Optimising clinical care through CDH1-specific germline variant curation: improvement of clinical assertions and updated curation guidelines.

BACKGROUND: Germline pathogenic variants in CDH1 are associated with increased risk of diffuse gastric cancer and lobular breast cancer. Risk reduction strategies include consideration of prophylactic surgery, thereby making accurate interpretation of germline CDH1 variants critical for physicians deciding on these procedures. The Clinical Genome Resource (ClinGen) CDH1 Variant Curation Expert Panel (VCEP) developed specifications for CDH1 variant curation with a goal to resolve variants of uncertain significance (VUS) and with ClinVar conflicting interpretations and continues to update these specifications. METHODS: CDH1 variant classification specifications were modified based on updated genetic testing clinical criteria, new recommendations from ClinGen and expert knowledge from ongoing CDH1 variant curations. The CDH1 VCEP reviewed 273 variants using updated CDH1 specifications and incorporated published and unpublished data provided by diagnostic laboratories. RESULTS: Updated CDH1-specific interpretation guidelines include 11 major modifications since the initial specifications from 2018. Using the refined guidelines, 97% (36 of 37) of variants with ClinVar conflicting interpretations were resolved to benign, likely benign, likely pathogenic or pathogenic, and 35% (15 of 43) of VUS were resolved to benign or likely benign. Overall, 88% (239 of 273) of curated variants had non-VUS classifications. To date, variants classified as pathogenic are either nonsense, frameshift, splicing, or affecting the translation initiation codon, and the only missense variants classified as pathogenic or likely pathogenic have been shown to affect splicing. CONCLUSIONS: The development and evolution of CDH1-specific criteria by the expert panel resulted in decreased uncertain and conflicting interpretations of variants in this clinically actionable gene, which can ultimately lead to more effective clinical management recommendations.

Phenotypic continuum between POLE-related recessive disorders: A case report and literature review.

POLE is a pleiotropic gene with phenotypic expression of pathogenic variants depending on the type of variant, impact on the protein, and mode of inheritance. Heterozygous missense variants located within the exonuclease domain have been shown to result in polymerase proofreading-associated polyposis (PPAP) which is characterized by an increased risk for colon polyps and colorectal cancer. Biallelic variants resulting in markedly reduced amounts of normal protein have been reported in two separate recessive pediatric syndromes: facial dysmorphism, immunodeficiency, livedo, and short stature as well as intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenital, and genital anomalies. Here we report two siblings identified to have POLE c.1686 + 32C > G in trans with POLE p.(Glu709*) via exome sequencing. A detailed review of the reported phenotypes in these two siblings and from available literature revealed that individuals with biallelic POLE pathogenic variants resulting in partial loss-of-function present with a similar phenotype: short stature and facial dysmorphism with or without immunodeficiency. These data suggest a phenotypic continuum between the previously reported POLE-related recessive disorders.

Patterns of germline and somatic testing after universal tumor screening for Lynch syndrome: A clinical practice survey of active members of the Collaborative Group of the Americas on Inherited Gastrointestinal Cancer.

Clinical guidelines recommend universal tumor screening (UTS) of colorectal and endometrial cancers for Lynch syndrome (LS). There are limited guidelines for how to integrate germline testing and somatic tumor testing after a mismatch repair deficient (dMMR) tumor is identified. We sought to characterize current practice patterns and barriers to preferred practice among clinical providers in high-risk cancer programs. A clinical practice survey was sent to 423 active members of the Collaborative Group of the Americas on Inherited Gastrointestinal Cancer (CGA-IGC) with a follow-up survey sent to 103 clinician responders. The survey outlined clinical vignettes and asked respondents their preferred next test. The survey intended to assess: (1) the role of patient age and family history in risk assessment and (2) barriers to preferred genetic testing. Genetic test options included targeted germline testing based on dMMR expression, germline testing for LS, germline testing with a multigene cancer panel including LS, or paired tumor/germline testing including LS. In October 2020, 117 of 423 (28%) members completed the initial survey including 103 (88%) currently active clinicians. In April 2021, a follow-up survey was sent to active clinicians, with 45 (44%) completing this second survey. After selecting their preferred next germline or paired tumor/germline tumor test based on the clinical vignette, 39% of respondents reported wanting to make a different choice for the initial genetic test without any testing barriers. The proportion of respondents choosing a different initial genetic test was dependent on the proband's age at diagnosis and specified family history. The reported barriers included patient's lack of insurance coverage, patient unable/unwilling to self-pay for proposed testing, and inadequate tumor tissue. Responders reported insurance, financial constraints, and limited tumor tissue as influencing preferred genetic testing in high-risk clinics, thus resulting in possible under-diagnosis of LS and impacting potential surveillance and cascade testing of at-risk relatives.

Ancestry-specific hereditary cancer panel yields: Moving toward more personalized risk assessment.

Healthcare disparities in genomic medicine are well described. Despite some improvements, we continue to see fewer individuals of African American, Asian, and Hispanic ancestry undergo genetic counseling and testing compared to those of European ancestry. It is well established that variant of uncertain significance (VUS) rates are higher among non-European ancestral groups undergoing multi-gene hereditary cancer panel testing. However, pathogenic variant (PV) yields, and genomic data in general, are often reported in aggregate and derived from cohorts largely comprised of individuals of European ancestry. We performed a retrospective review of clinical and ancestral data for individuals undergoing multi-gene hereditary cancer panel testing to determine ancestry-specific PV and VUS rates. An ancestry other than European was reported in 29,042/104,851 (27.7%) of individuals. Compared to Europeans (9.4%), individuals of Middle Eastern ancestry were more likely to test positive for one or more pathogenic variants (12.1%, p = .0025), while African Americans were less likely (7.9%, p < .0001). Asian and Middle Eastern individuals were most likely (34.8% and 33.2%, respectively) to receive a report with an overall classification of VUS, while individuals of Ashkenazi Jewish and European ancestry were least likely (17.1% and 20.4%, respectively). These data suggest that in addition to higher VUS rates, there may be ancestry-specific PV yields. Providing aggregate data derived from cohorts saturated with European individuals does not adequately reflect genetic testing outcomes in minority groups, and interrogation of ancestry-specific data is a step toward a more personalized risk assessment.

Determining the clinical validity of hereditary colorectal cancer and polyposis susceptibility genes using the Clinical Genome Resource Clinical Validity Framework.

PURPOSE: Gene-disease associations implicated in hereditary colorectal cancer and polyposis susceptibility were evaluated using the ClinGen Clinical Validity framework. METHODS: Forty-two gene-disease pairs were assessed for strength of evidence supporting an association with hereditary colorectal cancer and/or polyposis. Genetic and experimental evidence supporting each gene-disease relationship was curated independently by two trained biocurators. Evidence was reviewed with experts and assigned a final clinical validity classification. RESULTS: Of all gene-disease pairs evaluated, 14/42 (33.3%) were Definitive, 1/42 (2.4%) were Strong, 6/42 (14.3%) were Moderate, 18/42 (42.9%) were Limited, and 3/42 (7.1%) were either No Reported Evidence, Disputed, or Refuted. Of panels in the National Institutes of Health Genetic Testing Registry, 4/26 (~15.4%) contain genes with Limited clinical evidence. CONCLUSION: Clinicians and laboratory diagnosticians should note that <60% of the genes on clinically available panels have Strong or Definitive evidence of association with hereditary colon cancer or polyposis, and >40% have only Moderate, Limited, Disputed, or Refuted evidence. Continuing to expand the structured assessment of the clinical relevance of genes listed on hereditary cancer testing panels will help clinicians and diagnostic laboratories focus the communication of genetic testing results on clinically significant genes.

Age-adjusted association of homologous recombination genes with ovarian cancer using clinical exomes as controls.

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.

Specifications of the ACMG/AMP variant curation guidelines for the analysis of germline CDH1 sequence variants.

The variant curation guidelines published in 2015 by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) provided the genetics community with a framework to assess variant pathogenicity; however, these rules are not gene specific. Germline pathogenic variants in the CDH1 gene cause hereditary diffuse gastric cancer and lobular breast cancer, a clinically challenging cancer predisposition syndrome that often requires a multidisciplinary team of experts to be properly managed. Given this challenge, the Clinical Genome Resource (ClinGen) Hereditary Cancer Domain prioritized the development of the CDH1 variant curation expert panel (VCEP) to develop and implement rules for CDH1 variant classifications. Here, we describe the CDH1 specifications of the ACMG/AMP guidelines, which were developed and validated after a systematic evaluation of variants obtained from a cohort of clinical laboratory data encompassing ∼827,000 CDH1 sequenced alleles. Comparing previously reported germline variants that were classified using the 2015 ACMG/AMP guidelines to the CDH1 VCEP recommendations resulted in reduced variants of uncertain significance and facilitated resolution of variants with conflicted assertions in ClinVar. The ClinGen CDH1 VCEP recommends the use of these CDH1-specific guidelines for the assessment and classification of variants identified in this clinically actionable gene.

MSH6 and PMS2 germ-line pathogenic variants implicated in Lynch syndrome are associated with breast cancer.

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.

Utility of Expedited Hereditary Cancer Testing in the Surgical Management of Patients with a New Breast Cancer Diagnosis.

BACKGROUND: Knowledge of a germline pathogenic/likely pathogenic variant (PV) may inform breast cancer management. BRCA1/2 PV often impact surgical decisions, but data for multi-gene panel testing are lacking. Expedited genetic testing reduces turn-around times based on request for treatment-related decision making. This report aims to describe the clinical utility of expedited multi-gene panel testing for patients with newly diagnosed breast cancer. METHODS: Clinical and demographic information were reviewed for patients with newly diagnosed female breast cancer undergoing expedited panel testing between 2013 and 2017. The National Comprehensive Cancer Network guidelines (NCCN, version 1.2018) were evaluated in terms of published management recommendations for the genes in which PVs were identified. RESULTS: The overall PV yield was 9.5% (678/7127) for women undergoing expedited panel testing, with 700 PVs identified among 678 women. PVs were identified in genes other than BRCA1/2 in 55.9% (391/700) of cases. The NCCN guidelines recommend management for the genes in which 96.6% (676/700) of PVs are identified. The NCCN guidelines also recommend risk-reducing mastectomy for 46.0% (322/700) of PVs identified. An additional 45.6% (319/700) of PVs were identified in genes for which NCCN recommends mastectomy based on family history. In addition, 49.9% (349/700) of PVs were in genes with NCCN guidelines recommending prophylactic surgery for tissues other than breast. CONCLUSION: A majority of the patients with newly diagnosed breast cancer were candidates for surgical intervention according to the NCCN guidelines, and half of these patients would have been missed if only BRCA1/2 testing had been ordered. Expedited multi-gene hereditary cancer panel testing should be considered as a first-line approach to provide comprehensive information for breast cancer management.

High-grade endometrial stromal sarcoma as the initial presentation of an adult patient with Peutz-Jeghers Syndrome: a case report.

A 46-year-old female presents with a pelvic mass and is diagnosed as having a high-grade endometrial stromal sarcoma. During surgery, she is noted to have areas of intussusception of the small bowel secondary to large hamartomatous polyps. The patient had a previous history of small bowel obstruction secondary to what had been thought to be hyperplastic polyps but represented hamartomatous polyps on further review. Additional examination revealed the presence of subtle hyperpigmented macules on the fingers leading to a diagnosis of Peutz-Jeghers Syndrome (PJS). The diagnosis was confirmed by the presence of a germ-line STK11 mutation. Immunohistochemistry analysis of the tumor showed decreased expression of STK-11 as compared to one of the patient's hamartomatous polyps. Next generation sequencing of the tumor specimen failed to demonstrate a "second hit" somatic mutation in STK-11. This case represents the first case of endometrial stromal sarcoma associated with PJS and illustrates the importance of increased awareness of this condition among oncologists. PJS is associated with dysregulation of the mTOR pathway; treatment with an mTOR inhibitor was not effective in this case.

A clinical scoring system to identify patients with sebaceous neoplasms at risk for the Muir-Torre variant of Lynch syndrome.

PURPOSE: The Muir-Torre syndrome variant of Lynch syndrome is characterized by the presence of sebaceous neoplasms (adenoma, epithelioma/sebaceoma, carcinoma) and Lynch syndrome-associated cancers (colon, endometrial, and others). Several clinical scoring systems have been developed to identify patients with colon cancer at high risk of Lynch syndrome. However, no such system has been described for patients presenting with sebaceous neoplasms. METHODS: Based on logistic regression analysis, a scoring system was developed for patients with sebaceous neoplasm to identify those with the highest likelihood of having Muir-Torre syndrome. The final version of the scoring system included variables such as age at presentation of initial sebaceous neoplasm, total number of sebaceous neoplasms, personal history of a Lynch-related cancer, and family history of Lynch-related cancers. RESULTS: Patients with a score of 3 or more were more likely to have Muir-Torre syndrome (28 of 29 patients), those with a score of 2 had intermediate likelihood (12 of 20 patients), and no patient with a score of 0 or 1 was diagnosed with Muir-Torre syndrome. CONCLUSION: The Mayo Muir-Torre syndrome risk scoring system appears to identify whether patients who present with sebaceous neoplasms are in need of further Lynch syndrome evaluation using easily ascertained clinical information. Abnormal mismatch repair gene immunohistochemistry of a sebaceous neoplasm is a poor predictor in regard to diagnosing Lynch syndrome.

Gastric adenomas in familial adenomatous polyposis are common, but subtle, and have a benign course.

BACKGROUND: Patients with familial adenomatous polyposis (FAP) are known to have an increased risk for gastric adenomas. The clinical features of gastric adenomas in FAP have not been well characterized, and there is a lack of standardized approaches to the management of these lesions. AIMS: To study the endoscopic appearance, risk factors, clinical course, and response to therapy of gastric adenomas in patients with FAP. METHODS: We retrospectively reviewed the records of 97 patients with FAP who underwent esophagogastroduodenoscopy (EGD) at Mayo Clinic (Florida, Rochester and Arizona) between 2004 and 2013. RESULTS: Nine patients (9%) had biopsy-proven gastric adenomas. Adenomas were located in the antrum (five patients), in the body and fundus in the setting of background fundic gland polyps (FGP) (three patients), and in the body not associated with FGP (one patient). Adenoma size was 3-40 mm and the number of adenomas per patient ranged from one to 20. Adenomas in the antrum were flat and subtle, whereas those in the gastric body or fundus were polypoid and difficult to differentiate from the cystic FGPs seen in patients with FAP. The performing endoscopists reported difficulty with identifying adenomas, and six patients had at least one EGD within the previous three years where gastric adenomas were not reported. Adenomas were classified as tubular in eight patients and tubulovillous in one patient. High grade dysplasia was noted in one patient. After a median follow-up of 63 months (interquartile range: 20-149 months), no patient in our entire cohort (with or without gastric adenomas) developed gastric cancer. The patients in whom gastric adenoma developed, compared to those without gastric adenoma, were more likely to be younger [36 ± 12 vs. 48 ± 15 years, p = 0.02], have concomitant chronic gastritis [22% vs. 0%, p = 0.008], and have desmoid tumors [5 (56%) vs. 19 (22%), p = 0.04]. CONCLUSIONS: Gastric adenomas are not uncommon in patients with FAP and are often difficult to identify endoscopically. Endoscopists should have a high degree of suspicion for gastric adenomas in these patients and a low threshold to biopsy. Given the benign clinical course, recommended initial management is conservative with endoscopic therapy and periodic surveillance.

Screening for Muir-Torre syndrome using mismatch repair protein immunohistochemistry of sebaceous neoplasms.

Screening for the Muir-Torre variant of Lynch Syndrome (LS) using Mismatch Repair (MMR) gene immunohistochemistry (IHC) on sebaceous neoplasms (SNs) is technically feasible. To date, research into the clinical utility of MMR IHC for this indication is limited. We conducted a retrospective chart review of 90 patients with MMR IHC completed on at least one SN from January 2005 to May 2010. SNs included were adenomas, epitheliomas, carcinomas and basal and squamous cell carcinomas with sebaceous differentiation. Of the 90 patients, 13 (14 %) had genetically confirmed or fulfilled clinical criteria for a diagnosis of MTS and 51 patients (57 %) presented with an abnormal MMR IHC result (loss of one or more MMR proteins) on at least one SN. Abnormal IHC had a sensitivity of 85 %, specificity of 48 %, positive predictive value (PPV) of 22 % and negative predictive value (NPV) of 95 % when evaluating for MTS. When personal or family history of colorectal cancer (≥2 family members with a history of colorectal cancer) was taken into consideration, ignoring IHC results, sensitivity was 92 %, specificity was 99 %, PPV was 92 % and NPV was 99 %. MMR IHC on SNs when used to screen for MTS has poor diagnostic utility. We recommend that MMR IHC not be performed routinely on SNs when the patient does not have either personal or family history of colorectal cancer.

Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C.

Pathogenic protein-truncating variants of RAD51C, which plays an integral role in promoting DNA damage repair, increase the risk of breast and ovarian cancer. A large number of RAD51C missense variants of uncertain significance (VUS) have been identified, but the effects of the majority of these variants on RAD51C function and cancer predisposition have not been established. Here, analysis of 173 missense variants by a homology-directed repair (HDR) assay in reconstituted RAD51C-/- cells identified 30 nonfunctional (deleterious) variants, including 18 in a hotspot within the ATP-binding region. The deleterious variants conferred sensitivity to cisplatin and olaparib and disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes. Computational analysis indicated the deleterious variant effects were consistent with structural effects on ATP-binding to RAD51C. A subset of the variants displayed similar effects on RAD51C activity in reconstituted human RAD51C-depleted cancer cells. Case-control association studies of deleterious variants in women with breast and ovarian cancer and noncancer controls showed associations with moderate breast cancer risk [OR, 3.92; 95% confidence interval (95% CI), 2.18-7.59] and high ovarian cancer risk (OR, 14.8; 95% CI, 7.71-30.36), similar to protein-truncating variants. This functional data supports the clinical classification of inactivating RAD51C missense variants as pathogenic or likely pathogenic, which may improve the clinical management of variant carriers. SIGNIFICANCE: Functional analysis of the impact of a large number of missense variants on RAD51C function provides insight into RAD51C activity and information for classification of the cancer relevance of RAD51C variants.

Fibroblast testing can inform medical management in individuals with mosaic variants detected on hereditary cancer panels.

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 (p<0.0001) 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.

Monoallelic MUTYH pathogenic variants ascertained via multi-gene hereditary cancer panels are not associated with colorectal, endometrial, or breast cancer.

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.

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.