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Moody, Emily W., Jennie Vagher, Whitney Espinel, David Goldgar, Kelsi J. Hagerty, and Amanda Gammon. "Comparison of Somatic and Germline Variant Interpretation in Hereditary Cancer Genes." JCO Precision Oncology, no. 3 (December 2019): 1–8. http://dx.doi.org/10.1200/po.19.00144.
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PURPOSE To compare the classification of genetic variants reported on tumor genomic profiling (TGP) reports with germline classifications on clinical test results and ClinVar. Results will help to inform germline testing discussions and decisions in patients with tumor variants in genes that are relevant to hereditary cancer risk. PATIENTS AND METHODS This study compared somatic and germline classifications of small nucleotide variants in the following genes: BRCA1, BRCA2, CHEK2, PALB2, ATM, MLH1, MSH2, MSH6, and PMS2. Somatic classifications were taken from reports from a single commercial TGP laboratory of tests ordered by providers at Huntsman Cancer Institute between March 2014 and June 2018. Somatic variant interpretations were compared with classifications from germline test results as well as with ClinVar interpretations. RESULTS Of the 623 variants identified on TGP, 353 had a definitive classification in ClinVar, and 103 were assayed with a germline test, with 66 of the variants tested observed in germline. Analysis of somatic variants of uncertain significance listed on TGP reports determined that 22% had a different interpretation compared with ClinVar and that 32% differed from the interpretation on a germline test result. Pathogenic variants on TGP test results were found to differ 13% and 5% of the time compared with ClinVar interpretations and germline test results, respectively. CONCLUSION These results suggest that TGP variants are often classified differently in a germline context. Differences may be due to different processes in variant interpretation between somatic and germline laboratories. These results are important for health care providers to consider when making decisions about additional testing for hereditary cancer risks.
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Kraft, Ira L., and Lucy A. Godley. "Identifying potential germline variants from sequencing hematopoietic malignancies." Blood 136, no. 22 (November 26, 2020): 2498–506. http://dx.doi.org/10.1182/blood.2020006910.
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Abstract Next-generation sequencing (NGS) of bone marrow and peripheral blood increasingly guides clinical care in hematological malignancies. NGS data may help to identify single nucleotide variants, insertions/deletions, copy number variations, and translocations at a single time point, and repeated NGS testing allows tracking of dynamic changes in variants during the course of a patient’s disease. Tumor cells used for NGS may contain germline, somatic, and clonal hematopoietic DNA alterations, and distinguishing the etiology of a variant may be challenging. We describe an approach using patient history, individual variant characteristics, and sequential NGS assays to identify potential germline variants. Our current criteria for identifying an individual likely to have a deleterious germline variant include a strong family history or multiple cancers in a single patient, diagnosis of a hematopoietic malignancy at a younger age than seen in the general population, variant allele frequency > 0.3 of a deleterious allele in a known germline predisposition gene, and variant persistence identified on clinical NGS panels, despite a change in disease state. Sequential molecular testing of hematopoietic specimens may provide insight into disease pathology, impact patient and family members’ care, and potentially identify new cancer-predisposing risk alleles. Ideally, individuals should give consent at the time of NGS testing to receive information about potential germline variants and to allow future contact as research advances.
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Kraft, Ira L., and Lucy A. Godley. "Identifying potential germline variants from sequencing hematopoietic malignancies." Hematology 2020, no. 1 (December 4, 2020): 219–27. http://dx.doi.org/10.1182/hematology.2020006910.
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Abstract Next-generation sequencing (NGS) of bone marrow and peripheral blood increasingly guides clinical care in hematological malignancies. NGS data may help to identify single nucleotide variants, insertions/deletions, copy number variations, and translocations at a single time point, and repeated NGS testing allows tracking of dynamic changes in variants during the course of a patient’s disease. Tumor cells used for NGS may contain germline, somatic, and clonal hematopoietic DNA alterations, and distinguishing the etiology of a variant may be challenging. We describe an approach using patient history, individual variant characteristics, and sequential NGS assays to identify potential germline variants. Our current criteria for identifying an individual likely to have a deleterious germline variant include a strong family history or multiple cancers in a single patient, diagnosis of a hematopoietic malignancy at a younger age than seen in the general population, variant allele frequency > 0.3 of a deleterious allele in a known germline predisposition gene, and variant persistence identified on clinical NGS panels, despite a change in disease state. Sequential molecular testing of hematopoietic specimens may provide insight into disease pathology, impact patient and family members’ care, and potentially identify new cancer-predisposing risk alleles. Ideally, individuals should give consent at the time of NGS testing to receive information about potential germline variants and to allow future contact as research advances.
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Michalski, Scott T., Daniel Esteban Pineda Alvarez, Meaghan Russell, Shan Yang, Guru Sonpavde, and Edward D. Esplin. "Tumor sequencing with germline genetic testing: Identification of patients with hereditary cancer and precision treatment eligibility." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 1580. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.1580.
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1580 Background: Cancer is a fundamentally genetic disease, as such, somatic and germline mutation analysis is used in the comprehensive assessment of patients with cancer. Studies report that approximately 10% of patient’s tumors have clinically significant variants known to predispose to hereditary cancer, with medical implications for both patients and family members. We retrospectively reviewed a series of patients where providers suspected a somatic variant also existed in the germline and followed up with clinical germline genetic testing. We report the rate of concordance between germline and somatic results and their clinical impact. Methods: Our study used de-identified data from 1043 consecutive patients who underwent somatic genetic testing followed by germline testing with NGS-based hereditary cancer gene panels. Results: Somatic results most frequently prompting germline testing included variants in BRCA2 (290), BRCA1 (174), TP53 (158), ATM (70), MLH1 (65), APC (65), PMS2 (61), MSH6 (58), PTEN (54) and CDH1 (42). In 364/1043 cases (35%) the variant was detected as likely pathogenic or pathogenic (LP/P) in the germline. Genes confirmed as germline variants in 60-100% of cases included: FANCA, AXIN2, RAD50, MUTYH, BLM, PALB2, CHEK2, FANCD2, MITF, SDHB. Variants in: FH, BRCA2, RET, ATM, SDHA, BRIP1, MSH2, BRCA1, BAP1, EGFR and RAD51D confirmed in the germline in 25-60%. Variants were rarely germline for TP53 (3%), APC (3%), PTEN (2%) and none in CDKN2A, NF1 and STK11. In 24 (2%) cases a LP/P germline variant was detected but not reported in the tumor. Conclusions: Approximately ⅓ of patients suspected to have hereditary risk after tumor testing had LP/P germline variants. Notably, some genes had a high probability of variants occurring in the germline, while others were primarily seen in tumors. Interestingly, 6% of the germline variants were not included on the somatic report due to technical and gene content differences in either assays or due to differences of clinical classification between somatic and germline testing. Adding germline results to somatic testing may inform options for precision treatment, prevention, or early detection of, secondary malignancies and guide genetic counseling of family members.
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Fujita, Atsushi, Takefumi Higashijima, Hiroshi Shirozu, Hiroshi Masuda, Masaki Sonoda, Jun Tohyama, Mitsuhiro Kato, et al. "Pathogenic variants of DYNC2H1, KIAA0556, and PTPN11 associated with hypothalamic hamartoma." Neurology 93, no. 3 (June 13, 2019): e237-e251. http://dx.doi.org/10.1212/wnl.0000000000007774.
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ObjectiveIntensive genetic analysis was performed to reveal comprehensive molecular insights into hypothalamic hamartoma (HH).MethodsThirty-eight individuals with HH were investigated by whole exome sequencing, target capture-based deep sequencing, or single nucleotide polymorphism (SNP) array using DNA extracted from blood leukocytes or HH samples.ResultsWe identified a germline variant of KIAA0556, which encodes a ciliary protein, and 2 somatic variants of PTPN11, which forms part of the RAS/mitogen-activated protein kinase (MAPK) pathway, as well as variants in known genes associated with HH. An SNP array identified (among 3 patients) one germline copy-neutral loss of heterozygosity (cnLOH) at 6p22.3–p21.31 and 2 somatic cnLOH; one at 11q12.2–q25 that included DYNC2H1, which encodes a ciliary motor protein, and the other at 17p13.3–p11.2. A germline heterozygous variant and an identical somatic variant of DYNC2H1 arising from cnLOH at 11q12.2–q25 were confirmed in one patient (whose HH tissue, therefore, contains biallelic variants of DYNC2H1). Furthermore, a combination of a germline and a somatic DYNC2H1 variant was detected in another patient.ConclusionsOverall, our cohort identified germline/somatic alterations in 34% (13/38) of patients with HH. Disruption of the Shh signaling pathway associated with cilia or the RAS/MAPK pathway may lead to the development of HH.
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Aldubayan, Saud H., Jake Conway, Leora Witkowski, Eric Kofman, Brendan Reardon, Sabrina Camp, Seunghun Han, et al. "Expanding the diagnostic yield of germline genetic testing in cancer patients using deep learning." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 1518. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.1518.
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1518 Background: Germline genetic analysis is an essential tool for implementing precision cancer prevention and treatment. However, only a small fraction of cancer patients, even those with features suggestive of a cancer-predisposition syndrome, have detectable pathogenic germline events, which may in part reflect incomplete pathogenic variant detection by current gold-standard methods. Here, we leveraged deep learning approaches to expand the diagnostic utility of genetic analysis in cancer patients. Methods: Systematic analysis of the detection rate of pathogenic cancer-predisposition variants using the standard clinical variant detection method and a deep learning approach in germline whole-exome sequencing data of 2367 cancer patients (n = 1072 prostate cancer, 1295 melanoma). Results: Of 1072 prostate cancer patients, deep learning variant detection identified 16 additional prostate cancer patients with clinically actionable pathogenic cancer-predisposition variants that went undetected by the gold-standard method (198 vs. 182), yielding higher sensitivity (94.7% vs. 87.1%), specificity (64.0% vs. 36.0%), positive predictive value (95.7% vs. 91.9%), and negative predictive value (59.3% vs. 25.0%). Similarly, germline genetic analysis of 1295 melanoma patients showed that, compared with the standard method, deep learning detected 19 additional patients with validated pathogenic variants (93 vs. 74) with fewer false-positive calls (78 vs. 135) leading to a higher diagnostic yield. Collectively, deep learning identified one additional patient with a pathogenic cancer-risk variant, that went undetected by the standard method, for every 52 to 67 cancer patients undergoing germline analysis. Superior performance of deep learning, for detecting putative loss-of-function variants, was also seen across 5197 clinically relevant Mendelian genes in these cohorts. Conclusions: The gold-standard germline variant detection method, universally used in clinical and research settings, has significant limitations for identifying clinically relevant pathogenic disease-causing variants. We determined that deep learning approaches have a clinically significant increase in the diagnostic yield across commonly examined Mendelian gene sets.
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Rampersaud, Evadnie, David S. Ziegler, Ilaria Iacobucci, Debbie Payne-Turner, Michelle L. Churchman, Kasmintan A. Schrader, Vijai Joseph, et al. "Germline deletion of ETV6 in familial acute lymphoblastic leukemia." Blood Advances 3, no. 7 (April 2, 2019): 1039–46. http://dx.doi.org/10.1182/bloodadvances.2018030635.
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Abstract Recent studies have identified germline mutations in TP53, PAX5, ETV6, and IKZF1 in kindreds with familial acute lymphoblastic leukemia (ALL), but the genetic basis of ALL in many kindreds is unknown despite mutational analysis of the exome. Here, we report a germline deletion of ETV6 identified by linkage and structural variant analysis of whole-genome sequencing data segregating in a kindred with thrombocytopenia, B-progenitor acute lymphoblastic leukemia, and diffuse large B-cell lymphoma. The 75-nt deletion removed the ETV6 exon 7 splice acceptor, resulting in exon skipping and protein truncation. The ETV6 deletion was also identified by optimal structural variant analysis of exome sequencing data. These findings identify a new mechanism of germline predisposition in ALL and implicate ETV6 germline variation in predisposition to lymphoma. Importantly, these data highlight the importance of germline structural variant analysis in the search for germline variants predisposing to familial leukemia.
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Karantanos, Theodoros, Shruti Chaturvedi, Christopher D. Gocke, Donna Marie Williams, Alison R. Moliterno, and Evan M. Braunstein. "ATM Germline Variant Increases the Risk of MPN Progression." Blood 134, Supplement_1 (November 13, 2019): 835. http://dx.doi.org/10.1182/blood-2019-125362.
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Introduction: Chronic myeloproliferative neoplasms (MPN) share the same driver mutations but their disease course and prognosis varies significantly. Deficiency of DNA damage repair (DDR) due to germline mutation is known to predispose to certain cancer types and has been implicated in the biology of MPN. The aim of our study was to evaluate the impact of germline variants in DDR genes in the natural history and outcomes of MPN patients. Patients and Methods: 76 individuals who were diagnosed with MPN (essential thrombocytosis (ET), polycythemia vera (PV) and primary myelofibrosis (PMF)) at Johns Hopkins University Hospital were included in this study. Targeted sequencing of 63 genes implicated in myeloid malignancies was performed as part of a standard clinical evaluation. Germline variants were determined by a variant allele frequency between 40-60% in blood samples and presence in the dbSNP database. Only rare variants (minor allele frequency < 0.01) were included in this analysis. Regression analysis was used to determine the association of the presence of germline variants with disease phenotype, driver mutation, number of somatic mutations, age and sex. Cox regression and Kaplan-Meier were used to assess the implication of germline variants in the progression to MF. Results: Median time from diagnosis to enrollment and follow up were 6 and 11 years respectively. 22 patients (28.9%) had at least one variant in a DDR gene, with ATM the most frequent (11/76 patients, 14.5%). Other recurrently mutated DDR genes included RECQL4 (6/76 patients, 7.9%), ATRX and RAD50 (Figure 1A). Patients with an ATM germline variant had higher incidence of a second malignancy (OR 4.37, 95% CI 1.16 - 16.46, P=0.029), a non-significant trend toward positive family history of malignancy (OR 3.75, 95% CI 0.91 - 15.46, P=0.067) and higher incidence of both second malignancy and positive family history of cancer (OR 4.58, 95% CI 1.17 - 17.94, P=0.029) (Figure 1B). The presence of an ATM germline variant was associated with MF or AML as opposed to ET or PV at the time of sequencing (RRR 5.84, 95% CI 1.12 - 30.34, P=0.036) independently of driver mutation, number of additional somatic mutations, age and male sex (Figure 1C). We did not find a significant difference in the number of somatic mutations between patients with and without ATM variant, however there was a trend toward increased chromosomal abnormalities among patients with ATM variant (Figure 1D). Finally, the presence of ATM variant was associated with higher risk of MF transformation (HR 3.43, 95% CI 1.02 - 11.6, P=0.047) independently of driver mutation (JAK2 Ref, CALR - HR 0.79, 95% CI 0.21 - 2.94, P=0.73) and male sex (HR 1.4, 95% CI 0.52 - 3.76, P=0.68). Kaplan-Meier analysis confirmed that progression to MF-free survival was shorter in patients harboring an ATM variant (P=0.01) (Figure 1E). Conclusion: The presence of a DDR gene germline variant, particularly ATM, is relatively common among patients with MPN. Patients with ATM variants had higher incidence of additional cancers and family history of malignancy, as well as an association with MF/AML phenotype and early transformation to MF. These data suggest involvement of ATM signaling in the progression of MPN, potentially via accumulation of DNA damage and genomic instability. Figure 1. A. Frequency of germline variants in MPN patients. The graph includes the gene variants identified in at least 2 distinct patients. Of known DDR related genes (in red) ATM is the most frequent (11/76 patients, 14.5%), followed by RECQL4 (6/76 patients, 7.9%) and ATRX and RAD50. B. Personal and family history of cancer among patients with and without ATM variant. Patients with ATM germline variant have higher incidence of additional malignancy (OR 4.37, 95% CI 1.16 - 16.46, P=0.029), higher incidence of family history of malignancy (OR 3.75, 95% CI 0.91 - 15.46, P=0.067) and higher incidence of concurrent personal history of malignancy and family history of malignancy (OR 4.58, 95% CI 1.17 - 17.94, P=0.029). C. MPN subtype per ATM variant status. Patients with an ATM germline variant were more likely to have MF or AML at the time of sequencing independent of driver mutation, number of somatic mutations, age and sex. D. Patients with an ATM variant had higher number of chromosomal abnormalities. E. Kaplan-Meier analysis of progression to MF free survival(P=0.01). Disclosures Chaturvedi: Shire/Takeda: Research Funding; Alexion: Consultancy; Sanofi: Consultancy.
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Cardot Bauters, Catherine, Emmanuelle Leteurtre, Bruno Carnaille, Christine Do Cao, Stéphanie Espiard, Malo Penven, Evelyne Destailleur, et al. "Genetic predisposition to neural crest-derived tumors: revisiting the role of KIF1B." Endocrine Connections 9, no. 10 (October 2020): 1042–50. http://dx.doi.org/10.1530/ec-20-0460.
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Objective We previously described a family in which predisposition to pheochromocytoma (PCC) segregates with a germline heterozygous KIF1B nucleotide variant (c.4442G>A, p.Ser1481Asn) in three generations. During the clinical follow-up, one proband’s brother, negative for the KIF1B nucleotide variant, developed a bilateral PCC at 31 years. This prompted us to reconsider the genetic analysis. Design and methods Germline DNA was analyzed by next-generation sequencing (NGS) using a multi-gene panel plus MLPA or by whole exome sequencing (WES). Tumor-derived DNA was analyzed by SnapShot, Sanger sequencing or NGS to identify loss-of-heterozygosity (LOH) or additional somatic mutations. Results A germline heterozygous variant of unknown significance in MAX (c.145T>C, p.Ser49Pro) was identified in the proband’s brother. Loss of the wild-type MAX allele occurred in his PCCs thus demonstrating that this variant was responsible for the bilateral PCC in this patient. The proband and her affected grandfather also carried the MAX variant but no second hit could be found at the somatic level. No other pathogenic mutations were detected in 36 genes predisposing to familial PCC/PGL or familial cancers by WES of the proband germline. Germline variants detected in other genes, TFAP2E and TMEM214, may contribute to the multiple tumors of the proband. Conclusion In this family, the heritability of PCC is linked to the MAX germline variant and not to the KIF1B germline variant which, however, may have contributed to the occurrence of neuroblastoma (NB) in the proband.
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Garza-Rodríguez, María Lourdes, Víctor Treviño, Antonio Alí Pérez-Maya, Hazyadee Frecia Rodríguez-Gutiérrez, Moisés González-Escamilla, Miguel Ángel Elizondo-Riojas, Genaro A. Ramírez-Correa, Oscar Vidal-Gutiérrez, Carlos Horacio Burciaga-Flores, and Diana Cristina Pérez-Ibave. "Identification of a Novel Pathogenic Rearrangement Variant of the APC Gene Associated with a Variable Spectrum of Familial Cancer." Diagnostics 11, no. 3 (February 28, 2021): 411. http://dx.doi.org/10.3390/diagnostics11030411.
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Familial adenomatous polyposis (FAP) is an autosomal-dominant condition characterized by the presence of multiple colorectal adenomas, caused by germline variants in the adenomatous polyposis coli (APC) gene. More than 300 germline variants have been characterized. The detection of novel variants is important to understand the mechanisms of pathophysiology. We identified a novel pathogenic germline variant using next-generation sequencing (NGS) in a proband patient. The variant is a complex rearrangement (c.422+1123_532-577 del ins 423-1933_423-1687 inv) that generates a complete deletion of exon 5 of the APC gene. To study the variant in other family members, we designed an endpoint PCR method followed by Sanger sequencing. The variant was identified in the proband patient’s mother, one daughter, her brother, two cousins, a niece, and a second nephew. In patients where the variant was identified, we found atypical clinical symptoms, including mandibular, ovarian, breast, pancreatic, and gastric cancer. Genetic counseling and cancer prevention strategies were provided for the family. According to the American College of Medical Genetics (ACMG) guidelines, this novel variant is considered a PVS1 variant (very strong evidence of pathogenicity), and it can be useful in association with clinical data for early surveillance and suitable treatment.
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Amaral, Teresa, Martin Schulze, Tobias Sinnberg, Maike Nieser, Peter Martus, Florian Battke, Claus Garbe, Saskia Biskup, and Andrea Forschner. "Are Pathogenic Germline Variants in Metastatic Melanoma Associated with Resistance to Combined Immunotherapy?" Cancers 12, no. 5 (April 28, 2020): 1101. http://dx.doi.org/10.3390/cancers12051101.
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Background: Combined immunotherapy has significantly improved survival of patients with advanced melanoma, but there are still patients that do not benefit from it. Early biomarkers that indicate potential resistance would be highly relevant for these patients. Methods: We comprehensively analyzed tumor and blood samples from patients with advanced melanoma, treated with combined immunotherapy and performed descriptive and survival analysis. Results: Fifty-nine patients with a median follow-up of 13 months (inter quartile range (IQR) 11–15) were included. Interestingly, nine patients were found to have pathogenic or likely pathogenic (P/LP) germline variants in one of these genes: BRCA2, POLE, WRN, FANCI, CDKN2A, BAP1, PALB2 and RAD54B. Most of them are involved in DNA repair mechanisms. Patients with P/LP germline variants had a significantly shorter progression-free survival (PFS) and melanoma specific survival (MSS) compared to patients without P/LP germline variants (HR = 2.16; 95% CI: 1.01–4.64; p = 0.048 and HR = 3.21; 95% CI: 1.31–7.87; p = 0.011, respectively). None of the patients with a P/LP germline variant responded to combined immunotherapy. In the multivariate Cox-regression analysis, presence of a P/LP germline variant, S100B and lactate dehydrogenase (LDH) remained independently significant factors for MSS (p = 0.036; p = 0.044 and p = 0.001, respectively). Conclusions: The presence of P/LP germline variants was associated with resistance to combined immunotherapy in our cohort. As genes involved in DNA repair mechanisms are also involved in lymphocyte development and T-cell differentiation, a P/LP germline variant in these genes may preclude an antitumor immune response.
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Thoma, Clemens. "Subtype linked to germline variant." Nature Reviews Urology 14, no. 9 (July 25, 2017): 519. http://dx.doi.org/10.1038/nrurol.2017.122.
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Schwartz, Alison, Sophie Hyman, Samantha Stokes, Danielle Castillo, Jeffrey N. Weitzel, Huma Q. Rana, and Judy Ellen Garber. "Nearly half of TP53 variants are misattributed to Li-Fraumeni syndrome: A clinical evaluation of individuals with TP53 variants detected by hereditary cancer panel assays on blood or saliva." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): 10501. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.10501.
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10501 Background: Multigene panel testing (MGPT) has identified TP53 pathogenic or likely pathogenic (P/LP) variants in patients with diverse phenotypes from no cancer to classic Li-Fraumeni syndrome (LFS). There is increasing recognition of variants at low allelic fraction (VAF) for TP53 in particular, which can be suggestive of post-zygotic mosaicism or aberrant clonal expansion (ACE), comprising clonal hematopoiesis of indeterminate potential (CHIP) or occult hematologic neoplasia. Distinguishing among these categories is essential because of widely different cancer risk and management implications for patients and their relatives. We report an evaluation of TP53 positive probands to determine germline versus somatic status from a cancer genetics clinic. Methods: We reviewed probands with TP53 P/LP variants by MGPT on blood (N = 83) or saliva (N = 1) samples from 2012-2019. Available VAFs were collected from commercial testing laboratories. Probands positive for a known familial variant, who met LFS testing criteria without indication of low VAF, or who carried the Brazil founder p.R337H variant were considered germline. For those with uncertain germline status, data was obtained from ancillary testing of family members, cultured skin fibroblasts, and other somatic benign or tumor tissues. TP53 variants were further categorized based on all available data. Results: Of the 84 probands, 28 (33%) had germline TP53 P/LP variants determined by above initial criteria; 18 (21%) were confirmed germline through ancillary testing. Seven (8%) individuals were classified as having constitutional mosaicism. In eleven (13%) individuals, the TP53 variants were consistent with ACE, in 9 (11%) with CHIP and in 2 (2%) with a hematologic malignancy (1 CLL, 1 NHL). Five (6%) cases could not be categorized despite ancillary testing. Fifteen (18%) probands declined any further workup. Conclusions: A TP53 P/LP variant found on peripheral blood or saliva MGPT does not always originate in the germline. In a clinical cancer genetics cohort, only 54% of patients had TP53 P/LP germline variants; these patients plus those with constitutional mosaicism (8%) require intensified surveillance. Assessment of VAF, family member testing, and analysis of TP53 in cultured fibroblasts or other tissue samples may distinguish germline and constitutional mosaic variants from the ACE spectrum. Expanding use of MGPT will increase this clinical challenge, which may motivate the modification of lab reports to include VAF and possible non-germline explanations. The findings of this study support a framework of multiple strategies to discern true constitutional status of a TP53 P/LP variant.
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Kondrashova, Olga, Jannah Shamsani, Tracy A. O’Mara, Felicity Newell, Amy E. McCart Reed, Sunil R. Lakhani, Judy Kirk, John V. Pearson, Nicola Waddell, and Amanda B. Spurdle. "Tumor Signature Analysis Implicates Hereditary Cancer Genes in Endometrial Cancer Development." Cancers 13, no. 8 (April 7, 2021): 1762. http://dx.doi.org/10.3390/cancers13081762.
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Risk of endometrial cancer (EC) is increased ~2-fold for women with a family history of cancer, partly due to inherited pathogenic variants in mismatch repair (MMR) genes. We explored the role of additional genes as explanation for familial EC presentation by investigating germline and EC tumor sequence data from The Cancer Genome Atlas (n = 539; 308 European ancestry), and germline data from 33 suspected familial European ancestry EC patients demonstrating immunohistochemistry-detected tumor MMR proficiency. Germline variants in MMR and 26 other known/candidate EC risk genes were annotated for pathogenicity in the two EC datasets, and also for European ancestry individuals from gnomAD as a population reference set (n = 59,095). Ancestry-matched case–control comparisons of germline variant frequency and/or sequence data from suspected familial EC cases highlighted ATM, PALB2, RAD51C, MUTYH and NBN as candidates for large-scale risk association studies. Tumor mutational signature analysis identified a microsatellite-high signature for all cases with a germline pathogenic MMR gene variant. Signature analysis also indicated that germline loss-of-function variants in homologous recombination (BRCA1, PALB2, RAD51C) or base excision (NTHL1, MUTYH) repair genes can contribute to EC development in some individuals with germline variants in these genes. These findings have implications for expanded therapeutic options for EC cases.
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Lim, Tristan L., David B. Lieberman, Adam R. Davis, Ryan Hausler, Ashkan Bigdeli, Yimei Li, Jacquelyn Powers, et al. "Germline POT1 Variants Can Predispose to a Variety of Hematologic Neoplasms." Blood 136, Supplement 1 (November 5, 2020): 2–4. http://dx.doi.org/10.1182/blood-2020-134160.
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Germline mutations in the shelterin component protection of telomeres 1 (POT1) were recently found to be associated with familial chronic lymphocytic leukemia (CLL), melanoma, glioma, and several other familial cancer syndromes. The role of POT1 mutations in myeloid neoplasms and other hematologic malignancies, however, remains unknown. To explore the role of POT1 variants in hematologic neoplasms, we analyzed POT1 variants in 3323 consecutive patients who underwent next-generation sequencing (NGS) of a panel of hematologic malignancy-associated genes at our institution and characterized the clinical and pathological characteristics of patients with germline and somatic POT1 mutations. Of 3323 consecutive patients who underwent NGS, 2770 patients had a hematologic malignancy (lymphoid n = 1299, myeloid n = 934, and both lymphoid and myeloid n = 537), while 553 patients were evaluated for non-malignant cytopenias. All 57 patients (2.06%) carrying either a POT1 disease-associated variant or variant of uncertain significance had a hematologic malignancy compared to no identified POT1 variants in 553 patients with benign cytopenias (OR = 23.5, p < 0.001), suggesting that the presence of POT1 variants was predictive of a hematologic malignancy. Of 57 patients, 33 had lymphoid malignancies, 23 had myeloid neoplasms, and 2 had a lymphoid and myeloid neoplasm (Fig 1). Patient variants were classified as germline or somatic using constitutional DNA sequencing, POT1 emergence/disappearance over time, or POT1 maintenance in remission. In the absence of these data, likely germline or likely somatic designations were made by assessing variant allele frequencies against clinical/pathologic characteristics. 18 patients (33%) were found to have germline or likely germline POT1 variants (29% and 42% in the lymphoid and myeloid malignancy groups, respectively). Another 6 patients (11%) had variants whose germline status could not be determined. Of the 17 unique germline POT1 variants, 10 were missense and located within mapped functional protein domains, while 7 were classified as predicted loss-of-function (pLOF) due to a disruption of start, premature stop, frameshift, or spice site alteration. Patients with hematological malignancies had a ~5-8x increased odds of having a germline pLOF POT1 variant compared to cancer-free individuals in the Genome Aggregation Database (gnomAD, n = 113,108 exomes, OR = 7.5, p < 0.001) or in the Penn Medicine BioBank (PMBB, n = 7877, OR = 5.0, p = 0.010), with a prevalence of 0.25% compared to 0.03% and 0.05%, respectively. Germline pLOF POT1 variants were significantly more enriched in patients with myeloid (gnomAD: OR = 6.1, p = 0.02) and lymphoid (gnomAD: OR = 9.8, p < 0.001; PMBB: OR = 6.5, p = 0.004) malignancies. In 33 patients with lymphoid malignancies and POT1 variants, the most common diagnoses were CLL/SLL (n = 21, germline n = 6, somatic n = 12), CD5- CD10- indolent B cell neoplasms (n = 4, germline n = 1, somatic n = 3), and multiple myeloma (n = 3, all somatic) (Table 1). Lymphoid malignancies with a germline POT1 variant had a relative paucity of additional mutations; in contrast, somatic POT1 variants frequently co-occurred with other mutations, most commonly with TP53 (Fig 2, n = 5, 23%). Among 23 patients with myeloid malignancies, patients with germline POT1 variants developed malignancies at a significantly younger age compared to those whose POT1 variants were somatic (median age 59.5 vs 70.5 years, p = 0.04). The most common diagnosis in patients with myeloid neoplasms carrying germline POT1 variants was MPN (germline n = 5, somatic n = 1). AML, MDS/MPN, and MDS occurred in 4, 3, and 1 patients respectively. All patients with myeloid neoplasms had additional disease-associated mutations, with the most common co-occurring variants in TET2 (n = 7), JAK2 (n = 6, co-occurring with 50% of germline POT1 myeloid variants), and NRAS (n = 6). In conclusion, this is the first comprehensive analysis of POT1 variants in an unselected hospital-based population undergoing molecular testing for variants associated with hematologic malignancies. Our results show that the presence of POT1 variants is predictive of having a hematologic neoplasm and that over 30% of POT1 variants in hematologic malignancy patients are germline. Our study expands the spectrum of POT1-associated familial neoplasms and highlights the needs for better recognition of familial hematologic cancer syndromes. Disclosures No relevant conflicts of interest to declare.
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Schneider, Bryan P., Leigh Anne Stout, Santosh Philips, Courtney Schroeder, Susanna F. Scott, Cynthia Hunter, Nawal Kassem, Patrick J. Kiel, and Milan Radovich. "Implications of Incidental Germline Findings Identified In the Context of Clinical Whole Exome Sequencing for Guiding Cancer Therapy." JCO Precision Oncology, no. 4 (October 2020): 1109–21. http://dx.doi.org/10.1200/po.19.00354.
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PURPOSE Identification of incidental germline mutations in the context of next-generation sequencing is an unintended consequence of advancing technologies. These data are critical for family members to understand disease risks and take action. PATIENTS AND METHODS A retrospective cohort analysis was conducted of 1,028 adult patients with metastatic cancer who were sequenced with tumor and germline whole exome sequencing (WES). Germline variant call files were mined for pathogenic/likely pathogenic (P/LP) variants using the ClinVar database and narrowed to high-quality submitters. RESULTS Median age was 59 years, with 16% of patients ≤ 45 years old. The most common tumor types were breast cancer (12.5%), colorectal cancer (11.5%), sarcoma (9.3%), prostate cancer (8.4%), and lung cancer (6.6%). We identified 3,427 P/LP variants in 471 genes, and 84% of patients harbored one or more variant. One hundred thirty-two patients (12.8%) carried a P/LP variant in a cancer predisposition gene, with BRCA2 being the most common (1.6%). Patients with breast cancer were most likely to carry a P/LP variant (19.2%). One hundred ten patients (10.7%) carried a P/LP variant in a gene that would be recommended by the American College of Medical Genetics and Genomics to be reported as a result of clinical actionability, with the most common being ATP7B (2.7%), BRCA2 (1.6%), MUTYH (1.4%), and BRCA1 (1%). Of patients who carried a P/LP variant in a cancer predisposition gene, only 53% would have been offered correct testing based on current clinical practice guidelines. Of 471 mutated genes, 231 genes had a P/LP variant identified in one patient, demonstrating significant genetic heterogeneity. CONCLUSION The majority of patients undergoing clinical cancer WES harbor a pathogenic germline variation. Identification of clinically actionable germline findings will create additional burden on oncology clinics as broader WES becomes common.
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Khanna, Shivani, Steven Brad Maron, Leah Chase, Samantha Lomnicki, Sonia Kupfer, and Daniel V. T. Catenacci. "Suspected and confirmed germline variants from tumor-only somatic sequencing of 864 gastrointestinal malignancies." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e13131-e13131. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e13131.
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e13131 Background: Targeted tumor-only somatic sequencing informs therapies and is becoming a routine part of cancer care. It also identifies patients with possible germline variants who require confirmatory genetic testing. The aim was to identify patients with suspected and confirmed germline variants whose GI tumors underwent somatic sequencing. Methods: 864 patients with GI tumors who had Foundation One (FO) somatic sequencing from 4/2003-3/2018 were evaluated. Inclusion criteria for suspected germline variants were: a) allele frequency ≥ 35% in hereditary cancer genes and b) pathogenic variants by FO and/or ClinVar. Variants in commonly mutated somatic genes ( TP53, KRAS, CDKN2A) were excluded in patients over age 40. Recommendation of genetic evaluation and germline test results were recorded. Patient, family, and tumor characteristics were compared using univariate analysis. Results: 199 of 864 patients had suspected germline pathogenic variants. 50 patients were recommended genetic evaluation, and 26 patients underwent genetic testing. A germline pathogenic variant was confirmed in 15 patients. Among all patients suspected to have germline variants, 8% were confirmed by genetic testing. Patients under age 40 and those with family cancer history were more often referred for testing (Table). Patients with variants in BRCA1, MLH1, MSH2, PMS2, POLE and TP53 were more often referred for testing. Conclusions: A quarter of patients carried a somatic pathogenic variant with allele frequency ≥35% in a hereditary cancer gene. 25% of these patients were recommended for genetic evaluation. Younger patients and those with family history were more often referred. 8% of patients with suspected germline variants were confirmed by genetic testing. These results provide “real world” experience in using somatic only tumor testing to identify patients with germline pathogenic variants who then might benefit from future cancer screening and genetic testing in family members. Comparison of characteristics by recommendation to undergo genetic testing based on somatic tumor sequencing results. [Table: see text]
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Matzenbacher Bittar, Camila, Yasminne Marinho de Araújo Rocha, Igor Araujo Vieira, Clévia Rosset, Tiago Finger Andreis, Ivaine Tais Sauthier Sartor, Osvaldo Artigalás, et al. "Clinical and molecular characterization of patients fulfilling Chompret criteria for Li-Fraumeni syndrome in Southern Brazil." PLOS ONE 16, no. 9 (September 16, 2021): e0251639. http://dx.doi.org/10.1371/journal.pone.0251639.
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Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition syndrome caused by pathogenic germline variants in the TP53 gene, characterized by a predisposition to the development of a broad spectrum of tumors at an early age. The core tumors related to LFS are bone and soft tissue sarcomas, premenopausal breast cancer, brain tumors, adrenocortical carcinomas (ACC), and leukemias. The revised Chompret criteria has been widely used to establish clinical suspicion and support TP53 germline variant testing and LFS diagnosis. Information on TP53 germline pathogenic variant (PV) prevalence when using Chompret criteria in South America and especially in Brazil is scarce. Therefore, the aim of this study was to characterize patients that fulfilled these specific criteria in southern Brazil, a region known for its high population frequency of a founder TP53 variant c.1010G>A (p.Arg337His), as known as R337H. TP53 germline testing of 191 cancer-affected and independent probands with LFS phenotype identified a heterozygous pathogenic/likely pathogenic variant in 26 (13.6%) probands, both in the DNA binding domain (group A) and in the oligomerization domain (group B) of the gene. Of the 26 carriers, 18 (69.23%) were R337H heterozygotes. Median age at diagnosis of the first tumor in groups A and B differed significantly in this cohort: 22 and 2 years, respectively (P = 0.009). The present study shows the clinical heterogeneity of LFS, highlights particularities of the R337H variant and underscores the need for larger collaborative studies to better define LFS prevalence, clinical spectrum and penetrance of different germline TP53 pathogenic variants.
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Stout, Leigh Anne Anne, Nawal Kassem, Cynthia Hunter, Santosh Philips, Milan Radovich, and Bryan P. Schneider. "Identification of germline cancer predisposition variants during clinical ctDNA testing." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e15555-e15555. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e15555.
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e15555 Background: Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) is a non-invasive method to guide therapy selection for cancer patients. Identification of inherited germline cancer predisposition mutations that have significant implications for at-risk relatives may be missed during routine ctDNA testing. Allele frequency has the potential to enhance the likelihood that a mutation is germline; and is often reported in many NGS tests from ctDNA. Here, we report on the fidelity of allele frequency in ctDNA as a predictor for pathogenic germline variant carriage. Methods: ctDNA sequencing of patients with metastatic cancer from the Indiana University Health Precision Genomics Program was performed using the FoundationOne Liquid assay. All variants detected by the ctDNA assay report were considered. All patients also had germline testing information and pathogenicity of germline variants were determined using ClinVar. Germline variants with conflicting interpretations were manually reviewed to determine pathogenicity. Comparisons between ctDNA results with known germline status were performed. Results: Of 91 previously identified germline cancer predisposition variants, 36 (40%) were also identified by ctDNA analysis. All germline variants that were tested for in the ctDNA assay (n = 36, 100%) were identified. When detected, the allele frequencies of detected germline variants in the ctDNA ranged from 39-87.6% with an average of 52.1%. Conversely, 111 of 160 (69%) variants identified by ctDNA analysis with allele frequency between 40-60% in a cancer predisposition gene were found to be germline in origin (regardless of pathogenicity). Variants in the BRCA2, BRCA1, and CDH1 genes were most likely to be germline in origin (26/27 [96%], 20/22 [91%], 13/15 [87%], respectively). Variants in the TP53 and APC genes were least likely to be germline in origin (9/36 [25%] and 1/6 [17%], respectively). There was an 85% (95/111) concordance in actionability between the somatic testing lab and ClinVar germline classifications. Of the 16 discordant variants, 100% were determined to be actionable by the somatic testing lab but not actionable in ClinVar. Conclusions: ctDNA allele frequency can alter the likelihood that a variant is germline. Importantly, however, this testing is far from comprehensive and should not be used as a replacement for germline testing. Variants with allele frequency between 40-60% in cancer predisposition genes should induce a high level of suspicion for germline status.
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Martins Rodrigues, Fernanda, Qingsong Gao, Kuan-lin Huang, Adam David Scott, Steven M. Foltz, Justin King, Mark A. Fiala, et al. "Characterization of Germline Variants in Multiple Myeloma." Blood 132, Supplement 1 (November 29, 2018): 4499. http://dx.doi.org/10.1182/blood-2018-99-118673.
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Abstract Multiple myeloma (MM) is an incurable hematological malignancy characterized by the clonal proliferation of malignant plasma cells in the bone marrow. Like other cancers, MM is a genetically complex and heterogeneous disease. One of its distinctive characteristics is that it is preceded by a pre-malignant condition known as monoclonal gammopathy of undetermined significance (MGUS), which then progresses to asymptomatic (smoldering) multiple myeloma (SMM) and, ultimately, to late-stage MM. Its progression through these stages is determined by a sequence of genomic aberrations, starting with germline events that predispose to the disease, followed by early initiating events and the later acquisition of mutations that contribute to disease progression. Although considerable progress has been made in the past 6 years in cataloguing somatic events underlying MM development and progression, little is known about its genetic predisposition. Therefore, large-scale germline genomic variant studies are urgently needed. Recently, our group has published the largest-scale pan-cancer study of >10K adult and >1K pediatric cases that revealed new insights on germline predisposition variants across 33 cancer types (853 pathogenic or likely pathogenic variants) (Huang et al., 2018). Here, we aim to apply a similar strategy to MM cases. The CoMMpass study, promoted by MMRF (Multiple Myeloma Research Foundation) is a longitudinal, prospective observational study involving the collection and analysis of sequencing and clinical data from >1K MM patients at diagnosis and relapse. We performed germline variant calling on 808 normal samples from this dataset using GenomeVIP (https://github.com/ding-lab/GenomeVIP), which integrates multiple tools: VarScan2 and Genome Analysis ToolKit (GATK) for the identification of single nucleotide variants (SNVs) and indels; and Pindel for indel prediction. Variants were limited to coding regions of full length transcripts obtained from Ensembl release 70 plus the additional two base pairs flanking each exon that cover splice donor/acceptor sites. SNVs were based on the union of raw GATK and VarScan calls. Indels were required to be called by at least two out of the three callers (GATK, Pindel, VarScan). Variant calls from all tools were merged, filtered (allelic depth ≥ 5 for the alternative allele; rare variants with allele frequency ≤ 0.01 in 1000 Genomes and ExAC), and annotated using Variant Effect Predictor (VEP), resulting in an average of 1,653 variants per sample. Further, we applied CharGer (Characterization of Germline Variants, https://github.com/ding-lab/CharGer) to classify the identified germline variants as pathogenic, likely pathogenic, and prioritized variants of unknown significance (VUS). CharGer is an automatic variant classification pipeline developed by our group which adopts ACMG-AMP guidelines specifically for rare variants in cancer. Here, we were able to classify a total of 635 germline variants as pathogenic and 150 as likely pathogenic, affecting 90% of samples. Among pathogenic variants, 28 were found in known cancer predisposition genes including BRCA1 and BRCA2 - which have been previously associated with MM risk - BRIP1, CHEK2, TP53, TERT, and PMS2. Ongoing analyses include: functional characterization of these variants, identifying genes with enriched pathogenic or likely pathogenic variants in our dataset; investigation of LOH and two-hit (biallelic) events; gene and protein expression analyses in carriers of pathogenic germline variants of the respective gene; scanning for rare, germline copy number variations (CNVs); and identification of variants in post-translational modification sites that may affect protein signaling. Additionally, we are currently working on improving our CharGer tool by integrating new tumor associated data, such as DNA-Seq, RNA-Seq, Methyl-Seq and MS proteomics data, to improve variant classification. The preliminary results and analysis strategies described here will allow for efficient and cost-effective discovery of genetic changes relevant to MM etiology. Ultimately, we hope this work will impact our overall understanding of the genetics underlying MM predisposition, allowing for the development of better prevention and early detection strategies. Disclosures Vij: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees.
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Kraft, Ira Lignugaris, Amy M. Trottier, George F. Steinhardt, Nifang Niu, Pankhuri Wanjari, Wenjun Chen, Jeremy Segal, and Lucy A. Godley. "Using sequential next-generation sequencing assays to identify germline cancer predisposition variants." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 1581. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.1581.
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1581 Background: Next-generation sequencing (NGS) increasingly guides clinical care in hematological malignancies by identifying DNA mutations that change dynamically over time. Clinical samples contain variable numbers of malignant and non-malignant cells. So, careful interpretation is required to determine if a particular variant is somatic, germline, or clonal hematopoietic in origin. Methods: The University of Chicago uses a targeted NGS assay of ~1200 genes, reporting 150 as a clinical test. We aimed to identify individuals with hereditary predisposition by detecting persistent variants on sequential assays regardless of disease state. Results: 943 NGS assays from July 2017 – Feb. 2020 on 711 patients [ages 1 mo – 95 yrs, median 65 yrs] were included. 2,320 variants in 33 genes were identified with 144 patients having the same variant identified on more than one assay. Single nucleotide variants (SNVs) with variant allele frequency (VAF) ≥ 0.3 were prioritized. The first candidate gene identified with potential germline SNVs was CSF3R. 28 unique SNVs in CSF3R were found, 14 were confirmed as germline, 6 somatic, and 8 were unconfirmed due to lack of available tissue. At least 2 confirmed germline CSF3R variants were likely deleterious based on functional testing. Sequential SNVs were quantified using the coefficient of variation, characterizing each by change in VAF over time. Using a worst-case-scenario analysis, in which unconfirmed variants were not counted as germline, a computer algorithm was designed to identify potential germline variants (specificity 0.89, PPV 0.75). Via an iterative method, the algorithm compares new assays to a pool of previously reported tests, flagging patients with potential germline mutations so that biopsies may be studied in the lab, records reviewed, and referrals placed to genetic counselors. To date, 61 patients with 89 likely germline variants have been identified. Known hereditary hematological malignancy genes, such as ATM, ASXL1, CHEK2, DDX41, TSC1, and RUNX1, had the most variants identified. Limitations include the challenge in distinguishing variants that do not change over time, reliance on a targeted NGS panel, and normalizing VAF data prior to analysis. Conclusions: These data highlight the utility of NGS of bone marrow and peripheral blood samples to identify patients suspected of having germline DNA variants. In addition to identifying known predisposition syndromes, one may discover new inherited cancer syndromes and help guide clinical practice in real time.
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Bang, Lisa, Manu Shivakumar, Tullika Garg, and Dokyoon Kim. "Genetic Analysis Reveals Rare Variants in T-Cell Response Gene MR1 Associated with Poor Overall Survival after Urothelial Cancer Diagnosis." Cancers 13, no. 8 (April 14, 2021): 1864. http://dx.doi.org/10.3390/cancers13081864.
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Urothelial carcinoma of the bladder (UC) is the fifth most common cancer in the United States. Germline variants, especially rare germline variants, may account for a portion of the disparity seen among patients in terms of UC incidence, presentation, and outcomes. The objectives of this study were to identify rare germline variant associations in UC incidence and to determine its association with clinical outcomes. Using exome sequencing data from the DiscovEHR UC cohort (n = 446), a European-ancestry, North American population, the complex influence of germline variants on known clinical phenotypes were analyzed using dispersion and burden metrics with regression tests. Outcomes measured were derived from the electronic health record (EHR) and included UC incidence, age at diagnosis, and overall survival (OS). Consequently, key rare variant association genes were implicated in MR1 and ADGRL2. The Kaplan–Meier survival analysis reveals that individuals with MR1 germline variants had significantly worse OS than those without any (log-rank p-value = 3.46 × 10−7). Those with ADGRL2 variants were found to be slightly more likely to have UC compared to a matched control cohort (FDR q-value = 0.116). These associations highlight several candidate genes that have the potential to explain clinical disparities in UC and predict UC outcomes.
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Murphy, Jo-Ellen, Sara Sadan, Jessica Kim Lee, Jana Pruski-Clark, Rebecca Sutphen, and Kimberly McGregor. "Rare BAP1 variant of unknown significance (VUS) and analysis of BAP1 codon 146 genomics: Potential germline and therapeutic implications." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): 10533. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.10533.
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10533 Background: The BAP1 gene ( B RCA1-Associated Protein) encodes a protein ubiquitin carboxyl-terminal hydrolase (BAP1), which removes ubiquitin moieties and regulates various cellular functions including DNA repair. This association has driven interest in defining if BAP1 variants confer susceptibility to PARP inhibitors (PARPi). Germline and somatic BAP1 alterations are both rare, mostly unique, often classified as VUS’s, and associated with a broad range of overlapping tumor types. Based on the identification of a BAP1 R146K VUS variant in tumor, also previously identified as germline, an analysis of BAP1 codon 146 alterations was initiated to explore potential genetic and therapeutic implications. Methods: 394,756 tumor specimens were tested using hybrid capture-based genomic profiling (Foundation Medicine) for all variant types and Tumor Mutational Burden (TMB). BAP1 codon 146 cases were analyzed for clinicogenomic features and germline results when available. Results: BAP1 codon 146 variants were identified in 23 unique patients across the following tumor types: cholangiocarcinoma (CCA) (4), mesothelioma (4), NSCLC (3), RCC (2), ocular melanoma (2), and carcinoma of unknown primary (CUP) (3); many of which overlap with known and suspected germline associated BAP1 syndromic tumor types. BAP1 R146 mutations were classified as VUS in 16 patients and 7 were likely or known pathogenic. In 20 of the 23 cases where zygosity could be determined 16 (80%) were homozygous and 4 (20%) were heterozygous. In 2 of the 3 NSCLC cases, the BAP1 variant appeared likely somatic and/or associated with a high TMB. A previously reported germline BAP1 variant in a RCC patient, R146K, occurred 5 times in our tumor database. One case which was homozygous in tumor and confirmed in germline occurred in a patient who had both breast and CCA. She also had a sibling with RCC who shared the germline BAP1 R146K variant along with multiple 1st and 2nd degree relatives with RCC, mesothelioma, melanoma, liver cancer, colon cancer, and a cancer of unknown primary. Conclusions: Codon 146 of BAP1 localizes to the UCH (ubiquitin carboxyl hydrolase) domain, which includes the BARD1 interaction region. Loss of BAP1 activity as a consequence of germline or somatic mutation likely impacts ubiquitination status and activity of downstream proteins, such as those involved in DNA repair. For patients with suspected BAP1 inactivating alterations, often seen in non-homologous recombination deficiency related tumor types, PARPi therapy may be relevant. As demonstrated here, variants identified through tumor testing may also aid in re-classification of germline VUS’s. These results support the further investigation and validation of BAP1 alterations for germline risk stratification and therapeutic strategies with either PARPi and/or other therapies specific to tumors with impaired chromatin remodeling.
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Trottier, Amy M., Ira L. Kraft, Lawrence J. Druhan, Amanda Lance, Belinda R. Avalos, and Lucy A. Godley. "New Germline Syndrome Discovery: Heterozygous CSF3R Mutations May Predispose to Myeloid and Lymphoid Malignancies." Blood 134, Supplement_1 (November 13, 2019): 2543. http://dx.doi.org/10.1182/blood-2019-129492.
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Introduction Colony stimulating factor 3 receptor (CSF3R) plays an important role in granulocyte proliferation and differentiation. Acquired mutations in CSF3R are found in the majority of cases of chronic neutrophilic leukemia and in a minority of atypical chronic myeloid leukemia and acute myeloid leukemia patients, whereas inherited biallelic mutations have been identified as a cause of severe congenital neutropenia (SCN). CSF3R variants have also been detected in lymphoid cells in patients with SCN. However, whether germline monoallelic CSF3R variants predispose to myeloid and/or lymphoid malignancies is unknown. Methods We analyzed variants detected by a clinical next generation sequencing (NGS) panel of about 150 genes important in oncogenesis (OncoPlus) performed on peripheral blood and/or bone marrow of patients with hematopoietic malignancies at The University of Chicago Medical Center from June 2017 to February 2019. We prioritized genes for which variants were likely of germline origin based on variant allele fraction (VAF) > 0.4 and persistence over multiple tests. Variant germline status was determined by extracting DNA from cultured bone marrow-derived mesenchymal stromal cells and/or from cultured skin fibroblasts. Variants in CSF3R [transcript NM_000760.3] were ranked by predicted pathogenicity using standard American College of Medical Genetics criteria. Variants of uncertain significance (VUS) were evaluated using population frequency data, in silico functional prediction, and REVEL scores. Results A total of 620 OncoPlus tests were conducted on 496 patients (ages 1 month - 96 years; median 64 years), and 824 unique variants were identified within 33 genes. Among these patients, 89 (17.9%) had an invariant variant, one that was found at similar VAFs across multiple testing time points. These invariant variants involved 31 different genes with a total of 203 unique variants. Variants with a VAF less than 0.4 were excluded from further study, leaving 81 patients with 164 invariant variants among 30 genes (Figure 1). The genes with the most invariant variants included known germline predisposition genes, such as ATM, BRCA2, DDX41, ETV6, and TP53, as well as CSF3R, which was not previously recognized as an autosomal dominant germline cancer susceptibility gene. Among the 81 patients, 8 (9.9%) were found to have invariant variants in CSF3R, with 7 unique variants identified. Including patients for whom only a single OncoPlus test was conducted in addition to those with invariant variants, there were a total of 43 patients with 20 unique variants in CSF3R among the entire 496 patient cohort (Figure 2). Of these 20 CSF3R variants, 8 (40%) were found to be germline, and among those, one was classified as likely pathogenic, one was classified as a VUS, and six were classified as likely benign. We focused our attention on the likely pathogenic and VUS germline variants. These two variants were among those with the highest predicted pathogenicity scores. The likely pathogenic variant, W547*, is a nonsense mutation in the extracellular domain of CSF3R that was identified in a patient with a history of bladder cancer treated with surgery and chemotherapy who subsequently developed therapy-related myelodysplastic syndrome. W547* has previously been found to be pathogenic in a compound heterozygous state in an infant with SCN. The germline VUS, P784T, is a missense variant identified in a patient with multiple myeloma. This variant localizes to the cytoplasmic domain of CSF3R and is not listed in the COSMIC or gnomAD databases. Functional studies are underway to assess the oncogenic potential of the W547* and P784T variants. Conclusion Detection of invariant variants on clinical, tumor-based NGS panels can be used as a way to identify potential germline mutations to aid in new germline syndrome discovery. Using this approach, CSF3R was identified as a candidate gene for which monoallelic pathogenic variants may predispose to both myeloid and lymphoid malignancies. Future work is ongoing to assess the functional consequences of germline CSF3R variants and the frequency of such mutations. Disclosures Avalos: Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia; Juno: Membership on an entity's Board of Directors or advisory committees. Godley:Invitae, Inc.: Membership on an entity's Board of Directors or advisory committees; UpToDate, Inc.: Patents & Royalties: receives royalties from a coauthored article on inherited hematopoietic malignancies .
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Frebourg, Thierry, Svetlana Bajalica Lagercrantz, Carla Oliveira, Rita Magenheim, and D. Gareth Evans. "Guidelines for the Li–Fraumeni and heritable TP53-related cancer syndromes." European Journal of Human Genetics 28, no. 10 (May 26, 2020): 1379–86. http://dx.doi.org/10.1038/s41431-020-0638-4.
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Abstract Fifty years after the recognition of the Li–Fraumeni syndrome (LFS), our perception of cancers related to germline alterations of TP53 has drastically changed: (i) germline TP53 alterations are often identified among children with cancers, in particular soft-tissue sarcomas, adrenocortical carcinomas, central nervous system tumours, or among adult females with early breast cancers, without familial history. This justifies the expansion of the LFS concept to a wider cancer predisposition syndrome designated heritable TP53-related cancer (hTP53rc) syndrome; (ii) the interpretation of germline TP53 variants remains challenging and should integrate epidemiological, phenotypical, bioinformatics prediction, and functional data; (iii) the penetrance of germline disease-causing TP53 variants is variable, depending both on the type of variant (dominant-negative variants being associated with a higher cancer risk) and on modifying factors; (iv) whole-body MRI (WBMRI) allows early detection of tumours in variant carriers and (v) in cancer patients with germline disease-causing TP53 variants, radiotherapy, and conventional genotoxic chemotherapy contribute to the development of subsequent primary tumours. It is critical to perform TP53 testing before the initiation of treatment in order to avoid in carriers, if possible, radiotherapy and genotoxic chemotherapies. In children, the recommendations are to perform clinical examination and abdominal ultrasound every 6 months, annual WBMRI and brain MRI from the first year of life, if the TP53 variant is known to be associated with childhood cancers. In adults, the surveillance should include every year clinical examination, WBMRI, breast MRI in females from 20 until 65 years and brain MRI until 50 years.
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Mellid, Sara, Javier Coloma, Bruna Calsina, María Monteagudo, Juan M. Roldán-Romero, María Santos, Luis J. Leandro-García, et al. "Novel DNMT3A Germline Variant in a Patient with Multiple Paragangliomas and Papillary Thyroid Carcinoma." Cancers 12, no. 11 (November 9, 2020): 3304. http://dx.doi.org/10.3390/cancers12113304.
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Over the past few years, next generation technologies have been applied to unravel the genetics of rare inherited diseases, facilitating the discovery of new susceptibility genes. We recently found germline DNMT3A gain-of-function variants in two patients with head and neck paragangliomas causing a characteristic hypermethylated DNA profile. Here, whole-exome sequencing identifies a novel germline DNMT3A variant (p.Gly332Arg) in a patient with bilateral carotid paragangliomas, papillary thyroid carcinoma and idiopathic intellectual disability. The variant, located in the Pro-Trp-Trp-Pro (PWWP) domain of the protein involved in chromatin targeting, affects a residue mutated in papillary thyroid tumors and located between the two residues found mutated in microcephalic dwarfism patients. Structural modelling of the variant in the DNMT3A PWWP domain predicts that the interaction with H3K36me3 will be altered. An increased methylation of DNMT3A target genes, compatible with a gain-of-function effect of the alteration, was observed in saliva DNA from the proband and in one independent acute myeloid leukemia sample carrying the same p.Gly332Arg variant. Although further studies are needed to support a causal role of DNMT3A variants in paraganglioma, the description of a new DNMT3A alteration in a patient with multiple clinical features suggests a heterogeneous phenotypic spectrum related to DNMT3A germline variants.
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Li, Shuwei, Dajun Qian, Bryony A. Thompson, Stephanie Gutierrez, Sitao Wu, Tina Pesaran, Holly LaDuca, Hsiao-Mei Lu, Elizabeth C. Chao, and Mary Helen Black. "Tumour characteristics provide evidence for germline mismatch repair missense variant pathogenicity." Journal of Medical Genetics 57, no. 1 (August 7, 2019): 62–69. http://dx.doi.org/10.1136/jmedgenet-2019-106096.
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BackgroundPathogenic variants in mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2) increase risk for Lynch syndrome and related cancers. We quantified tumour characteristics to assess variant pathogenicity for germline MMR genes.MethodsAmong 4740 patients with cancer with microsatellite instability (MSI) and immunohistochemical (IHC) results, we tested MMR pathogenic variant association with MSI/IHC status, and estimated likelihood ratios which we used to compute a tumour characteristic likelihood ratio (TCLR) for each variant. Predictive performance of TCLR in combination with in silico predictors, and a multifactorial variant prediction (MVP) model that included allele frequency, co-occurrence, co-segregation, and clinical and family history information was assessed.ResultsCompared with non-carriers, carriers of germline pathogenic/likely pathogenic (P/LP) variants were more likely to have abnormal MSI/IHC status (p<0.0001). Among 150 classified missense variants, 73.3% were accurately predicted with TCLR alone. Models leveraging in silico scores as prior probabilities accurately classified >76.7% variants. Adding TCLR as quantitative evidence in an MVP model (MVP +TCLRPred) increased the proportion of accurately classified variants from 88.0% (MVP alone) to 98.0% and generated optimal performance statistics among all models tested. Importantly, MVP +TCLRPred resulted in the high yield of predicted classifications for missense variants of unknown significance (VUS); among 193 VUS, 62.7% were predicted as P/PL or benign/likely benign (B/LB) when assessed according to American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines.ConclusionOur study demonstrates that when used separately or in conjunction with other evidence, tumour characteristics provide evidence for germline MMR missense variant assessment, which may have important implications for genetic testing and clinical management.
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Caggiari, Laura, Mara Fornasarig, Mariangela De Zorzi, Renato Cannizzaro, Agostino Steffan, and Valli De Re. "Family’s History Based on the CDH1 Germline Variant (c.360delG) and a Suspected Hereditary Gastric Cancer Form." International Journal of Molecular Sciences 21, no. 14 (July 11, 2020): 4904. http://dx.doi.org/10.3390/ijms21144904.
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Hereditary diffuse gastric cancer (HDGC) is a cancer susceptibility syndrome caused by germline pathogenic variant in CDH1, the gene encoding E-cadherin. The germline loss-of-function variants are the only proven cause of the cancer syndrome HDGC, occurring in approximately 10–18% of cases and representing a helpful tool in genetic counseling. The current case reports the family history based on a CDH1 gene variant, c.360delG, p.His121Thr in a suspected family for hereditary gastric cancer form. This frameshift deletion generates a premature stop codon at the amino acid 214, which leads to a truncated E-cadherin protein detecting it as a deleterious variant. The present study expands the mutational spectra of the family with the CDH1 variant. Our results highlight the clinical impact of the reported CDH1 variant running in gastric cancer families.
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Modlin, Leslie A., Jessica Flynn, Zhigang Zhang, Oren Cahlon, Boris Mueller, Atif J. Khan, Erin F. Gillespie, et al. "Tolerability of Breast Radiotherapy Among Carriers of ATM Germline Variants." JCO Precision Oncology, no. 5 (January 2021): 227–34. http://dx.doi.org/10.1200/po.20.00334.
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PURPOSE ATM, a gene that controls repair of DNA double-strand breaks, confers an excess lifetime risk of breast cancer among carriers of germline pathogenic variants (PV). ATM PV homozygotes are particularly sensitive to DNA damage caused by ionizing radiation. Consequently, there is concern that adjuvant radiotherapy (RT) may cause excess morbidity among heterozygous carriers of ATM PV. We evaluated the tolerability of breast RT among carriers of ATM germline variants. METHODS Of 167 patients with ATM germline variants presenting to our institution with breast cancer, 91 received RT. Treatment-related toxicity was ascertained from medical records and graded across organ systems. Toxicities grade > 2 were recorded from the end of treatment to last evaluable follow-up and were analyzed according to ATM variant pathogenicity. RESULTS Of 91 evaluable carriers of ATM variants, with a median follow-up of 32 months following RT, 25% (n = 23) harbored a PV, whereas 75% (n = 68) harbored a variant of uncertain significance (VUS). Prevalence of grade ≥ 2 toxicity unrelated to post-mastectomy reconstruction among patients with ATM PV was: 32% at the end of treatment ( v 34% for VUS carriers), 11% at 1 year of follow-up ( v 4% for VUS carriers), and 8% at the last follow-up ( v 13% for VUS carriers), consistent with previous studies of RT among unselected populations. No grade 4 or 5 toxicities were observed. ATM variant pathogenicity was not associated with local toxicity, contralateral breast cancer, or secondary malignancy in this limited cohort of patients who received breast RT. CONCLUSION We found no evidence of excess RT-associated toxicity among carriers of pathogenic ATM germline variants. Breast-conserving therapy and adjuvant RT may be safely considered among appropriately selected carriers of ATM germline variants.
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Lo, Winifred, Bin Zhu, Arvind Sabesan, Ho-Hsiang Wu, Astin Powers, Rebecca A. Sorber, Sarangan Ravichandran, et al. "Associations of CDH1 germline variant location and cancer phenotype in families with hereditary diffuse gastric cancer (HDGC)." Journal of Medical Genetics 56, no. 6 (February 11, 2019): 370–79. http://dx.doi.org/10.1136/jmedgenet-2018-105361.
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IntroductionHereditary diffuse gastric cancer (HDGC) is a cancer syndrome associated with variants in E-cadherin (CDH1), diffuse gastric cancer and lobular breast cancer. There is considerable heterogeneity in its clinical manifestations. This study aimed to determine associations between CDH1 germline variant status and clinical phenotypes of HDGC.MethodsOne hundred and fifty-two HDGC families, including six previously unreported families, were identified. CDH1 gene-specific guidelines released by the Clinical Genome Resource (ClinGen) CDH1 Variant Curation Expert Panel were applied for pathogenicity classification of truncating, missense and splice site CDH1 germline variants. We evaluated ORs between location of truncating variants of CDH1 and incidence of colorectal cancer, breast cancer and cancer at young age (gastric cancer at <40 or breast cancer <50 years of age).ResultsFrequency of truncating germline CDH1 variants varied across functional domains of the E-cadherin receptor gene and was highest in linker (0.05785 counts/base pair; p=0.0111) and PRE regions (0.10000; p=0.0059). Families with truncating CDH1 germline variants located in the PRE-PRO region were six times more likely to have family members affected by colorectal cancer (OR 6.20, 95% CI 1.79 to 21.48; p=0.004) compared with germline variants in other regions. Variants in the intracellular E-cadherin region were protective for cancer at young age (OR 0.2, 95% CI 0.06 to 0.64; p=0.0071) and in the linker regions for breast cancer (OR 0.35, 95% CI 0.12 to 0.99; p=0.0493). Different CDH1 genotypes were associated with different intracellular signalling activation levels including different p-ERK, p-mTOR and β-catenin levels in early submucosal T1a lesions of HDGC families with different CDH1 variants.ConclusionType and location of CDH1 germline variants may help to identify families at increased risk for concomitant cancers that might benefit from individualised surveillance and intervention strategies.
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Skopelitou, Diamanto, Beiping Miao, Aayushi Srivastava, Abhishek Kumar, Magdalena Kuświk, Dagmara Dymerska, Nagarajan Paramasivam, et al. "Whole Exome Sequencing Identifies APCDD1 and HDAC5 Genes as Potentially Cancer Predisposing in Familial Colorectal Cancer." International Journal of Molecular Sciences 22, no. 4 (February 12, 2021): 1837. http://dx.doi.org/10.3390/ijms22041837.
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Germline mutations in predisposition genes account for only 20% of all familial colorectal cancers (CRC) and the remaining genetic burden may be due to rare high- to moderate-penetrance germline variants that are not explored. With the aim of identifying such potential cancer-predisposing variants, we performed whole exome sequencing on three CRC cases and three unaffected members of a Polish family and identified two novel heterozygous variants: a coding variant in APC downregulated 1 gene (APCDD1, p.R299H) and a non-coding variant in the 5′ untranslated region (UTR) of histone deacetylase 5 gene (HDAC5). Sanger sequencing confirmed the variants segregating with the disease and Taqman assays revealed 8 additional APCDD1 variants in a cohort of 1705 familial CRC patients and no further HDAC5 variants. Proliferation assays indicated an insignificant proliferative impact for the APCDD1 variant. Luciferase reporter assays using the HDAC5 variant resulted in an enhanced promoter activity. Targeting of transcription factor binding sites of SNAI-2 and TCF4 interrupted by the HDAC5 variant showed a significant impact of TCF4 on promoter activity of mutated HDAC5. Our findings contribute not only to the identification of unrecognized genetic causes of familial CRC but also underline the importance of 5’UTR variants affecting transcriptional regulation and the pathogenesis of complex disorders.
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Hahn, Christopher N., Milena Babic, Andreas W. Schreiber, Monika M. Kutyna, L. Amilia Wee, Anna L. Brown, Michelle Perugini, et al. "Rare and Common Germline Variants Contribute to Occurrence of Myelodysplastic Syndrome." Blood 126, no. 23 (December 3, 2015): 1644. http://dx.doi.org/10.1182/blood.v126.23.1644.1644.
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Abstract Background: Majority of MDS cases appear to be sporadic in nature, but 10-15% have clear familial basis due to predisposing mutations in genes such as RUNX1, GATA2, CEBPA and DDX41. Contribution of germline variants in sporadic MDS is not studied. This study attempts to address the contribution of germline variants in MDS pathogenesis. Methods: We performed amplicon-based massively parallel sequencing (AmpliSeq custom panel adapted for Illumina HiSeq2500 sequencing) on all coding regions of 29 myeloid genes for 144 MDS samples. After identifying the variants in five genes (TET2, MET, GATA2, ASXL1, NOTCH1), we tested an additional 96 MDS samples including therapy-related myeloid neoplasm (T-MN) using a Sequenom assay. We also analyzed WES data for these variants in 178 AML samples and 758 normal controls and AmpliSeq data for ASXL1 and TET2 variants in 655 CML samples. Results: Collation of all coding variants in the 29 myeloid genes sequenced identified germline variants occurring in primary MDS at frequencies significantly higher than expected when compared to the normal population (ExAC and matched cohort were similar) (Table 1). These variants occurred in 5 genes (TET2, MET, GATA2, ASXL1 and NOTCH1) at increased frequencies of 1.5-16.6 fold. Numerous MDS samples had multiple variants (4 with 4 variants, 4 with 3 variants, 18 with 2 variants) while 70 had 1 variant. The 3 germline MET variants have been previously investigated in solid tumorigenesis and likely generate MET variant proteins that contribute to numerous cancer types including MDS. Interestingly, 7/17 (41%) MDS cases with germline MET variants also had other cancers including pancreatic, gastric and laryngeal cancers. Of the TET2 variants, Y867H and P1723S were concurrent in 5 MDS, 5 AML and 6 CML samples indicative of them being on the same allele (i.e. a haplotype). They were seen at higher than normal frequency in MDS and AML, but were not significantly enriched in CML. We are currently confirming their coexistence on the same allele and assaying for decreased TET2 activity to determine whether one or both variants contribute to the phenotype. Other variants identified in MDS include the rare GATA2 (P161A) variant which is present in 1% of the population and the nearby common GATA2 (A164T) allele (~20%). These were mutually exclusive in our cohort and were seen at 3.9 and 1.5-fold, respectively, above the expected population frequency. We generated the P161A variant using site-directed mutagenesis and assayed for GATA2 transactivation activity in HEK293 cells with a GATA2-responsive LYL1 promoter-Luciferase construct (Figure 1). We also included empty vector (EV), wildtype (WT) GATA2 and T354M which is the most common highly penetrant autosomal dominant mutation leading to familial MDS/AML. As expected, T354M displayed a marked decrease in transactivation ability when compared to WT. The P161A variant similarly displayed loss-of-function in this assay, but not to the same magnitude as T354M. This is consistent with the hypothesis that reduced GATA2 function predisposes to myeloid malignancy where decreasing GATA2 activity correlates with increasing risk of developing malignancy. In our study 10/36 (28%) cases harboring these variants were T-MN cases. Apart from MET (E168D) (11.4-fold), the 2 rare variants with highest frequency in MDS versus controls were ASXL1 (N986S) (16.6-fold) and NOTCH1 (R912W) (6.5-fold). ASXL1 is an epigenetic regulator often mutated in hematopoietic malignancy and aberrant NOTCH1 function has been associated with myeloid and lymphoid malignancies. Conclusions: We have identified common and rare germline variants in genes involved in myeloid malignancy that may contribute to MDS pathogenesis. It remains to be seen whether they contribute to initiation, maintenance and/or progression of MDS and other hematopoietic malignancies. This is the first study reporting higher frequency of germline variants in sporadic MDS cases. Table 1. Frequency of germline variants in MDS, AML and CML in comparison to ExAC. Table 1. Frequency of germline variants in MDS, AML and CML in comparison to ExAC. Disclosures Hiwase: Celgene Corporation: Research Funding.
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Imizcoz-Fabra, Teresa, Eva Cañada-Higueras, Nerea García de Vicuña-Bilbao, Beatriz Ramírez-Horcada, Arancha Bielsa-Colás, Maria Isabel Mora, Javier Ursúa-Noain, et al. "Tumor sequencing aids to identify individuals with hereditary cancer." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e13678-e13678. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e13678.
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e13678 Background: Early identification of individuals with hereditary cancer significantly improves survival in patients and relatives. New genomic tools can identify germline pathogenic variants via tumor-only genomic sequencing in addition to treatment guiding biomarkers. Methods: Our tumor sequencing protocol in medical practice since 2018 via Oncomine Comprehensive Assay (OCA) interrogates several hereditary cancer associated genes; therefore, we aimed to evaluate OCA’s capacity to point out individuals’ with hereditary cancer. But first, OCA’s technical validity was evaluated by testing up to 20 year old FFPE block isolated tumor genomes of individuals known to carry 19 pathogenic variants. Results: All variants were present in the OCA raw dataset but 26% were not called by the commercial software due to specificity of the variant calling algorithm and bias caused by amplicon-based enrichment and limited coverage. Even with this limitation, OCA’s clinical utility was evaluated next. A pathogenic variant candidate to be germline was identified in 5% (26/510) of tumor genomes routinely tested looking for treatment guiding biomarkers, but up to 8% (26/317) when excluding lung cancer patients. Importantly, 73% (14/19) of the ones that were followed-up by peripheral blood testing were confirmed to be germline, so inherited nature of the cancer was confirmed in 9% of ovarian, 8% of endometrium, 5% of biliary tract, 4% of breast, 1% of colorectal and 1% of lung cancer patients whose tumor genome was tested for routine treatment guiding biomarker identification. Conclusions: OCA is a useful genomic tool for identifying individuals with hereditary cancer that opens a new era in clinical practice in oncology. An increased awareness among physicians about this new genomic tool includes understanding the importance of peripheral blood confirmation to define the germline nature of the OCA-identified candidate variant; and proper genetic counseling and germline testing when OCA does not find a candidate variant in a patient with strong family history due to the lack of comprehensive identification of every type of pathogenic variants in all known genes associated with inherited cancer via tumor sequencing tools like OCA.
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Schlussel, Andrew T., Susan S. Donlon, Faye A. Eggerding, and Ronald A. Gagliano. "Identification of an APC Variant in a Patient with Clinical Attenuated Familial Adenomatous Polyposis." Case Reports in Medicine 2014 (2014): 1–3. http://dx.doi.org/10.1155/2014/432324.
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Introduction.The objective of this case report is to discuss an unclassified germline variant of the adenomatous polyposis coli (APC) gene identified in an older patient with attenuated familial adenomatous polyposis syndrome (AFAP).Methods.We present a case report of a 66-year-old man diagnosed with AFAP. Colonoscopy found multiple polyps and invasive adenocarcinoma arising in the transverse colon. Samples were tested for mutations in the APC gene.Results.DNA sequencing of germline DNA identified a cytosine (C) to thymine (T) transition at nucleotide 1240, heterozygous. The C to T transition at codon 414 is predicted to convert an arginine residue to a cysteine that is possibly pathogenic. Analysis of the patient’s colon tumor DNA indicated that the tumor had lost the mutant variant allele and retained only the normal allele, suggesting that the variant may not be significant.Conclusions.The p.R414C variant has been described previously as a germline mutation of probable pathogenicity. This substitution should be considered an unclassified variant and possibly not pathogenic. These findings support the need for further genetic testing of tissue, as well as for developing a mechanism for testing all variants, as this could significantly impact the lives of patients and their family members.
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Yannakou, Costas K., Kate Jones, Georgina L. Ryland, Ella R. Thompson, Gareth Reid, Michelle McBean, Alison Trainer, David Westerman, and Piers Blombery. "Incidental detection of germline variants of potential clinical significance by massively parallel sequencing in haematological malignancies." Journal of Clinical Pathology 71, no. 1 (August 11, 2017): 84–87. http://dx.doi.org/10.1136/jclinpath-2017-204481.
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Massively parallel sequencing (MPS) technology has become routinely available for diagnosis, prognostication and therapeutic decision-making in haematological malignancies. However, increased throughput and wider coverage of genes can have unintended consequences. Germline variants of potential clinical significance (GVPCSs) detected during cancer testing may have implications for patients and families beyond the biological evaluation of a specific tumour. 721 reports generated from MPS panels used in the routine testing of myeloid and lymphoid malignancies were reviewed and variants within genes of potential germline relevance (TP53, RUNX1, GATA2 and WT1 in all contexts and CBL, KRAS and NRAS in the setting of juvenile myelomonocytic leukaemia) were analysed. A variant allele fraction threshold of ≥33.09% for considering germline origin of variants within cancer samples was established. The detection rate of incidental, pathogenic germline variants was 0.42%. Patient education and confirmatory germline sample testing of GVPCSs in appropriate circumstances are recommended.
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Chasseloup, Fanny, Nathan Pankratz, John Lane, Fabio R. Faucz, Margaret F. Keil, Prashant Chittiboina, Denise M. Kay, et al. "Germline CDKN1B Loss-of-Function Variants Cause Pediatric Cushing’s Disease With or Without an MEN4 Phenotype." Journal of Clinical Endocrinology & Metabolism 105, no. 6 (March 31, 2020): 1983–2005. http://dx.doi.org/10.1210/clinem/dgaa160.
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Abstract Context Germline loss-of-function CDKN1B gene variants cause the autosomal dominant syndrome of multiple endocrine neoplasia type 4 (MEN4). Even though pituitary neuroendocrine tumors are a well-known component of the syndrome, only 2 cases of Cushing’s disease (CD) have so far been described in this setting. Aim To screen a large cohort of CD patients for CDKN1B gene defects and to determine their functional effects. Patients We screened 211 CD patients (94.3% pediatric) by germline whole-exome sequencing (WES) only (n = 157), germline and tumor WES (n = 27), Sanger sequencing (n = 6), and/or germline copy number variant (CNV) analysis (n = 194). Sixty cases were previously unpublished. Variant segregation was investigated in the patients’ families, and putative pathogenic variants were functionally characterized. Results Five variants of interest were found in 1 patient each: 1 truncating (p.Q107Rfs*12) and 4 nontruncating variants, including 3 missense changes affecting the CDKN1B protein scatter domain (p.I119T, p.E126Q, and p.D136G) and one 5’ untranslated region (UTR) deletion (c.-29_-26delAGAG). No CNVs were found. All cases presented early (10.5 ± 1.3 years) and apparently sporadically. Aside from colon adenocarcinoma in 1 carrier, no additional neoplasms were detected in the probands or their families. In vitro assays demonstrated protein instability and disruption of the scatter domain of CDKN1B for all variants tested. Conclusions Five patients with CD and germline CDKN1B variants of uncertain significance (n = 2) or pathogenic/likely pathogenic (n = 3) were identified, accounting for 2.6% of the patients screened. Our finding that germline CDKN1B loss-of-function may present as apparently sporadic, isolated pediatric CD has important implications for clinical screening and genetic counselling.
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Cunha, Renato, Priscila Nejo, Sandra Bento, and Fátima Vaz. "ATM germline variants and male breast cancer." BMJ Case Reports 14, no. 1 (January 2021): e238100. http://dx.doi.org/10.1136/bcr-2020-238100.
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Male breast cancer is rare and has been frequently associated with cancer predisposing variants, particularly in BRCA 1 and BRCA 2 genes. ATM pathogenic variants may also increase risk for breast and other cancers. However, less than 10 cases relating ATM mutations and male breast cancer have been previously reported. Therefore, risk estimates and surveillance recommendations are not well established. We report a case of a male patient with breast cancer found to be heterozygous for a pathogenic ATM variant after multigene testing. We also review the literature regarding increased cancer risk associated with ATM germline variants, with emphasis on potential recommendations for surveillance and follow-up.
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DeRoin, Lia, Marcela Cavalcante De Andrade Silva, Kristin Petras, Kelly Arndt, Nathaniel Phillips, Pankhuri Wanjari, Hari Prasanna Subramanian, et al. "Assessing the Feasibility and Limitations of Cultured Skin Fibroblasts for Germline Genetic Testing in Hematologic Disorders." Blood 136, Supplement 1 (November 5, 2020): 35–36. http://dx.doi.org/10.1182/blood-2020-138431.
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Introduction Peripheral blood is the standard tissue source for germline genetic testing in most scenarios. In patients with hematologic malignancies, however, peripheral blood frequently contains tumor- or clonal hematopoiesis-related acquired genetic variants, often occurring in genes that can also cause inherited cancer susceptibility if present in the germline. Thus, an alternative tissue source is necessary. Cultured skin fibroblasts have been used as a potentially ideal alternative because they are free from blood contamination and provide ample DNA yields, advantages that other alternatives such as saliva or nail clippings lack. However, optimal culture methods, expected time from biopsy to sufficient DNA yield, culture failure rate, and limitations of this technique, including the possibility of variants being acquired solely due to the culturing process, are not yet known. Methods We conducted a retrospective cohort study of subjects with cytopenias or hematologic malignancies who underwent skin biopsy and fibroblast culture for germline genetic testing from April 2014 to June 2018. Skin biopsy culture technical data, including time from biopsy to culture set-up, shipment from an outside institution, culture failure, and biopsy size, were abstracted from tissue culture logs. Patient demographics, comorbidities, medication history, and hematologic diagnosis and treatment were abstracted from medical records. Next generation sequencing data from targeted capture of 144 inherited cancer and bone marrow failure predisposition genes obtained for clinical genetic testing purposes were analyzed to identify variants at both germline (40-60%) and subclonal (10-40%) variant allele frequencies (VAF). Pathogenicity was interpreted according to ACMG/AMP guidelines. Fisher's exact tests and logistic regression models were used to assess associations with culture failure. T-tests and linear regression models were used to assess factors associated with mean time to confluency. Results In total, we studied 350 samples from unique patients, including 61 (24%) who carried one or more pathogenic or likely pathogenic cancer susceptibility gene variant(s). Overall, 16 of the 350 (5%) biopsies failed to grow in culture. The median time from skin biopsy to sufficient growth to extract DNA for genetic testing was 27 days (IQR 22-29 days). Culture failure was significantly more likely in samples with a delay in culture initiation for 24 hours post biopsy (OR=4.32; p<0.01), and a pathogenic germline variant in a gene associated with telomere maintenance (OR=64.50; p<0.01). Factors associated with an increased mean time to sufficient growth included prior allogeneic stem cell transplant (32.1 days versus 27.2 days; p<0.01) and prior intravenous (IV) steroid exposure (29.9 days versus 26.4 days; p<0.01). Among samples cultured successfully, carriers of any pathogenic germline variant had a significantly decreased mean time to sufficient growth (25.4 days versus 28.6 days; p<0.01). A pathogenic or likely pathogenic subclonal variant was identified in 11 (4%) subjects at a median VAF of 20%. Among eight of these with additional tissue available, the presence of the variant was confirmed in four (50%). In individual cases, we found evidence of loss of a pathogenic variant in the hematopoietic malignancy. In one patient with a pathogenic variant with a 50% VAF in the original skin culture, the variant was not present in a skin culture from a second, fresh skin biopsy done due to discordant phenotype. Conclusions Culturing of skin fibroblasts for germline genetic testing in patients with hematologic disorders has a high success rate, especially when cultures are initiated within 24 hours of collection, and adds on average 27 days to genetic testing turnaround time. From patients with a hereditary syndrome, most skin biopsies will culture with the exception of individuals with a short telomere syndrome. For this subset, a direct skin biopsy without culture may be necessary. Subclonal variants at VAFs relevant to interpretation of a germline test were found in 4% of cases. Half were confirmed in an alternative tissue. Etiology of the subclonal variants, whether acquired during the culturing process or due to mosaicism or sequencing biases was not always clear. Careful assessment of the clinical phenotype in interpreting and applying germline genetic results to patient care will always be warranted. Disclosures Godley: UptoDate, Inc.: Honoraria; Invitae, Inc.: Membership on an entity's Board of Directors or advisory committees. Segal:BMS: Consultancy, Research Funding; AbbVie: Consultancy; Merck: Consultancy; Astra Zeneca: Consultancy. Churpek:UpToDate, Inc: Honoraria.
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Golubeva, Volha A., Thales C. Nepomuceno, and Alvaro N. A. Monteiro. "Germline Missense Variants in BRCA1: New Trends and Challenges for Clinical Annotation." Cancers 11, no. 4 (April 12, 2019): 522. http://dx.doi.org/10.3390/cancers11040522.
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Genetic testing allows for the identification of germline DNA variations, which are associated with a significant increase in the risk of developing breast cancer (BC) and ovarian cancer (OC). Detection of a BRCA1 or BRCA2 pathogenic variant triggers several clinical management actions, which may include increased surveillance and prophylactic surgery for healthy carriers or treatment with the PARP inhibitor therapy for carriers diagnosed with cancer. Thus, standardized validated criteria for the annotation of BRCA1 and BRCA2 variants according to their pathogenicity are necessary to support clinical decision-making and ensure improved outcomes. Upon detection, variants whose pathogenicity can be inferred by the genetic code are typically classified as pathogenic, likely pathogenic, likely benign, or benign. Variants whose impact on function cannot be directly inferred by the genetic code are labeled as variants of uncertain clinical significance (VUS) and are evaluated by multifactorial likelihood models that use personal and family history of cancer, segregation data, prediction tools, and co-occurrence with a pathogenic BRCA variant. Missense variants, coding alterations that replace a single amino acid residue with another, are a class of variants for which determination of clinical relevance is particularly challenging. Here, we discuss current issues in the missense variant classification by following a typical life cycle of a BRCA1 missense variant through detection, annotation and information dissemination. Advances in massively parallel sequencing have led to a substantial increase in VUS findings. Although the comprehensive assessment and classification of missense variants according to their pathogenicity remains the bottleneck, new developments in functional analysis, high throughput assays, data sharing, and statistical models are rapidly changing this scenario.
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DiNardo, Courtney D., Larissa A. Korde, and Matthew B. Yurgelun. "A Case-Based Approach to Understanding Complex Genetic Information in an Evolving Landscape." American Society of Clinical Oncology Educational Book, no. 41 (June 2021): e328-e338. http://dx.doi.org/10.1200/edbk_321041.
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The rapid integration of highly sensitive next-generation sequencing technologies into clinical oncology care has led to unparalleled progress, and yet these technological advances have also made genetic information considerably more complex. For instance, accurate interpretation of genetic testing for germline/inherited cancer predisposition syndromes and somatic/acquired pathogenic variants now requires a more nuanced understanding of the presence and incidence of clonal hematopoiesis and circulating tumor cells, with careful evaluation of pathogenic variants occurring at low variant allele frequency required. The interplay between somatic and germline pathogenic variants and awareness of distinct genotype-phenotype manifestations in various inherited cancer syndromes are now increasingly appreciated and can impact patient management. Through a case-based approach, we focus on three areas of particular relevance to the treating clinician oncologist: (1) understanding clonal hematopoiesis and somatic mosaicism, which can be detected on germline sequencing and lead to considerable confusion in clinical interpretation; (2) implications of the detection of a potentially germline pathogenic variant in a high-penetrance cancer susceptibility gene during routine tumor testing; and (3) a review of gene-specific risks and surveillance recommendations in Lynch syndrome. A discussion on the availability and difficulties often associated with direct-to-consumer genetic testing is also provided.
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Vaske, Charles Joseph, Chad Garner, Tara Elisabeth Seery, Christopher Szeto, and Sandeep K. Reddy. "Clinical trial screening of CDKN2A genomic alterations in patients with pancreatic cancer and hepatobiliary cancers requires greater precision than somatic sequencing alone." Journal of Clinical Oncology 37, no. 4_suppl (February 1, 2019): 287. http://dx.doi.org/10.1200/jco.2019.37.4_suppl.287.
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287 Background: The TAPUR Study is a phase II multi‐basket study that evaluates the anti‐tumor activity of commercially available targeted agents in pts with advanced cancers with genomic alterations known to be drug targets. Results in two cohorts of PC and GBC pts each with CDKN2A loss or mutation were reported at ASCO 2018. The conclusion was that monotherapy with palbocicilib is not associated with clinical activity in these patients. This may be a false conclusion if the genomic targets were absent in these patients. Methods: A total of 158 GI pts (P = 123, GB = 20, Bile Duct = 15) with deep whole exome sequencing (WES) of tumor and blood samples, and whole transcriptomic sequencing (RNA-Seq) (∼200x106 reads per tumor) were available for this analysis from a commercial database. Variant calling was performed through joint probabilistic analysis of tumor and normal DNA reads, with germline status of variants being determined by heterozygous or homozygous alternate allele fraction in the germline sample. Results: 26 somatic variants and 12 germline variants were detected, with one sample overlapping with a germline and a somatic variant (p.A148T and p.A76Rfs∗44). Counting all 11 discrete germline variants as false positives, a total 37 of 158 samples would be positive for CDKN2A mutant status, a rate of 23% (17%-31% CI). However, if the 8 common germline variants are excluded, the call rate is 29/158 = 18% (12%-25% CI). The false positive rate is 4/158 = 14% (4%-31% CI). By RNAseq, true somatic CDKN2A variants had significantly higher TPM counts than germline variants (T-test p = 0.0002). RB expression was not significantly different between the two groups. Conclusions: The failure of palbociclib to show benefit in CDKN2A mutated PC and GBC patients in the 20 patient cohort of the TAPUR study could possibly be explained by patient selection rather than solely drug failure. It is unlikely related to RB loss.
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Ricker, Charité, Erika Amundson, Sandra Algaze, Marcia Ciccone, Stephen Dong, Anishka D'souza, Kimberly Felicetti, et al. "Assessing somatic and germline variants in cancer patients." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): 10601. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.10601.
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10601 Background: The increasing integration of somatic and germline testing into oncology practice allows physicians to target oncologic therapy and identify those with cancer predisposition. We explored the impact of a somatic assay (liquid biopsy, LB) on the identification of patients appropriate for germline genetic testing. Methods: We identified a cohort of diverse cancer patients with LB to assess for targetable somatic gene variants at LAC+USC Medical Center between 2016 and 2020 (n= 467). To enrich the cohort for variants that may reflect germline findings, we focused on the 46 patients (9.9%) who had at least one variant identified with a cell-free DNA (cfDNA) fraction of 25.00% or greater. Retrospective chart review extracted demographics and medical history with variables related to cancer history and treatment. LB variants were classified based on whether germline confirmation was indicated and the results of germline tests, when done, were reviewed. Results: Table summarizes the characteristics of the 46 patients identified to have at least one variant on LB in ≤ 25% of the cfDNA. The most frequently mutated genes on LB were TP53 (n=18, 39%), KRAS (n=11, 24%), APC (n=8, 17%), BRCA2 (n=7, 15%), PIK3CA (n=6, 13.0%), and BRCA1 (n=4, 9%). Seventeen patients (40%) were referred for genetic counseling and 13 (30%) underwent germline testing of whom 10 (77%) carried pathogenic variants (PV). All germline PV were concordant with LB variants identified. Four patients with PV BRCA2 on LB and confirmed to be germline, had lung or biliary tract diagnoses, which are not part of the typical BRCA-tumor spectrum. Thirty-three patients were not referred for genetic counseling, though 24 (72%) had LB-identified variants in cancer predisposition genes and 18 (54%) merited a genetics referral. Among patients with germline mutations, three (23%) had targeted therapy and two (15%) had preventive surgery to address second primary cancer risk. Among the 467 patients with LB results, there were an additional 13 patients (not included in the enriched group) known to have a cancer predisposition gene PV. Only two (15%) had findings on LB reports that suggested germline testing would be indicated. Conclusions: While the purpose of somatic testing is to identify targeted therapy, it can provide germline insight, especially for patients not typically referred for genetic assessment. Further education and guidance is needed to assure that this aspect of somatic testing is appreciated by oncology providers and acted upon.[Table: see text]
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Braunstein, Evan M., Hang Chen, Felicia Juarez, Fanghan Yang, Lindsay Tao, Igor Makhlin, Donna M. Williams, et al. "Germline ERBB2/HER2 Coding Variants Are Associated with Increased Risk of Myeloproliferative Neoplasms." Cancers 13, no. 13 (June 29, 2021): 3246. http://dx.doi.org/10.3390/cancers13133246.
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Familial cases of myeloproliferative neoplasms (MPN) are relatively common, yet few inherited risk factors have been identified. Exome sequencing of a kindred with a familial cancer syndrome characterized by both MPN and melanoma produced a germline variant in the ERBB2/HER2 gene that co-segregates with disease. To further investigate whether germline ERBB2 variants contribute to MPN predisposition, the frequency of ERBB2 variants was analyzed in 1604 cases that underwent evaluation for hematologic malignancy, including 236 cases of MPN. MPN cases had a higher frequency of rare germline ERBB2 coding variants compared to non-MPN hematologic malignancies (8.9% vs. 4.1%, OR 2.4, 95% CI: 1.4 to 4.0, p = 0.0028) as well as cases without a blood cancer diagnosis that served as an internal control (8.9% vs. 2.7%, OR 3.5, 95% CI: 1.4 to 8.3, p = 0.0053). This finding was validated via comparison to an independent control cohort of 1587 cases without selection for hematologic malignancy (8.9% in MPN cases vs. 5.2% in controls, p = 0.040). The most frequent variant identified, ERBB2 c.1960A > G; p.I654V, was present in MPN cases at more than twice its expected frequency. These data indicate that rare germline coding variants in ERBB2 are associated with an increased risk for development of MPN. The ERBB2 gene is a novel susceptibility locus which likely contributes to cancer risk in combination with additional risk alleles.
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Schneider, Bryan P., Leigh Anne Stout, Santosh Philips, Cynthia Hunter, Patrick Kiel, and Milan Radovich. "Implications of incidental germline findings identified in the context of clinical whole exome sequencing (WES) for guiding cancer therapy." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 1581. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.1581.
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1581 Background: The identification of incidental germline mutations in the context of next-generation sequencing to guide therapy for cancer patients has become an unintended consequence of advancing technologies. While not the primary goal, additional information buried within the genomic data generated could be important for family members to better understand inherited disease risks and take action. While targeted gene panels are predominantly used in practice, comprehensive WES is becoming increasingly more common. The broader use of WES increases the complexity and scope of potential cancer and non-cancer pathogenic germline variation. Methods: 582 adult metastatic cancer patients from the Indiana University Health Precision Genomics Program were sequenced with tumor and germline CLIA-based WES. The primary intent was for guiding cancer therapy. Germline variant call files were mined for pathogenic/likely pathogenic (P/LP) variants using the ClinVar database. Clinvar classifications were narrowed only to high quality submitters. Results: We identified 1,454 P/LP variants in 209 genes. 92% of patients harbored at least one variant. 72 P/LP variants were identified in 19 cancer predisposition genes with BRCA2 being the most common. 63 patients (10.8%) carried a P/LP variant in a gene that would be recommended by the ACMG to be reported due to clinical actionability with the most common being ATP7B (N = 17), BRCA2 (n = 13), MUTYH (n = 8), and BRCA1 (n = 5). We observed a preponderance of patients who carried autosomal recessive non-cancer pathogenic mutations of varying penetrance. Notable mutated genes included CFTR (cystic fibrosis, n = 16), BTD (biotinidase deficiency, n = 43), and CBS (homocystinuria, n = 90). Of 209 mutated genes, 173 genes had mutations present in less than 1% of our population, demonstrating significant genetic heterogeneity. Conclusions: The majority of patients undergoing clinical cancer WES harbor pathogenic germline variation. Identification of clinically actionable germline findings will create additional burden on oncology clinics as broader WES becomes commonplace.
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Schrader, Kasmintan A., Kelly Lynn Stratton, Rajmohan Murali, Yael Laitman, Luca Cavallone, Lily Offit, Yong Hannah Wen, et al. "Genome sequencing of multiple primary tumors to reveal underlying germline cancer susceptibility." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): 1552. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.1552.
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1552 Background: Apart from germline mutations in BRCA1 and BRCA2, the basis for genetic susceptibility to breast and ovarian cancer is heterogeneous, and can necessitate sequential or multiplex genetic testing. In addition, examination of germline DNA alone may not be conclusive. Information regarding both primary and secondary genetic events can be obtained from genomic analysis of tumors in conjunction with germline DNA. Methods: To determine the underlying cause of multiple primary malignancies in an Ashkenazi Jewish individual, whole genome sequencing was performed on DNA from the patient’s germline, invasive ductal carcinoma of the breast, and ovarian high-grade serous carcinoma. After identifying a structural variant of interest in this patient, germline DNA of 1846 Ashkenazi Jewish individuals who had a personal history of either breast, pancreatic, or ovarian cancer or a history of both breast and/or ovarian cancer and a similar family history, were screened using a TaqMan copy number assay or a specific PCR breakpoint assay to determine if this structural variant is a founder mutation. Results: A novel germline complex structural variant of PALB2 creating a 3790 base-pair deletion encompassing exon 11 associated with a 68 base-pair insertion was identified and confirmed by Sanger sequencing and a TaqMan copy number assay. The germline deletion was retained in both tumors. In addition, both tumors acquired second hits that led to inactivation of the wild-type allele of PALB2. The germline PALB2 structural variant was not identified in any of the additional 1846 Ashkenazi Jewish individuals genotyped. Conclusions: Whole genome sequencing of multiple primary tumors enabled identification and characterization of a novel germline structural variant in PALB2 as the basis for the individual’s susceptibility to breast and ovarian cancer. The variant does not appear to be a founder mutation in Ashkenazim.
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Hernández-Ramírez, Laura C., Nathan Pankratz, John Lane, Mingming Hu, Fabio R. Faucz, Prashant Chittiboina, Kay M. Denise, James L. Mills, and Constantine A. Stratakis. "Potential Role for the RASD1 Glucocorticoid-Responsive Gene in Corticotroph Tumorigenesis." Journal of the Endocrine Society 5, Supplement_1 (May 1, 2021): A549. http://dx.doi.org/10.1210/jendso/bvab048.1118.
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Abstract Introduction: Originally identified due to its dexamethasone inducibility in mouse corticotropinoma AtT20 cells, RASD1 is a receptor-independent activator of G-proteins, via guanine nucleotide exchange factor (GEF) activity. It remains unclear, however, whether, and if so, how RASD1 mediates the effects of glucocorticoids on corticotroph cells. We identified a rare germline RASD1 variant and investigated its functional effects in vitro. Methods: We screened 209 CD patients (94.3% pediatric) studied at the at the National Institutes of Health Clinical Research Center between 1997 and 2018 by germline whole-exome sequencing (WES) only (n=157), germline and tumor WES (n=27), and/or RASD1 droplet digital PCR germline copy number variant (CNV) analysis (n=201). Corticotropinoma DNA was available in 72 patients to screen for USP8 hotspot variants by Sanger sequencing. A RASD1 variant was identified and functionally characterized. Results: We studied 119 female (56.9%) and 90 (43.1%) male CD cases, including 197 pediatric (≤18 years at disease onset) and 12 adult patients. USP8 defects were present in 19.4% (14/72) of cases. No RASD1 CNVs were found. A rare (with a minor allele frequency of 0.0022% in gnomAD v3) heterozygous germline missense RASD1 variant, c.580A>C, p.M194L was detected in one male sporadic case. Neither USP8 variants nor loss of heterozygosity at the RASD1 variant position were observed in the patient’s microadenoma. The wild type and p.M194L RASD1 transiently overexpressed proteins displayed similar short half-lives (<1 h) by cycloheximide chase in HEK293 cells, as well as cytoplasmic localization by immunocytofluorescence in AtT20 cells. A CRISPR/Cas9 Rasd1 knockout AtT20 cell line displayed reduced Pomc expression compared with the parental cell line at the mRNA level (Actb-normalized absolute quantification 5.80±0.92 vs 9.62±0.7, P=0.005). Viability of the cell lines did not differ significantly by MTT assay. Overexpression of p.M194L resulted in increased accumulation of phospho-CREB S133 (1.83±0.8 vs 1±0.2 in empty vector control, P=0.0390) as well as a non-significant increase in Pomc expression in wild type, but not in Rasd1 knockout AtT20 cells by immunoblot band densitometry. Conclusions: We found an infrequent RASD1 variant in one CD patient. Rasd1 seems to have a role within the intracellular signaling pathways controlling Pomc expression. Overexpression of the p.M194L variant caused phospho-CREB S133 activation, suggesting increased GEF activity for this variant. Interestingly, another variant at the same position, p.M194I, was found in the COSMIC database (COSS2121715) as a somatic change in cutaneous malignant melanoma. Further studies are required to better define the role of RASD1 in corticotroph physiology and its possible involvement in tumorigenesis.
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Bettini, Laura Rachele, Claudia Saitta, Barbara Buldini, Geertruij Te Kronnie, Stefano Rebellato, Daniela Silvestri, Elena Barisone, et al. "Incidence and Therapeutic Implications of Germline TP53 Mutations in Hypodiploid Childhood Acute Lymphoblastic Leukemia: A Retrospective Analysis of the Italian Cohort." Blood 136, Supplement 1 (November 5, 2020): 43–44. http://dx.doi.org/10.1182/blood-2020-142729.
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Background. Hypodiploid Acute Lymphoblastic Leukemia (ALL) is a rare subtype of childhood ALL known to be associated with a poor prognosis. Currently, intensive chemotherapy and haematopoietic stem cell transplantation are recommended as the main therapeutic strategy for these patients. Previous studies showed that low-hypodiploid ALL frequently carry TP53 variants, the majority of which are germline mutations. This finding suggests that hypodiploid ALL may be a manifestation of underlying Li-Fraumeni syndrome, with an associated high risk for secondary tumors. We analyzed TP53 variants in the Italian cohort of hypodiploid pediatric ALL patients diagnosed between 2000 and 2019, to explore frequency and characteristics, as well as the potential role of germline variants in the therapeutic strategy. Patients were treated according to the AIEOP-BFM ALL2000 (n=16), ALL2009 (n=16) and ALL2017 (n=8) protocols. Methods. We performed a targeted Next Generation Sequencing (NGS) Nextera Flex DNA panel of 40 genes, including TP53, in a retrospective series of hypodiploid paediatric ALL Italian patients enrolled in four nationwide frontline protocols. Ploidy was defined based on DNA index (DNAi) and/or cytogenetics/FISH, and cases with DNAi<0.8 were included in this study. Only TP53 variants with VF >5% and coverage 500X were considered. Bioinformatics analysis has been carried out by Sophia DDM software. NGS has been performed both in disease (hematopoietic) and germline (remission or buccal brush) tissues samples. Results. Nineteen TP53 variants were observed in 20/40 (50%) hypodiploid ALL patients, with 19/20 being low-hypodiploid ALL cases (DNAi between 0,6 and 0,8); 13/19 TP53 variants were known to be pathogenic whereas 6/19 were classified as Variant of Unknown Significance (VUS). Considering the VUS variants, 3/6 were missense, 1/6 frameshift and 2/6 in-frame ins/del. Notably, 13 patients out of 20 (65%) were found to carry a germline variant, while 5 patients presented a somatic variant; in 2 cases the remission sample was not available, but the VF<20% was indicative of a somatic variant. Among the germline variants, 1 was found to be a germline mosaicism, presenting a nonsense pathogenic TP53 variant both in remission (VF 10%) and buccal brush sample at remission (VF 8.5%). 16/19 variants reside in the p53 core DNA binding domain, known to be a fundamental site that mediates the transcription of p53 regulated genes, where most of the pathogenic mutations in cancer cells occur. The 3 variants outside the DNA binding domain were 1 nonsense, 1 frameshift and 1 missense, and were localized in the nuclear localization signal region (NLS) or in the oligomerization domain (OD). Considering the 32 patients with at least 5 years of follow-up, EFS was not significantly different between TP53 wild type (47.1% ± 12.1) vs TP53 variant (51.9% ± 17,8) subgroups. According to MRD stratification criteria, the patients carrying a TP53 variant were classified as medium risk in 14/20, standard risk in 5/20 and HR in 1 case, not statistically different from TP53 wild type. Collection of data on secondary neoplasms in patients and their relatives in ongoing. Conclusion. Our data represent the largest low hypodiploid patient cohort tested so far, confirming the high frequency of deleterious TP53 mutations. Mutational testing of TP53 in patients with hypodiploid ALL is warranted, in order to ensure a proper genetic counseling for patients with germline mutations and their families, with tailored clinical surveillance (according to Li-Fraumeni Syndrome guidelines). The best therapeutic regimen for hypodiploid patients is still a matter of debate, with MRD at the end of induction and TP53 germline mutations potentially indicating a highest relevance in the indication to hematopoietic stem cell transplantation. Chemotherapy-only treatment could be indicated for MRD-low patients and HSCT for MRD-High, potentially considering a reduced conditioning regimen for TP53 mutated cases. Disclosures Locatelli: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharmaceeutical: Speakers Bureau. Rizzari:Sobi: Consultancy, Other: Advisory Board.
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Stadler, Zsofia K., Anna Maio, Debyani Chakravarty, Yelena Kemel, Margaret Sheehan, Erin Salo-Mullen, Kaitlyn Tkachuk, et al. "Therapeutic Implications of Germline Testing in Patients With Advanced Cancers." Journal of Clinical Oncology 39, no. 24 (August 20, 2021): 2698–709. http://dx.doi.org/10.1200/jco.20.03661.
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PURPOSE Tumor mutational profiling is increasingly performed in patients with advanced cancer. We determined the extent to which germline mutation profiling guides therapy selection in patients with advanced cancer. METHODS Patients with cancer undergoing tumor genomic profiling were prospectively consented for germline cancer predisposition gene analysis (2015-2019). In patients harboring germline likely pathogenic or pathogenic (LP/P) alterations, therapeutic actionability was classified using a precision oncology knowledge base. Patients with metastatic or recurrent cancer receiving germline genotype–directed therapy were determined. RESULTS Among 11,947 patients across > 50 malignancies, 17% (n = 2,037) harbored a germline LP/P variant. By oncology knowledge base classification, 9% (n = 1042) had an LP/P variant in a gene with therapeutic implications (4% level 1; 4% level 3B; < 1% level 4). BRCA1/2 variants accounted for 42% of therapeutically actionable findings, followed by CHEK2 (13%), ATM (12%), mismatch repair genes (11%), and PALB2 (5%). When limited to the 9,079 patients with metastatic or recurrent cancer, 8% (n = 710) harbored level 1 or 3B genetic findings and 3.2% (n = 289) received germline genotype–directed therapy. Germline genotype–directed therapy was received by 61% and 18% of metastatic cancer patients with level 1 and level 3B findings, respectively, and by 54% of BRCA1/2, 75% of mismatch repair, 43% of PALB2, 35% of RAD51C/D, 24% of BRIP1, and 19% of ATM carriers. Of BRCA1/2 patients receiving a poly(ADP-ribose) polymerase inhibitor, 45% (84 of 188) had tumors other than breast or ovarian cancer, wherein the drug, at time of delivery, was delivered in an investigational setting. CONCLUSION In a pan-cancer analysis, 8% of patients with advanced cancer harbored a germline variant with therapeutic actionability with 40% of these patients receiving germline genotype–directed treatment. Germline sequence analysis is additive to tumor sequence analysis for therapy selection and should be considered for all patients with advanced cancer.
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Baer, Constance Regina, Niroshan Nadarajah, Claudia Haferlach, Wolfgang Kern, and Torsten Haferlach. "A Study on Paired Tissue Sequencing in Hematologic Diseases to Distinguish Somatic from Germline Sequence Variants in Routine Diagnostics." Blood 128, no. 22 (December 2, 2016): 5511. http://dx.doi.org/10.1182/blood.v128.22.5511.5511.
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Abstract Background: Genetic testing is an integral part of modern diagnostics. Sequencing genomes or exomes in different consortia revealed novel aberrations with importance to hematologic classification, prognosis or therapy. However, a high number of low frequency variants were also found in healthy populations. This challenged the distinction between population variation (polymorphism) and disease associated changes based on early databases with limited extent. For diagnostic purposes, the distinction of somatic (acquired) variants from rare germline variants allows moving towards personalized genetic characterization including molecular markers for individual follow-up. Aim: 1) present an approach to distinguish between somatic or germline variants by comparison with matched tissue (buccal swap, nails), 2) define diagnostically relevant patterns for variant classification or database use. Patients and Methods: Variants were initially classified in a three-tier system: (A) Protein truncating variants (PTV) or changes with strong evidence in literature (e.g. JAK2V617F) were defined as actionable/disease associated. (B) Criteria for polymorphism were met, if population frequencies were available from two sources (1000 Genomes, ExAC). (C) Remaining, critical variants were sequenced in germline DNA (ACMG guidelines, Richards, 2015). We selected 88 patients with critical variants in peripheral blood (PB, n=29) or bone marrow (BM, n=59) and available DNA from buccal swaps (n=40), nails (n=31) or both (n=17). Samples were received for routine diagnostic assessment (suspected diagnosis: myelodysplastic syndrome or chronic myelomonocytic leukemia [n=56], myeloproliferative neoplasm [n=8], acute myeloid leukemia [n=6] or B cell malignancy [n=18]). From PB or BM, 829 analysis by Sanger-, 454- (Roche, Branford, CT) or Illumina sequencing (San Diego, CA) were performed (1-49 [median 6] genes/patient). Results: In 88 patients, we identified 74 actionable/disease associated changes, 67 polymorphisms and one or two variants (n=96) per patient that could not be classified in the previous categories, requiring matched germline DNA sequencing. We found that 35% (34/96) of these variants were also present in germline, although not listed in common polymorphism databases. Consequently, theses variants do not qualify as markers for clonality and follow-up. Of note, 19% of nails and 14% of all buccal swabs received in our laboratory were not analyzable due to low DNA amounts (not included in cohort). Importantly, DNA from both sources can contain low levels of somatic mutations. Next, we compared somatic and germline variants in terms of predicted effects on function, variant burden and population frequency, to identify patterns with relevance to future categorization. Firstly, predicted as damaging by PolyPhen algorithm were significantly more somatic (92%, 49/53) than germline variants (61%, 19/31, p<0.001). Most variants (excluding PTVs) were found in TET2 (n=25). Of 11 confirmed somatic variants, 10 were located in conserved domains, while none of the germline variants was located in these domains. Secondly, germline variants had a median burden of 50% (40-59%) or 98% in one case, which is the expected result for variants derived from either one or both alleles. For somatic variants, burdens were observed between 2% and 100% (median 40%), representing the varying degree of malignant cells in PB or BM. For comparison, disease associated variants showed a similar distribution: 3-90% (median: 40%). Thirdly, we compared variants to ExAC data, the largest available set of exonic variants in healthy individuals (over 60,000). Only 14/34 (41%) germline variants were found in the ExAC data (population frequencies <0.1%). However, 3/62 (5%) of our somatic variants also occurred in the ExAC set. Conclusions: A growing number of sequencing data outdated the traditional distinction between polymorphism and mutation. By comparison with DNA from buccal swabs or nails, we showed that somatic and germline variants have different global patterns (e.g. variant burden, predicted function), but the decision in individual cases based on in silico data can be misleading. Only sequencing germline DNA distinguishes somatic from germline variants on a personalized level and allows strategies to define germline variants potentially contributing to tumorigenesis in future studies. Disclosures Baer: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Other: Part Owner MLL Munich Leukemia Laboratory.
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Lin, Yunting, Yanna Cai, Jianan Xu, Chunhua Zeng, Huiying Sheng, Yang Yu, Xiuzhen Li, and Li Liu. "‘Isolated’ germline mosaicism in the phenotypically normal father of a girl with X-linked hypophosphatemic rickets." European Journal of Endocrinology 182, no. 1 (January 2020): K1—K6. http://dx.doi.org/10.1530/eje-19-0472.
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Objective X-linked hypophosphatemic rickets (XLHR) is the most common form of inherited rickets caused by pathogenic variants of PHEX gene with an X-linked dominant inheritance pattern. Precise molecular diagnosis of pathogenic variant will benefit the genetic counseling and prenatal diagnosis for the family with XLHR. Here, we presented an ‘isolated’ germline mosaicism in the phenotypically normal father of a girl with XLHR. Methods and results For the initial molecular screen of PHEX gene, DNA samples of the proband and her parents were extracted from their peripheral blood samples respectively. Sanger sequencing found a ‘de novo’ novel heterozygous variant, c.1666C>T(p.Q556X), at the PHEX gene in the proband, but not in her phenotypically healthy parents. Due to an occasional abnormality of his serum phosphate previously, further examinations for the father were taken to exclude the possibility of paternal mosaicism. Eight samples from different tissues were analyzed for PHEX gene by Sanger sequencing. Surprisingly, one ‘isolated’ germline mosaicism was detected only in his sperm with an estimated frequency of 26.67%. The mosaic allele was identical to the c.1666C>T(p.Q556X) variant in the proband. Conclusions This is the first case of ‘isolated’ germline mosaicism with pathogenic PHEX variant. Our study provides accurate diagnosis and valuable counseling for this family. This report also alerts clinicians and geneticists to exclude the possibility of the isolated germline mosaicism and prevent intrafamilial recurrences of inherited diseases.