A patient with newly diagnosed breast cancer found to have mosaic TP53 likely pathogenic variant.

Familial adenomatous polyposis is most frequently caused by pathogenic variants in either the APC gene or the MUTYH gene. The detection rate of pathogenic variants depends on the severity of the phenotype and sensitivity of the screening method, including sensitivity for mosaic variants. For 171 patients with multiple colorectal polyps without previously detectable pathogenic variant, APC was reanalyzed in leukocyte DNA by one uniform technique: high-resolution melting (HRM) analysis. Serial dilution of heterozygous DNA resulted in a lowest detectable allelic fraction of 6 % for the majority of variants. HRM analysis and subsequent sequencing detected pathogenic fully heterozygous APC variants in 10 (6 %) of the patients and pathogenic mosaic variants in 2 (1 %). All these variants were previously missed by various conventional scanning methods. In parallel, HRM APC scanning was applied to DNA isolated from polyp tissue of two additional patients with apparently sporadic polyposis and without detectable pathogenic APC variant in leukocyte DNA. In both patients a pathogenic mosaic APC variant was present in multiple polyps. The detection of pathogenic APC variants in 7 % of the patients, including mosaics, illustrates the usefulness of a complete APC gene reanalysis of previously tested patients, by a supplementary scanning method. HRM is a sensitive and fast pre-screening method for reliable detection of heterozygous and mosaic variants, which can be applied to leukocyte and polyp derived DNA.Electronic supplementary materialThe online version of this article (doi:10.1007/s10689-015-9780-5) contains supplementary material, which is available to authorized users.

P3-09-05: Clinical outcome of patients with advanced triple negative breast cancer with germline and somatic variants in homologous recombination gene

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 <35%. Mean age at first breast cancer diagnosis was 49.2 years. Twenty children of 13 women with mosaic TP53 PV pursued targeted testing; none were positive for their mother's TP53 PV. Six of the 48 women (16.7%) pursued cultured fibroblast testing for the mosaic TP53 PV. In five, the PV was not found in fibroblasts. All five were diagnosed with breast cancer >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.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small vessel disease caused predominantly by pathogenic variants in NOTCH3 gene. Neither germline nor somatic mosaicism has been previously published in NOTCH3 gene. CADASIL is inherited in an autosomal dominant manner; only rare cases have been associated with de novo pathogenic variants. Mosaicism is more common than previously thought because mosaic variants often stay unrevealed. An apparently de novo variant might actually be a consequence of a parental mosaicism undetectable with Sanger sequencing, especially in the case of low grade mosaicism. Parental testing by sensitive tools like deep targeted next-generation sequencing (NGS) analysis could detect cases of unrevealed medium or low level mosaicism in patients tested by Sanger sequencing. Here, we report the first patient with mosaic NOTCH3 gene pathogenic variant to our knowledge; the allelic fraction in the leucocyte DNA was low (13%); the pathogenic variant was inhered by his two daughters. The patient was diagnosed by deep targeted NGS analysis after studying his two affected daughters. This report highlights the importance of parental testing by sensitive tools like deep targeted NGS analysis. Detection of mosaicism is of great importance for diagnosis and adequate family genetic counseling.

Blaschko linear hypopigmentation has been attributed to multiple mosaic genetic causes including chromosomal abnormalities and single gene variants. However, many cases remain unsolved. Two unrelated female children presented to our service for assessment of abnormal pigmentation. The first, aged 2 years, had extensive Blaschko linear hypopigmentation on the left upper limb, left trunk and left lower limb but no other symptoms or signs. The second, aged 12 years, had Blaschko linear hypopigmentation affecting the left lower limb, in addition to four café au lait macules and a small capillary malformation under the left lower eyelid, but no other symptoms or signs. Both patients were consented for genetic testing and a skin biopsy was taken from a representative area of hypopigmentation. DNA was extracted directly from the whole biopsy and underwent targeted next-generation sequencing. Patient 1 was found to have a pathogenic variant in GNA11 c.547C>T p.(Arg183Cys) at 1.5% variant allele frequency. Patient 2 was found to have a pathogenic variant in PIK3CA c.1633G>A p.(Glu545Lys) at 4% variant allele frequency. Neither GNA11 nor PIK3CA has been described previously to cause Blaschko linear hypopigmentation. However, the exact variants described here are well known in the context of other cutaneous mosaic diseases. The mosaic GNA11 variant can cause port-wine stains with or without Sturge–Weber syndrome, phakomatosis pigmentovascularis with dermal melanocytosis, and extensive dermal melanocytosis. The mosaic PIK3CA variant can cause overgrowth, vascular or lymphatic malformations and epidermal naevi. This is therefore highly supportive of the pathogenicity of these variants in this new clinical presentation. As Blaschko linear patterning is a sign of genetic disease affecting the epidermal lineage, we propose that this new phenotype is caused by the mosaic variant affecting that lineage. These cases expand our current knowledge of mosaic variants causing Blaschko linear hypopigmentation, and of the disease phenotypes caused by mosaicism for GNA11 and PIK3CA. GNA11 and PIK3CA testing should be considered in children presenting with Blaschko linear hypopigmentation.

Blaschko linear hypopigmentation has been attributed to multiple mosaic genetic causes including chromosomal abnormalities and single-gene variants. However, many cases remain unsolved. Two unrelated female children presented to our service for assessment of abnormal pigmentation. The first, aged 2 years, had extensive Blaschko linear hypopigmentation on the left upper limb, left trunk and left lower limb but no other symptoms or signs. The second, aged 12 years, had Blaschko linear hypopigmentation affecting the left lower limb, in addition to four café au lait macules and a small capillary malformation under the left lower eyelid, but no other symptoms or signs. Both patients were consented for genetic testing and a skin biopsy was taken from a representative area of hypopigmentation. DNA was extracted directly from the whole biopsy and underwent targeted next-generation sequencing. Patient 1 was found to have a pathogenic variant in GNA11 c.547C>T p.(Arg183Cys) at 1.5% variant allele frequency. Patient 2 was found to have a pathogenic variant in PIK3CA c.1633G>A p.(Glu545Lys) at 4% variant allele frequency. Neither GNA11 nor PIK3CA has been described previously to cause Blaschko linear hypopigmentation. However, the exact variants described here are well known in the context of other cutaneous mosaic diseases. The mosaic GNA11 variant can cause port-wine stains with or without Sturge–Weber syndrome, phakomatosis pigmentovascularis with dermal melanocytosis, and extensive dermal melanocytosis. The mosaic PIK3CA variant can cause overgrowth, vascular or lymphatic malformations and epidermal naevi. This is therefore highly supportive of the pathogenicity of these variants in this new clinical presentation. As Blaschko linear patterning is a sign of genetic disease affecting the epidermal lineage, we propose that this new phenotype is caused by the mosaic variant affecting that lineage. These cases expand our current knowledge of mosaic variants causing Blaschko linear hypopigmentation, and of the disease phenotypes caused by mosaicism for GNA11 and PIK3CA. GNA11 and PIK3CA testing should be considered in children presenting with Blaschko linear hypopigmentation.

CHEK2 pathogenic and likely pathogenic variants (PVs) are common, and low-risk (LR) variants, p.I157T, p.S428F, and p.T476M, are even more common. Biallelic CHEK2 PVs are associated with specific cancer phenotypes, including early age at onset of breast cancers. Whether biallelic LR variants are associated with cancer predisposition is unknown. To characterize the cancer phenotype among individuals with biallelic CHEK2 variants, specifically those that have been associated with lower cancer risk in the heterozygous state. This retrospective observational cohort study examining cancer phenotype by CHEK2 genotype was conducted at a single diagnostic genetic testing laboratory. Of 36 821 individuals who underwent genetic testing, 3783 (10.3%) with CHEK2 PVs or LR variants were ascertained from July 1, 2012, to September 30, 2019. Analyses were conducted from September 2022 to January 2024. Cancer phenotype among individuals with 2 LR variants and those with 1 PV and 1 LR variant was compared with cancer phenotype among individuals with wild type (WT) (n = 33 034), single LR variant (n = 1566), single PV controls (n = 2167), and 2 PVs (n = 21). Cancer phenotypes were investigated for any cancer, multiple primary cancers, female breast cancer, and bilateral female breast cancers. Cancer phenotype of CHEK2 2 LRs and 1 PV and 1 LR. Of 36 821 individuals, 92.1% were female, and the median age at testing was 53 years (IQR, 44-63 years); 3787 (10.3%) were identified as having a CHEK2 PV or LR variant. There were 13 individuals with 2 LR variants and 20 with 1 PV and 1 LR variant. Among those with 2 LR variants, prevalence of any cancer (76.9%) and breast cancer (60.0%) were similar to those with WT (any cancer, 69.8%; breast cancer, 52.7%) and those with a single LR variant (any cancer, 70.9%; breast cancer, 57.5%). Among participants with 1 PV and 1 LR variant, 95.0% had a prior cancer diagnosis, a higher rate than among those with a single PV (76.8%), but the difference was not statistically significant. Among female individuals with 1 PV and 1 LR variant, 86.7% had a breast cancer diagnosis, compared with 67.1% with a single PV, although these differences were not statistically significant. In this cohort study, individuals with 2 LR variants in CHEK2 had a cancer phenotype similar to those with a single LR variant and similar to WT controls. Individuals with 1 PV and 1 LR variant may have a more penetrant cancer phenotype than individuals with a single PV. Future studies focused on CHEK2 LR variants will aid in better understanding whether these variants are genetic modifiers associated with cancer risk.

Introduction:HBOPC syndrome is mostly associated with germline pathogenic and likely pathogenic variants (PV) in BRCA1 and BRCA2 genes. Other high and moderate penetrance genes are increasingly diagnosed in these families, especially after implementation of NGS methodologies in clinical practice. In this study we analyze the global and regional mutational patterns of Portuguese HBOPC families, looking for associated phenotypes and possible clusters of specific PV. Methods:Observational study including all index patients (pts) with identified PV in genes associated with breast, ovarian and prostate cancers in our Centre, between 2000 and 2020. Geographical mapping of variants was done regarding residency and birth origin. Main cancer diagnoses of pts were also registered. Results:Between January 2000 and December 2020, 5233 index pts consented in genetic testing and 658 (12.6%) tested positive for a PV associated with HBOPC syndrome (84 were non-Portuguese residents). Regarding these 658 positive pts, 537 (81.6%) had breast (BC), 113 (17.2%) had ovarian and 9 (1.4%) had prostate cancer. BRCA2 was the most mutated gene (302 pts, 45.9%), followed by BRCA1 (179 pts, 27.2%), while PV in other genes were diagnosed in 177 (26.9%) pts (CHEK2, ATM, PALB2, RAD51C, TP53, RAD51D, MUTYH, BRIP1, RAD50, BLM and PTEN PV were identified in 54 (8.2%), 27 (4.1%), 21 (3.2%), 17 (2.6%), 16 (2.4%), 14 (2.1%), 11 (1.7%), 7 (1.1%), 4 (0.6%), 4 (0.6%) and 2 (0.3%) pts, respectively). PV in MSH2, MLH1, FAM175A and MRE11A were each described in 1 (0.15%) pt, all diagnosed with BC. Double heterozygosity was identified in 7 (1.1%) pts: BRCA1/BRCA2, BRCA1/CHEK2, BRCA2/PALB2, BRCA2/CHEK2, ATM/PALB2, ATM/RAD51C and MUTYH/MSH2. BRCA2 PV were more frequent than BRCA1 in all Portuguese regions except Algarve, where BRCA2 frequency was lower (BRCA1:BRCA2 > 1). The known BRCA2 founder c.156_157insAlu was the most recurrently diagnosed PV: 36.4% of all BRCA2 families including 8 non-Portuguese families (6 from Angola, 1 from France and 1 from Brazil). Further analysis revealed 3 possible regional clusters: 1- Duplication of exon 12 (previously described as ins6KbEx13) was the only BRCA1 identified in Algarve, with 90.9% of all families with this PV originating in this region; 2- 75% of all BRCA2 c.6405_6409del; p.(Asn2135LysfsTer3) families have origin in Alentejo, being the third most diagnosed BRCA event in the region, after c.156_157insAlu and c.5266dup; p.(Gln1756ProfsTer74); 3- c.3331_3334del; (p.Gln1111AsnfsTer5) was the only BRCA1 PV identified in Madeira (6 families). When comparing Portuguese and foreign pts, similar rates of breast, ovarian and prostate cancers were found (81.5%, 17.2% and 1.2% versus 82.1%, 16.7% and 2.4%, respectively). Most foreign residents lived in Lisboa and Vale do Tejo (82,1%) and their BRCA1:BRCA2 ratio was 0,87 (versus 0,46 in Portuguese pts), while in Algarve (13,5%) their BRCA1:BRCA2 was even higher (1.5 versus 0,69 in Portuguese pts). Discussion and conclusion:Our study confirms that BRCA2 PV are more frequent than BRCA1 PV in Portugal, with the exception of the Algarve region. Our data suggests that foreign pts accessing genetic testing in this region may have imbalanced the BRCA1:BRCA2 ratio, while for the whole population the imbalance towards BRCA2 is associated with the known founder BRCA2 c.156_157insAlu. Our data identified 2 BRCA1 (Algarve and Madeira) and 1 BRCA2 (Alentejo) geographical clusters. Citation Format: Inês Calvinho de Oliveira, Sofia Fragoso, Sidónia Santos, Teresa Duarte, Catarina Bexiga, Beatriz Mira, Isália Miguel, Ana Luís, Fátima Vaz. Geographical patterns of pathogenic genetic variants associated with hereditary breast, ovarian and prostate cancer (HBOPC) in Portugal [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-07-04.

In addition to classic germline APC gene variants, APC mosaicism and deep intronic germline APC variants have also been reported to be causes of adenomatous polyposis. In this study, we investigated 80 unexplained colorectal polyposis patients without germline pathogenic variants in known polyposis predisposing genes to detect mosaic and deep intronic APC variants. All patients developed more than 50 colorectal polyps, with adenomas being predominantly observed. To detect APC mosaicism, we performed next-generation sequencing (NGS) in leukocyte DNA. Furthermore, using Sanger sequencing, the cohort was screened for the following previously reported deep intronic pathogenic germline APC variants: c.1408 + 731C > T, p.(Gly471Serfs*55), c.1408 + 735A > T, p.(Gly471Serfs*55), c.1408 + 729A > G, p.(Gly471Serfs*55) and c.532-941G > A, p.(Phe178Argfs*22). We did not detect mosaic or intronic APC variants in the screened unexplained colorectal polyposis patients. The results of this study indicate that the deep intronic APC variants investigated in this study are not a cause of colorectal polyposis in this Dutch population. In addition, NGS did not detect any further mosaic variants in our cohort.

Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. However, mosaic variants in the MMR genes have been rarely described. We identified a likely de novo mosaic MSH6:c.1135_1139del p.Arg379* pathogenic variant in a patient diagnosed with suspected Lynch syndrome/Lynch-like syndrome. The patient developed MSH6-deficient EC and CRC at 54 and 58 years of age, respectively, without a detectable germline MMR pathogenic variant. Multigene panel sequencing of tumor and blood-derived DNA identified an MSH6 somatic mutation (MSH6:c.1135_1139del p.Arg379*) common to both the EC and CRC, raising suspicion of mosaicism. A droplet digital polymerase chain reaction (ddPCR) assay detected the MSH6 variant at 5.34% frequency in normal colonic tissue, 3.49% in saliva and 1.64% in blood DNA, demonstrating the presence of the MSH6 variant in all three germ layers. This study highlights the utility of tumor sequencing to guide sensitive ddPCR testing to detect low-level mosaicism in the MMR genes. Further investigation of the prevalence of MMR mosaicism is needed to inform routine diagnostic approaches and genetic counselling.

ACMG SF v3.0 list for reporting of secondary findings in clinical exome and genome sequencing: a policy statement of the American College of Medical Genetics and Genomics (ACMG)

Women diagnosed with breast cancer (BC) at an older age are less likely to undergo genetic cancer risk assessment and genetic testing since the guidelines and referrals are biased toward earlier age at diagnosis. Thus, we determined the prevalence and type of pathogenic cancer predisposition variants among women with a history of BC diagnosed at the age of 65 years or older vs younger than 65 years. Prospective registration cohort. The Clinical Cancer Genomics Community Research Network, including 40 community-based clinics in the United States and 5 in Latin America. Women with BC and genetic testing results. Sociodemographic characteristics, clinical variables, and genetic profiles were compared between women aged 65 years and older and those younger than 65 years at BC diagnosis. Among 588 women diagnosed with BC and aged 65 years and older and 9412 diagnosed at younger than 65 years, BC-associated pathogenic variants (PVs) were detected in 5.6% of those aged 65 years and older (n = 33) and 14.2% of those younger than 65 years (n = 1340) (P < .01). PVs in high-risk genes (eg, BRCA1 and BRCA2) represented 81.1% of carriers among women aged 65 years and older (n = 27) and 93.1% of those younger than 65 years (n = 1248) (P = .01). BRCA2 PVs represented 42.4% of high-risk gene findings for those aged 65 years and older, whereas BRCA1 PVs were most common among carriers younger than 65 years (49.7%). PVs (n = 7) in moderate-risk genes represented 21.2% for carriers aged 65 years and older and 7.3% of those younger than 65 years (n = 98; P < .01). CHEK2 PVs were the most common moderate-risk gene finding in both groups. Clinically actionable BC susceptibility PVs, particularly in BRCA2 and CHEK2, were relatively prevalent among older women undergoing genetic testing. The significant burden of PVs for older women with BC provides a critical reminder to recognize the full spectrum of eligibility and provide genetic testing for older women, rather than exclusion based on chronological age alone. J Am Geriatr Soc 67:884-888, 2019.

10584 Background: 5-10% of breast cancers are associated with germline pathogenic variants in breast cancer predisposition genes. 2-5% of these variants are in moderate penetrance (MP) genes, conferring a relative risk to develop breast cancer of 2-5. Various personal history (PH) and family history (FH)-based criteria for testing high penetrance genes, such as BRCA1 and BRCA2, have been proposed, but it is unknown to what extent women with pathogenic variants in MP genes may meet such criteria. We evaluated PH and FH of cancer among women with pathogenic variants in MP genes in a diverse and unselected patient cohort in New York City to determine how often they met well-established genetic testing criteria from the National Comprehensive Cancer Network (NCCN). Methods: Exome sequence data from ~16,000 female Bio Me Biobank participants were evaluated for expected pathogenic (per ClinVar, or predicted loss-of-function) variants in ATM, BARD1, BRIP1, CHEK2, NF1, PALB2, RAD51C, and RAD51D. We extracted demographic information, PH and FH of breast, ovarian, and/or pancreatic cancer, and PH and FH of genetic testing from participant questionnaires and electronic medical record review. We then determined which participants met current NCCN criteria (version 1.2023) for high penetrance breast, ovarian, and pancreatic cancer genetic testing. Results: We identified 252 women with expected pathogenic variants in MP genes: 105 CHEK2, 58 ATM, 35 BRIP1, 24 PALB2, 9 BARD1, 8 NF1, 8 RAD51D, and 5 RAD51C. 30% met NCCN criteria, including 30% of those with CHEK2 variants, 36% ATM, 14% BRIP1, 33% PALB2, and 30% with variants in BARD1, NF1, RAD51D, or RAD51C. 36% of those who met NCCN criteria had previously undergone clinical genetic testing. 29 of 33 (88%) women with a PH of breast cancer and 52 of 81 (64%) women with a FH of breast cancer met criteria. Conclusions: Current genetic testing criteria focus on identifying individuals harboring pathogenic variants in high penetrance cancer genes. However, in our study, a substantial portion of women with a pathogenic variant in a MP breast cancer gene also met such criteria, particularly when PH or FH of breast cancer were present. Only a third of those who met NCCN criteria had undergone clinical genetic testing. These findings suggest that the majority of women with genetic risk for breast cancer may be missed in clinical practice. Studies have shown that most physicians would change management strategies for patients with pathogenic variants, including variants in MP genes. As our understanding of genetic risk for breast cancer continues to evolve, we must explore strategies to improve identification and management of individuals with variants in MP breast cancer genes.

The objective of this study was to investigate the pathogenesis and inheritance pattern of a Chinese Han family with hereditary spastic paraplegia and to retrospectively analyze the characteristics of KIF1A gene variants and related clinical manifestations. High-throughput whole-exome sequencing was performed on members of a Chinese Han family with a clinical diagnosis of hereditary spastic paraplegia, and the sequencing results were validated by Sanger sequencing. Deep high-throughput sequencing was performed on subjects with suspected mosaic variants. The previously reported pathogenic variant loci of the KIF1A gene with complete data were collected, and the clinical manifestations and characteristics of the pathogenic KIF1A gene variant were analyzed. A pathogenic heterozygous variant located in the neck coil of the KIF1A gene (c.1139G>C, p.Arg380Pro) was identified in the proband and four additional members of the family. It was derived from the de novo low-frequency somatic-gonadal mosaicism of the proband's grandmother and had a rate of 10.95%. This study helps us to better understand the pathogenic mode and characteristics of mosaic variants, and to understand the location and clinical characteristics of pathogenic variants in KIF1A.

BackgroundCutaneous epidermal nevi are genotypically diverse mosaic disorders. Pathogenic hotspot variants in HRAS, KRAS, and less frequently, NRAS and BRAF may cause isolated keratinocytic epidermal nevi and sebaceous nevi or...