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Mosaic variants are regularly detected on hereditary cancer genetic tests. While some of these variants may be constitutional, the majority are likely limited to blood lineages. In the present study, we correlate clinical histories from individuals with mosaic findings identified on hereditary cancer testing and the outcomes of follow-up fibroblast (FB) testing. We observed 620 mosaic variants, including 339 pathogenic or likely pathogenic variants (PVs) occurring most often in TP53, CHEK2, ATM, and NF1. About half of individuals with NF1 mosaic PVs did not report any clinical features of NF1 and were older at testing (p0.0001) compared to those with an NF1-related phenotype. Among 42 mosaic PVs evaluated by FB testing, 17 (40.5%) were confirmed in FB and were mostly identified in individuals with phenotypes consistent with the gene disease spectrum. Our data show that FB testing is helpful for identifying those with likely constitutional mosaicism benefitting from increased screening and follow-up vs. those with blood-limited variants potentially not requiring intense surveillance but warranting further hematologic work-up.

Purpose:The identification of variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes by hereditary cancer testing poses great challenges for the clinical management of variant carriers. The ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) variant classification framework, which incorporates multiple sources of evidence, has the potential to establish the clinical relevance of many VUS. We sought to classify the clinical relevance of 133 single-nucleotide substitution variants encoding missense variants in the DNA-binding domain (DBD) of BRCA2 by incorporating results from a validated functional assay into an ACMG/AMP-variant classification model from a hereditary cancer–testing laboratory.Experimental Design:The 133 selected VUS were evaluated using a validated homology-directed double-strand DNA break repair (HDR) functional assay. Results were combined with clinical and genetic data from variant carriers in a rules-based variant classification model for BRCA2.Results:Of 133 missense variants, 44 were designated as non-functional and 89 were designated as functional in the HDR assay. When combined with genetic and clinical information from a single diagnostic laboratory in an ACMG/AMP-variant classification framework, 66 variants previously classified by the diagnostic laboratory were correctly classified, and 62 of 67 VUS (92.5%) were reclassified as likely pathogenic (n = 22) or likely benign (n = 40). In total, 44 variants were classified as pathogenic/likely pathogenic, 84 as benign/likely benign, and 5 remained as VUS.Conclusions:Incorporation of HDR functional analysis into an ACMG/AMP framework model substantially improves BRCA2 VUS re-classification and provides an important tool for determining the clinical relevance of individual BRCA2 VUS.

Supplementary Table from Classification of BRCA2 Variants of Uncertain Significance (VUS) Using an ACMG/AMP Model Incorporating a Homology-Directed Repair (HDR) Functional Assay

Purpose: The identification of variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes by hereditary cancer testing poses great challenges for the clinical management of variant carriers. The ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) variant classification framework, which incorporates multiple sources of evidence, has the potential to establish the clinical relevance of many VUS. We sought to classify the clinical relevance of 133 single-nucleotide substitution variants encoding missense variants in the DNA-binding domain (DBD) of BRCA2 by incorporating results from a validated functional assay into an ACMG/AMP-variant classification model from a hereditary cancer–testing laboratory. Experimental Design: The 133 selected VUS were evaluated using a validated homology-directed double-strand DNA break repair (HDR) functional assay. Results were combined with clinical and genetic data from variant carriers in a rules-based variant classification model for BRCA2. Results: Of 133 missense variants, 44 were designated as non-functional and 89 were designated as functional in the HDR assay. When combined with genetic and clinical information from a single diagnostic laboratory in an ACMG/AMP-variant classification framework, 66 variants previously classified by the diagnostic laboratory were correctly classified, and 62 of 67 VUS (92.5%) were reclassified as likely pathogenic (n = 22) or likely benign (n = 40). In total, 44 variants were classified as pathogenic/likely pathogenic, 84 as benign/likely benign, and 5 remained as VUS. Conclusions: Incorporation of HDR functional analysis into an ACMG/AMP framework model substantially improves BRCA2 VUS re-classification and provides an important tool for determining the clinical relevance of individual BRCA2 VUS.

We report the first case of blood chimerism involving a pathogenic RB1 variant in naturally conceived monochorionic-dizygotic twins (MC/DZ) with the twin-twin-transfusion syndrome (TTTS), presumably caused by the exchange of stem-cells. Twin A developed bilateral retinoblastoma at 7 months of age. Initial genetic testing identified a de novo RB1 pathogenic variant, with a 20% allelic ratio in both twins' blood. Subsequent genotyping of blood and skin confirmed dizygosity, with the affected twin harboring the RB1 pathogenic variant in skin and blood, and the unaffected twin carrying the variant only in blood.

We aimed to determine whether monoallelic MUTYH pathogenic and likely pathogenic variants (PVs) are associated with colorectal, breast, and endometrial cancer. Cases were individuals with colorectal, female breast, or endometrial cancer who reported European ancestry alone and underwent a multi-gene hereditary cancer panel at a large reference laboratory. Controls were individuals of European (non-Finnish) descent from GnomAD with cancer cohorts removed. We performed a Fisher's exact test to generate odds ratios (ORs) with 95% confidence intervals (CI). Prevalence of single MUTYH PVs in cancer cohorts versus controls, respectively, was: colorectal cancer, 2.1% vs. 1.8% (OR 1.2, 95% CI 0.99-1.5, p = 0.064); breast cancer 1.9% vs. 1.7% (OR 1.1, 95% CI 0.96-1.3, p = 0.15); and endometrial cancer, 1.7% vs. 1.7% (OR 0.98; 95% CI 0.70-1.3, p = 0.94). Using the largest colorectal and endometrial cancer cohorts and one of the largest breast cancer cohorts from a single case-control study, we did not observe a significant difference in the prevalence of monoallelic MUTYH PVs in these cohorts compared to controls. Additionally, frequencies among cancer cohorts were consistent with the published MUTYH carrier frequency of 1-2%. These findings suggest there is no association between colorectal, endometrial, or breast cancer and MUTYH heterozygosity in individuals of European ancestry.

Background: PARP inhibitors (PARPi) are FDA approved for a subset of metastatic human epidermal growth factor receptor 2-negative (H2N) breast cancer patients who harbor a germline pathogenic or likely pathogenic variant (PV) in BRCA1/2, two of the most well-described breast cancer susceptibility genes in the homologous recombination (HR) pathway. While the NCCN guidelines recommend consideration of BRCA1/2 testing for patients with H2N disease that are eligible for single-agent therapy, there are currently clinical trials available for women with advanced H2N breast cancer who have PVs in HR genes beyond BRCA1/2 to investigate outcomes of receiving PARPi. The yield of germline PVs in other HR genes in the H2N population is not well-described. Methods: Clinical histories and test results were reviewed for women with a diagnosis of H2N breast cancer who underwent multi-gene hereditary cancer panel testing that included a minimum of 10 homologous recombination (HR) genes in addition to BRCA1/2 (ATM, BARD1, BRIP1, CHEK2, FANCC, NBN, PALB2, PTEN, RAD51C, RAD51D). Those with prior BRCA1/2 testing were excluded. We assessed the yield of PVs in non-BRCA1/2 HR genes in the H2N breast cancer population. In addition, we compared the yield of PVs in non-BRCA1/2 HR genes in a “low risk” population (probands with H2N breast cancer with no reported personal history of ovarian or pancreatic cancer and no reported family history of breast, ovarian, pancreatic, or prostate cancer) to a “high risk” population (probands with H2N breast cancer who also have a personal history of ovarian or pancreatic cancer and/or a reported family history of breast, ovarian, pancreatic, or prostate cancer) via a two-tailed Fisher's exact test. Results: A total of 6179 women with H2N breast cancer were identified. Of these, BRCA1/2 PVs were identified in 4.8% (299/6179), while 5.7% (351/6179) carried PVs in HR genes other than BRCA1/2. These included CHEK2 (145), ATM (62), PALB2 (59), BRIP1 (26), FANCC (18), BARD1 (17), RAD51C (12), NBN (11), RAD51D (6), and PTEN (2). No statistically significant difference in the likelihood to harbor a PV in one of the 10 non-BRCA1/2 HR genes was observed between those with a “low risk” presentation (4.8% (53/1096) as compared to those with a “high risk” presentation (5.9% (298/5083)) (p=0.1956). Conclusions: Our findings show that the yield of PVs in HR genes other than BRCA1/2 is appreciable in the H2N breast cancer population. As such, it may be beneficial to include all HR genes when testing H2N breast cancer patients, regardless of other personal or family history, if and/or when a patient develops metastatic disease. Citation Format: Vogel Postula KJ, McGill AK, Sutcliffe E, Murphy PD, Klein RT, Hruska KS. Germline variants in non-BRCA homologous recombination genes detected in HER2-negative breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-09-08.

Purpose Although CHEK2 is a well-established cancer gene, questions remain including whether risks vary substantially between different variants and whether biallelic carriers have higher risks than heterozygotes. We report on a cohort of individuals with CHEK2 pathogenic and likely pathogenic variants (collectively, PV) in order to better characterize this gene. Methods We retrospectively queried samples submitted for multi-gene hereditary cancer testing to identify individuals with CHEK2 PVs and assessed differences in phenotypes among various genotypes. Results CHEK2 PVs were identified in 2,508 individuals, including 32 individuals with biallelic CHEK2 PVs. Breast (female, 59.9% and male, 11.8%), prostate (20.1%), and colorectal (3.5%), were among the most frequently reported cancers. Select missense PVs showed similar cancer prevalence to truncating PVs while some others showed lower prevalence. No significant differences were observed between biallelic carriers and heterozygotes. Conclusions Our data support that some, but not all, CHEK2 missense PVs demonstrate lower cancer prevalence; further studies are needed to continue characterizing possible variant specific risks. In addition, biallelic CHEK2 PVs do not appear to be associated with a more severe phenotype than single CHEK2 PVs. Furthermore, co-occurrences with PVs in other cancer risk genes are common among CHEK2 heterozygotes and often warrant additional management.

The ClinGen PTEN Expert Panel was organized by the ClinGen Hereditary Cancer Clinical Domain Working Group to assemble clinicians, researchers, and molecular diagnosticians with PTEN expertise to develop specifications to the 2015 ACMG/AMP Sequence Variant Interpretation Guidelines for PTEN variant interpretation. We describe finalized PTEN-specific variant classification criteria and outcomes from pilot testing of 42 variants with benign/likely benign (BEN/LBEN), pathogenic/likely pathogenic (PATH/LPATH), uncertain significance (VUS), and conflicting (CONF) ClinVar assertions. Utilizing these rules, classifications concordant with ClinVar assertions were achieved for 14/15 (93.3%) BEN/LBEN and 16/16 (100%) PATH/LPATH ClinVar consensus variants for an overall concordance of 96.8% (30/31). The variant where agreement was not reached was a synonymous variant near a splice donor with non-canonical sequence for which in silico models cannot predict the native site. Applying these rules to six VUS and five CONF variants, adding shared internal laboratory data enabled one VUS to be classified as LBEN and two CONF variants to be as classified as PATH and LPATH. This study highlights the benefit of gene-specific criteria and the value of sharing internal laboratory data for variant interpretation. Our PTEN-specific criteria and expertly reviewed assertions should prove helpful for laboratories and others curating PTEN variants.

Background: Germline TP53 pathogenic variants are indicative of Li-Fraumeni syndrome (LFS), a dominantly inherited hereditary cancer syndrome with high lifetime risks for female breast and other cancers. Genetic testing for LFS may reveal mosaicism, indicating a variant is present in some, but not all, of the cells tested. While these findings may represent a true constitutional event, mosaicism for TP53 variants also has been reported as a somatic event in lymphoblastoid cells from individuals with hematologic malignancy, previous chemotherapy exposure, or due to age-related clonal hematopoietic expansion. Understanding if a mosaic variant is constitutional can influence the patient's management and impact familial risk assessment. We present clinical history and follow-up testing from a series of female breast cancer patients with blood or oral rinse testing revealing a mosaic TP53 pathogenic or likely pathogenic variant (collectively, PV). Methods: We retrospectively reviewed clinical history and genetic testing results to identify women with a personal history of breast cancer and mosaicism for one or more TP53 PV identified on multi-gene hereditary cancer testing at our clinical diagnostic laboratory. Descriptive statistics were employed. Results: Forty-eight women with breast cancer were identified as having a mosaic TP53 PV, defined as an allelic fraction of 40 years of age and three had other primary cancer diagnoses, including one with sarcoma. In one patient with early-onset and HER2-positive breast cancer, testing of fibroblasts also identified mosaicism for the TP53 PV, indicating constitutional mosaicism. Conclusions: For individuals with a mosaic TP53 PV, identification of the variant in a second tissue is necessary to confirm constitutional mosaicism and heightened risk for other LFS-associated cancers. In this series, additional testing confirmed one of six patients with mosaic TP53 PV pursuing fibroblast testing to have constitutional mosaicism. This individual's breast cancer was HER2-positive and her age at diagnosis was younger than those whose mosaic PV was not identified in fibroblasts. Additional follow-up testing data are needed to understand whether confirmatory testing in a second tissue is indicated for any breast cancer patient with a mosaic TP53 PV, or would be most likely to reveal constitutional mosaicism in individuals whose breast cancers are HER2-positive or early-onset. Citation Format: Mester JL, Postula K, Bissonnette J, Klein RT, Hruska K. Mosaic TP53 variants in women with breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-06.

Background/Statement of Purpose: The role of cancer susceptibility genes in the male breast cancer population beyond BRCA1 and BRCA2 (BRCA) is not well defined. While breast cancer has been documented in men with pathogenic variants in a number of other breast cancer susceptibility genes (e.g. CHEK2, PALB2, PTEN), the yield of testing is not well documented nor are predictive clinical features of those likely to harbor causative variants. This study assesses the yield of pathogenic/likely pathogenic variants (collectively, PV) in male breast cancer patients who underwent multi-gene hereditary cancer panel testing. In addition, we aim to examine predictors of identifying a PV in this population. Methods: Clinical histories and test results were reviewed for men with a diagnosis of breast cancer who underwent panel testing that included a minimum of eight well-described breast cancer susceptibility genes (ATM, BRCA1, BRCA2, CDH1, CHEK2, PALB2, PTEN, TP53) and up to 24 additional genes. Using t-test and two-tailed Fisher's exact test, we assessed whether age at diagnosis, family history of breast cancer, or the presence of selected second primary cancers (second breast, prostate, pancreatic, colon, or melanoma cancers) were associated with a greater likelihood of identifying a PV. Results: The clinical histories and test results of 381 men with breast cancer were reviewed, of whom 12.1% had at least one PV (46/381). When we limited our assessment to men who had not had prior negative BRCA testing, 13.3% had at least one PV (42/315). Variants were most commonly detected in BRCA2 (21) and CHEK2 (17), followed by PALB2 (4), BRCA1 (4), and ATM (2). A two sample t-test showed no significant difference (p=0.39) in the average age of diagnosis for those with a PV (62.5y, n=47) compared to those without a PV (60.9y, n=334). Two-tailed Fisher's exact test showed no association between having a PV and a history of a selected second primary (SP) cancer [10.8% (7/65) w/SP vs 12.3% (39/316) w/out SP; p=0.84]. Lastly, two-tailed Fisher's exact test showed those with a family history of breast cancer (fhx br) were more likely to have a PV [15.1% (32/212) fhx br vs. 8.3% (14/169) no fhx br], although this did not reach statistical significance (p=0.06). Conclusions: While BRCA2 remains the most common gene in which PVs are identified in men with breast cancer, a significant proportion of patients will have a PV in another well-described breast cancer susceptibility gene, particularly CHEK2, PALB2, and ATM. Therefore, it is reasonable to utilize a panel that is inclusive of these genes when testing male breast cancer patients. As the likelihood to harbor a PV was not significantly associated with age of onset, family history of breast cancer, or presence of a second primary, all men with breast cancer could consider genetic testing. Further study is warranted as the current sample size may limit the power to detect associations. Citation Format: Vogel Postula KJ, Andolina LM, Theobald K, McGill AK, Sutcliffe E, Arvai KJ, Murphy PD, Klein RT, Hruska KS. The role of multi-gene hereditary cancer panels in male patients with breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD7-11.

Purpose An association of Lynch syndrome (LS) with breast cancer has been long suspected; however, there have been insufficient data to address this question for each of the LS genes individually. Methods We conducted a retrospective review of personal and family history in 423 women with pathogenic or likely pathogenic germ-line variants in MLH1 (N = 65), MSH2 (N = 94), MSH6 (N = 140), or PMS2 (N = 124) identified via clinical multigene hereditary cancer testing. Standard incidence ratios (SIRs) of breast cancer were calculated by comparing breast cancer frequencies in our study population with those in the general population (Surveillance, Epidemiology, and End Results 18 data). Results When evaluating by gene, the age-standardized breast cancer risks for MSH6 (SIR = 2.11; 95% confidence interval (CI), 1.56–2.86) and PMS2 (SIR = 2.92; 95% CI, 2.17–3.92) were associated with a statistically significant risk for breast cancer whereas no association was observed for MLH1 (SIR = 0.87; 95% CI, 0.42–1.83) or MSH2 (SIR = 1.22; 95% CI, 0.72–2.06). Conclusion Our data demonstrate that two LS genes, MSH6 and PMS2, are associated with an increased risk for breast cancer and should be considered when ordering genetic testing for individuals who have a personal and/or family history of breast cancer.

Birt-Hogg-Dube (BHD) disease is an autosomal dominant cancer syndrome characterized by benign skin tumors, renal cancer and spontaneous pneumothorax and is caused by mutations in the Folliculin (FLCN) gene. Benign skin tumors and pneumothorax occur in the majority of patients affected by BHD syndrome, but only 30-45% of them develop renal cell carcinoma (RCC) with a median age of diagnosis at 48. The earliest onset of RCC in a BHD patient has been reported at age 20. Here we report a case of a 14 year-old patient with germline FLCN mutation leading to an early-onset bulky RCC that could not be classified strictly according to existing histological types. Germline genetic testing revealed a deletion at FLCN exon 5. The father of the patient was identified as the asymptomatic carrier. We report the youngest patient with BHD-related RCC. This early onset presentation supports genetic testing of at-risk patients and initiation of imaging surveillance for RCC in early adolescence. In addition, future studies are necessary to understand the determinants of reduced penetrance in BHD disease.

Background Genes in the homologous recombination pathway have shown varying results in the literature regarding ovarian cancer (OC) association. Recent case-control studies have used allele counts alone to quantify genetic associations with cancer. Methods A retrospective case-control study was performed on 6,182 women with OC referred for hereditary cancer multi-gene panel testing (cases) and 4,690 mothers from trios who were referred for whole-exome sequencing (controls). We present age-adjusted odds ratios (ORAdj) to determine association of OC with pathogenic variants (PVs) in homologous recombination genes. Results Significant associations with OC were observed in BRCA1, BRCA2, RAD51C and RAD51D. Other homologous recombination genes, BARD1, NBN, and PALB2, were not significantly associated with OC. ATM and CHEK2 were only significantly associated with OC by crude odds ratio (ORCrude) or by ORAdj, respectively. However, there was no significant difference between ORCrude and ORAdj for these two genes. The significant association of PVs in BRIP1 by ORCrude (2.05, CI = 1.11 to 3.94, P = 0.03) was not observed by ORAdj (0.87, CI = 0.41 to 1.93, P = 0.73). Interestingly, the confidence intervals of the two effect sizes were significantly different (P = 0.04). Conclusion The lack of association of PVs in BRIP1 with OC by ORAdj is inconsistent with some previous literature and current management recommendations, highlighted by the significantly older age of OC onset for BRIP1 PV carriers compared to non-carriers. By reporting ORAdj, this study presents associations that reflect more informed genetic contributions to OC when compared to traditional count-based methods. Electronic supplementary material The online version of this article (10.1186/s13053-019-0119-3) contains supplementary material, which is available to authorized users.

Cancer genetics: Original article., [Background] PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2—without incurring overprediction—is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines., [Methods] Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2 c.212–1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant., [Results] We identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies., [Conclusions] PVS1 is not necessarily warranted for splice site variants targeting four PALB2 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenic without further evidences. Our study puts a warning in up to five PALB2 genetic variants that are currently reported as pathogenic/likely pathogenic in ClinVar., Received funding from the NHMRC, the National Breast Cancer Foundation, Cancer Australia and the National Institute of Health [USA].

IMPORTANCE: CDH1 pathogenic variants have been estimated to confer a 40% to 70% and 56% to 83% lifetime risk for gastric cancer in men and women, respectively. These are likely to be overestimates owing to ascertainment of families with multiple cases of gastric cancer. To our knowledge, there are no penetrance estimates for CDH1 without this ascertainment bias. OBJECTIVE: To estimate CDH1 penetrance in a patient cohort not exclusively ascertained based on strict hereditary diffuse gastric cancer (HDGC) criteria. DESIGN, SETTING, AND PARTICIPANTS: Retrospective review of 75 families found to have pathogenic variants in CDH1 through clinical ascertainment and multigene panel testing at a large commercial diagnostic laboratory from August 5, 2013, to June 30, 2018. CDH1 pathogenic variants were identified in 238 individuals from 75 families. Pedigrees from those families included cancer status for 1679 relatives. Penetrance estimates are based on 41 families for which completed pedigrees were available. MAIN OUTCOMES AND MEASURES: Gastric cancer standardized incidence ratio estimates relative to Surveillance, Epidemiology, and End Results (SEER) Program incidence for pathogenic CDH1 variants from families ascertained without regard to HDGC criteria. RESULTS: Among the 238 individuals with a CDH1 pathogenic variant, mean (SD) age was 49.3 (18.1) years and 63.4% were female. Ethnicity was reported for 67 of 75 (89%) families; of these 67 families, 51 (76%) reported European ancestry, whereas Asian, African, Latino, and 2 or more ancestries were reported for 4 families (6%) each. Standardized incidence ratios for gastric and breast cancer were significantly elevated above SEER incidence. Extrapolated cumulative incidence of gastric cancer at age 80 years was 42% (95% CI, 30%-56%) for men and 33% (95% CI, 21%-43%) for women with pathogenic variants in CDH1, whereas cumulative incidence of female breast cancer was estimated at 55% (95% CI, 39%-68%). International Gastric Cancer Linkage Consortium criteria were met in 25 of the 75 (33%) families; however, dispensing with the requirement of confirmation of HDGC histologic subtype, 43 (57%) would meet criteria. CONCLUSIONS AND RELEVANCE: The cumulative incidence of gastric cancer for individuals with pathogenic variants in CDH1 is significantly lower than previously described. Because prophylactic gastrectomy can have bearing upon both physical and psychological health, further discussion is warranted to assess whether this surgical recommendation is appropriate for all individuals with pathogenic variants in CDH1.

The recognition of genes implicated in ovarian cancer risk beyond BRCA1, BRCA2, and the Lynch syndrome genes has increased the variety of testing options available to providers and patients. We report the frequency of pathogenic variants identified among individuals with ovarian cancer undergoing clinical genetic testing via a multi-gene hereditary cancer panel.Genetic testing of up to 32 genes using a hereditary cancer panel was performed on 4439 ovarian cancer cases, and results were analyzed for frequency of pathogenic variants. Statistical comparisons were made using t-tests and Fisher's exact tests.The positive yield was 13.2%. While BRCA1/2 pathogenic variants were most frequent, one third (33.7%) of positive findings were in other homologous recombination genes, and accounted for over 40.0% of findings in endometrioid and clear cell cases. Women with a personal history of breast cancer (22.1%), who reported a family history of ovarian cancer (17.7%), and/or serous histology (14.7%) were most likely to harbor a pathogenic variant. Those with very early onset (30 years) and late onset (≥70 years) ovarian cancer had low positive yields.Our study highlights the genetic heterogeneity of ovarian cancer, showing that a large proportion of cases are not due to BRCA1/2 and the Lynch syndrome genes, but still have an identifiable hereditary basis. These findings substantiate the utility of multi-gene panel testing in ovarian cancer care regardless of age at diagnosis, family history, or histologic subtype, providing evidence for testing beyond BRCA1/2 and the Lynch syndrome genes.

Objectives Large deletions and duplications are recognized as a cause of many inherited cancer syndromes; however, few studies have examined the frequency. In one study, large rearrangements accounted for 11-14% of pathogenic variants in BRCA1 and 1-3% of pathogenic variants in BRCA2 (Judkins 2012). Additionally, in a study of invasive ovarian cancer patients, large deletions and duplications accounted for 6% of BRCA1 and 3% of BRCA2 pathogenic variants (Zhang 2011). The frequency of large deletions and duplications in moderate penetrance genes, such as ATM, CHEK2, and PALB2, has not been carefully studied. We therefore sought to determine the frequency of pathogenic and likely pathogenic (P/LP) large deletions and duplications in BRCA1, BRCA2, ATM, CHEK2, and PALB2. Methods We analyzed a consecutive case series of patients who had BRCA1/2 and cancer panel testing. The data were analyzed to determine the percentage of deletions or duplications of greater than 250 bp identified on array comparative genome hybridization as a percentage of all pathogenic variants (PVs)/likely pathogenic variants (LPVs) in each of BRCA1, BRCA2, ATM, CHEK2, and PALB2. Results Large deletions/duplications accounted for 10.1% (55/545) of cases with a BRCA1 PV/LPV and 1.8% (10/556) of cases with a BRCA2 PV/LPV. By ethnicity, BRCA1/2 large deletions/duplications accounted for a higher fraction of PV/LPVs in Asians (11.3%) and Hispanics (8.1%). In Hispanic individuals, all of the large deletions/duplications were identified in BRCA1, accounting for 14.6% of all BRCA1 PV/LPV in this group. In the moderate penetrance genes, large deletions/duplications accounted for 4.6% (9/195), 5.1% (22/436), and 9.9% (13/132) of cases with a PV/LPV in ATM, CHEK2, and PALB2, respectively. There were two recurrent deletions in our cohort: the CHEK2 founder Slavic deletion of exons 9-10 observed in 16 of our cases, and a deletion of exon 11 in PALB2 observed in 6 cases. Conclusions Large deletions/duplications account for a substantial proportion of clinically significant variants in hereditary cancer genes. The proportion of large deletions/duplications in BRCA1 and BRCA2 reported here is similar to previous data. However, our cohort suggests that Asian individuals may have a higher proportion of mutations due to large deletions/duplications than previously reported. Among moderate risk cancer genes, PALB2 has the highest proportion of large deletions/duplications. Citation Format: Bissonnette J, Doonanco K, Xu Z, Klein RT, Hruska KS. Frequency of large rearrangements in BRCA1, BRCA2, ATM, CHEK2, and PALB2 in hereditary cancer testing. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-09-17.

Purpose: Germ-line testing for panels of cancer genes using next-generation sequencing is becoming more common in clinical care. We report our experience as a clinical laboratory testing both well-established, high-risk cancer genes (e.g., BRCA1/2, MLH1, MSH2) as well as more recently identified cancer genes (e.g., PALB2, BRIP1), many of which have increased but less well-defined penetrance. Genet Med 18 8, 823–832. Methods: Clinical genetic testing was performed on over 10,000 consecutive cases referred for evaluation of germ-line cancer genes, and results were analyzed for frequency of pathogenic or likely pathogenic variants, and were stratified by testing panel, gene, and clinical history. Genet Med 18 8, 823–832. Results: Overall, a molecular diagnosis was made in 9.0% of patients tested, with the highest yield in the Lynch syndrome/colorectal cancer panel. In patients with breast, ovarian, or colon/stomach cancer, positive yields were 9.7, 13.4, and 14.8%, respectively. Approximately half of the pathogenic variants identified in patients with breast or ovarian cancer were in genes other than BRCA1/2. Genet Med 18 8, 823–832. Conclusion: The high frequency of positive results in a wide range of cancer genes, including those of high penetrance and with clinical care guidelines, underscores both the genetic heterogeneity of hereditary cancer and the usefulness of multigene panels over genetic tests of one or two genes. Genet Med 18 8, 823–832.

Background: Hereditary breast and ovarian cancer (HBOC) is a common indication for referral to cancer genetic counselors. Next generation sequencing panels allow for the efficient evaluation of many genes associated with increased risk of these cancers. The purpose of this study is to compare clinical histories of those with pathogenic variants in high risk versus low/moderate risk genes in order to determine which patients might benefit from more extensive testing by a panel approach. Methods: We queried the results of patients tested at GeneDx for a panel of 21 genes causing increased breast and/or ovarian cancer risk. Data regarding personal and family history of cancer provided on the test requisition forms were analyzed and classified according NCCN guidelines for testing criteria for HBOC syndrome. Results: Of 1709 individuals referred for testing, 146 (8.5%) tested positive for a pathogenic variant. Of these, 33% percent were found to carry a pathogenic BRCA1/2 variant while 67% tested positive for a pathogenic variant in a gene other than BRCA1/2 (CHEK2: 17%; ATM: 12%; PALB2: 10%; BRIP1: 7%; BARD1: 3%; PTEN: 3%; each of FANCC, MSH2, MSH6, NBN, PMS2, RAD51C, RAD51D: 2%; MLH1: 1%). Eighty-six percent of these individuals were affected with cancer. In the probands with a pathogenic BRCA1/2 variant, 66% were diagnosed with breast cancer and 22% with ovarian cancer compared to the probands with a pathogenic variant in a gene other than BRCA1/2 of whom 84% had a history of breast cancer and 17% had ovarian cancer. The highest number of breast cancer diagnoses were found, in decreasing order, in association with pathogenic variants in BRCA1 (20), CHEK2 (20), ATM (17), PALB2 (14), BRCA2 (13), and BRIP1 (12). The greatest number of ovarian cancers were identified, in decreasing order, in association with pathogenic variants in BRCA2 (8), BRCA1 (3), CHEK2 (3), and ATM (3). Furthermore, all of the individuals with pathogenic variants met NCCN guidelines for HBOC. Conclusion: A high yield of pathogenic variants were found in genes other than BRCA1/2. Data analysis also shows that individuals with a pathogenic variant in genes other than BRCA1/2 did not have a notably less severe clinical history than those pathogenic variants in BRCA1/2. As all individuals tested meet NCCN guidelines for HBOC testing, panel testing should be considered in this population. Citation Format: Erica Vaccari, Lauren Yackowski, Melanie Hussong, Patricia Murphy, Maria L Cremona, Jessica Booker, Kathleen Hruska. Characterizing the clinical presentation of individuals with pathogenic variants in a breast/ovarian cancer gene panel [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-01.

Introduction: Expansion of genetic testing technologies has brought multi-gene panels for cancer susceptibility into the clinic; however, the clinical utility of these next-generation sequencing (NGS) panels is largely unknown. Hypothesis: We hypothesized that the use of multi-gene panels would yield a significant number of cases with more than one pathogenic variant. Methods: We retrospectively queried oncology tests reported at GeneDx from August 2013 to April 2014 for individuals with more than one pathogenic variant. Next, we calculated the proportion of individuals with more than one pathogenic variant among all positive reports, excluding familial and stand-alone BRCA1/2 tests. We then extracted personal and family histories, including available segregation data, to categorize the phenotypes. Results: Of 406 unique, unrelated individuals with pathogenic or likely pathogenic findings, 11 (2.7%) had more than one pathogenic variant. This total includes nine individuals with a mutation in more than one gene, as well as two individuals with two mutations in trans in the same gene. Ten of these 11 individuals were identified by multi-gene panel tests, one individual by step-wise (tiered) testing. Seven of 11 individuals were positive for a pathogenic variant in a traditional, highly-penetrant cancer susceptibility gene and another pathogenic variant in a gene with moderate cancer susceptibility, such as CHEK2 and ATM. Three of 11 probands had more than one primary tumor. Several of the families were significant for bilineal cancer risk. Conclusions: Our data suggest that a traditional single gene approach to cancer testing may fail to identify all pathogenic variants related to the clinical presentation. The identification of a second risk factor for inherited susceptibility to cancer allows for appropriate testing and management considerations for family members. In conclusion, we provide early evidence for the consideration of multi-gene panel testing in a clinical oncology setting. Citation Format: Rachel Nusbaum, Lisa Susswein, Kathleen Hruska, Melanie Hussong, Windy Berkofsky-Fessler, Mingjuan Liao, Erica Rinella, Nina Sanapareddy, Joaquin Villar, Haiyan Wan, Zhixiong Xu, Rebecca Y Bassett, Elisabeth McKeen, Constance Murphy, Deborah Pencarinha, Jessica Booker, Maria L Cremona, Patricia Murphy, Rachel T Klein. Individuals with more than one pathogenic variant: Rationale for considering multi-gene panel testing for cancer susceptibility [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-05.

Mendelian disorders are most commonly caused by mutations identifiable by DNA sequencing. Exonic deletions and duplications can go undetected by sequencing, and their frequency in most Mendelian disorders is unknown.We designed an array comparative genomic hybridization (CGH) test with probes in exonic regions of 589 genes. Targeted testing was performed for 219 genes in 3,018 patients. We demonstrate for the first time the utility of exon-level array CGH in a large clinical cohort by testing for 136 autosomal dominant, 53 autosomal recessive, and 30 X-linked disorders.Overall, 98 deletions and two duplications were identified in 53 genes, corresponding to a detection rate of 3.3%. Approximately 40% of positive findings were deletions of only one or two exons. A high frequency of deletions was observed for several autosomal dominant disorders, with a detection rate of 2.9%. For autosomal recessive disorders, array CGH was usually performed after a single mutation was identified by sequencing. Among 138 individuals tested for recessive disorders, 10.1% had intragenic deletions. For X-linked disorders, 3.5% of 313 patients carried a deletion or duplication.Our results demonstrate that exon-level array CGH provides a robust option for intragenic copy number analysis and should routinely supplement sequence analysis for Mendelian disorders.

1580Background: Li-Fraumeni Syndrome (LFS) is an autosomal dominant cancer susceptibility syndrome due to germline pathogenic variants in TP53 and is associated with a significant risk for sarcomas...

Gaucher disease (GD) is an autosomal recessive disorder caused by the deficiency of glucocerebrosidase, a lysosomal enzyme that catalyses the hydrolysis of the glycolipid glucocerebroside to ceramide and glucose. Lysosomal storage of the substrate in cells of the reticuloendothelial system leads to multisystemic manifestations, including involvement of the liver, spleen, bone marrow, lungs, and nervous system. Patients with GD have highly variable presentations and symptoms that, in many cases, do not correlate well with specific genotypes. Almost 300 unique mutations have been reported in the glucocerebrosidase gene (GBA), with a distribution that spans the gene. These include 203 missense mutations, 18 nonsense mutations, 36 small insertions or deletions that lead to either frameshifts or in-frame alterations, 14 splice junction mutations, and 13 complex alleles carrying two or more mutations in cis. Recombination events with a highly homologous pseudogene downstream of the GBA locus also have been identified, resulting from gene conversion, fusion, or duplication. In this review we discuss the spectrum of GBA mutations and their distribution in the patient population, evolutionary conservation, clinical presentations, and how they may affect the structure and function of glucocerebrosidase.

Methoxychlor (MXC) is an organochlorine pesticide that increases the rate of ovarian atresia. To date, little is known about the mechanism by which MXC induces atresia. Because Bcl-2 (an antiapoptotic factor), Bax (a proapoptotic factor), gonadotropins, and estradiol are important regulators of atresia in the ovary, the purpose of this study was first to examine whether MXC-induced atresia occurred through alterations in Bcl-2 or Bax, and second, to examine the effect of MXC on gonadotropins, estradiol, and their receptors. CD-1 mice were dosed with 8–64 mg kg−1 day−1 MXC or vehicle (sesame oil). Ovaries were subjected to analysis of antral follicle numbers, Bcl-2, Bax, estrogen receptor, and follicle-stimulating hormone receptor levels. Blood was used to measure gonadotropins and estradiol. In some experiments, mice that overexpressed Bcl-2 or mice that were deficient in Bax were dosed with MXC or vehicle and their ovaries were analyzed for atresia. MXC caused a dose-dependent increase in the pe...

As an initial step towards enhancing mastitis resistance in dairy animals, we generated BLG-Lys transgenic mice that secrete lysostaphin, a potent antistaphylococcal protein, in their milk. In the current study, we continue our assessment of lysostaphin as a suitable antimicrobial protein for mastitis resistance and have investigated mammary gland development and function in three lines of transgenic mice. As the lines were propagated, there was a tendency for fewer BLG-Lys litters to survive to weaning (51% as compared to 90% for nontransgenic lines, p = 0.080). Nontransgenic pups fostered on dams from these three lines exhibited diminished growth rates during the first week of lactation. Rates of gain became comparable to pups on nontransgenic dams at later time points. Initial slow growth also resulted in decreased weaning weights for pups nursed by transgenic dams (15.35±0.27 g) when compared to pups delivered and nursed by nontransgenic dams (18.61 ± 0.61 g; p < 0.001), but the effect was temporary, as similar weights were attained by adulthood. Milk yield at peak lactation was not different between BLG-Lys (0.79 ± 0.33 g) and nontransgenic (0.91 ± 0.38 g; p = 0.166) dams. Histological examination of the transgenic mammary glands during gestation revealed no differences when compared to control glands; however, at early lactational stages, the BLG-Lys glands exhibited less alveolar area than control glands and a delay in lobulo-alveolar maturation. The results clearly demonstrate reduced growth of neonates on BLG-Lys dams; whether the poor pup performance can be attributed to delayed mammary development or the gland development merely reflects reduced suckling stimuli from the pups remains to be determined.

668 Background: Approximately 25% of pheochromocytomas (PCC) have a hereditary basis, and germline variants in the SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, VHL, FH, RET, MEN1, and NF1 genes have been associated with a predisposition to PCC and paraganglioma (PGL). Multi-gene hereditary cancer panel testing for PCC has become increasingly more common than single-gene testing algorithms. Identification of a pathogenic or likely pathogenic variant (PV/LPV) in one of these genes has important implications for surveillance in patients and their family members. Here we describe the spectrum of PV/LPV variants identified in individuals with PCC. Methods: We performed a retrospective review of clinical and molecular data for all individuals diagnosed with PCC who underwent panel testing through BioReference Laboratories that included at least SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, VHL, FH, RET, MEN1, and NF1 between January 2016 and February 2017. Results: Seventy-nine individuals underwent testing due to a personal (n = 76) or family (n = 3) history of PCC. The positive yield was 14% (11/79). The majority of PV/LPV were in SDHB (n = 4; 36%), followed by RET (n = 2, 18%), with the remaining variants being identified in SDHA (1), SDHC (1), VHL (1), TMEM127 (1), and MAX (1). Approximately half (6/11) of those with a PV/LPV had a non-syndromic presentation of a unilateral PCC with no reported family history of PCC or PGL. The average age at tumor diagnosis was lower for probands testing positive than those without PV/LPV (34y±14 vs 44y±16). Conclusions: Our data support previous recommendations that patients with apparently sporadic, non-syndromic PCC be considered for genetic testing. Panel testing is a useful tool for identifying individuals with hereditary PCC.

596 Background: Lynch syndrome (LS), also known as hereditary non-polyposis colorectal cancer (HNPCC), is a hereditary colorectal cancer syndrome thought to present with few or no polyps. It has been suggested that the majority of LS patients with colon polyps will develop 1-9 cumulative adenomatous polyps in their lifetime, while approximately 10% will develop ≥10. Our aim was to describe polyp burden in a cohort of LS patients who were ascertained via multi-gene cancer panel testing. Methods: We retrospectively reviewed the personal history for all individuals with a pathogenic or likely pathogenic variant (collectively, PV) in MLH1, MSH2 (including 3’ EPCAM deletions), MSH6, or PMS2. Individuals with more than one PV in a LS-associated or other gene were excluded. All polyp data was obtained from provided test requisition forms and/or pathology reports. Results: A total of 131 individuals reported to have colon polyps were molecularly confirmed as having LS. Of these individuals, 55% (72/131) reported a history of colorectal cancer. Overall, polyp burden was reported as follows: 83% (109/131) with 1-10 polyps, 13% (17/131) with 10-19 polyps, and 4% (5/131) with 20-50 polyps. Of the 5 individuals reporting 20-50 polyps, the average was 31.2 polyps (range 21-50). Adenomatous pathology was reported for one or more polyps in 69% (91/131) of individuals. When limited to those with adenomatous polyps, 85% (77/91) reported 1-10 polyps, 11% (10/91) reported 10-19 polyps, and 4% (4/91) reported 20-50 polyps. When breaking down by presence and absence of colorectal cancer, 18% (13/72) and 15% (9/59), respectively, reported a polyp burden ≥10. Overall, 17% (22/131) of all LS patients reported ≥10 polyps. Conclusions: Similar to previously published data, the majority of LS patients with colon polyps report a polyp burden of < 10. However, our data suggest that the proportion of LS patients with a polyp burden of ≥10 may be higher than previously reported (17% vs. 10%) stressing the importance of including the LS-associated genes for these patients. While LS has historically been referred to as a “non-polyposis” syndrome, a subset of patients actually present with an attenuated polyposis-like phenotype.

576 Background: Young age at onset is a hallmark feature of an inherited predisposition to cancer. Recent evidence suggests that the incidence of pathogenic/likely pathogenic variants (PV) in cancer predisposition genes among individuals diagnosed with colorectal cancer (CRC) ≤ age 35 is high, but few studies have examined the frequency identified by multi-gene hereditary cancer panel testing. We report on PV yield and clinical presentation of individuals diagnosed with CRC ≤ 35 years (y). Methods: We retrospectively reviewed test requisition forms and provided pathology reports for 4,727 individuals with CRC who underwent panel testing of, at a minimum, MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH. Two-tailed Fisher’s exact tests were used to determine statistically significant differences between groups. Results: Of the 691 individuals diagnosed with CRC ≤35y, 137 PV were identified in 126 individuals (126/691, 18.2%), including 72 with Lynch syndrome, 16 with familial adenomatous polyposis (FAP), five with MUTYH-associated polyposis, four with constitutional mismatch repair deficiency, one with juvenile polyposis syndrome, and one with Peutz-Jeghers syndrome. Thirty-eight additional PVs were identified in other non-CRC genes. Microsatellite instability (MSI) and immunohistochemistry (IHC) results were reported for 277 individuals and were reportedly abnormal in 63. The yield of PV in individuals with abnormal tumor studies was 52.4% (33/63); PV in genes other than the Lynch syndrome-associated genes were identified, commonly in the MSI-H group. While statistical significance was not observed, the positive rate was higher for individuals with non-rectal cancers (colon cancer: 104/534, 19.5%; rectal cancer: 22/157, 14.0%, p = 0.13). Conclusions: In our cohort, 18.2% of individuals diagnosed with CRC ≤35y were found to harbor a PV. Yield increased in those with abnormal MSI/IHC as well as non-rectal tumor location. Hereditary predispositions in patients diagnosed with very early-onset CRC are not limited to Lynch syndrome and FAP. Therefore, individuals with very early-onset CRC may benefit from multi-gene panel testing rather than syndromic-based testing.

The effect of pregnancy on postweaning mammary gland involution was investigated in mice. On the third day after forced weaning at Lactation Day 10, the apoptotic index was 56% lower in mammary tissue of mice that were pregnant at the time of weaning than in nonpregnant mice. Conversely, the bromodeoxyuridine-labeling index was increased sevenfold in pregnant mice compared to nonpregnant controls (3.5% vs. 0.5%, respectively). Structure of mammary alveoli was largely maintained in postweaning pregnant mice. The effect of pregnancy on three specific mammary epithelial cell survival pathways was also examined. First, pregnancy blocked the loss of Stat5a phosphorylation during involution. Significantly, loss of Stat5a phosphorylation during involution was not correlated with loss of Stat5a nuclear localization. Second, pregnancy maintained nuclear-localized progesterone receptor during lactation. Third, pregnancy was associated with increased expression of bfl-1 during involution but had little effect on the expression of other bcl-2 family members. The data indicate that pregnancy inhibits mammary cell apoptosis after weaning while permitting proliferation of the mammary epithelium, and they support the hypothesis that Stat5a and progesterone-signaling pathways act in concert to mediate this effect.

Attempts to delineate the mechanisms of estrogen action have promoted the creation of several estrogen receptor alpha (ERα) mouse models in the past decade. These traditional models are limited by the fact that the receptors are either absent or present throughout all stages of development. The purpose of this work was to develop a conditional transgenic model that would provide an in vivo method of controlling the spatial and temporal regulation of ERα expression. The tetracycline responsive system was utilized. Three lines of transgenic mice carrying a transgene composed of the coding sequence for murine ERα placed under the regulatory control of a tet operator promoter (tet-op) were generated. These three lines of tet-op-mERα mice were each mated to an established line of transgenic mice expressing a tetracycline-dependent transactivator protein (tTA) from the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Double transgenic MMTV-tTA/tet-op-mERα mice were produced. All three lines demonstrated dominant gain of ERα shown by RT-PCR, immunoprecipitation, and immunohistochemistry. Transgene-specific ERα was expressed in numerous tissues including the mammary gland, salivary gland, testis, seminal vesicle, and epididymis. Expression was silenced by administration of doxycycline in the drinking water. This model can be utilized to evaluate the consequences of ERα dominant gain in targeted tissues at specific times during development. In this study dominant gain of ERα was associated with a reduction in epididymal/vas deferens and seminal vesicle weights consistent with the proposed action of ERα on fluid transport in the male reproductive tract. Combining this model with other dominant gain and gene knockout mouse models will be useful for testing effects of ERα action in combination with specific gene products and to evaluate if developmental and stage-specific expression of ERα can rescue identified phenotypes in gene knockout mice.

1518 Background: In multi-gene hereditary cancer testing, multiple syndromes may be interrogated simultaneously, increasing the likelihood of detecting an underlying cancer predisposition. We characterized the yield and distribution of pathogenic or likely pathogenic variants (PV/LPV) in patients with a personal history of sarcoma undergoing panel testing through our clinical diagnostic laboratory. Methods: We retrospectively reviewed panel test results, demographic data, and personal/family history information from patients reporting a personal history of sarcoma. Patients included in this study underwent panel testing (NGS and del/dup) of up to 61 genes at this laboratory and concurrent or prior TP53 analysis at this or an outside laboratory. Chi-square and Fisher’s exact tests were used to compare groups. Results: Among 374 sarcoma patients, 53 (14.2%) harbored one or more PV/LPV in 14 genes: TP53, BRCA2, CHEK2, BRCA1, ATM, MSH6, MLH1, NBN, BAP1, BRIP1, FLCN, MSH2, PTEN, and RB1. Thirty-nine (10.4%) individuals reported both a personal and family history of sarcoma; however, PV/LPV were not more likely to be detected among this group than those with personal history alone (p = 1.0). PV/LPV were most often detected in BRCA1/2 (15/374, 4.0%) or TP53 (12/374, 3.2%). Notably, four probands with BRCA1/2 PV/LPV reported a family history of sarcoma, including one kindred in which the variant was present in two brothers, both affected with sarcoma. Additionally, seven patients were found to have PV/LPV (1.9%) in genes causative for Lynch syndrome. Among patients with PV/LPV in genes other than TP53, nearly half (17/41, 41.5%) met National Comprehensive Cancer Network TP53 testing criteria. Conclusions: The majority of PV/LPV were identified in genes for which association with sarcoma risk is not well-established. While several of these genes have been implicated in somatic pathways related to sarcoma development, it is unclear whether these germline findings are causative, play no role, or modify sarcoma risk. Although these data do not inherently associate non- TP53 genes with sarcoma risk, they suggest a potential clinical benefit can be gained from performing hereditary cancer risk assessment and multi-gene panel testing in sarcoma patients.

Uniparental disomy (UPD) can be the cause of atypical disease manifestations in patients with Mendelian disorders. Consideration of this disease mechanism proved essential in our evaluation of a 3-year-old boy, referred to the NIH Clinical Center for further evaluation of Gaucher disease (GD). ### Case report. The patient was born at term to non-consanguineous parents with a paternal family history of autosomal dominant Charcot-Marie-Tooth disease type I (CMT1). His medical history included moderate sensorineural hearing loss, Wolff-Parkinson-White arrhythmia, bicuspid aortic valve, and motor delay, ambulating at 26 months. At 17 months, hepatosplenomegaly was noted and a bone marrow biopsy led to the diagnosis of type 3 GD. Molecular analysis revealed homozygosity for the NM_000157.2:c.1448T>C (L444P) mutation in the glucocerebrosidase gene ( GBA ). He began enzyme replacement therapy (Imiglucerase, 120 U/kg biweekly), with significant resolution of his organomegaly. On examination, he had mild hepatosplenomegaly, bilateral pes cavus, externally rotated hips, genu valgus, hammertoes, decreased muscle tone, and mild foot drop. Reflexes were depressed in the upper extremities and absent in knees and ankles. Ophthalmologic evaluation revealed nystagmus, dysconjugate gaze, slowed, hypometric horizontal saccades, and decreased pupillary constriction (Adie-like). Nerve conduction studies were 13 …

Alteration G2019S in the leucine-rich repeat kinase 2 gene (LRRK2) has been identified in several populations of patients with parkinsonism, including Ashkenazi Jewish subjects with Parkinson disease. Mutations in glucocerebrosidase (GBA), the enzyme deficient in Gaucher disease, are also identified at an increased frequency among Parkinson probands, including those of Ashkenazi Jewish ancestry. A Taqman Assay-by-Design SNP genotyping strategy was utilized to establish whether G2019S was found in association with GBA mutations. Among 37 subjects with parkinsonism who were heterozygous for a GBA mutation, none carried G2019S. Furthermore, G2019S was not found in 18 patients with Gaucher disease who developed parkinsonian manifestations and 11 other Gaucher probands with parkinsonism in a first degree relative. Among 45 patients with Gaucher disease without a history of parkinsonism, one G2019S carrier was found. These findings suggest that GBA and LRRK2 mutations are discrete risk factors for parkinsonism in both Ashkenazi Jewish and non-Jewish subjects.

244 Background: Pancreatic ductal adenocarcinoma (PC) is associated with multiple hereditary cancer syndromes. Genes implicated in hereditary PC include ATM, BRCA1, BRCA2, CDKN2A, EPCAM, MLH1, MSH2, MSH6, PALB2 and PMS2. The advent of multi-gene hereditary cancer panel testing streamlines diagnoses and medical management for clinicians and patients. Our objective was to assess the yield of pathogenic/likely pathogenic variants (PV/LPV) in individuals with PC undergoing panel testing as an initial test at GeneDx. Methods: We retrospectively reviewed panel test results of 605 individuals reporting a personal history of PC. Panel testing evaluated up to 32 genes associated with hereditary cancer. Individuals reporting neuroendocrine pathology or previous BRCA1/BRCA2 testing were excluded. Results: In this cohort, 61 PV/LPV were detected in 57 individuals in the following genes: ATM (17), BRCA2 (14), BRCA1 (5), CDKN2A (5), PALB2 (5), CHEK2 (4), MLH1 (2), MUTYH (2), PMS2 (2), BARD1 (1), FANCC (1), MSH2 (1), RAD51D (1) and TP53 (1), corresponding to a positive yield of 9.4% (57/605). Fifty-one of 61 PV/LPV were detected in genes associated with PC (84%) while 10 PV/LPV (16%) were identified in other genes including BARD1, CHEK2, FANCC, MUTYH, and RAD51D. The diagnostic yield among those reporting a family history of PC (33/294, 11.2%) was not statistically different from those without a reported family history (24/311, 7.7%). However, PV/LPV in ATM were detected more often in individuals reporting a family history of PC compared to those without a family history (4.1% vs. 1.6%, p=0.018). Conclusions: In total, 9.4% of patients with PC tested positive for PV/LPV in 14 different genes by panel testing. Although the majority of PV/LPV were identified in known PC genes, 16% of positive findings occurred in genes not typically associated with PC. ATM was most commonly implicated and more frequently reported in patients reporting family histories of PC. Assessing whether these genes are indeed causally related to PC and/or are possibly associated with other cancer types requires further investigation. Based on our results we conclude multi-gene panel testing may be considered as a first option for patients with PC regardless of their family history.

In Gaucher disease (GD), the inherited deficiency of glucocerebrosidase results in the accumulation of glucocerebroside within lysosomes. Although almost 300 mutations in the glucocerebrosidase gene (GBA) have been identified, the ability to predict phenotype from genotype is quite limited. In this study, we sought to examine potential GBA transcriptional regulatory elements for variants that contribute to phenotypic diversity. Specifically, we generated the genomic sequence for the orthologous genomic region ( approximately 39.4kb) encompassing GBA in eight non-human mammals. Computational comparisons of the resulting sequences, using human sequence as the reference, allowed the identification of multi-species conserved sequences (MCSs). Further analyses predicted the presence of two putative clusters of transcriptional regulatory elements upstream and downstream of GBA, containing five and three transcription factor-binding sites (TFBSs), respectively. A firefly luciferase (Fluc) reporter construct containing sequence flanking the GBA gene was used to test the functional consequences of altering these conserved sequences. The predicted TFBSs were individually altered by targeted mutagenesis, resulting in enhanced Fluc expression for one site and decreased expression for seven others sites. Gel-shift assays confirmed the loss of nuclear-protein binding for several of the mutated constructs. These identified conserved non-coding sequences flanking GBA could play a role in the transcriptional regulation of the gene contributing to the complexity underlying the phenotypic diversity seen in GD.

Background An association between glucocerebrosidase, the enzyme deficient in Gaucher disease, and the synucleinopathies has been suggested both by the development of parkinsonism in Gaucher probands and carriers, as well as by the presence of mutations in the gene for glucocerebrosidase (GBA) in different series of subjects with synucleinopathies. In this study, an open access Parkinson repository was used to establish the incidence of GBA alterations in a different ethnic cohort with sporadic Parkinson disease (PD). Methods The glucocerebrosidase gene was sequenced in samples collected from 92 Chinese Parkinson disease patients from Taiwan along with 92 clinically screened controls, matched for age and ethnicity. Findings The frequency of GBA mutations among the Chinese PD probands was 4.3%, in contrast to 1.1% in Chinese controls. Mutant alleles identified included two known mutations, L444P and D409H, and two novel mutations, L174P and Q497R. Interpretation These results, ascertained in subjects from Taiwan collected in a standardized and clinically rigorous open access Parkinson disease repository and screened by direct sequencing of GBA, demonstrate that GBA mutations are also encountered in Chinese subjects with sporadic PD at a higher frequency than many other known PD genes. The study demonstrates that the association of GBA mutations with the development of parkinsonian pathology is not related to ethnic origin.

Several recent observations suggest a connection between Gaucher disease, the inherited deficiency of glucocerebrosidase, and the synucleinopathies. Rare patients have been observed who develop both Gaucher disease and parkinsonism. Autopsy studies on these subjects reveal synuclein-positive Lewy bodies and inclusions. An increased incidence of synucleinopathies also has been noted in relatives of Gaucher probands. In complementary studies, screening of patients with parkinsonism has identified a greater than expected frequency of glucocerebrosidase mutations. These glucocerebrosidase mutation carriers have a wide spectrum of associated parkinsonian phenotypes, ranging from classic L-dopa-responsive Parkinson disease to a phenotype more characteristic of Lewy body dementia. Despite this association, the vast majority of Gaucher carriers and patients with Gaucher disease never develop parkinsonism. However, mutations in this gene are likely to be a contributing risk factor in subjects otherwise prone to developing synucleinopathies.

Background: Gaucher disease is classified into neuronopathic and non-neuronopathic forms with wide phenotypic variation among patients sharing the same genotype. While homozygosity for the common L444P allele usually correlates with the neuronopathic forms, how a defined genotype leads to a phenotype remains unknown. Methods: The genetic and epigenetic factors causing phenotypic differences were approached by a clinical association study in 32 children homozygous for the point mutation L444P. Direct sequencing and Southern blots were utilised to establish the genotype and exclude recombinant alleles. Glucocerebrosidase activity was measured in lymphoblast and fibroblast cell lines. Results: Residual enzyme activity was highly variable and did not correlate with the observed clinical course. There was also a wide spectrum of phenotypes. Average age at diagnosis was 15 months, and slowed saccadic eye movements were the most prevalent finding. The most severe systemic complications and highest mortality occurred in splenectomised patients before the advent of enzyme replacement therapy (ERT). On ERT, as morbidity and mortality decreased, developmental and language deficits emerged as a major issue. Some trends related to ethnic background were observed. Conclusion: The wide clinical spectrum observed in the L444P homozygotes implicates the contribution of genetic modifiers in defining the phenotype in Gaucher disease.

Methoxychlor (MXC) is an organochlorine pesticide that increases the rate of ovarian atresia. To date, little is known about the mechanism by which MXC induces atresia. Because Bcl-2 (an antiapoptotic factor), Bax (a proapoptotic factor), gonadotropins, and estradiol are important regulators of atresia in the ovary, the purpose of this study was first to examine whether MXC-induced atresia occurred through alterations in Bcl-2 or Bax, and second, to examine the effect of MXC on gonadotropins, estradiol, and their receptors. CD-1 mice were dosed with 8-64 mg kg(-1) day(-1) MXC or vehicle (sesame oil). Ovaries were subjected to analysis of antral follicle numbers, Bcl-2, Bax, estrogen receptor, and follicle-stimulating hormone receptor levels. Blood was used to measure gonadotropins and estradiol. In some experiments, mice that overexpressed Bcl-2 or mice that were deficient in Bax were dosed with MXC or vehicle and their ovaries were analyzed for atresia. MXC caused a dose-dependent increase in the percentage of atretic antral follicles compared with controls at the 32 and 64 mg kg(-1) day(-1) doses of MXC. MXC treatment did not result in changes in Bcl-2 levels, but it did result in an increase in Bax levels in antral follicles. MXC treatment did not affect gonadotropin or estradiol levels, nor did it affect the levels of follicle-stimulating hormone or estrogen receptors. Mice that overexpressed Bcl-2 or mice that were deficient in Bax were protected from MXC-induced atresia. These data suggest that MXC induces atresia through direct effects on the Bax and Bcl-2 signaling pathways in the ovary.

As an initial step towards enhancing mastitis resistance in dairy animals, we generated BLG-Lys transgenic mice that secrete lysostaphin, a potent antistaphylococcal protein, in their milk. In the current study, we continue our assessment of lysostaphin as a suitable antimicrobial protein for mastitis resistance and have investigated mammary gland development and function in three lines of transgenic mice. As the lines were propagated, there was a tendency for fewer BLG-Lys litters to survive to weaning (51% as compared to 90% for nontransgenic lines, p = 0.080). Nontransgenic pups fostered on dams from these three lines exhibited diminished growth rates during the first week of lactation. Rates of gain became comparable to pups on nontransgenic dams at later time points. Initial slow growth also resulted in decreased weaning weights for pups nursed by transgenic dams (15.35 +/- 0.27 g) when compared to pups delivered and nursed by nontransgenic dams (18.61 +/- 0.61 g; p0.001), but the effect was temporary, as similar weights were attained by adulthood. Milk yield at peak lactation was not different between BLG-Lys (0.79 +/- 0.33 g) and nontransgenic (0.91 +/- 0.38 g; p = 0.166) dams. Histological examination of the transgenic mammary glands during gestation revealed no differences when compared to control glands; however, at early lactational stages, the BLG-Lys glands exhibited less alveolar area than control glands and a delay in lobulo-alveolar maturation. The results clearly demonstrate reduced growth of neonates on BLG-Lys dams; whether the poor pup performance can be attributed to delayed mammary development or the gland development merely reflects reduced suckling stimuli from the pups remains to be determined.

Conditional expression of estrogen receptor (ER)-alpha) was introduced into tetracycline-responsive MMTV-tTA/tetop-TAg mice to develop a mouse model of estrogen-responsive ER-alpha-positive mammary adenocarcinoma. Mammary adenocarcinomas developed in the mice with a mean latency of 11 months. Precursor lesions including ductal hyperplasia and hyperplastic alveolar nodules were present by the age of 4 months. The mammary adenocarcinomas exhibited histological features similar to human breast cancers. ER steroid-binding studies conducted on adenocarcinoma lysates demonstrated binding to estradiol. Tumor explant studies in the presence and absence of estradiol in ovariectomized athymic nude mice revealed that growth of mammary tumors was stimulated by estrogen. In addition, the presence of ER-alpha altered the tumor spectrum in other MMTV-targeted tissues in the tTA/TAg female mice. Lymphomas, which develop in 40% of tTA/TAg female mice, were found in only 4% of tTA/TAg/ER-alpha mice (P = 0.014, chi-square test). These experiments demonstrate that the introduction of an ER-alpha transgene targeted to mammary epithelial cells can be used to develop mouse models of ER-alpha-responsive mammary cancer.

Attempts to delineate the mechanisms of estrogen action have promoted the creation of several estrogen receptor alpha (ERalpha) mouse models in the past decade. These traditional models are limited by the fact that the receptors are either absent or present throughout all stages of development. The purpose of this work was to develop a conditional transgenic model that would provide an in vivo method of controlling the spatial and temporal regulation of ERalpha expression. The tetracycline responsive system was utilized. Three lines of transgenic mice carrying a transgene composed of the coding sequence for murine ERalpha placed under the regulatory control of a tet operator promoter (tet-op) were generated. These three lines of tet-op-mERa mice were each mated to an established line of transgenic mice expressing a tetracycline-dependent transactivator protein (tTA) from the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Double transgenic MMTV-tTA/tet-op-mERalpha mice were produced. All three lines demonstrated dominant gain of ERalpha shown by RT-PCR, immunoprecipitation, and immunohistochemistry. Transgene-specific ERalpha was expressed in numerous tissues including the mammary gland, salivary gland, testis, seminal vesicle, and epididymis. Expression was silenced by administration of doxycycline in the drinking water. This model can be utilized to evaluate the consequences of ERalpha dominant gain in targeted tissues at specific times during development. In this study dominant gain of ERalpha was associated with a reduction in epididymal/vas deferens and seminal vesicle weights consistent with the proposed action of ERalpha on fluid transport in the male reproductive tract. Combining this model with other dominant gain and gene knockout mouse models will be useful for testing effects of ERalpha action in combination with specific gene products and to evaluate if developmental and stage-specific expression of ERalpha can rescue identified phenotypes in gene knockout mice.