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Current clinical approaches for mutation discovery are based on short sequence reads (100-300 bp) of exons and flanking splice sites targeted by multigene panels or whole exomes. Short-read sequencing is highly accurate for detection of single nucleotide variants, small indels and simple copy number differences but is of limited use for identifying complex insertions and deletions and other structural rearrangements. We used CRISPR-Cas9 to excise complete BRCA1 and BRCA2 genomic regions from lymphoblast cells of patients with breast cancer, then sequenced these regions with long reads (>10 000 bp) to fully characterise all non-coding regions for structural variation. In a family severely affected with early-onset bilateral breast cancer and with negative (normal) results by gene panel and exome sequencing, we identified an intronic SINE-VNTR-Alu retrotransposon insertion that led to the creation of a pseudoexon in the BRCA1 message and introduced a premature truncation. This combination of CRISPR-Cas9 excision and long-read sequencing reveals a class of complex, damaging and otherwise cryptic mutations that may be particularly frequent in tumour suppressor genes replete with intronic repeats., Competing Interests: Competing interests: TW discloses consulting fees from Color Genomics outside the submitted work. M-CK is an American Cancer Society Research Professor., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: We hypothesized that mutations in homologous recombination repair (HRR) genes beyond BRCA1 and BRCA2 improve outcomes for ovarian carcinoma patients treated with platinum therapy and would impact the relative benefit of adding prolonged bevacizumab. Experimental Design: We sequenced DNA from blood and/or neoplasm from 1,195 women enrolled in GOG-0218, a randomized phase III trial in advanced ovarian carcinoma of bevacizumab added to carboplatin and paclitaxel. Defects in HRR were defined as damaging mutations in 16 genes. Proportional hazards models were used to estimate relative hazards for progression-free survival (PFS) and overall survival (OS). Results: Of 1,195 women with ovarian carcinoma, HRR mutations were identified in 307 (25.7%). Adjusted hazards for progression and death compared with those without mutations were lower for women with non- BRCA HRR mutations [HR = 0.73; 95% confidence interval (CI), 0.57-0.94; P = 0.01 for PFS; HR = 0.67; 95% CI, 0.50-0.90; P = 0.007 for OS] and BRCA1 mutations (HR = 0.80; 95% CI, 0.66-0.97; P = 0.02 for PFS; HR = 0.74; 95% CI, 0.59-0.94; P = 0.01 for OS) and were lowest for BRCA2 mutations (HR = 0.52; 95% CI, 0.40-0.67; P < 0.0001 for PFS; HR = 0.36; 95% CI, 0.25-0.53; P < 0.0001 for OS). A test of interaction showed no difference in the effect of bevacizumab on PFS between cases with and without mutations. Conclusions: HRR mutations, including non- BRCA genes, significantly prolong PFS and OS in ovarian carcinoma and should be stratified for in clinical trials. The benefit of adding bevacizumab was not significantly modified by mutation status. Clin Cancer Res; 24(4); 777-83. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Importance: Among Ashkenazi Jewish women, 3 mutations in BRCA1 and BRCA2 severely increase the risk of breast and ovarian cancer. However, among Ashkenazi Jewish patients with breast cancer who do not carry one of these founder mutations, the likelihood of carrying another pathogenic mutation in BRCA1 or BRCA2 or another breast cancer gene is not known. This information would be valuable to the patient and family for cancer prevention and treatment., Objective: To determine the frequency of cancer-predisposing mutations other than the BRCA1 and BRCA2 founder alleles among patients of Ashkenazi Jewish ancestry with breast cancer., Design, Setting, and Participants: In this cohort study, genomic DNA of women from 12 major cancer centers with a first diagnosis of invasive breast cancer who identified themselves and all 4 grandparents as Ashkenazi Jewish and participated in the New York Breast Cancer Study (NYBCS) from 1996 to 2000 was sequenced for known and candidate breast cancer genes. Data analysis was performed from July 10, 2014, to March 10, 2017., Main Outcomes and Measures: Genomic DNA from all 1007 NYBCS probands was sequenced for 23 known and candidate breast cancer genes using BROCA, a targeted multiplexed gene panel., Results: Of the 1007 probands in the study, 903 probands had no founder mutations in BRCA1 or BRCA2; of these probands, 7 (0.8%) carried another pathogenic mutation in BRCA1 or BRCA2, and 31 (3.4%) carried a pathogenic mutation in another breast cancer gene (29 in CHEK2, and 1 each in BRIP1 and NBN). Of all inherited predispositions to breast cancer in the NYBCS, 73.8% (104 of 142) were due to a BRCA1 or BRCA2 founder allele, 4.9% (7 of 142) to another BRCA1 or BRCA2 mutation, and 21.8% (31 of 142) to a mutation in another gene. Overall, 14.1% (142 of 1007) of Ashkenazi Jewish patients with breast cancer in the NYBCS carried a germline mutation responsible for their disease: 11.0% (111 of 1007) in BRCA1 or BRCA2, and 3.1% (31 of 1007) in CHEK2 or another breast cancer gene. Of the 111 patients with BRCA1 or BRCA2 mutations, 57 (51.4%) had a mother or sister with breast or ovarian cancer and 54 patients (48.6%) did not., Conclusions and Relevance: Comprehensive sequencing would provide complete relevant genetic information for Ashkenazi Jewish patients with breast cancer.

Purpose: Breast cancer-predisposing mutations PALB2 c.1027C>T (p.Gln343*) and PALB2 c.2167&#95;2168delAT have each been observed multiple times in breast cancer families of Italian ancestry. More recently, the c2167&#95;2168delAT mutation was identified in unrelated breast cancer cases of various ancestries. For each mutation, we investigated whether the origin was multiple mutational events (a "hot-spot") or a single event (a founder allele)., Methods: We genotyped and reconstructed haplotypes for 36 participants of Italian, Italian-American, Hispanic, and Nigerian ancestries, using seven short tandem repeat (STR) markers that covered 3 Megabases within and flanking PALB2 on chromosome 16., Results: For PALB2 c.1027C>T, a shared haplotype with a minimum size of 150 kb was shared by all 19 carriers investigated, all of Italian ancestry. This result suggests that this allele arose as a single event in a shared ancestor. For PALB2 c.2167&#95;2168delAT, all 12 carriers from American-Italian and Italian families shared a 1-Mb haplotype, the 3 Hispanic carriers shared a different haplotype of size 2 Mb, and the Nigerian carrier had different alleles at all 7 STR markers. These results suggest that PALB2 c.2167&#95;2168delAT arose multiple times, but that within each population, PALB2 c.2167&#95;2168delAT likely represents a single mutational event., Conclusion: We identified two PALB2 mutations that are founder alleles in Italian families, one of which is, independently, also a founder mutation in American-Hispanic breast cancers.

Purpose: Hallmarks of germline BRCA1/2-associated ovarian carcinomas include chemosensitivity and improved survival. The therapeutic impact of somatic BRCA1/2 mutations and mutations in other homologous recombination DNA repair genes is uncertain., Experimental Design: Using targeted capture and massively parallel genomic sequencing, we assessed 390 ovarian carcinomas for germline and somatic loss-of-function mutations in 30 genes, including BRCA1, BRCA2, and 11 other genes in the homologous recombination pathway., Results: Thirty-one percent of ovarian carcinomas had a deleterious germline (24%) and/or somatic (9%) mutation in one or more of the 13 homologous recombination genes: BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, MRE11A, NBN, PALB2, RAD51C, and RAD51D. Nonserous ovarian carcinomas had similar rates of homologous recombination mutations to serous carcinomas (28% vs. 31%, P = 0.6), including clear cell, endometrioid, and carcinosarcoma. The presence of germline and somatic homologous recombination mutations was highly predictive of primary platinum sensitivity (P = 0.0002) and improved overall survival (P = 0.0006), with a median overall survival of 66 months in germline homologous recombination mutation carriers, 59 months in cases with a somatic homologous recombination mutation, and 41 months for cases without a homologous recombination mutation., Conclusions: Germline or somatic mutations in homologous recombination genes are present in almost one third of ovarian carcinomas, including both serous and nonserous histologies. Somatic BRCA1/2 mutations and mutations in other homologous recombination genes have a similar positive impact on overall survival and platinum responsiveness as germline BRCA1/2 mutations. The similar rate of homologous recombination mutations in nonserous carcinomas supports their inclusion in PARP inhibitor clinical trials., (©2013 AACR.)

Genes disrupted in schizophrenia may be revealed by de novo mutations in affected persons from otherwise healthy families. Furthermore, during normal brain development, genes are expressed in patterns specific to developmental stage and neuroanatomical structure. We identified de novo mutations in persons with schizophrenia and then mapped the responsible genes onto transcriptome profiles of normal human brain tissues from age 13 weeks gestation to adulthood. In the dorsolateral and ventrolateral prefrontal cortex during fetal development, genes harboring damaging de novo mutations in schizophrenia formed a network significantly enriched for transcriptional coexpression and protein interaction. The 50 genes in the network function in neuronal migration, synaptic transmission, signaling, transcriptional regulation, and transport. These results suggest that disruptions of fetal prefrontal cortical neurogenesis are critical to the pathophysiology of schizophrenia. These results also support the feasibility of integrating genomic and transcriptome analyses to map critical neurodevelopmental processes in time and space in the brain., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The c.156&#95;157insAlu BRCA2 mutation has so far only been reported in hereditary breast/ovarian cancer (HBOC) families of Portuguese origin. Since this mutation is not detectable using the commonly used screening methodologies and must be specifically sought, we screened for this rearrangement in a total of 5,443 suspected HBOC families from several countries. Whereas the c.156&#95;157insAlu BRCA2 mutation was detected in 11 of 149 suspected HBOC families from Portugal, representing 37.9% of all deleterious mutations, in other countries it was detected only in one proband living in France and in four individuals requesting predictive testing living in France and in the USA, all being Portuguese immigrants. After performing an extensive haplotype study in carrier families, we estimate that this founder mutation occurred 558 ± 215 years ago. We further demonstrate significant quantitative differences regarding the production of the BRCA2 full length RNA and the transcript lacking exon 3 in c.156&#95;157insAlu BRCA2 mutation carriers and in controls. The cumulative incidence of breast cancer in carriers did not differ from that of other BRCA2 and BRCA1 pathogenic mutations. We recommend that all suspected HBOC families from Portugal or with Portuguese ancestry are specifically tested for this rearrangement.

Inherited loss-of-function mutations in the tumor suppressor genes BRCA1, BRCA2, and multiple other genes predispose to high risks of breast and/or ovarian cancer. Cancer-associated inherited mutations in these genes are collectively quite common, but individually rare or even private. Genetic testing for BRCA1 and BRCA2 mutations has become an integral part of clinical practice, but testing is generally limited to these two genes and to women with severe family histories of breast or ovarian cancer. To determine whether massively parallel, "next-generation" sequencing would enable accurate, thorough, and cost-effective identification of inherited mutations for breast and ovarian cancer, we developed a genomic assay to capture, sequence, and detect all mutations in 21 genes, including BRCA1 and BRCA2, with inherited mutations that predispose to breast or ovarian cancer. Constitutional genomic DNA from subjects with known inherited mutations, ranging in size from 1 to >100,000 bp, was hybridized to custom oligonucleotides and then sequenced using a genome analyzer. Analysis was carried out blind to the mutation in each sample. Average coverage was >1200 reads per base pair. After filtering sequences for quality and number of reads, all single-nucleotide substitutions, small insertion and deletion mutations, and large genomic duplications and deletions were detected. There were zero false-positive calls of nonsense mutations, frameshift mutations, or genomic rearrangements for any gene in any of the test samples. This approach enables widespread genetic testing and personalized risk assessment for breast and ovarian cancer.

Context: Genetic testing for inherited mutations in BRCA1 and BRCA2 has become integral to the care of women with a severe family history of breast or ovarian cancer, but an unknown number of patients receive negative (ie, wild-type) results when they actually carry a pathogenic BRCA1 or BRCA2 mutation. Furthermore, other breast cancer genes generally are not evaluated., Objective: To determine the frequency and types of undetected cancer-predisposing mutations in BRCA1, BRCA2, CHEK2, TP53, and PTEN among patients with breast cancer from high-risk families with negative (wild-type) genetic test results for BRCA1 and BRCA2., Design, Setting, and Participants: Between 2002-2005, probands from 300 US families with 4 or more cases of breast or ovarian cancer but with negative (wild-type) commercial genetic test results for BRCA1 and BRCA2 were screened by multiple DNA-based and RNA-based methods to detect genomic rearrangements in BRCA1 and BRCA2 and germline mutations of all classes in CHEK2, TP53, and PTEN., Main Outcome Measures: Previously undetected germline mutations in BRCA1, BRCA2, CHEK2, TP53, and PTEN that predispose to breast cancer; frequencies of these mutations among families with negative genetic test results., Results: Of the 300 probands, 52 (17%) carried previously undetected mutations, including 35 (12%) with genomic rearrangements of BRCA1 or BRCA2, 14 (5%) with CHEK2 mutations, and 3 (1%) with TP53 mutations. At BRCA1 and BRCA2, 22 different genomic rearrangements were found, of sizes less than 1 kb to greater than 170 kb; of these, 14 were not previously described and all were individually rare. At CHEK2, a novel 5.6-kb genomic deletion was discovered in 2 families of Czechoslovakian ancestry. This deletion was found in 8 of 631 (1.3%) patients with breast cancer and in none of 367 healthy controls in the Czech and Slovak Republics. For all rearrangements, exact genomic breakpoints were determined and diagnostic primers validated. The 3 families with TP53 mutations included cases of childhood sarcoma or brain tumors in addition to multiple cases of breast cancer., Conclusions: The mutational spectra of BRCA1 and BRCA2 include many high-penetrance, individually rare genomic rearrangements. Among patients with breast cancer and severe family histories of cancer who test negative (wild type) for BRCA1 and BRCA2, approximately 12% can be expected to carry a large genomic deletion or duplication in one of these genes, and approximately 5% can be expected to carry a mutation in CHEK2 or TP53. Effective methods for identifying these mutations should be made available to women at high risk.