Identified Germline Mutations Research Articles

Radiomics for differentiation of somatic BAP1 mutation on CT scans of patients with pleural mesothelioma.

The BRCA1-associated protein 1 (BAP1) gene is of great interest because somatic (BAP1) mutations are the most common alteration associated with pleural mesothelioma (PM). Further, germline mutation of the BAP1 gene has been linked to the development of PM. This study aimed to explore the potential of radiomics on computed tomography scans to identify somatic BAP1 gene mutations and assess the feasibility of radiomics in future research in identifying germline mutations. A cohort of 149 patients with PM and known somatic BAP1 mutation status was collected, and a previously published deep learning model was used to first automatically segment the tumor, followed by radiologist modifications. Image preprocessing was performed, and texture features were extracted from the segmented tumor regions. The top features were selected and used to train 18 separate machine learning models using leave-one-out cross-validation (LOOCV). The performance of the models in distinguishing between BAP1-mutated (BAP1+) and BAP1 wild-type (BAP1-) tumors was evaluated using the receiver operating characteristic area under the curve (ROC AUC). A decision tree classifier achieved the highest overall AUC value of 0.69 (95% confidence interval: 0.60 and 0.77). The features selected most frequently through the LOOCV were all second-order (gray-level co-occurrence or gray-level size zone matrices) and were extracted from images with an applied transformation. This proof-of-concept work demonstrated the potential of radiomics to differentiate among BAP1+/- in patients with PM. Future work will extend these methods to the assessment of germline BAP1 mutation status through image analysis for improved patient prognostication.

8332 Genotype/Phenotype Profile Of Pheochromocytoma/Paraganglioma Patients In A Single Tertiary Referral Center Of South Texas

Abstract Disclosure: V. Desai: None. M.A. Escobar Vasco: None. C. Estrada-Zuniga: None. B. Landry: None. H. Gonzalez-Cantu: None. D. Pruthi: None. F. Tozzi: None. J. Ferrell: None. M. Kitano: None. C. Solis-Herrera: None. J.M. Bruder: None. S. Raghunathan: None. P.L. Dahia: None. Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors. Nearly 40% of patients with PPGLs have a heritable germline mutation and around 30% carry somatic mutations in one of >20 known susceptibility genes. 15-20% of cases can progress to metastatic disease. Recently, ethnic differences have been suggested in PPGLs and support further evaluation of ethnically diverse cohorts which can offer guidance for surveillance and patient care. Goal: The objective of this study is to identify genetic and clinical features of patients with a diagnosis of PPGL in predominant ethnic groups located in a single tertiary referral center of South Texas that is a designated Pheo-Para Alliance Clinical and Research Center of Excellence. Methods: Medical records were utilized to obtain demographic and clinical data, including age at diagnosis, gender, ethnicity, family history, tumor location, metastasis, clinical disease vs. carrier status, and genotype from patients enrolled in an IRB-approved repository. Results: 62 index patients who were diagnosed with PPGL or had a pathogenic germline mutation with no clinical disease were enrolled between 2006 to 2023. Patients were predominantly Caucasian (n=30/59, 50.8%) and Hispanic (n=24/59, 40.6%). In the Hispanic group, twenty-one were diagnosed with PPGL and three were asymptomatic carriers. This group had an average age of 38.3 years (range, 13-77), 85.7% had pheochromocytomas (11.1% were bilateral), 14.3% were metastatic, 16.7% had a positive family history, and 65% had an identifiable germline mutation. The most commonly mutated genes in Hispanics were VHL (n=5), SDHx (n=5), and EPAS1 (n=3). In the Caucasian group, thirty patients were diagnosed with PPGL and three were asymptomatic carriers. The average age for Caucasians was 46.4 years (range, 19-80). 23% had a positive family history, 66.7% had pheochromocytoma, 22.2% had metastatic disease, and 77.3% had a germline mutation. The most common genotype in Caucasians was SDHx (n=12) followed by NF1 (n=4). Conclusion: In this PPGL cohort, Hispanic patients tended to be younger at diagnosis, have lower proportion of SDH germline mutations and higher frequency of somatic mutations compared to Caucasians. This preliminary observation should inform further investigation into PPGL genotype-phenotype associations across ethnic groups toward improving surveillance and patient care. Presentation: 6/2/2024

Shared germline genomic variants in two patients with double primary gastrointestinal stromal tumours (GISTs)

BackgroundGastrointestinal stromal tumours (GISTs) are prevalent mesenchymal tumours of the gastrointestinal tract, commonly exhibiting structural variations in KIT and PDGFRA genes. While the mutational profiling of somatic tumours is well...

Abstract 7334: Germline mutations in cancer susceptibility genes in patients with mucosal melanoma

Abstract Background: Mucosal melanoma (MM) is the rarest subtype of melanoma with markedly worse outcomes compared to cutaneous melanoma (CM). Unlike CM which is driven by somatic mutations due to UV exposure, risk factors for MM are less defined. While germline mutations in cancer susceptibility genes are well-studied in CM, they are less understood in MM. Herein, we aimed to identify the prevalence of germline mutations in cancer susceptibility genes in a large MM cohort. Methods: A cohort of 247 MM patients (pts) from MD Anderson Cancer Center with available blood for germline sequencing was identified. Pt characteristics were reflective of U.S. MM population with median age of 63, 60% females and 84.6% white. Clinicodemographic features of pts including tumor site, key somatic mutations (BRAF, NRAS, KIT) and personal history of other malignancies were abstracted, and their frequencies were compared in pts with and w/o germline mutation. Frequency of MITF and CHEK2 alleles were compared to expected population control rate based on TOPMED by Fisher’s exact test. Results: Among 247 pts, 10.9% had germline mutations, primarily in MITF (c.G952A:p.E318K) (2.4%) and CHEK2 (c.1100delC:p.T367fs) (1.6%) at OR of 13.9 (95%CI 5.0, 30.9) and 16.4 (4.4, 42.9) compared to population control rate (Table 1). MM sites of origin were gastrointestinal (36%), genitourinary (34%) and head & neck (30%). In terms of somatic mutations, KIT (26%) was most common, followed by NRAS (15%), non-V600E BRAF (6.5%) and BRAF V600E (2.4%). These variables did not differ between those with and w/o germline mutation. Pts with germline mutation had a higher incidence of non-melanoma malignancies (48% vs 29%; p=0.044). Table 1. Summary of identified germline mutations in the mucosal melanoma cohort. Germline mutation Protein MITF (N= 6) p.E318K CHEK2 (N=4) p.T367fs MUYTH p.G368D ATM splice ATM p.L263fs ATM p.R1875X ATM p.L1327X ATM p.A2067D ATM p.R2034X BRCA2 p.A1689fs CHEK2 p.S400T ERCC3 p.R109X MITF p.C29Y NF1 p.L1183R PALB2 p.V221X POLE p.D287E TP53 p.R306X TP53 p.V73Wfs*50 TSC1 p.Q844X Conclusions: This study reports a high prevalence of germline cancer susceptibility mutations in the largest cohort of MM to date, including mutations which are actionable. There is overlap with known MITF risk alleles for CM. We are currently performing a GWAS to comprehensively understand the genetic architecture of risk in MM. Citation Format: Afsaneh Amouzegar, Xiaogang Wu, James Long, Sabitha Prabhakaran, Latasha Little, Curtis Gumbs, Jared Malke, Julie Simon, Jianhua Zhang, Jennifer Wargo, Paul Scheet, Justin W. Wong, Priyadharsini Nagarajan, Jennifer L. McQuade, Andrew Futreal. Germline mutations in cancer susceptibility genes in patients with mucosal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7334.

Genetic profiling of azoospermic men to identify the etiology and predict reproductive potential

PurposeTo identify germline mutations related to azoospermia etiology and reproductive potential of surgically retrieved spermatozoa, and to investigate the feasibility of predicting seminiferous tubule function of nonobstructive azoospermic men by transcriptomic profiling of ejaculates.Materials and methodsSperm specimens were obtained from 30 men (38.4 ± 6 years) undergoing epididymal sperm aspiration for obstructive azoospermia (OA, n = 19) acquired by vasectomy, or testicular biopsy for nonobstructive azoospermia (NOA, n = 11). To evaluate for a correlation with azoospermia etiology, DNAseq was performed on surgically retrieved spermatozoa, and cell-free RNAseq on seminal fluid (n = 23) was performed to predict spermatogenesis in the seminiferous tubule.ResultsOverall, surgically retrieved sperm aneuploidy rates were 1.7% and 1.8% among OA and NOA cohorts, respectively. OA men carried housekeeping-related gene mutations, while NOA men displayed mutations on genes involved in crucial spermiogenic functions (AP1S2, AP1G2, APOE). We categorized couples within each cohort according to ICSI clinical outcomes to investigate genetic causes that may affect reproductive potential. All OA-fertile men (n = 9) carried mutations in ZNF749 (sperm production), whereas OA-infertile men (n = 10) harbored mutations in PRB1, which is essential for DNA replication. NOA-fertile men (n = 8) carried mutations in MPIG6B (stem cell lineage differentiation), whereas NOA-infertile individuals (n = 3) harbored mutations in genes involved in spermato/spermio-genesis (ADAM29, SPATA31E1, MAK, POLG, IFT43, ATG9B) and early embryonic development (MBD5, CCAR1, PMEPA1, POLK, REC8, REPIN1, MAPRE3, ARL4C). Transcriptomic assessment of cell-free RNAs in seminal fluid from NOA men allowed the prediction of residual spermatogenic foci.ConclusionsSperm genome profiling provides invaluable information on azoospermia etiology and identifies gene-related mechanistic links to reproductive performance. Moreover, RNAseq assessment of seminal fluid from NOA men can help predict sperm retrieval during testicular biopsies.

A MALDI-TOF mass spectrometry-based method for detection of copy number variations in BRCA1 and BRCA2 genes.

Background: Identifying germline mutations in BRCA1 and BRCA2 genes (BRCAs) would benefit the carriers in multiple aspects. In addition to single-nucleotide variations and small indels, copy number variations (CNVs) is also an indispensable component of identifiable mutations in BRCAs. A sensitive, rapid and throughput-flexible method to detect CNVs would be preferred to meet the rising clinical requirements for BRCAs testing. Methods: We developed a MALDI-TOF-MS-based method (MS assay) which included three steps: first, multiplex end-point PCR followed by a single base extension reaction; second, automated analyte transfer and data acquisition; third, data analysis. We applied MS assay to detect CNVs in BRCAs in 293 Chinese patients with ovarian or pancreatic cancer. All the samples were examined by targeted next-generation sequencing (TS) simultaneously. Samples were further cross-validated by multiplex ligation-dependent probe amplification (MLPA) if the results from MS assay and TS were inconsistent. Long range PCR was then applied to identify the exact breakpoints in BRCAs. Results: MS assay introduced highly multiplexed panels to detect CNVs of BRCAs semi-quantitatively. Simplified on-board data analysis was available for MS assay and no complex bioinformatics was needed. The turnaround time of MS assay was less than 8hours with a hands-on time of only 40min. Compared to TS, MS assay exhibited higher sensitivity (100% vs. 75%) and was more flexible in throughput, with the reagent cost per sample remaining constant no matter how many samples were examined per assay. A total of eight CNVs in BRCAs were detected from the 293 samples, and the molecular breakpoints were successfully identified in five samples through long-range PCR followed by Sanger sequencing. Conclusion: Our results suggested that MS assay might be an effective method in primary screening for CNVs in genes such as BRCAs, especially when short turnaround time and/or high sensitivity is a top priority.

Spectrum of histopathologic findings in risk-reducing bilateral prophylactic mastectomy in patients with and without BRCA mutations

Many germline mutations have been implicated in breast cancer pathogenesis and despite several studies on occult atypical lesions in prophylactic mastectomy specimens from patients with BRCA1/2 mutations, there are very limited data on other genes associated with increased breast cancer risk and the distribution of lesions in patients with hereditary breast cancer. We identified 207 patients who underwent bilateral prophylactic mastectomy due to germline mutations in BRCA1/2, PALB2, CHEK2, ATM, CDH1, PTEN, BARD1, or strong family history between 2015 and 2023. Patients with biopsy-proven past or current invasive breast carcinoma or carcinoma in-situ preoperatively were excluded. In addition to multiple benign lesions, the following atypical lesions were identified: flat epithelial atypia (16.9%), atypical ductal hyperplasia (14.0%), lobular neoplasia (14.0%), ductal carcinoma in-situ (4.3%), invasive ductal carcinoma (0.4%). Both low-grade and high-grade pathway lesions were identified in this cohort, and in a subset of patients, they co-occurred. The frequency of atypical lesions identified in patients with strong family history were comparable to those with proven germline mutation. PTEN immunohistochemistry showed loss of expression in ductal carcinoma in-situ and tubular adenomas in PTEN-mutant patients. Overall, findings from this cohort support the benefit of prophylactic mastectomy in patients with germline mutations and/or strong family history. Additionally, this is the first demonstration that PTEN immunohistochemistry may be helpful in identifying germline mutations in patients with atypical or neoplastic proliferations.

Liquid biopsy: an innovative and reliable method for detecting not only somatic, but also germline mutations in patients with colorectal and non-small cell lung carcinoma

Liquid biopsy is a non-invasive method of detecting cancer-related mutations from circulating cell-free DNA (cfDNA) and has emerged as a promising alternative to traditional tissue biopsies. However, its utility has so far been limited to the detection of somatic mutations in cancer cells. Our study examined germline mutations that are associated with pharmacogenetics in 19 patients diagnosed with colorectal cancer and 12 patients with non-small cell lung carcinoma, utilizing the highly advanced and non-invasive technique of liquid biopsy, followed by subsequent next-generation sequencing for comprehensive analysis. Despite the relatively modest sample size of patients under consideration, our results indicated a noteworthy correlation between the presence of adverse effects and the identified germline mutations. We have identified the following mutations with significant implications for pharmacogenetics: MTHFR c.1286A > C, MTHFR c.665C > T, DPYD c.2194G > A, DPYD c.85C > T, XPC c.2815C > A, UMPS c.638G > C, SLC22A2 c.808T > G, EGFR c.1562G > A, ABCB1 2677 T > G, GSTP1 c.313A > G, ERCC2 c.2251A > C, and SLC19A1 c.80A > G. In addition, we observed various adverse drug reactions in our patient cohort, encompassing myelosuppression, hepatotoxicity, neurotoxicity and gastrointestinal toxicity. Liquid biopsy has the potential to revolutionize cancer diagnosis and treatment by detecting both somatic and germline mutations. The preliminary results of studies on germline mutations are promising, but further research is needed to address the remaining challenges and to establish the utility of liquid biopsy as a reliable diagnostic tool for germline mutations.

Identifying childhood leukemia with an excess of hematological malignancies in first-degree relatives in Brazil.

Familial aggregation in childhood leukemia is associated with epidemiological and genomic factors. Albeit epidemiological studies on the familial history of hematological malignancies (FHHMs) are scarce, genome-wide studies have identified inherited gene variants associated with leukemia risk. We revisited a dataset of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) patients to explore the familial aggregation of malignancies among their relatives. A series of 5,878 childhood leukemia (≤21 years of age) from the EMiLI study (2000-2019) were assessed. Lack of well-documented familial history of cancer (FHC) and 670 cases associated with genetic phenotypic syndromes were excluded. Leukemia subtypes were established according to World Health Organization recommendations. Logistic regression-derived odds ratios (ORs) and 95% confidence intervals (CIs) were performed and adjusted by age as a continuous variable, where ALL was the reference group for AML and conversely. The pedigree of 18 families with excess hematological malignancy was constructed. FHC was identified in 472 of 3,618 eligible cases (13%). Ninety-six of the 472 patients (20.3%) had an occurrence of FHHMs among relatives. Overall, FHC was significantly associated with AML (OR, 1.36; 95% CI, 1.01-1.82; p = 0.040). Regarding the first-degree relatives, the OR, 2.92 95% CI,1.57-5.42 and the adjOR, 1.16 (1.03-1.30; p0.001) were found for FHC and FHHM, respectively. Our findings confirmed that AML subtypes presented a significant association with hematological malignancies in first-degree relatives. Genomic studies are needed to identify germline mutations that significantly increase the risk of developing myeloid malignancies in Brazil.

Frequency of pathogenic germline variants in cancer susceptibility genes in 841 renal cell carcinoma cases.

e16551 Background: Hereditary Renal cell carcinoma (RCC) syndrome accounts for approximately 5-10% of all kidney cancers. However, the genetic architecture of susceptibility to RCC is not well defined. In this study, we investigated the prevalence and spectrum of pathogenic/likely pathogenic germline variants of cancer susceptibility genes in unselected RCC patients in the real-world Chinese population. Methods: We retrospectively profiled the germline mutations of 841 unselected RCC patients using a next-generation sequencing panel consisting of 808 cancer-related genes. Interpretation of sequence variations was classified according to the American College of Medical Genetics and Genomics guidelines. Genomic alterations include single nucleotide variations (SNV), short insertions/deletions (INDEL) and copy number variations (CNV). Results: Within 841 unselected RCC patients included in this study, 132 germline pathogenic/likely pathogenic (P/LP) variants were identified in 125 patients (14.9%), of which 114 (13.6%) were mutations of SNV/INDEL and 11 (1.3%) were CNV. Overall, 46 patients (5.5%) carried mutations of hereditary RCC syndrome-associated genes such as VHL (13, 1.5%), FH (13, 1.5%) and FLCN (8, 1.0%). Interestingly, variants in other cancer-predisposition genes not traditionally associated with RCC occurred in 9.4% of the unselected series, including 55 (6.5%) patients carried mutations in DNA damage (DDR)-related genes (e.g., MUTYH, CHEK2 and BRCA1/2) and the other 24 (2.9%) had germline mutations of non-DDR-related genes (e.g., SPINK1 and APC). The median age of patients with mutations of RCC syndrome-associated genes was 38 (range 19-77), while the median age of patients with mutations of other cancer-predisposition genes was 52(range 22-82). Furthermore, in patients older than 46 years, the prevalence of P/LP variants in RCC-associated and non–RCC-associated genes were 1.9% and 6.7%, respectively. Notably, among 125 patients with identified germline mutations, nearly half (56, 44.8%) did not meet the recommended criteria for genetic testing. Conclusions: Our findings confirm a high prevalence of pathogenic germline variants in genes associated with both hereditary RCC and other cancer predispositions in the unselected Chinese RCC population. Current guidelines recommendation for genetic testing seemed not sensitive enough to identify patients with hereditary RCC susceptibility. It is rational to promote genetic testing and expand the range of genes in RCC population with advanced age.

Abstract A056: Landscape of the molecular changes in the most frequent tumors in a safety net hospital

Abstract INTRODUCTION The advent of deep molecular analysis using next generation sequencing (NGS) leads to a better understanding of carcinogenesis and the ability to identify and target specific mutations. METHOD After obtaining IRB approval, patients were approached in several oncology clinics in Grady Health System and explained the risk and benefit of being tested for molecular testing to better guide clinical management of their disease. After informed consent, we carried out Clinical Laboratory Improvement Amendments of 1988 (CLIA) NGS for a large number of targetable genes, tumor mutation burden (TMB), microsatellite instability (MSI), and the somatic transcriptome. We identified germline mutations, actionable genes, biologically significant genes, pertinent negative, variants of unknown significance, tumor profile-matched clinical trials and the recommended therapies based on CLIA NGS findings. All data and medical reports were made available to treating physicians. RESULTS We tested 280 patients, of which 206 were women and 74 men. 95% are represented by African American population. The majority of our patients had endometrial cancer (33%), breast cancer (21%), lung (9%), colon (8&), ovary (7.5%), prostate (3.5%) and head and neck (3.5%). Fewer carcinomas were of thyroid, other gastrointestinal and genitourinary origin. Of the most frequent cancers in our pilot study, endometrial carcinomas, 26% displayed significant tumor burden with 53% demonstrating > 50 mutations. In additional, endometrial cancers were found to be MSI stable in 84% of cases, 11% had a germline mutation, 63% had actionable mutations (range of 1-9), 68% with biologically relevant mutations (range 2-5) and 100% had VUS (range 1 to 21). Importantly, 18% of the cases studied had novel treatment indications. Of the breast carcinomas, 44% of cases displayed a significant tumor burden with 29% demonstrating > 50 mutations. In additional, 76% of breast cancer case were found to be MSI stable in, 10% had a germline mutations, 71% had actionable mutations, 90% with biologically relevant mutations (range 1-7), and 86% had VUS (range 1 to 14). Clinical trials were available in 66% and 76% cases had unique treatment indications. Of the lung carcinomas, 89% had tumor mutation burden with > 50 mutations. 100% had VUS (range 2-15) and 44% had treatment indication. CONCLUSION Providing access to CLIA NGS to safety net hospital cancer patients revealed unique somatic changes and treatment options. Taken together, this approach is an important step to democratize precision medicine and improve health outcomes to advance health equity. Citation Format: Gabriella M. Oprea-Ilies, LaTrisha L. Horne, Eddie R. Stanley, Regina Lee, Joel Okoli, Giuseppe Del Priore, Pooja Mishra, Roland Matthew, Eric L. Flenaugh, Brian Rivers, James W. Lillard, Sam Sirotnikov, Mallik Matthews. Landscape of the molecular changes in the most frequent tumors in a safety net hospital [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A056.

Incidence of Hereditary Gastric Cancer May Be Much Higher than Reported.

Hereditary gastric cancers (HGCs) are supposed to be rare and difficult to identify. Nonetheless, many cases of young patients with gastric cancer (GC) fulfill the clinical criteria for considering this diagnosis but do not present the defined pathogenic mutations necessary to meet a formal diagnosis of HGC. Moreover, GC in young people is a challenging medical situation due to the usual aggressiveness of such cases and the potential risk for their relatives when related to a germline variant. Aiming to identify additional germline alterations that might contribute to the early onset of GC, a complete exome sequence of blood samples from 95 GC patients under 50 and 94 blood samples from non-cancer patients was performed and compared in this study. The number of identified germline mutations in GC patients was found to be much higher than that from individuals without a cancer diagnosis. Specifically, the number of high functional impact mutations, including those affecting genes involved in medical diseases, cancer hallmark genes, and DNA replication and repair processes, was much higher, strengthening the hypothesis of the potential causal role of such mutations in hereditary cancers. Conversely, classically related HGC mutations were not found and the number of mutations in genes in the CDH1 pathway was not found to be relevant among the young GC patients, reinforcing the hypothesis that existing alternative germline contributions favor the early onset of GC. The LILRB1 gene variants, absent in the world's cancer datasets but present in high frequencies among the studied GC patients, may represent essential cancer variants specific to the Amerindian ancestry's contributions. Identifying non-reported GC variants, potentially originating from under-studied populations, may pave the way for additional discoveries and translations to clinical interventions for GC management. The newly proposed approaches may reduce the discrepancy between clinically suspected and molecularly proven hereditary GC and shed light on similar inconsistencies among other cancer types. Additionally, the results of this study may support the development of new blood tests for evaluating cancer risk that can be used in clinical practice, helping physicians make decisions about strategies for surveillance and risk-reduction interventions.

Rare POLN mutations confer risk for familial nasopharyngeal carcinoma through weakened Epstein-Barr virus lytic replication.

SummaryBackgroundNasopharyngeal carcinoma (NPC) exhibits significant familial aggregation; however, its susceptibility genes are largely unknown. Thus, this study aimed to identify germline mutations that might contribute to the risk of familial NPC, and explore their biological functions.MethodsWhole-exome sequencing was performed in 13 NPC pedigrees with multiple cases. Mutations co-segregated with disease status were further validated in a cohort composed of 563 probands from independent families, 2,953 sporadic cases, and 3,175 healthy controls. Experimental studies were used to explore the functions of susceptibility genes and their disease-related mutations.FindingsThe three rare missense mutations in POLN (DNA polymerase nu) gene, P577L, R303Q, and F545C, were associated with familial NPC risk (5/576 [0·87%] in cases vs. 2/3374 [0·059%] in healthy controls with an adjusted OR of 44·84 [95% CI:3·91-514·34, p = 2·25 × 10−3]). POLN was involved in Epstein-Barr virus (EBV) lytic replication in NPC cells in vitro. POLN promoted viral DNA replication, immediate-early and late lytic gene expression, and progeny viral particle production, ultimately affecting the proliferation of host cells. The three mutations were located in two pivotal functional domains and were predicted to alter the protein stability of POLN in silico. Further assays demonstrated that POLN carrying any of the three mutations displayed reduced protein stability and decreased expression levels, thereby impairing its ability to promote complete EBV lytic replication and facilitate cell survival.InterpretationWe identified a susceptibility gene POLN for familial NPC and elucidated its function.FundingThis study was funded by the National Key Research and Development Program of China (2021YFC2500400); the National Key Research and Development Program of China (2020YFC1316902); the Basic and Applied Basic Research Foundation of Guangdong Province, China (2021B1515420007); the National Natural Science Foundation of China (81973131); the National Natural Science Foundation of China (82003520); the National Natural Science Foundation of China (81903395).

Frequency of actionable germline mutations identified through tumor next generation sequencing in a gynecologic cancer cohort (394)

Frequency of actionable germline mutations identified through tumor next generation sequencing in a gynecologic cancer cohort (394)

Novel Germline Mutations in a Cohort of Men with Familial Prostate Cancer.

Simple SummaryIn this study, we identified germline mutations that contribute to prostate cancer development in men who have multiple relatives with prostate cancer (and other cancers). We correlated the genetic mutations found in each patient with the resulting prostate cancer characteristics. Mutations found in ATM and CHEK2 genes were associated with aggressive prostate cancer. More data is needed to confirm the prevalence and impact of these germline mutations in prostate cancer.Background: Germline mutations in BRCA2 are associated with aggressive prostate cancer. Additional information regarding the clinical phenotype of germline pathogenic variants in other prostate cancer predisposition genes is required. Clinical testing has been limited by evidence, further restricting knowledge of variants that contribute to prostate cancer development. Objective: Prostate cancer patients who were first- and second-degree relatives from multi-case prostate cancer families underwent a gene panel screen to identify novel (non-BRCA) germline pathogenic variants in cancer predisposition genes and define clinical phenotypes associated with each gene. Methods: The germline genomic DNA (gDNA) of 94 index cases with verified prostate cancer from families with a minimum of two verified prostate cancer cases was screened with an 84-cancer-gene panel. Families were recruited for multi-case breast/ovarian cancer (n = 66), or multi-case prostate cancer (n = 28). Prostate cancer characteristics associated with each gene were compared with prostate cancer cases of confirmed non-mutation carriers (BRCAX), also from multi-case prostate cancer families (n = 111), and with data from the Prostate Cancer Outcomes Registry (PCOR). Results: Ninety-four prostate cancer index cases underwent gene panel testing; twenty-two index cases (22/94; 23%) were found to carry a class 4–5 (C4/5) variant. Six of twenty-two (27%) variants were not clinically notifiable, and seven of twenty-two (31.8%) variants were in BRCA1/2 genes. Nine of twenty-two (40.9%) index cases had variants identified in ATM (n = 4), CHEK2 (n = 2) and HOXB13G84 (n = 3); gDNA for all relatives of these nine cases was screened for the corresponding familial variant. The final cohort comprised 15 confirmed germline mutation carriers with prostate cancer (ATM n = 9, CHEK2 n = 2, HOXB13G84 n = 4). ATM and CHEK2-associated cancers were D’Amico intermediate or high risk, comparable to our previously published BRCA2 and BRCAX prostate cancer cohort. HOXB13G84 carriers demonstrated low- to intermediate-risk prostate cancer. In the BRCAX cohort, 53.2% of subjects demonstrated high-risk disease compared with 25% of the PCOR cohort. Conclusions: ATM and CHEK2 germline mutation carriers and the BRCAX (confirmed non-mutation carriers) cohort demonstrated high risk disease compared with the general population. Targeted genetic testing will help identify men at greater risk of prostate-cancer-specific mortality. Data correlating rare variants with clinical phenotype and familial predisposition will strengthen the clinical validity and utility of these results and establish these variants as significant in prostate cancer detection and management.

O-310 Browsing the Male Genome to Unravel Its Reproductive Potential

Abstract Study question Can whole genome profiling of sperm DNA be used to identify aspects of ART failure and predict embryo developmental competence? Summary answer Whole genome profiling of sperm DNA identifies germline gene mutations associated with reproductive failure and helps characterize subtle male factor infertility. What is known already A routine semen analysis provides limited information on the characteristics of the male gamete and is unable to predict spermatozoa performance in ART. Therefore, ancillary tests may be used to further assess the male gamete’s reproductive potential. Whole exome sequencing (WES) of the male genome carried out on somatic cells has proven to be a powerful technique capable of identifying the genetic roots of infertility. Here, we aim to preferentially detect germline mutations by exclusively sequencing spermatozoal DNA to identify genes related to the different underlying etiologies of reproductive failure. Study design, size, duration Over a 6-year period, 31 consenting couples with negative female infertility workups and normal semen parameters were included in this study. These couples were divided according to whether they reported a successful pregnancy with ART (fertile; n = 10) or not (infertile; n = 21). Sperm aneuploidy assessment by copy number variant (CNV) analysis with WES were carried out on ejaculated spermatozoa. Gene mutation profiles were enlisted and compared between the two patient cohorts to identify genes involved. Participants/materials, setting, methods DNA was extracted and amplified from at least 500 spermatozoa (DNA concentration, 760±486 ng/ul; quality, 1.7±0.1 nm) for CNV analyses by WES. Mutations corresponding to the CNV were then annotated and assessed using the CLC Genomic Server 9.0. Genes were considered duplicated or deleted when their read depth was >1.5 or < 0.5 times the median read depth in the control, respectively. Main results and the role of chance All couples (n = 31) (maternal age, 37.6±3yrs; paternal age, 39.7±5yrs) had adequate semen parameters (concentration, 59.2±30x106/mL, 44.8±18% motility, normal morphology) and normal peripheral karyotypes. The fertile cohort (n = 10) underwent 12 ICSI cycles, achieving an 82.6% (57/69) fertilization rate with 10/12 (83.3%) cycles resulting in live births. The infertile cohort (n = 21) underwent 25 ICSI cycles, achieving a 68.4% (91/133) fertilization rate and 6/14 (42.9%) clinical pregnancies, all resulting in pregnancy loss. CNV analysis indicated lower sperm aneuploidy in the fertile (4.0% vs. 8.4%) cohort (P < 0.00001). In both cohorts, mutations associated with sperm–egg fusion (ADAM3A) and acrosomal development (SPACA1, SPATA16) were identified, justifying ICSI utilization. The infertile cohort included complete fertilization failure, poor early embryo development, implantation failure, or pregnancy loss. Couples with complete fertilization failure (n = 4) had gene deletions (PLCZ1, PIWIL1, ADAM15) indicating sperm-related oocyte-activating deficiency. Those with poor early embryo development (n = 5) had mutations essential for centrosome integrity (HAUS1) and spindle/microtubular stabilization (KIF4A, XRN1). Couples who failed to achieve pregnancy (n = 7) had mutations commonly implicated in embryonic implantation (IL9R) and microtubule/centrosomal integrity (MAP1S). Those with pregnancy losses (n = 5) displayed mutations related to trophoblast development (NLRP7), cell cycle regulation (MARK4, TRIP13, DAB2IP, KIF1C), and a gene linked to recurrent miscarriage (TP53). Limitations, reasons for caution Using WES, we were able to identify germline mutations that appear to be involved in various aspects of human reproduction. These findings are new and should be validated in a larger study population. Moreover, although we attempted to control for maternal age, we still cannot exclude confounding female factors. Wider implications of the findings Evaluating the sperm genome can help identify elusive genetic factors associated with reproductive competence and help guide treatment options for couples unable to conceive who undergo ART. Therefore, screening spermatozoal DNA may serve as an additional tool in precision medicine to identify and treat subtle male factor infertility. Trial registration number N/A

Localized islet nuclear enlargement hyperinsulinism (LINE-HI) due to ABCC8 and GCK mosaic mutations.

Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in children. In addition to typical focal or diffuse HI, some cases with diazoxide-unresponsive congenital HI have atypical pancreatic histology termed Localized Islet Nuclear Enlargement (LINE) or mosaic HI, characterized by histologic features similar to diffuse HI, but confined to only a region of pancreas. Our objective was to characterize the phenotype and genotype of children with LINE-HI. The phenotype and genotype features of 12 children with pancreatic histology consistent with LINE-HI were examined. We compiled clinical features of 12 children with LINE-HI and performed next-generation sequencing on specimens of pancreas from eight of these children to look for mosaic mutations in genes known to be associated with diazoxide-unresponsive HI (ABCC8, KCNJ11, and GCK). Children with LINE-HI had lower birth weights and later ages of presentation compared to children with typical focal or diffuse HI. Partial pancreatectomy in LINE-HI cases resulted in euglycemia in 75% of cases; no cases have developed diabetes. Low-level mosaic mutations were identified in the pancreas of six cases with LINE-HI (three in ABCC8, three in GCK). Expression studies confirmed that all novel mutations were pathogenic. These results indicate that post-zygotic low-level mosaic mutations of known HI genes are responsible for some cases of LINE-HI that lack an identifiable germ-line mutation and that partial pancreatectomy may be curative for these cases.

Lung cancer patients with germline mutations have a unique fusion molecular spectrum.

e21009 Background: In contrast to numerous studies of somatic mutations, the researches of lung cancer germline mutations remain smaller. Some certain germline mutations are assumed to increase susceptibility to lung cancer. However, the details of such mechanism remain to be established. In this study, DNA profiles were performed by targeted next-generation sequencing (NGS). Methods: A retrospective study of 3,421 lung cancer patients with available NGS data was performed to identify germline mutations. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, germline mutations were categorized as being pathogenic (P), likely pathogenic (LP) and other significance (Non_P). Thus, the patients were divided into two groups: patients with P or LP mutation(P&LP), and patients with Non_P mutation. Results: Of 3421 patients with lung cancer, 174 (5.09%) patients were identified to carry pathogenic or likely pathogenic germline mutations, with a median age of 62.5 years old. BRCA2, ATM, MUTYH, BRCA1 and ERCC2 were the top five genes with germline alterations, and their mutation rates were 11.5, 6.90, 6.32, 5.17 and 4.60%, respectively. Homozygous mutations were identified in only two patients, one was TP53( p.Ser215Ile), and the other was TSC2(splice site alteration). No statistically significant difference in age and sex between P&LP and Non_P group. Somatic mutation data was also involved in our study. The frequency of gene fusion events was same between these two groups. Interestingly, fusion molecular spectrum of P&LP group was different. Among all the 25 fusion events of P&LP group, 20 were unique and 19 fusion events couldn't be retrieved in the PubMed database. In total, 44 genes involved in fusion events and 5 genes were located on chromosome 17. Frequency of FAT1 single nucleotide variant (SNV) mutation was significantly higher in P&LP group (9.77% vs 5.57%, p = 0.03). Most copy number variations (CNV) were amplification, and frequency of EGFR copy number amplification was higher in P&LP group (19.5% vs 12.6%, p = 0.01). Conclusions: These data implied that genomic profile of lung patients who carried germline gene mutations could be different. Associations between germline mutation and lung cancer susceptibility, treatment will be studied further.

Identification of Germline Mutations in Genes Involved in Classic FAP in Patients from Northern Brazil.

Colorectal cancer is a common cancer worldwide, with 5-10% of cases being hereditary. Familial adenomatous polyposis syndrome (FAP) is caused by germline mutations in the APC gene or rarely in the MUTYH gene. This work did not identify germline mutations in the MUTYH, NTHL1, POLD1 and POLE genes in 15 individuals belonging to five families with classic FAP, who had the mutation in the APC gene confirmed in a previous study. Our results support mutations in the APC gene as the main genetic contribution of classical FAP with severe phenotype. In the family that had the most aggressive form of the disease, we performed an array-based Comparative Genomic Hybridization analysis and identified the germinal loss of an allele of the NOTCH2 and BMPR2 genes in the mother (proband) and daughter. In order to validate the involvement of these genes in the other four families of this study, we analyzed the DNA copy number variation in the peripheral blood of the 15 participants. FAP is a syndrome with considerable genetic and phenotypic heterogeneity and this phenomenon may explain the presence of secondary genetic alterations, such as the allelic loss of NOTCH2 and BMPR2 genes, found only in one family in this study. The CNV analysis confirmed that only the two members of the FAP2 family (patient 02H and 02F) had a deletion of these two genes, as the aCGH methodology had found. The other study participants did not show allelic loss for these two genes. Validation in a larger number of families could confirm the presence of these new genetic alterations in classic FAP and improve understanding of the different types of aggressiveness of the disease.

The Mutationathon highlights the importance of reaching standardization in estimates of pedigree-based germline mutation rates.

In the past decade, several studies have estimated the human per-generation germline mutation rate using large pedigrees. More recently, estimates for various nonhuman species have been published. However, methodological differences among studies in detecting germline mutations and estimating mutation rates make direct comparisons difficult. Here, we describe the many different steps involved in estimating pedigree-based mutation rates, including sampling, sequencing, mapping, variant calling, filtering, and appropriately accounting for false-positive and false-negative rates. For each step, we review the different methods and parameter choices that have been used in the recent literature. Additionally, we present the results from a 'Mutationathon,' a competition organized among five research labs to compare germline mutation rate estimates for a single pedigree of rhesus macaques. We report almost a twofold variation in the final estimated rate among groups using different post-alignment processing, calling, and filtering criteria, and provide details into the sources of variation across studies. Though the difference among estimates is not statistically significant, this discrepancy emphasizes the need for standardized methods in mutation rate estimations and the difficulty in comparing rates from different studies. Finally, this work aims to provide guidelines for computational and statistical benchmarks for future studies interested in identifying germline mutations from pedigrees.