provided complementary information. The protein pattern of the LV reflects metabolic changes and an impaired energy production, which is associated with the degree of LV systolic dysfunction and remodelling and may yield important information about the disease status in DCM. On the other hand, at this disease stage of DCM with still preserved RV function, RV alterations in structural proteins may reflect myocardial compensatory protective mechanisms for maintenance of structure and cellular function. The study highlight particular proteins being of interest as heart failure biomarkers in both ventricles which seem to reflect the severity of the disease. Further comparative studies between different HF aetiologies have to evaluate those proteins as markers specific for DCM.

bcRep: R Package for Comprehensive Analysis of B Cell Receptor Repertoire Data.

Immunoglobulins, as well as T cell receptors, play a key role in adaptive immune responses because of their ability to recognize antigens. Recent advances in next generation sequencing improved also the quality and quantity of individual B cell receptors repertoire sequencing. Unfortunately, appropriate software to exhaustively analyze repertoire data from NGS platforms without limitations of the number of sequences are lacking. Here we introduce a new R package, bcRep, which offers a platform for comprehensive analyses of B cell receptor repertoires, using IMGT/HighV-QUEST formatted data. Methods for gene usage statistics, clonotype classification, as well as diversity measures, are included. Furthermore, functions to filter datasets, to do summary statistics about mutations, as well as visualization methods, are available. To compare samples in respect of gene usage, diversity, amino acid proportions, similar sequences or clones, several functions including also distance measurements, as well as multidimensional scaling methods, are provided.

Mitochondrial gene polymorphisms alter hepatic cellular energy metabolism and aggravate diet-induced non-alcoholic steatohepatitis.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is associated with an enhanced risk for liver and cardiovascular diseases and mortality. NAFLD can progress from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH). However, the mechanisms predisposing to this progression remain undefined. Notably, hepatic mitochondrial dysfunction is a common finding in patients with NASH. Due to a lack of appropriate experimental animal models, it has not been evaluated whether this mitochondrial dysfunction plays a causative role for the development of NASH. METHODS: To determine the effect of a well-defined mitochondrial dysfunction on liver physiology at baseline and during dietary challenge, C57BL/6J-mt(FVB/N) mice were employed. This conplastic inbred strain has been previously reported to exhibit decreased mitochondrial respiration likely linked to a non-synonymous gene variation (nt7778 G/T) of the mitochondrial ATP synthase protein 8 (mt-ATP8). RESULTS: At baseline conditions, C57BL/6J-mt(FVB/N) mice displayed hepatic mitochondrial dysfunction characterized by decreased ATP production and increased formation of reactive oxygen species (ROS). Moreover, genes affecting lipid metabolism were differentially expressed, hepatic triglyceride and cholesterol levels were changed in these animals, and various acyl-carnitines were altered, pointing towards an impaired mitochondrial carnitine shuttle. However, over a period of twelve months, no spontaneous hepatic steatosis or inflammation was observed. On the other hand, upon dietary challenge with either a methionine and choline deficient diet or a western-style diet, C57BL/6J-mt(FVB/N) mice developed aggravated steatohepatitis as characterized by lipid accumulation, ballooning of hepatocytes and infiltration of immune cells. CONCLUSIONS: We observed distinct metabolic alterations in mice with a mitochondrial polymorphism associated hepatic mitochondrial dysfunction. However, a second hit, such as dietary stress, was required to cause hepatic steatosis and inflammation. This study suggests a causative role of hepatic mitochondrial dysfunction in the development of experimental NASH.

Effect of Experimental Thyrotoxicosis onto Blood Coagulation: A Proteomics Study.

BACKGROUND: Hyperthyroidism is known to induce a hypercoagulable state. It stimulates plasma levels of procoagulative factors and reduces fibrinolytic activity. So far most of the data have been derived from patients with endogenous hyperthyroidism with a wide variability in the underlying pathogenesis and severity of the disease. OBJECTIVES: In this study we experimentally induced thyrotoxicosis in healthy volunteers to explore the effects of thyroxine excess on the plasma proteome. Using a shotgun proteomics approach, the abundance of plasma proteins was monitored before, during and after thyrotoxicosis. METHODS: Sixteen healthy male subjects were sampled at baseline, 4 and 8 weeks under 250 µg/day thyroxine p.o., as well as 4 and 8 weeks after stopping the application. Plasma proteins were analyzed after depletion of 6 high-abundance proteins (MARS6) by LC-ESI-MS/MS mass spectrometry. Mass spectrometric raw data were processed using a label-free, intensity-based workflow. Subsequently, the linear dependence between protein abundances and fT4 levels were calculated using a Pearson correlation. RESULTS: All subjects developed biochemical thyrotoxicosis, and this effect was reversed within the first 4 weeks of follow-up. None of the volunteers noticed any subjective symptoms. Levels of 10 proteins involved in the coagulation cascade specifically correlated with fT4, supporting an influence of thyroid hormone levels on blood coagulation even at nonpathological levels. CONCLUSIONS: The results suggest that experimental thyrotoxicosis exerts selective and specific thyroxine-induced effects on coagulation markers. Our study design allows assessment of thyroid hormone effects on plasma protein levels without secondary effects of other diseases or therapies.

Quantitative Trait Locus Analysis Implicates CD4⁺/CD44high Memory T Cells in the Pathogenesis of Murine Autoimmune Pancreatitis.

The mouse strain MRL/MpJ is prone to spontaneously develop autoimmune pancreatitis (AIP). To elucidate the genetic control towards the development of the phenotype and to characterize contributions of immunocompetent cell types, MRL/MpJ mice were interbred with three additional strains (BXD2/TYJ, NZM2410/J, CAST/EIJ) for four generations in an advanced intercross line. Cellular phenotypes were determined by flow cytometric quantification of splenic leukocytes and complemented by the histopathological evaluation of pancreatic lesions. An Illumina SNP array was used for genotyping. QTL analyses were performed with the R implementation of HAPPY. Out of 41 leukocyte subpopulations (B cells, T cells and dendritic cells), only three were significantly associated with AIP: While CD4+/CD44high memory T cells and CD4+/CD69+ T helper (Th) cells correlated positively with the disease, the cytotoxic T cell phenotype CD8+/CD44low showed a negative correlation. A QTL for AIP on chromosome 2 overlapped with QTLs for CD4+/CD44high and CD8+/CD44high memory T cells, FoxP3+/CD4+ and FoxP3+/CD8+ regulatory T cells (Tregs), and CD8+/CD69+ cytotoxic T cells. On chromosome 6, overlapping QTLs for AIP and CD4+/IL17+ Th17 cells and again FoxP3+/CD8+ Tregs were observed. In conclusion, CD4+/CD44high memory T cells are the only leukocyte subtype that could be linked to AIP both by correlation studies and from observed overlapping QTL. The potential role of this cell type in the pathogenesis of AIP warrants further investigations.