Mutations In Other Genes Research Articles

Common and Rare Variants in TMEM175 Gene Concur to the Pathogenesis of Parkinson’s Disease in Italian Patients

Parkinson’s disease (PD) represents the most common neurodegenerative movement disorder. We recently identified 16 novel genes associated with PD. In this study, we focused the attention on the common and rare variants identified in the lysosomal K+ channel TMEM175. The study includes a detailed clinical and genetic analysis of 400 cases and 300 controls. Molecular studies were performed on patient-derived fibroblasts. The functional properties of the mutant channels were assessed by patch-clamp technique and co-immunoprecipitation. We have found that TMEM175 was highly expressed in dopaminergic neurons of the substantia nigra pars compacta and in microglia of the cerebral cortex of the human brain. Four common variants were associated with PD, including two novel variants rs2290402 (c.-10C > T) and rs80114247 (c.T1022C, p.M341T), located in the Kozak consensus sequence and TM3II domain, respectively. We also disclosed 13 novel highly penetrant detrimental mutations in the TMEM175 gene associated with PD. At least nine of these mutations (p.R35C, p. R183X, p.A270T, p.P308L, p.S348L, p. L405V, p.R414W, p.P427fs, p.R481W) may be sufficient to cause the disease, and the presence of mutations of other genes correlated with an earlier disease onset. In vitro functional analysis of the ion channel encoded by the mutated TMEM175 gene revealed a loss of the K+ conductance and a reduced channel affinity for Akt. Moreover, we observed an impaired autophagic/lysosomal proteolytic flux and an increase expression of unfolded protein response markers in patient-derived fibroblasts. These data suggest that mutations in TMEM175 gene may contribute to the pathophysiology of PD.

Thrombosis in hereditary hemorrhagic telangiectasia

Thrombosis in hereditary hemorrhagic telangiectasia

Co-mutation of ASXL1 and SF3B1 Predicts Poorer Overall Survival Than Isolated ASXL1 or SF3B1 Mutations.

Mutations in the ASXL transcriptional regulator 1 (ASXL1) and splicing factor 3b subunit 1(SF3B1) genes are commonly observed in myeloid neoplasms and are independent predicative factors for overall survival (OS). Only a few contradictory reports exist on the clinical significance of concurrent ASXL1 and SF3B1 mutations. Previous studies also did not exclude patients with mutations of other genes, which could be confounding factors. We identified 69 patients with mutation of only ASXL1, 89 patients with mutation of only SF3B1, and 17 patients with mutations exclusively of both ASXL1 and SF3B1 from our database of 8,285 patients and compared their clinical features and outcomes. Patients with ASXL1 mutations more frequently had acute myeloid leukemia (22.47%) or clonal cytopenia of unknown significance than patients with SF3B1 mutations (1.45%) or with ASXL1/SF3B1 mutations (11.76%). Patients with SF3B1 or ASXL1/SF3B1 mutations were more frequently diagnosed with myelodysplastic syndrome (75.36% and 64.71%, respectively) than patients with ASXL1 mutations (24.72%). Patients with ASXL1/SF3B1 (23.53%) mutations more frequently had myelodysplastic/myeloid proliferative neoplasm than did patients with ASXL1 mutations (5.62%) or with SF3B1 mutations (15.94%). OS of the ASXL1 mutation-only group was worse than that of the SF3B1 mutation-only group with a hazard ratio of 5.83 (p=0.017). Finally, and most importantly, the OS of the ASXL1/SF3B1 co-mutation group was poorer than that of both single-mutation groups (p=0.005). ASXL1/SF3B1 co-mutations portend worse OS than isolated ASXL1 or SF3B1 mutations, which might be due to abnormalities in both the epigenetic-regulatory and RNA-splicing pathways or because two genes instead of one are mutated.

Applying bioinformatic analysis for prognostic assessment of the <i>HS3ST6</i> missense mutations clinical significance in the development of hereditary angioedema

Hereditary angioedema (HAE) is a genetically determined disease characterized by recurrent attacks of edema affecting the subcutaneous and/or submucosal layers of tissue, face, lips, neck, extremities of the body, oral cavity, intestine and/or larynx. In the latter case, the disease becomes life-threatening. The majority of HAE cases are associated with decreased levels of C1 (C1-esterase inhibitor), there are also descriptions of HAE with dysfunctional C1 inhibitor and HAE with normal C1 inhibitor. In the first and second variants, mutations in the C1NH gene are the cause of the disease. HAE with normal quantitative and functional levels of C1-inhibitor has the same clinical manifestations but with mutations in other genes, including F12, PLG, ANGPT1, KNG1, MYOF, and HS3ST6. Currently, mutations in the HS3ST6 gene remain poorly understood; only one missense mutation (p.Thr144Ser, rs746467957) associated with the development of HAE has been described.The aim of our work was to study new mutations in the HS3ST6 gene and analyze in silico their prognostic nature and clinical significance for the development of hereditary angioedema.The material was whole blood samples obtained from 13 patients with symptoms of hereditary angioedema without reduced levels and function of C1-INH.Whole exome sequencing of patients, bioinformatic analysis of HS3ST6 gene mutations using a number of databases and Web resources to predict the effect of mutations on the protein and assess the conservatism of the positions of the mutations detected was involved in study methods.Mutations in the HS3ST6 gene were identified in four patients, including two cases with two mutations simultaneously. Application of bioinformatic analysis allowed us to obtain new data on four missense mutations in the studied gene. Potential pathogenetic significance was determined for three of them. The mutation NC_000016.9:g.1962132G>A (p.A163V) is most likely to be involved in pathogenesis of HAE by indirect disruption of heparan sulfate O-sulfation directly within the protein. The NC_000016.9:g.1962024G>A mutation (p.P199L) appears to lead to the development of the disease through disruption of docking with SDC2 heparan sulfate. In the NC_000016.9:g.1962046C>T (p.A192T) mutation, destabilization of the 192 amino acid position next to PAPS, may contribute to disruption of heparan sulfate O-sulfation through disruption of protein functional activity and, therefore, catalysis transfer of sulfo group to heparan sulfate syndecan-2. Thus, in all three cases, the formation of HAE appears to be possible due to disruption of the O-sulfation steps of heparan sulfate syndecan-2.Considering that in silico methods offer new opportunities to assess the pathogenetic significance of mutations, the application of bioinformatic analysis can contribute to a detailed investigation of the causes of hereditary angioedema. The present work convincingly demonstrates that rare mutations in the HS3ST6 gene may be involved in the pathogenesis of HAE and provoke edema due to increased bradykinin release.

Co-Existing ASXL1 and SF3B1 Mutations Carried Worse Outcome Than ASXL1 or SF3B1 Mutations Alone

Introduction: Somatic mutations in the ASXL1 gene (additional sex combs like 1) are frequently observed in myeloid malignancies, including Clonal cytopenia of unknown significance (CCUS), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS/MPN, and acute myeloid leukemia (AML), and have been found to be independent predicative factor for poor overall survival. Mutations in SF3B1 (Splicing factor 3B subunit 1) were also frequently detected in the above hematopoietic disorders. It represents distinct molecular and clinical patient groups and shows particular association with ringed sideroblasts. Most studies have associated SF3B1 mutations with good clinical prognosis. Both ASXL1 and SF3B1 mutations have been reported to frequently coexist with the mutations in other genes. Co-mutation of ASXL1 with SF3B1 is less frequent and has even been reported to be mutually exclusive by some authors. There are very few and contradictory reports about the clinical significance of co-existing ASXL1 and SF3B1 mutations, which is the goal of this study. Previous studies were also confounded by patients with mutations in some other genes. Method: We identified 175 patients with only ASXL1 mutations, only SF3B1 mutations, or only ASXL1/SF3B1 co-mutations from our database of 8285 patients with NextGen sequencing (NGS) results, to study and compare their clinical features and outcomes. Baseline characteristics and other somatic mutation distribution were compared by chi square (categorical variables) and t-test (continuous variables). Overall survival was calculated using the Kaplan-Meier method log-rank test. Results: Approximately 8285 patients with diagnosed or suspected myeloid neoplasm were tested by NGS. There were1515 patients with ASXL1 mutations with or without other genes mutated, and 583 patients with SF3B1 mutations with or without other genes mutated. There were also 97 patients with concurrent ASXL1 and SF3B1 mutations, most with additional genes mutated. Out of these patients, we identified 69 patients with only ASXL1 mutations (ASXL1 group), 89 patients with only SF3B1 mutations (SF3B1 group), and 17 patients with only ASXL1 plus SF3B1 mutations (ASXL1/SF3B1 group). The median age of ASXL1 group (69) was much younger than SF3B1 (76) and ASXL1/SF3B1 group (p values 0.01208 and 0.0001, respectively). The female to male ratios were similar among these groups. ASXL1 mutations were more frequent in AML and CCUS (22.47% and 33.71%) than ASXL1/SF3B1 (11.76% and 0%) and SF3B1 (1.45% and 7.25%) (p values < 0.003) mutations. On the contrary, SF3B1 and ASXL1/SF3B1 mutations were more frequent in MDS patients (75.36% and 64.71%) than ASXL1 mutations. In MDS/MPN, ASXL1/SF3B1 mutations (23.53%) were more frequent than SF3B1 (15.94%) and ASXL1 mutations (5.62%, p value 0.03513). The three groups of patients showed similar cytogenetic changes, with similar percentages of patients with normal karyotype, del(5q), del(7/7q), del(17p/TP53), del(20q), or trisomy 8. Finally, we compared the overall survival of the three groups of patients with normal karyotype by Kaplan-Meier survival analysis. As expected, ASXL1 group showed worse overall survival than SF3B1 group with a hazard ratio of 5.834 (p value 0.0175). However, it is interesting to found that ASXL1/SF3B1 group showed poorer overall survival than both SF3B1 and ASXL1 groups with a hazard ratio of 9.840 (p value 0.0052) (Figure 1). Conclusion: Co-existing ASXL1 and SF3B1 mutations carried worse outcome than ASXL1 or SF3B1 mutation alone, likely due to abnormalities in both epigenetic regulatory pathway and RNA splicing pathway. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular Characterization of Clinical Response and Relapse in Patients with IDH1m ND-AML Treated with Ivo+AZA in the AGILE Study

Background: Mutations in isocitrate dehydrogenase 1 (mIDH1) occur in approximately 6-10% of patients (pts) with AML. Ivosidenib (IVO), a potent, oral, targeted inhibitor of mIDH1, is FDA approved as monotherapy for the treatment of relapsed/refractory AML and newly-diagnosed AML (ND-AML) in adults >75 years of age or with comorbidities that preclude the use of intensive chemotherapy. In addition, the combination of IVO with azacitidine (AZA) was recently FDA approved in older or unfit AML pts with mIDH1 as a result of the global Phase 3 AGILE trial (NCT03173248). Compared to placebo (PBO) + AZA, the combination of IVO + AZA extended event-free survival (EFS; hazard ratio 0.33, 95% CI [0.16 - 0.69]; P = 0.002), overall survival (OS; median 24.0 months vs. 7.9 months; hazard ratio 0.44; 95% CI [0.27 - 0.73]; P = 0.001), and increased the rates of complete remission or complete remission with partial hematologic recovery (CR/CRh; 52.8% vs. 17.6%). To date, biomarker analysis has shown that IVO+AZA is associated with deep and durable clearance of IDH1m clones with improved clinical responses and EFS/OS in several pt subgroups, including those with mutations in receptor tyrosine kinase (RTK) pathway genes, a subgroup refractory to IVO monotherapy. Aim: To characterize clonal evolution and relapse mechanisms in pts with mIDH1 ND-AML treated with IVO+AZA. Methods: Patients received oral IVO + AZA per study protocol. Next-generation DNA sequencing (DNA-seq) on baseline and longitudinal samples was performed on bone marrow aspirates (BMA), bone marrow mononuclear cells (BMMCs), or peripheral blood mononuclear cells (PBMCs) depending on sample availability using the ACE Extended Cancer Panel (Personalis, Inc); limit of detection (LOD) 2% variant allele frequency (VAF). This analysis was focused on variants in 95 AML-associated genes with known or likely oncogenic potential. Results: Among 72 pts enrolled on the IVO+AZA arm, 46 had both baseline and longitudinal DNA-seq data available, with a median follow-up of 366 days on study (range 16 - 1210). The median number of baseline mutations was 3 (range 1 - 10). Suppression of all baseline non-DTA (DNMT3A / TET2 / ASXL1) mutations to below the LOD ("mutation clearance") was observed in 28 patients, including 22/34 (65%) pts who achieved CR/CRh [Table 1]. To identify potential mechanisms of acquired resistance, on-treatment samples were analyzed for the emergence of mutations not detected at baseline. Overall, emerging mutations were identified in 22/46 (48%) pts (median 2 emerging mutations; range 1-6). 2/46 (4%) pts had emerging mutations in IDH2, but discontinued treatment due to adverse events and stable disease, respectively. No second-site IDH1 mutations were observed across any response category. Longitudinal samples were available for 8/10 (80%) pts who discontinued treatment due to relapse (RL) following an objective response (4 CR, 2 CRh, 1 CRi, 1 MLFS). Emerging mutations were detected in 6/8 (75%) of RL pts [Table 2]. Genes with emerging mutations recurrently associated with RL included ASXL1, FLT3, RUNX1, and TET2 (2 pts each). The IDH1 mutation was not detected at RL in 6/8 (75%) pts. Among the two RLs that occurred in the absence of emerging mutations, one was associated with expansion of a baseline FLT3-ITD subclone, and one was not associated with any new emerging or expanding mutations. To further evaluate clonal evolution and disease progression of these pts, scDNA-seq is being performed and will be presented. Conclusion: IVO+AZA combination therapy leads to deep and durable remissions associated with clearance of mIDH1 and baseline co-mutations. To date, no second-site IDH1 mutations have been observed, but mutations in other genes, including IDH2, can emerge and may provide an opportunity for relapse. Relapses were observed in a subset of responders and were generally associated with expansion or emergence of high-risk mutations occurring independently of mIDH1. Further study is needed to better characterize mechanisms of RL to IVO+AZA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clonal Hematopoiesis in Vexas Syndrome

Background VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a recently described systemic inflammatory disease of older men, due to somatic mutations in UBA1 (UBA1mut). VEXAS predisposes to myelodysplastic syndrome (MDS) and plasma cell dyscrasia (PCD). A few studies have reported concomitant somatic mutations in genes associated with myeloid neoplasia but their true prevalence and the clonal landscape is unknown in UBA1mut cases. We hypothesized that clonal hematopoiesis in myeloid-related genes (typical CH) may be linked to heterogenous clinical phenotypes and progression to MDS seen in VEXAS. Methods We performed error-corrected DNA sequencing (ECS) in 80 VEXAS patients and correlated findings with clinical outcomes in 77 patients. Targeted single-cell proteogenomic sequencing (scDNA) was performed on three patients to define clonal architectures. DNA methylation was also investigated in 38 cases. ResultsUBA1mut p.M41T c.122T>C (n=52; 65%) was most frequent followed by p.M41V c.121A>G (n=15;18%), p.M41L c.121A>C (n=10,12%), and three (4%) splice site mutations (c.118-1G>C); one patient had two UBA1mut in independent clones: a p.M41T and c.118-2A>C. Median variant allele frequency (VAF) of UBA1 mut variant was 75%. Forty-eight patients (60%) had one (n=28; 35%) or ≥2 typical CH mutations (n=20; 23%) concomitant with UBA1mut, a frequency much higher than in healthy age-matched controls (>50% in VEXAS patients ³40 years vs. 18% in controls). Mutations in DNMT3A and TET2 were most frequent, DNMT3A mutations at high VAF (median 27%), in contrast to TET2 (median 1.5%; Figure 1). Patients with typical CH were divided into 4 subgroups: with DNMT3A or TET2 mutations alone (D/T, n=19), concomitant DNMT3A and TET2 mutations (D&T; n=7), mutations in other genes regardless of DNMT3A/TET2 mutations (Others; n=20), and no typical CH mutations (n=31). UBA1mut genotype and co-occurring CH mutations did not predict inflammatory or hematologic manifestations (MDS, PCD, thrombosis, cytopenia). An MDS diagnosis was only associated with transfusion-dependent anemia and thrombocytopenia but not with typical CH mutation subgroups, UBA1 VAF or abnormal karyotype. Overall survival was 60% at 10 years. MDS diagnosis did not confer poorer prognosis but transfusion-dependent anemia, thrombocytopenia, and the presence of typical CH mutation at VAF>2% (particularly DNMT3A/TET2) increased the risk of mortality (Figure 2). VEXAS patients had marked epigenetic age acceleration with a median of 11 years (range 7-23), especially in those with DNMT3A or TET2 mutations. By scDNA, >90% of myeloid but not lymphoid cells were UBA1mut. The majority (60-90%) of HSPC in the marrow were also UBA1mut. There was a shift towards increased neutrophils (with typically high CD16/CD62L/CD10 expression), plasmacytoid DC and classical monocytes in PB, compared to a healthy control. In UPN1, a TET2 and DNMT3A mutations preceded UBA1mut in the same clone, resulting in striking clonal expansion after acquisition of UBA1mut; all variants were at VAF >45% by ECS. In UPN2, UBA1mut was a driver of a clone that dominated the clonal landscape; subclones with SF3B1 and TET2 mutations in the same cell remained stable at lower VAF by ECS (<5%). In UPN3, UBA1mut itself resulted in clonal expansion of mutated cells. Conclusions In VEXAS, UBA1mut cells are the primary cause of inflammatory and hematological manifestations, defining a new molecularly defined somatic entity that is associated with MDS. scDNA data demonstrate that UBA1mut clones have competitive proliferative advantage, therefore, the expansion of clones with typical CH mutations is dependent on co-occurrence with UBA1mut. In VEXAS, typical CH mutations may serve as a biomarker of shortened overall survival, without an increased rate of myeloid neoplastic transformation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A novel nonsense mutation in PPP2R5D is associated with neurodevelopmental disorders and shows incomplete penetrance in a Chinese pedigree

Background and purposePPP2R5D mutations are known to cause neurodevelopmental disorders in an autosomal-dominant manner. To date, the vast majority of the reported cases in the literature have been sporadic with de novo mutations. There are no data regarding PPP2R5D mutation penetrance. We aimed to unravel the underlying genetic defects in 3 generations of a family affected by intellectual disability, neurodevelopmental delay, and facial dysmorphology. MethodsWe performed detailed clinical examinations and whole-exome sequencing in the family. ResultsWe identified a novel mutation, c.1321 C>T (p.Arg441*), in PPP2R5D. The mutation cosegregated with affected family members I-2 and II-7 but not II-3, who bears the mutation but is phenotypically healthy. Our whole-exome sequencing also excluded the involvement of pathogenic mutations in other genes known to be related to neurodevelopmental disorders. The mutation was predicted to introduce a premature stop codon at position 441, thereby truncating the 162 amino acids at the C-terminus of the encoded protein. ConclusionsWe report a familial transmitted PPP2R5D-related neurodevelopmental disorder as well as a novel nonsense pathogenic mutation and its incomplete penetrance. Our study expands the mutational and phenotypic spectra of PPP2R5D-related neurodevelopmental disorders, broadens our understanding of these disorders, and will thus be valuable for mutation-based pre- and postnatal screening and genetic diagnosis of neurodevelopmental disorders.

Osteogenesis Imperfecta, a case report

Introduction: osteogenesis imperfecta is a rare genetic disease mainly caused by a dominant mutation in COL1A1 y COL1A2 collagen type I genes which codes for collagen type I ???? chains. The OI may also be the result of recessive mutations in other genes. Its clinical spectrum that goes from milder form to lethal perinatal forms. Clinical diagnosis might be established in a patient that presents repeated fractures, blue scleras, short stature, teething disorders and deafness. Case report: the patient was 24 years old, third gestation, with an obstetric history of a dead child with osteogenesis imperfecta, who was supervised and controlled in a public institution. Several laboratory tests corroborated that the current gestation presented osteogenesis imperfecta, thus, a surgical procedure was scheduled at 38.2 weeks of gestation. During the transoperative period, we found the newborn, with the described characteristics, alive. The child was admitted to the neonatology service, where we confirmed multiple fractures in extremities and thorax. In view of this reserved prognosis, she died within 24 hours due to pulmonary complications. Conclusions: an early diagnosis within the first three months of pregnancy is essential in order to establish a prognosis and timely treatment whenever it is necessary

Evolutionary rescue of phosphomannomutase deficiency in yeast models of human disease.

The most common cause of human congenital disorders of glycosylation (CDG) are mutations in the phosphomannomutase gene <i>PMM2,</i> which affect protein <i>N</i>-linked glycosylation. The yeast gene <i>SEC53</i> encodes a homolog of human <i>PMM2</i>. We evolved 384 populations of yeast harboring one of two human-disease-associated alleles, <i>sec53-</i>V238M and <i>sec53</i>-F126L, or wild-type <i>SEC53</i>. We find that after 1000 generations, most populations compensate for the slow-growth phenotype associated with the <i>sec53</i> human-disease-associated alleles. Through whole-genome sequencing we identify compensatory mutations, including known <i>SEC53</i> genetic interactors. We observe an enrichment of compensatory mutations in other genes whose human homologs are associated with Type 1 CDG, including <i>PGM1</i>, which encodes the minor isoform of phosphoglucomutase in yeast. By genetic reconstruction, we show that evolved <i>pgm1</i> mutations are dominant and allele-specific genetic interactors that restore both protein glycosylation and growth of yeast harboring the <i>sec53</i>-V238M allele. Finally, we characterize the enzymatic activity of purified Pgm1 mutant proteins. We find that reduction, but not elimination, of Pgm1 activity best compensates for the deleterious phenotypes associated with the <i>sec53</i>-V238M allele. Broadly, our results demonstrate the power of experimental evolution as a tool for identifying genes and pathways that compensate for human-disease-associated alleles.

CML-288 Impact of ASXL1 Mutations on Prognosis of Chronic Phase Chronic Myeloid Leukemia

Mutations in ABL1 are established risk factors for therapy resistance in chronic phase chronic myeloid leukemia (CML-CP). However, much less is known about the impact of other cancer-associated gene mutations. Study the prevalence and clinical impact of cancer-associated gene mutations in CML-CP. We screened our databases for CML-CP patients who were tested with a targeted next-generation sequencing panel that included 81 genes recurrently mutated in hematologic malignancies. Event-free survival (EFS) was measured from treatment initiation to the time of an event defined as loss of hematologic response, loss of major cytogenetic response (MCyR), lack of MCyR after 12 months, or transformation to accelerated/blast phase. Failure-free survival (FFS) was measured similarly, with the additional events of drug discontinuation due to adverse effects or no response. We subsequently performed univariate and multivariate analyses. The median age of patients was 60 years (18-80) and 46% were female. The most common first-line therapy was dasatinib (52%), followed by imatinib (19%), dasatinib + venetoclax (16%), and other TKIs (13%). The distribution of evaluable patients to low, intermediate, and high Sokal groups was 21%, 61%, and 18%, respectively. Cancer mutations were found in 23/68 patients (34%). The most common mutations involved ASXL1 (10/68 patients; 15%), DNMT3A (5/68 patients; 7%), and RUNX1 (3/68 patients; 4%). Mutations were also detected in ASXL2, CALR, EZH2, GNAS, IDH2, SF3B1, SMC3, SRSF2, STAG2, and SUZ12. Unlike mutations in other genes, patients with ASXL1 mutations had worse EFS compared to the no-mutation group (median of 33 vs. 88 months; P=0.002) and worse FFS (median of 14 vs. 58 months; P=0.04). There was no difference in overall survival by mutational status. In a multivariate analysis, mutated ASXL1 was independently associated with worse EFS (HR of 4.42; 95% CI 1.63-11.99). There was a non-statistically significant trend of lower molecular response rates and longer times to response (TTR) in ASXL1-mutated patients compared to wild-type (major molecular response rate of 78% vs. 82%; P=0.7 with median TTR of 17.5 vs. 9.2 months; P=0.5). Mutations in ASXL1 are associated with delayed responses and worse outcomes when detected in CML-CP.

Investigating a Genetic Link Between Alzheimer’s Disease and CADASIL-Related Cerebral Small Vessel Disease

Monogenic forms of Alzheimer’s disease (AD) have been identified through mutations in genes such as APP, PSEN1, and PSEN2, whilst other genetic markers such as the APOE ε carrier allele status have been shown to increase the likelihood of having the disease. Mutations in these genes are not limited to AD, as APP mutations can also cause an amyloid form of cerebral small vessel disease (CSVD) known as cerebral amyloid angiopathy, whilst PSEN1 and PSEN2 are involved in NOTCH3 signalling, a process known to be dysregulated in the monogenic CSVD, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). The overlap between AD genes and causes of CSVD led to the hypothesis that mutations in other genes within the PANTHER AD–presenilin pathway may be novel causes of CSVD in a cohort of clinically suspicious CADASIL patients without a pathogenic NOTCH3 mutation. To investigate this, whole exome sequencing was performed on 50 suspected CADASIL patients with no NOTCH3 mutations, and a targeted gene analysis was completed on the PANTHER. ERN1 was identified as a novel candidate CSVD gene following predicted pathogenic gene mutation analysis. Rare variant burden testing failed to identify an association with any gene; however, it did show a nominally significant link with ERN1 and TRPC3. This study provides evidence to support a genetic overlap between CSVD and Alzheimer’s disease.

Immunohistochemical loss of enhancer of Zeste Homolog 2 (EZH2) protein expression correlates with EZH2 alterations and portends a worse outcome in myelodysplastic syndromes

AbstractEZH2 coding mutation (EZH2MUT), resulting in loss-of-function, is an independent predictor of overall survival in MDS. EZH2 function can be altered by other mechanisms including copy number changes, and mutations in other genes and non-coding regions of EZH2. Assessment of EZH2 protein can identify alterations of EZH2 function missed by mutation assessment alone. Precise evaluation of EZH2 function and gene-protein correlation in clinical MDS cohorts is important in the context of upcoming targeted therapies aimed to restore EZH2 function. In this study, we evaluated the clinicopathologic characteristics of newly diagnosed MDS patients with EZH2MUT and correlated the findings with protein expression using immunohistochemistry. There were 40 (~6%) EZH2MUT MDS [33 men, seven women; median age 74 years (range, 55–90)]. EZH2 mutations spanned the entire coding region. Majority had dominant EZH2 clone [median VAF, 30% (1–92)], frequently co-occurring with co-dominant TET2 (38%) and sub-clonal ASXL1 (55%) and RUNX1 (43%) mutations. EZH2MUT MDS showed frequent loss-of-expression compared to EZH2WT (69% vs. 27%, p = 0.001). Interestingly, NINE (23%) EZH2WT MDS also showed loss-of-expression. EZH2MUT and loss-of-expression significantly associated with male predominance and chr(7) loss. Further, only EZH2 loss-of-expression patients showed significantly lower platelet counts, a trend for higher BM blast% and R-IPSS scores. Over a 14-month median follow-up, both EZH2MUT (p = 0.027) and loss-of-expression (p = 0.0063) correlated with poor survival, independent of R-IPSS, age and gender. When analyzed together, loss-of-expression showed a stronger correlation than mutation (p = 0.061 vs. p = 0.43). In conclusion, immunohistochemical assessment of EZH2 protein, alongside mutation, is important for prognostic workup of MDS.

The Genomic Environment of BRAF Mutated and BRAF/PIK3CA Double Mutated Colorectal Cancers.

Background: Colorectal cancer represents the most prevalent gastrointestinal malignancy. Prognosis of metastatic disease has improved in recent years with the introduction of effective systemic therapies, but mean survival remains in the range of two to three years. Targeted therapies based on specific molecular alterations in sub-sets of colorectal cancers have the potential of contributing to therapeutic progress. BRAF and PIK3CA are oncogenic kinases commonly mutated in colorectal cancers and can be targeted through small molecule kinase inhibitors. Methods: Clinical and genomic data from two extensive series of colorectal cancers were interrogated to define the molecular characteristics of cancers with BRAF mutations with and without concomitant mutations in PIK3CA. Results: Colorectal cancers that are BRAF and PIK3CA double mutants represent a small minority of about 5% of colorectal cancers in the two examined series of mostly localized disease. They also represent about one third of all BRAF mutated colorectal cancers. Most mutations in BRAF are classic V600E mutations. A high prevalence of MSI and CIMP is observed in BRAF mutated colorectal cancers with or without PIK3CA mutations. Mutations in tumor suppressors FBXW7 and ATM display a higher prevalence in BRAF mutated cancers. The prognosis of BRAF mutated colorectal cancers with or without PIK3CA mutations is not significantly different than counterparts with wild type BRAF. This contrasts with the known adverse prognostic effect of BRAF in metastatic disease and relates to the different prevalence of MSI in mutant BRAF localized versus metastatic colorectal cancers. Conclusions: BRAF mutations are the defining molecular alterations in double mutant BRAF and PIK3CA colorectal cancers as determined by increased MSI and CIMP in BRAF subsets with and without PIK3CA mutations. Moreover, BRAF mutated cancers with and without PIK3CA mutations are characterized by the absence of KRAS mutations and a lower prevalence of APC mutations than BRAF wild type counterparts. Mismatch-repair-associated gene mutations display higher frequencies in BRAF mutated colorectal cancers. Despite the absence of prognosis implications of BRAF mutations in the studied cohorts of mostly localized cancers, such mutations could be prognostic in certain subsets. The presence of mutations in other genes, such as ATM and high MSI status present opportunities for combination therapies.

MAP kinase and mammalian target of rapamycin are main pathways of gallbladder carcinogenesis: results from bioinformatic analysis of next generation sequencing data from a hospital-based cohort (NCT05404347).

Gallbladder Cancer (GBC) is one of the most common cancers of the biliary tract and the third commonest gastrointestinal (GI) malignancy worldwide. The disease is characterized by the late presentation and poor outcome despite treatment, and hence, newer therapies and targets need to be identified. The current study investigated various functionally enriched pathways in GBC pathogenesis involving the genes identified through Next Generation Sequencing (NGS) in a hospital-based cohort. The Pathway enrichment analysis and Gene Ontology (GO) were carried out after NGS, followed by the construction of the protein-protein interaction (PPI) network to discover associations among the genes. Of the thirty-three patients with GBC who were screened through next-generation sequencing (NGS), 27somatic mutations were identified. These mutations involved a total of 14 genes. The p53 and KRAS were commonly found to be mutated, while mutations in other genes were seen in one case each, the mean number of mutations were 1.2, and maximum mutation in a single case (eight) was seen in one case. The bioinformatics analysis identified MAP kinase, PI3K-AKT, EGF/EGFR, and Focal Adhesion PI3K-AKT-mTOR signaling pathways and cross-talk between these. The results suggest that the complex crosstalk between the mTOR, MAPK, and multiple interacting cell signaling cascades can promote GBC progression, and hence, mTOR-MAPK targeted treatment will be an attractive option.

A stepwise haematological screening and whole-exome sequencing reveal multiple mutations from SUPT5H causing an elevation of Hb A2 from a cohort of 47336 individuals.

Though an increase in Hb A2 is one of the most key markers of β-thal carriers, a few independent cases are reported to show elevated Hb A2 levels caused by mutations in other genes beyond β-globin gene. We reviewed the haematological indices of 47336 individuals to analyse the phenotype-genotype correlation and identified 1439 individuals (3.04%) positive in the elevation of Hb A2 . Globin and KLF1 genes analysis was performed, and further whole-exome sequencing was carried to dissect the genetic causes of those positive samples without β-thalassemic or KLF1 mutations. Of these 1439 individuals with elevated Hb A2 , 1381 had a molecular defect in globin genes, and most were β-thalassemic mutation; 10 had a molecular defect in KLF1 gene. Finally, among the 38 individuals without β-thalassemic or KLF1 mutations, 7 were identified to carried a loss-of-function mutation in SUPT5H. This study has provided a mutation spectrum of SUPT5H in a cohort screening leading to the elevation of Hb A2 . According to the previous observations that individuals with a combination of β-thal mutation and a SUPT5H variant might present moderate β-thaelassemia, these findings emphasized the importance of comprehensive molecular diagnosis to prevent birth defects of β-thaelassemia caused by rare mutations from modifier genes.

Natural mutation in the regulatory gene (srrG) influences virulence-associated genes and enhances invasiveness in Streptococcus dysgalactiae subsp. equisimilis strains isolated from cases of streptococcal toxic shock syndrome.

SummaryBackgroundStreptococcus dysgalactiae subspecies equisimilis (SDSE) has emerged as an important cause of severe invasive infections including streptococcal toxic shock syndrome (STSS). The present study aimed to identify genes involved in differences in invasiveness between STSS and non-invasive SDSE isolates.MethodsSTSS and non-invasive SDSE isolates were analysed to identify csrS/csrR mutations, followed by a comparative analysis of genomic sequences to identify mutations in other genes. Mutant strains were generated to examine changes in gene expression profiles and altered pathogenicity in mice.FindingsOf the 79 STSS-SDSE clinical isolates, 15 (19.0%) harboured csrS/csrR mutations, while none were found in the non-invasive SDSE isolates. We identified a small RNA (sRNA) that comprised three direct repeats along with an inverted repeat and was transcribed in the same direction as the sagA gene. The sRNA was referred to as srrG (streptolysin S regulatory RNA in GGS). srrG mutations were identified in the STSS-SDSE strains and were found to be associated with elevated expression of the streptolysin S (SLS) gene cluster and enhanced pathogenicity in mice.InterpretationThe csrS/csrR and srrG mutations that increased virulence gene expression in STSS-SDSE isolates were identified, and strains carrying these mutations caused increased lethality in mice. A significantly higher frequency of mutations was observed in STSS-SDSE isolates, thereby highlighting their importance in STSS.FundingJapan Agency for Medical Research and Development, the Japan Society for the Promotion of Science (JSPS), and the Ministry of Health, Labor, and Welfare of Japan.

Abstract 5774: Investigating the characteristics of BRAF Mutations and its potential relationship with therapy in a large Chinese Lung Cancer cohort

Abstract Background: In lung cancer, the proportion of BRAF mutations is low. Rare specific studies have evaluated the BRAF mutation characteristics in lung cancer. Currently, there are some studies suggesting that patients with BRAF mutations may be benefit from immunotherapy, while patients with BRAF V600E may benefit from targeted therapy. Here, the characteristics of BRAF mutation, including BRAF V600E, in a lung cancer cohort of Chinese were analyzed. The characteristics of BRAF mutation with PD-L1 expression were also analyzed. Method: BRAF mutant samples were extracted from 12,051 our cohort samples of lung cancer that from Onco Panscan࣪ (Genetron Health) based sequencing of tissue. The characteristics of them were then investigated. Co-mutations of PD-L1 expressed BRAF samples were also analyzed. Results: 684 samples (5.68%) in our cohort had BRAF mutations. Only 134 (19.59%) of them were V600E mutation, maybe good response to BRAF inhibitors. 550 (80.41%) of samples were BRAF non-V600E mutation, maybe benefit from immunotherapy. BRAF mutations were more happened in male (58.3%), while female patients were increased in V600E mutation group when compared with non-V600E group (49.25% vs. 39.82%, P=0.0509). BRAF mutations were more likely to occur in LUAD than in LUSC (6.37% vs. 3.89%, P=0.0022), especially BRAF V600E mutation. The mean age of BRAF mutant patients was 61.56 ± 10.32 years. BRAF V600E patients was significantly older than BRAF non-V600E patients on average (63.01 ± 9.76 years vs. 61.20 ±10.43, P=0.0129). BRAF mutations were distributed across all exons but concentrated on exon 15 (269/684, 39.33%). Moreover, BRAF V600E mutation accounted for 49.81% (134/269) of exon 15. In our data, there were 108 samples with BRAF mutation and PD-L1 positive (TPS≥1%). Among them, 95 samples (87.96%), including samples with BRAF V600E or non-V600E mutation, had no co-mutations or without other medication targets, may be benefit from BRAF inhibitors, or may be good response to immunotherapy, especially the 20 samples with TMB-high. Interestingly, 13 samples had EGFR sensitive co-mutation or EML4-ALK fusion. These 13 patients may be benefit from TKI firstly, then immunotherapy can be considered. Conclusions: Most of BRAF mutations in Chinese lung cancer samples were non-V600E mutations (80.26%). BRAF mutations were more occurred in male and LUAD patients whose age around 61.56 ± 10.32 years. BRAF V600E patients were much older. Among PD-L1 positive and BRAF mutant samples, most BRAF mutations (87.96%), including V600E, were mutually exclusive with driver mutations of other genes. These patients will most likely benefit from BRAF inhibitors or immunotherapy. For the samples with co-mutation of BRAF and EGFR or ALK, may be treated EGFR or ALK TKI firstly. Citation Format: Jianqing Zhang, Jiagang Feng, Ling Liu, Man Jia, Tong Liu, Jiaqiang Zhang, Zehua Liao, Jianmei Zhao, Yu Fang, Jing Chen, Xiaoyan Zhang, Tonghui Ma. Investigating the characteristics of BRAF Mutations and its potential relationship with therapy in a large Chinese Lung Cancer cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5774.

Abstract LB039: VHL-deficient renal cell carcinoma displays defective ATM activation and sensitivity to ATR inhibition

Abstract Introduction: Clear cell renal cell carcinoma (ccRCC) displays genomic instability, but lacks mutations in canonical DNA damage response (DDR) genes. Bi-allelic loss of VHL is a near ubiquitous, truncal event in ccRCC development. Herein, we performed preclinical and clinical analysis to understand how VHL loss impacts DDR pathway activity and how specific DDR deficiencies (DDR-D) may be targeted in ccRCC. Methods: We performed DNA whole exome sequencing (WES) and reverse phase protein array (RPPA) early stage ccRCC tumors. All patients were consented under an IRB-approved protocol for the use of tissue. Transcriptomic and protein analyses of select DDR pathways was performed on in silico data (TCGA). DDR functional assays and ATR inhibitor treatment were performed on preclinical cell line models, and in vivo ATR inhibitor treatment in ccRCC xenografts with varying VHL status. Results: Bi-allelic loss of VHL via mutation in one allele and copy number loss of chromosome 3p was observed in 13/13 patient tumors. Mutations in other genes associated with ccRCC and/or canonical DDR genes were not observed. Despite VHL loss being the only driver mutation, these early stage tumors displayed tumor mutational burden (TMB) similar to ccRCC across all tumor stages (1.1 ± 0.5 mut/Mb). Proteomic analysis of patient tumor versus normal tissue (N=12) revealed significantly different (P&amp;lt;0.05) mean expression level of 218 proteins, including significantly lower phospho-ATM at the autophosphorylated S1981 site and lower phospho-CHK2 at the downstream T68 site. VHL-/- cells display defective binding to and repressed activation of Tip60 (KAT5), a key acetyltransferase that activates ATM kinase and aids in the selection of DNA repair pathways following damage. Lastly, treatment with ATRi (VE-822) of 786-O and 786-VHL overexpressing tumor xenografts showed ATRi was safe and significantly inhibited tumor growth preferentially in the 786-O VHL null model. Conclusions: Here we show in patient tissue and preclinical models that VHL biallelic loss results in genomic instability and functional impairment of ATM kinase activity; furthermore, we provide preclinical evidence for selectively targeting the ATR signaling cascade in patients with VHL-deficient kidney cancer. Clinical trials of ATR inhibitor based therapy in patients with advanced cancer, including ccRCC, are underway (e.g. NCT04266912). Citation Format: Patrick G. Pilie, Lijun Zhou, Yan-Ting Zhang, Christine Peterson, Daniel McGrail, Yang Peng, Guang Peng, Xiande Liu, Xuesong Zhang, Eric Jonasch. VHL-deficient renal cell carcinoma displays defective ATM activation and sensitivity to ATR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB039.

P-210 Germline testing in pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains a challenging disease to treat. Despite advances in medical therapies, the 5-year survival rate remains below 9%. BRCA mutations have emerged as a target for the development of more effective therapies. Alterations in germline BRCA and PALB2 are detected in approximately 5-9% of patients with PDAC and can lead to homologous repair deficiency (HRD). PDAC with HRD is more susceptible to cytotoxic agents, that cause DNA damage and PARP inhibitors have emerged as an effective non-cytotoxic approach. Germline mutations in other genes involved in the homologous DNA repair pathway -such as ATM and RAD51- are potential targets. The aim of our study is to analyse the frequency of germline mutations in patients with PDAC and to evaluate the role of family history as a supportive criterion for genetic testing. Descriptive and retrospective study of the germline study of pathogenic variants (PV) and variants of unknown significance (VUS) in patients (p) with PDAC and those with 1st degree family history of pancreatic cancer studied at the Salamanca Genetic Counselling Unit (UCGS) from 2004 to 2022. The determination of BRCA1/2 and other genes in peripheral blood was carried out using Next-Generation Sequencing (NGS) techniques. We included 30 patients (p) with PDAC, studied as index case. Males (36.7%). Age (median): 68.5 years [28-84]. BRCA1/2 was performed in 26p and multiplex-gene panel (MGP) in 4p. Four PV were finding: 13.3% (2 in BRCA2 and 2 in PALB2) and 2 VUS in BRCA1 and BRCA 2. 1p carrier of the PALB2 mutation suffered pancreatic and gastric cancer. In patients carrying PV, 4 cases of breast cancer, 1 pancreatic cancer, 1 prostate cancer and 1 ovarian cancer were recorded as first-degree antecedents. Other oncological antecedents described in the family (2nd and 3rd degree): 3 breast cancer, 1 gastric cancer and 2 prostate cancer. In patients carrying VUS in BRCA1/2, familial aggregation was also described with 1 case of pancreatic cancer, 1 breast cancer and 1 breast cancer in immediate family. We collected 96p with cancer: breast cancer 64p (66.7%) followed by ovarian cancer 19p (19.8%), with 1st degree family history of pancreatic cancer. Age (median): 61.5 years [30-93]. BRCA1/2 was performed in 89p and MGP in 7p. Seven PV (7.3%) were found: 5 in BRCA1, 1 BRCA2 and 1 CHEK2. 16 VUS (16.7%) were found: 6 BRCA2, 5 BRCA1, 1 ATM, 1 MSH3, 1 POLE, 1 MLH1, 1 PABL2. In 2p, two variants were found in the same patient (1 of them 1PV in CHEK2 and 1 VUS in MSH1, and the other, 1 VUS in ATM and 1VUS in POLE). PDAC could be an inherited condition in around 10% of cases. Germline testing BRCA and other HRD genes will improve therapeutic strategies and precision medicine in PDAC. In addition, familial involvement due to the identification of a hereditary syndrome should be taken into account.