Background: Somatic mutations, copy-number variations, and genome instability of mitochondrial DNA (mtDNA) have been reported in different types of cancers and are suggested to play important roles in cancer development and metastasis. However, there is scarce information about pheochromocytomas and paragangliomas (PCCs/PGLs) formation.

Material: To determine the potential roles of mtDNA alterations in sporadic PCCs/PGLs, we analyzed a panel of 26 nuclear susceptibility genes and the entire mtDNA sequence of seventy-seven human tumors, using next-generation sequencing, and compared the results with normal adrenal medulla tissues. We also performed an analysis of copy-number alterations, large mtDNA deletion, and gene and protein expression.

Results: Our results revealed that 53.2% of the tumors harbor a mutation in at least one of the targeted susceptibility genes, and 16.9% harbor complementary mitochondrial mutations. More than 50% of the mitochondrial mutations were novel and predicted pathogenic, affecting mitochondrial oxidative phosphorylation. Large deletions were found in 26% of tumors, and depletion of mtDNA occurred in more than 87% of PCCs/PGLs. The reduction of the mitochondrial number was accompanied by a reduced expression of the regulators that promote mitochondrial biogenesis (PCG1α, NRF1, and TFAM). Further, P62 and LC3a gene expression suggested increased mitophagy, which is linked to mitochondrial dysfunction.

Conclusion: The pathogenic role of these finding remains to be shown, but we suggest a complementarity and a potential contributing role in PCCs/PGLs tumorigenesis.

INTRODUCTION: Genetic variants in the Beta-glucocerebrosidase gene (GBA1) is a known risk factor for Parkinson's disease. The GBA1 mutations L444P, N370S and many other have been shown to associate with the disease in populations with diverse background. Some GBA1 polymorphisms have a less pronounced effect, and their pathogenicity has been debated. We have previously found associations with L444P, N370S and E326K and Parkinson's disease in Sweden.

METHOD: In this study we used pyrosequencing to genotype the T369M variant in a large Swedish cohort consisting of 1,131 patients with idiopathic Parkinson's disease, and 1,594 control subjects to evaluate the possibility of this variant conferring an increased risk for Parkinson's disease.

RESULTS: The minor allele frequency was 2.15% in patients and 1.76% in controls. Statistical analysis showed that there was no significant difference in allele frequency between patients and control subjects, p-value 0.37, Odds Ratio 1.23 with a 95% confidence interval of 0.82-1.83.

CONCLUSION: Our results suggest that T369M is not a risk factor for Parkinson's disease in the Swedish population.

Objective To identify and characterize genetic loci associated with the risk of developing ANCA-associated vasculitides (AAV).

Methods Genetic association analyses were performed after Illumina sequencing of 1853 genes and subsequent replication with genotyping of selected single nucleotide polymorphisms in a total cohort of 1110 Scandinavian cases with granulomatosis with polyangiitis or microscopic polyangiitis, and 1589 controls. A novel AAV-associated single nucleotide polymorphism was analysed for allele-specific effects on gene expression using luciferase reporter assay.

Results PR3-ANCA⁺ AAV was significantly associated with two independent loci in the HLA-DPB1/HLA-DPA1 region [rs1042335, P = 6.3 x 10^-61, odds ratio (OR) 0.10; rs9277341, P = 1.5 x 10^-44, OR 0.22] and with rs28929474 in the SERPINA1 gene (P = 2.7 x 10^-10, OR 2.9). MPO-ANCA⁺ AAV was significantly associated with the HLA-DQB1/HLA-DQA2 locus (rs9274619, P = 5.4 x 10^-25, OR 3.7) and with a rare variant in the BACH2 gene (rs78275221, P = 7.9 x 10^-7, OR 3.0), the latter a novel susceptibility locus for MPO-ANCA⁺ granulomatosis with polyangiitis/microscopic polyangiitis. The rs78275221-A risk allele reduced luciferase gene expression in endothelial cells, specifically, as compared with the non-risk allele.

Conclusion We identified a novel susceptibility locus for MPO-ANCA⁺ AAV and propose that the associated variant is of mechanistic importance, exerting a regulatory function on gene expression in specific cell types.

Objectives: To further elucidate the role of the MHC in ankylosing spondylitis by typing 17 genes, searching for HLA-B∗27 independent associations and assessing the impact of sex on this male biased disease.

Methods: High-confidence two-field resolution genotyping was performed on 310 cases and 2196 controls using an n-1 concordance method. Protein-coding variants were called from next-generation sequencing reads using up to four software programs and the consensus result recorded. Logistic regression tests were applied to the dataset as a whole, and also in stratified sets based on sex or HLA-B∗27 status. The amino acids driving association were also examined.

Results: Twenty-five HLA protein-coding variants were significantly associated to disease in the population. Three novel protective associations were found in a HLA-B∗27 positive population, HLA-A∗24:02 (OR = 0.4, CI = 0.2–0.7), and HLA-A amino acids Leu95 and Gln156. We identified a key set of seven loci that were common to both sexes, and robust to change in sample size. Stratifying by sex uncovered three novel risk variants restricted to the female population (HLA-DQA1∗04.01, -DQB1∗04:02, -DRB1∗08:01; OR = 2.4–3.1). We also uncovered a set of neutral variants in the female population, which in turn conferred strong effects in the male set, highlighting how population composition can lead to the masking of true associations.

Conclusion: Population stratification allowed for a nuanced investigation into the tightly linked MHC region, revealing novel HLA-B∗27 signals as well as replicating previous HLA-B∗27 dependent results. This dissection of signals may help to elucidate sex biased disease predisposition and clinical progression.

Glioma O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status informs clinical decision making. Worldwide different methods and cutoff levels are used, which can lead to discordant methylation results.We conducted an international survey to clarify which methods are regularly used and why. We also explored opinions regarding international consensus on methods and cutoff.The survey had 152 respondents from 25 countries. MGMT methylation status is determined for all glioblastomas in 37% of laboratories. The most common methods are methylation-specific polymerase chain reaction (msPCR) (37%) and pyrosequencing (34%). A method is selected for simplicity (56%), cost-effectiveness (50%), and reproducibility of results (52%). For sequencing, the number of CpG sites analyzed varies from 1–3 up to more than 16. For 50% of laboratories, the company producing the kit determines which CpG sites are examined, whereas 33% select the sites themselves. Selection of cutoff is equally distributed among a cutoff defined in the literature, by the local laboratory, or by the outside laboratory performing the analysis. This cutoff varies, reported from 1% to 30%, and in 1 laboratory tumor is determined as methylated in case of 1 methylated CpG site of 17 analyzed. Some report tumors as unmethylated or weakly vs highly methylated. An international consensus on MGMT methylation method and cutoff is warranted by 66% and 76% of respondents, respectively. The method preferred would be msPCR (45%) or pyrosequencing (42%), whereas 18% suggest next-generation sequencing.Although analysis of MGMT methylation status is routine, there is controversy regarding laboratory methods and cutoff level. Most respondents favor development of international consensus guidelines.

Objective: To screen for CLCN2 mutations in apparently sporadic cases of aldosterone-producing adenomas (APAs). Description: Recently, CLCN2, encoding for the voltage-gated chloride channel protein 2 (CIC-2), was identified to be mutated in familial hyperaldosteronism II (FH II). So far, somatic mutations in CLCN2 have not been reported in sporadic cases of APAs. We screened 80 apparently sporadic APAs for mutations in CLCN2. One somatic mutation was identified at p.Gly24Asp in CLCN2. The male patient had a small adenoma in size but high aldosterone levels preoperatively. Postoperatively, the patient had normal aldosterone levels and was clinically cured. Conclusion: In this study, we identified a CLCN2 mutation in a sporadic APA comprising about 1% of all APAs investigated. This mutation was complementary to mutations in other susceptibility genes for sporadic APAs and may thus be a driving mutation in APA formation.

Introduction: Venous thromboembolism (VTE) is a common problem among patients with glioblastoma multi-forme (GBM) and with some other cancers. Here, we evaluated genetic and non-genetic potential risk factors for VTE among GBM patients. Materials and methods: A cohort of 139 patients treated with concomitant radiotherapy and temozolomide were included in the study. Next generation sequencing and genotyping approaches were applied to assess genetic risk factors in the haemostatic system. Clinical data including surgery, reoperation as well as blood group and patient information such as age and gender were available from patient records. Logistic regression analysis was performed to asses VTE risk. Results: In the study 47 patients (34%) were diagnosed for VTE during the course of their disease. When genetic and non-genetic potential risk factors were evaluated, only B blood group was found to be significantly associated with VTE incidence (odds ratio [OR] = 6.91; confidence interval [CI] = 2.19-24.14; P = 0.001). In contrast, A and O blood groups did not correlate with VTE risk. Frontal lobe tumor location also seemed to slightly increase VTE risk compared to other brain sites (OR = 3.14; CI = 1.1-10.7) although the significance level was at borderline (P = 0.05). Current study identified B blood group as the component in non-O blood groups that is responsible for increased VTE risk. Conclusion: In conclusion, these results suggest for the first time that B blood group is predictive for VTE incidence among patients with glioblastoma, information that may be potentially valuable when selecting GBM patients who are at risk for VTE for anticoagulant prophylaxis.

Thiopurines are widely used in the treatment of leukemia and inflammatory bowel diseases. Thiopurine metabolism varies among individuals because of differences in the polymorphic enzyme thiopurine methyltransferase (TPMT, EC 2.1.1.67), and to avoid severe adverse reactions caused by incorrect dosing it is recommended that the patients TPMT status be determined before the start of thiopurine treatment. This study describes the concordance between genotyping for common TPMT alleles and phenotyping in a Swedish cohort of 12,663 patients sampled before or during thiopurine treatment. The concordance between TPMT genotype and enzyme activity was 94.5%. Compared to the genotype, the first measurement of TPMT enzyme activity was lower than expected for 4.6% of the patients. Sequencing of all coding regions of the TPMT gene in genotype/phenotype discrepant individuals led to the identification of rare and novel TPMT alleles. Fifteen individuals (0.1%) with rare or novel genotypes were identified, and three TPMT alleles (TPMT*42, *43, and *44) are characterized here for the first time. These 15 patients would not have been detected as carrying a deviating TPMT genotype if only genotyping of the most common TPMT variants had been performed. This study highlights the benefit of combining TPMT genotype and phenotype determination in routine testing. More accurate dose recommendations can be made, which might decrease the number of adverse reactions and treatment failures during thiopurine treatment.

Pheochromocytomas (PCCs) and abdominal paragangliomas (PGLs), collectively abbreviated PPGL, are believed to exhibit malignant potential-but only subsets of cases will display full-blown malignant properties. The Pheochromocytoma of the Adrenal Gland Scaled Score (PASS) algorithm is a proposed histologic system to detect potential for aggressive behavior, but little is known regarding the coupling to underlying molecular genetics. In this study, a total of 92 PPGLs, previously characterized for susceptibility gene status and mRNA expressional profiles, were histologically assessed using the PASS criteria. A total of 32/92 PPGLs (35%) exhibited a PASS score amp;gt;= 4, including all 8 cases with malignant behavior (7 with known metastases and 1 with extensively infiltrative local recurrence). Statistical analyzes between expressional data and clinical parameters as well as individual PASS criteria yielded significant associations to Chromogranin B (CHGB), BRCA2, HIST1H3B, BUB1B, and RET to name a few, and CHGB had the strongest correlation to both PASS and metastasis/local recurrence of all analyzed genes. Evident CHGB downregulation was observed in PPGLs with high PASS and overtly malignant behavior, and was also associated with shorter disease-related survival. This finding was validated using quantitative real-time polymerase chain reaction, in which CHGB expression correlated with both PASS and metastasis/local recurrence with consistent findings obtained in the TCGA cohort. Moreover, immunohistochemical analyses of subsets of tumors showed a correlation between high PASS scores and negative or weak CHGB protein expression. Patients with PPGLs obtaining high PASS scores postoperatively, also exhibited low preoperative plasma levels of CHGB. These data collectively point out CHGB as a possible preoperative and postoperative marker for PPGLs with potential for aggressive behavior.