Alison H. Trainer

Exome sequencing identifies rare deleterious mutations in DNA repair genes FANCC and BLM as potential breast cancer susceptibility alleles.

Despite intensive efforts using linkage and candidate gene approaches, the genetic etiology for the majority of families with a multi-generational breast cancer predisposition is unknown. In this study, we used whole-exome sequencing of thirty-three individuals from 15 breast cancer families to identify potential predisposing genes. Our analysis identified families with heterozygous, deleterious mutations in the DNA repair genes FANCC and BLM, which are responsible for the autosomal recessive disorders Fanconi Anemia and Bloom syndrome. In total, screening of all exons in these genes in 438 breast cancer families identified three with truncating mutations in FANCC and two with truncating mutations in BLM. Additional screening of FANCC mutation hotspot exons identified one pathogenic mutation among an additional 957 breast cancer families. Importantly, none of the deleterious mutations were identified among 464 healthy controls and are not reported in the 1,000 Genomes data. Given the rarity of Fanconi Anemia and Bloom syndrome disorders among Caucasian populations, the finding of multiple deleterious mutations in these critical DNA repair genes among high-risk breast cancer families is intriguing and suggestive of a predisposing role. Our data demonstrate the utility of intra-family exome-sequencing approaches to uncover cancer predisposition genes, but highlight the major challenge of definitively validating candidates where the incidence of sporadic disease is high, germline mutations are not fully penetrant, and individual predisposition genes may only account for a tiny proportion of breast cancer families.

The molecular mechanism underlying Roberts syndrome involves loss of ESCO2 acetyltransferase activity

Roberts syndrome/SC phocomelia (RBS) is an autosomal recessive disorder with growth retardation, craniofacial abnormalities and limb reduction. Cellular alterations in RBS include lack of cohesion at the heterochromatic regions around centromeres and the long arm of the Y chromosome, reduced growth capacity, and hypersensitivity to DNA damaging agents. RBS is caused by mutations in ESCO2, which encodes a protein belonging to the highly conserved Eco1/Ctf7 family of acetyltransferases that is involved in regulating sister chromatid cohesion. We identified 10 new mutations expanding the number to 26 known ESCO2 mutations. We observed that these mutations result in complete or partial loss of the acetyltransferase domain except for the only missense mutation that occurs in this domain (c.1615T>G, W539G). To investigate the mechanism underlying RBS, we analyzed ESCO2 mutations for their effect on enzymatic activity and cellular phenotype. We found that ESCO2 W539G results in loss of autoacetyltransferase activity. The cellular phenotype produced by this mutation causes cohesion defects, proliferation capacity reduction and mitomycin C sensitivity equivalent to those produced by frameshift and nonsense mutations associated with decreased levels of mRNA and absence of protein. We found decreased proliferation capacity in RBS cell lines associated with cell death, but not with increased cell cycle duration, which could be a factor in the development of phocomelia and cleft palate in RBS. In summary, we provide the first evidence that loss of acetyltransferase activity contributes to the pathogenesis of RBS, underscoring the essential role of the enzymatic activity of the Eco1p family of proteins.

The role of BRCA mutation testing in determining breast cancer therapy

Landmark discoveries in the field of breast cancer research include the identification of germline BRCA mutations as a cause of hereditary disease, and the use of gene-expression profiling to identify distinct subtypes of breast cancer. These findings, coupled with the availability of rapid germline testing, make it possible to identify a BRCA mutation carrier contemporaneous with a diagnosis of breast cancer. For the first time, testing for a germline mutation that predisposes to cancer has the potential to influence the immediate surgical, radiotherapeutic, and drug treatment choices of an individual with a new diagnosis of breast cancer. In this Review, we examine the implications of moving germline BRCA mutation testing from highly specialized family cancer clinics to mainstream settings.

Panel Testing for Familial Breast Cancer: Calibrating the Tension Between Research and Clinical Care

PURPOSE Gene panel sequencing is revolutionizing germline risk assessment for hereditary breast cancer. Despite scant evidence supporting the role of many of these genes in breast cancer predisposition, results are often reported to families as the definitive explanation for their family history. We assessed the frequency of mutations in 18 genes included in hereditary breast cancer panels among index cases from families with breast cancer and matched population controls. PATIENTS AND METHODS Cases (n = 2,000) were predominantly breast cancer-affected women referred to specialized Familial Cancer Centers on the basis of a strong family history of breast cancer and BRCA1 and BRCA2 wild type. Controls (n = 1,997) were cancer-free women from the LifePool study. Sequencing data were filtered for known pathogenic or novel loss-of-function mutations. RESULTS Excluding 19 mutations identified in BRCA1 and BRCA2 among the cases and controls, a total of 78 cases (3.9%) and 33 controls (1.6%) were found to carry potentially actionable mutations. A significant excess of mutations was only observed for PALB2 (26 cases, four controls) and TP53 (five cases, zero controls), whereas no mutations were identified in STK11. Among the remaining genes, loss-of-function mutations were rare, with similar frequency between cases and controls. CONCLUSION The frequency of mutations in most breast cancer panel genes among individuals selected for possible hereditary breast cancer is low and, in many cases, similar or even lower than that observed among cancer-free population controls. Although multigene panels can significantly aid in cancer risk management and expedite clinical translation of new genes, they equally have the potential to provide clinical misinformation and harm at the individual level if the data are not interpreted cautiously.

Genome-wide association study identifies a common variant in RAD51B associated with male breast cancer risk

We conducted a genome-wide association study of male breast cancer comprising 823 cases and 2,795 controls of European ancestry, with validation in independent sample sets totaling 438 cases and 474 controls. A SNP in RAD51B at 14q24.1 was significantly associated with male breast cancer risk (P = 3.02 × 10−13; odds ratio (OR) = 1.57). We also refine association at 16q12.1 to a SNP within TOX3 (P = 3.87 × 10−15; OR = 1.50).

Analysis of RAD51C germline mutations in high‐risk breast and ovarian cancer families and ovarian cancer patients

There is strong evidence that overtly inactivating mutations in RAD51C predispose to hereditary breast and ovarian cancer but the prevalence of such mutations, and whether they are associated with a particular clinical phenotype, remains unclear. Resolving these questions has important implications for the implementation of RAD51C into routine clinical genetic testing. Consequently, we have performed a large RAD51C mutation screen of hereditary breast and ovarian cancer families, and the first study of unselected patients diagnosed with ovarian cancer. Our data confirm a consistent but low frequency (2/335 families) of inactivating RAD51C mutations among families with a history of both breast and ovarian cancer and an absence of mutations among breast cancer only families (0/1,053 families). Our data also provide support for the designation of the missense variant p.Gly264Ser as a moderate penetrance allele. Hum Mutat 33:95–99, 2012.

Familial cardiological and targeted genetic evaluation: Low yield in sudden unexplained death and high yield in unexplained cardiac arrest syndromes

BACKGROUND It has been reported that cardiological screening and genetic evaluation in relatives of families with sudden unexplained death syndrome and unexplained cardiac arrest (UCA) may uncover a heritable etiology in a significant proportion of families. OBJECTIVE To evaluate the yield of a comprehensive evaluation protocol of a large unselected cohort of consecutive families with autopsy-negative sudden unexplained death syndrome (termed sudden arrhythmic death syndrome [SADS]) and UCA. METHODS We studied (1) 109 consecutive families (411 relatives) referred with 1 or more sudden deaths in the family and (2) 52 consecutive probands with UCA (91 relatives) referred by cardiologists between January 2007 and December 2012. A comprehensive cardiological screening was performed followed by targeted genetic evaluation if a clinical phenotype was proven or suspected. Diagnosis was made by a multidisciplinary team using published clinical criteria. RESULTS A diagnosis was made in 19 of 109 families with SADS (yield 18%), with the majority having long QT syndrome (LQTS). Diagnosis varied according to proband age, with LQTS most common in the very young (≤20 years) and Brugada syndrome in the older age probands (≥40 years) (P = .03). In contrast, a diagnosis was made in 32 of 52 families with UCA (yield 62%), the majority of which had LQTS and Brugada syndrome. No clinical or circumstantial factors increased the likelihood of diagnosis in families with either SADS or UCA. CONCLUSIONS In contrast to previously published series, a comprehensive strategy of cardiological evaluation and targeted genetic testing in more than 100 families with SADS was found to have a lower diagnostic yield (18%). Diagnostic yield in families with UCA was approximately 4 times higher (62%), which is consistent with the published literature.

A Role for Common Genomic Variants in the Assessment of Familial Breast Cancer

PURPOSE Genome-wide association studies have identified common genomic variants associated with increased susceptibility to breast cancer. In the general population, the risk associated with these known variants seems insufficient to inform clinical management. Their contribution to the development of familial breast cancer is less clear. PATIENTS AND METHODS We studied 1,143 women with breast cancer who had completed BRCA1 and BRCA2 mutation screening as a result of a high risk for hereditary breast cancer. Genotyping of 22 breast cancer-associated genomic variants was performed. A polygenic risk score (PRS), calculated as the sum of the log odds ratios for each allele, was compared with the same metric in 892 controls from the Australian Ovarian Cancer Study. The clinical features associated with the high and low ends of the polygenic risk distribution were compared. RESULTS Women affected by familial breast cancer had a highly significant excess of risk alleles compared with controls (P = 1.0 × 10(-16)). Polygenic risk (measured by the PRS) was greater in women who tested negative for a BRCA1 or BRCA2 mutation compared with mutation carriers (P = 2.3 × 10(-6)). Non-BRCA1/2 women in the top quartile of the polygenic risk distribution were more likely to have had early-onset breast cancer (< 30 years of age, odds ratio [OR]= 3.37, P = .03) and had a higher rate of second breast cancer (OR 1.96, P = .02) compared with women with low polygenic risk. CONCLUSION Genetic testing for common risk variants in women undergoing assessment for familial breast cancer may identify a distinct group of high-risk women in whom the role of risk-reducing interventions should be explored.

Phenotypic variability in 49 cases of ESCO2 mutations, including novel missense and codon deletion in the acetyltransferase domain, correlates with ESCO2 expression and establishes the clinical criteria for Roberts syndrome

Background Roberts syndrome (RBS) and SC phocomelia are caused by mutations in ESCO2, which codes for an acetyltransferase involved in the regulation of sister chromatid cohesion. Of 26 mutations described to date, only one missense mutation has been reported and all others are predicted to be truncating mutations. Genotype–phenotype analysis has been hampered by limited numbers of patients with clinical information available. Objective To provide unpublished clinical data for 31 patients with proven ESCO2 mutations and combine this series with previously reported clinical and mutation data on 18 cases. Methods Genotype–phenotype correlations and functional effects of two novel ESCO2 mutations were analysed. In situ hybridisation on human embryos at Carnegie stages 14, 17 and 21 was performed to study ESCO2 expression during development. Results and conclusions Using the cohort of 49 patients, the clinical criteria for RBS were delineated to include: growth retardation; symmetric mesomelic shortening of the limbs in which the upper limbs are more commonly and severely affected than the lower limbs; characteristic facies with microcephaly. The severity of malformations of the facies correlates with the severity of limb reduction. The occurrence of corneal opacities may be associated with specific mutations. Two new mutations, both in the ESCO2 acetyltransferase domain, are described and their acetylation effects in vitro demonstrated. In situ hybridisation on human embryos showed ESCO2 expression in the brain, face, limb, kidney and gonads, which corresponds to the structures affected in RBS.

The genomic landscape of phaeochromocytoma

Phaeochromocytomas (PCCs) and paragangliomas (PGLs) are rare neural crest‐derived tumours originating from adrenal chromaffin cells or extra‐adrenal sympathetic and parasympathetic tissues. More than a third of PCC/PGL cases are associated with heritable syndromes involving 13 or more known genes. These genes have been broadly partitioned into two groups based on pseudo‐hypoxic and receptor tyrosine kinase (RTK) signalling pathways. Many of these genes can also become somatically mutated, although up to one third of sporadic cases have no known genetic driver. Furthermore, little is known of the genes that co‐operate with known driver genes to initiate and drive tumourigenesis. To explore the genomic landscape of PCC/PGL, we applied exome sequencing, high‐density SNP‐array analysis, and RNA sequencing to 36 PCCs and four functional PGL tumours. All tumours displayed low mutation frequency, in contrast to frequent large segmental copy‐number alterations, aneuploidy, and evidence for chromothripsis in one case. Multi‐region sampling of one benign familial PCC tumour provided evidence for the timing of mutations during tumourigenesis and ongoing clonal evolution. Thirty‐one of 40 (77.5%) cases could be explained by germline or somatic mutations or structural alterations affecting known PCC/PGL genes. Deleterious somatic mutations were also identified in known tumour‐suppressor genes associated with genome maintenance and epigenetic modulation. A multitude of other genes were also found mutated that are likely important for normal neuroendocrine cell function. We revisited the gene‐expression subtyping of PCC/PGL by integrating published microarray data with our RNA‐seq data, enabling the identification of six robust gene‐expression subtypes. The majority of cases in our cohort with no identifiable driver mutation were classified into a gene‐expression subtype bearing similarity to MAX mutant PCC/PGL. Our data suggest there are yet unknown PCC/PGL cancer genes that can phenocopy MAX mutant PCC/PGL tumours. This study provides new insight into the molecular diversity and genetic origins of PCC/PGL tumours. Copyright