Evaluation of AlphaFold structure-based protein stability prediction on missense variations in cancer.

Identifying pathogenic missense variants in hereditary cancer is critical to the efforts of patient surveillance and risk-reduction strategies. For this purpose, many different gene panels consisting of different number and/or set of genes are available and we are particularly interested in a panel of 26 genes with a varying degree of hereditary cancer risk consisting of ABRAXAS1, ATM, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MEN1, MLH1, MRE11, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, and XRCC2. In this study, we have compiled a collection of the missense variations reported in any of these 26 genes. More than a thousand missense variants were collected from ClinVar and the targeted screen of a breast cancer cohort of 355 patients which contributed to this set with 160 novel missense variations. We analyzed the impact of the missense variations on protein stability by five different predictors including both sequence- (SAAF2EC and MUpro) and structure-based (Maestro, mCSM, CUPSAT) predictors. For the structure-based tools, we have utilized the AlphaFold (AF2) protein structures which comprise the first structural analysis of this hereditary cancer proteins. Our results agreed with the recent benchmarks that computed the power of stability predictors in discriminating the pathogenic variants. Overall, we reported a low-to-medium-level performance for the stability predictors in discriminating pathogenic variants, except MUpro which had an AUROC of 0.534 (95% CI [0.499-0.570]). The AUROC values ranged between 0.614-0.719 for the total set and 0.596-0.682 for the set with high AF2 confidence regions. Furthermore, our findings revealed that the confidence score for a given variant in the AF2 structure could alone predict pathogenicity more robustly than any of the tested stability predictors with an AUROC of 0.852. Altogether, this study represents the first structural analysis of the 26 hereditary cancer genes underscoring 1) the thermodynamic stability predicted from AF2 structures as a moderate and 2) the confidence score of AF2 as a strong descriptor for variant pathogenicity.

Analysis of Mitochondrial DNA Control Region D-Loop in Gliomas: Result of 52 Patients.

AIM: To investigate the effect of mitochondrial DNA (mtDNA) variants mainly in D-loop on glioma biology. MATERIAL AND METHODS: Sanger sequencing of D-loop (15971?16451 bp) for 52 glioma patients was performed and the variations were statistically analyzed for gender, WHO classification, morphological grade, IDH/TERT status. RESULTS: Total of 122 variations (51 unique) were identified in 52 patients. C16223T, T16189C, T16311C and T16126C variants were frequently detected. The total variation number was statistically non-significant among the analyzed categories. When individual variants were considered, T16311C and T16224C were statistically significant for WHO classification (p=0.033), morphological grade (p=0.036) and gender (p=0.039), respectively. CONCLUSION: Total variation number in D-loop was not found to be related with clinical variables. Our data suggests that individual variants may play a critical role in glioma biology.

Whole exome sequencing-based analysis to identify DNA damage repair deficiency as a major contributor to gliomagenesis in adult diffuse gliomas.

OBJECTIVE: Processes that cause or contribute to cancer, such as aging, exposure to carcinogens, or DNA damage repair deficiency (DDRd), create predictable and traceable nucleotide alterations in one's genetic code (termed "mutational signatures"). Large studies have previously identified various such mutational signatures across cancers that can be attributed to the specific causative processes. To gain further insight into the processes in glioma development, the authors analyzed mutational signatures in adult diffuse gliomas (DGs). METHODS: Twenty-five DGs and paired blood samples were whole exome sequenced. Somatic mutational signatures were identified using 2 different methods. Associations of the signatures with age at diagnosis, molecular subset, and mutational load were investigated. As DDRd-related signatures were frequently observed, germline and somatic DDR gene mutations as well as microsatellite instability (MSI) status were determined for all samples. For validation of signature prevalence, publicly available data from The Cancer Genome Atlas (TCGA) were used. RESULTS: Each tumor had a unique combination of signatures. The most common signatures were signature 1 (88%, aging related), signature 3 (52%, homologous recombination related), and signature 15 (56%, mismatch repair related). Eighty-four percent of the tumors contained at least 1 DDRd signature. The findings were validated using public TCGA data. The weight of signature 1 positively correlated with age (r = 0.43) while cumulative weight of DDRd signatures negatively correlated with age (r = -0.16). Each subject had at least 1 germline/somatic alteration in a DDR gene, the most common being the risk single nucleotide polymorphism rs1800734 in MLH1. The rs1800734-AA genotype had a higher cumulative DDRd weight as well as higher mutational load; TP53 was the most common somatically altered DDR gene. MSI was observed in 24% of the tumors. No significant associations of MSI status with mutational load, rs1800734, or the cumulative weight of DDRd signatures were identified. CONCLUSIONS: Current findings suggest that DDRd may act as a fundamental mechanism in gliomagenesis rather than being a random, secondary event.

Use of telomerase promoter mutations to mark specific molecular subsets with reciprocal clinical behavior in IDH mutant and IDH wild-type diffuse gliomas.

OBJECTIVE Recent studies have established that hemispheric diffuse gliomas may be grouped into subsets on the basis of molecular markers; these subsets are loosely correlated with the histopathological diagnosis but are strong predictors of clinical tumor behavior. Based on an analysis of molecular and clinical parameters, the authors hypothesized that mutations of the telomerase promoter (TERTp-mut) mark separate oncogenic programs among isocitrate dehydrogenase 1 and/or 2 (IDH) mutant (IDH-mut) and IDH wild-type (IDH-wt) diffuse gliomas independent of histopathology or WHO grade. METHODS Four molecular subsets of the combined statuses of IDH and TERT-promoter mutations (double mutant, IDH only, TERT only, and double negative) were defined. Differences in age, anatomical location, molecular genetics, and survival rates in a surgical cohort of 299 patients with a total of 356 hemispheric diffuse gliomas (WHO Grade II, III, or IV) were analyzed. RESULTS TERTp-mut were present in 38.8% of IDH-mut and 70.2% of IDH-wt gliomas. The mutational status was stable in each patient at 57 recurrence events over a 2645-month cumulative follow-up period. Among patients with IDH-mut gliomas, those in the double-mutant subset had better survival and a lower incidence of malignant degeneration than those in the IDH-only subset. Of patients in the double-mutant subset, 96.3% were also positive for 1p/19q codeletions. All patients with 1p/19q codeletions had TERTp-mut. In patients with IDH-mut glioma, epidermal growth factor receptor or phosphatase and tensin homolog mutations were not observed, and copy-number variations were uncommon. Among IDH-wt gliomas, the TERT-only subset was associated with significantly higher age, higher Ki-67 labeling index, primary glioblastoma-specific oncogenic changes, and poor survival. The double-negative subset was genetically and biologically heterogeneous. Survival analyses (Kaplan-Meier, multivariate, and regression-tree analyses) confirmed that patients in the 4 molecular subsets had distinct prognoses. CONCLUSIONS Molecular subsets result in different tumor biology and clinical behaviors in hemispheric diffuse gliomas.

IDH-mutant glioma specific association of rs55705857 located at 8q24.21 involves MYC deregulation.

The single nucleotide polymorphism rs55705857, located in a non-coding but evolutionarily conserved region at 8q24.21, is strongly associated with IDH-mutant glioma development and was suggested to be a causal variant. However, the molecular mechanism underlying this association has remained unknown. With a case control study in 285 gliomas, 316 healthy controls, 380 systemic cancers, 31 other CNS-tumors, and 120 IDH-mutant cartilaginous tumors, we identified that the association was specific to IDH-mutant gliomas. Odds-ratios were 9.25 (5.17-16.52; 95% CI) for IDH-mutated gliomas and 12.85 (5.94-27.83; 95% CI) for IDH-mutated, 1p/19q co-deleted gliomas. Decreasing strength with increasing anaplasia implied a modulatory effect. No somatic mutations were noted at this locus in 114 blood-tumor pairs, nor was there a copy number difference between risk-allele and only-ancestral allele carriers. CCDC26 RNA-expression was rare and not different between the two groups. There were only minor subtype-specific differences in common glioma driver genes. RNA sequencing and LC-MS/MS comparisons pointed to significantly altered MYC-signaling. Baseline enhancer activity of the conserved region specifically on the MYC promoter and its further positive modulation by the SNP risk-allele was shown in vitro. Our findings implicate MYC deregulation as the underlying cause of the observed association.

Endothelial progenitor cells display clonal restriction in multiple myeloma.

BACKGROUND: In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. METHODS: A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (VH). RESULTS: In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (> or = 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (> or = 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with VH primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI. CONCLUSION: Our results suggest that EPCs in at least a substantial subpopulation of MM patients are related to the neoplastic clone and that this is an important mechanism for upregulation of tumor neovascularization in MM.

Evidence from autoimmune thyroiditis of skewed X-chromosome inactivation in female predisposition to autoimmunity.

The etiologic factors in the development of autoimmune thyroid diseases (AITDs) are not fully understood. We investigated the role of skewed X-chromosome inactivation (XCI) mosaicism in female predisposition to AITDs. One hundred and ten female AITDs patients (81 Hashimoto's thyroiditis (HT), 29 Graves' disease (GD)), and 160 female controls were analyzed for the androgen receptor locus by the HpaII/polymerase chain reaction assay to assess XCI patterns in DNA extracted from peripheral blood cells. In addition, thyroid biopsy, buccal mucosa, and hair follicle specimens were obtained from five patients whose blood revealed an extremely skewed pattern of XCI, and the analysis was repeated. Skewed XCI was observed in DNA from peripheral blood cells in 28 of 83 informative patients (34%) as compared with 10 of 124 informative controls (8%, P<0.0001). Extreme skewing was present in 16 patients (19%), but only in three controls (2.4%, P<0.0001). The buccal mucosa, and although less marked, the thyroid specimens also showed skewing. Analysis of two familial cases showed that only the affected individuals demonstrate skewed XCI patterns. Based on these results, skewed XCI mosaicism may play a significant role in the pathogenesis of AITDs.