Ramūnas Janavičius

Founder BRCA1/2 mutations in the Europe: implications for hereditary breast-ovarian cancer prevention and control

Detection of mutations in hereditary breast and ovarian cancer-related BRCA1 and BRCA2 genes is an effective method of cancer prevention and early detection. Different ethnic and geographical regions have different BRCA1 and BRCA2 mutation spectrum and prevalence. Along with the emerging targeted therapy, demand and uptake for rapid BRCA1/2 mutations testing will increase in a near future. However, current patients selection and genetic testing strategies in most countries impose significant lag in this practice. The knowledge of the genetic structure of particular populations is important for the developing of effective screening protocol and may provide more efficient approach for the individualization of genetic testing. Elucidating of founder effect in BRCA1/2 genes can have an impact on the management of hereditary cancer families on a national and international healthcare system level, making genetic testing more affordable and cost-effective. The purpose of this review is to summarize current evidence about the BRCA1/2 founder mutations diversity in European populations.

Comprehensive BRCA1 and BRCA2 mutational profile in Lithuania

There is limited knowledge about the BRCA1/2 mutational profile in Lithuania. We aimed to define the full BRCA1 and BRCA2 mutational spectrum and the clinically relevant prevalence of these gene mutations in Lithuania. A data set of 753 unrelated probands, recruited through a clinical setting, was used and consisted of 380 female breast cancer cases, 213 epithelial ovarian cancer cases, 20 breast and ovarian cancer cases, and 140 probands with positive family history of breast or ovarian cancer. A comprehensive mutation analysis of the BRCA1/2 genes by high resolution melting analysis coupled with Sanger sequencing and multiplex ligation-dependent probe amplification analysis was performed. Genetic analysis revealed 32 different pathogenic germline BRCA1/2 mutations: 20 in the BRCA1 gene and 12 in the BRCA2 gene, including four different large genomic rearrangements in the BRCA1 gene. In all, 10 novel BRCA1/2 mutations were found. Nine different recurrent BRCA1 mutations and two recurrent BRCA2 mutations were identified, which comprised 90.4% of all BRCA1/2 mutations. BRCA1 exon 1-3 deletion and BRCA2 c.658_659del are reported for the first time as recurrent mutations, pointing to a possible Baltic founder effect. Approximately 7% of breast cancer and 22% of ovarian cancer patients without family history and an estimated 0.5-0.6% of all Lithuanian women were found to be carriers of mutations in the BRCA1 or BRCA2 gene.

Novel and common BRCA1 mutations in familial breast/ovarian cancer patients from Lithuania

The germline mutations in the BRCA1 (MIM 113705) and BRCA2 (MIM600185) genes account for the majority of hereditary breast and ovarian cancers cases. Founder mutations in BRCA1 gene have been recently described in Eastern Baltic Sea region, with variable distribution in Baltic (Latvian [1] and Lithuanian [2]) and Slavic (Polish [3], Belorussian [4], Russian [5]) populations, accounting for 80–90% of all mutations. However, data about its incidence in Lithuania are inconclusive and based on one study where only three mutations were tested in BRCA1 gene [2]. Here we present the complexity of BRCA1 gene mutation profile in familial breast and/or ovarian cancer index cases referred to Vilnius University Hospital Santariskiu Clinics from 1 year period (2006–2007).

Identification and Functional Testing of ERCC2 Mutations in a Multi-national Cohort of Patients with Familial Breast- and Ovarian Cancer.

The increasing application of gene panels for familial cancer susceptibility disorders will probably lead to an increased proposal of susceptibility gene candidates. Using ERCC2 DNA repair gene as an example, we show that proof of a possible role in cancer susceptibility requires a detailed dissection and characterization of the underlying mutations for genes with diverse cellular functions (in this case mainly DNA repair and basic cellular transcription). In case of ERCC2, panel sequencing of 1345 index cases from 587 German, 405 Lithuanian and 353 Czech families with breast and ovarian cancer (BC/OC) predisposition revealed 25 mutations (3 frameshift, 2 splice-affecting, 20 missense), all absent or very rare in the ExAC database. While 16 mutations were unique, 9 mutations showed up repeatedly with population-specific appearance. Ten out of eleven mutations that were tested exemplarily in cell-based functional assays exert diminished excision repair efficiency and/or decreased transcriptional activation capability. In order to provide evidence for BC/OC predisposition, we performed familial segregation analyses and screened ethnically matching controls. However, unlike the recently published RECQL example, none of our recurrent ERCC2 mutations showed convincing co-segregation with BC/OC or significant overrepresentation in the BC/OC cohort. Interestingly, we detected that some deleterious founder mutations had an unexpectedly high frequency of > 1% in the corresponding populations, suggesting that either homozygous carriers are not clinically recognized or homozygosity for these mutations is embryonically lethal. In conclusion, we provide a useful resource on the mutational landscape of ERCC2 mutations in hereditary BC/OC patients and, as our key finding, we demonstrate the complexity of correct interpretation for the discovery of “bonafide” breast cancer susceptibility genes.

Cumulative Small Effect Genetic Markers and the Risk of Colorectal Cancer in Poland, Estonia, Lithuania, and Latvia.

The continued identification of new low-penetrance genetic variants for colorectal cancer (CRC) raises the question of their potential cumulative effect among compound carriers. We focused on 6 SNPs (rs380284, rs4464148, rs4779584, rs4939827, rs6983267, and rs10795668), already described as risk markers, and tested their possible independent and combined contribution to CRC predisposition. Material and Methods. DNA was collected and genotyped from 2330 unselected consecutive CRC cases and controls from Estonia (166 cases and controls), Latvia (81 cases and controls), Lithuania (123 cases and controls), and Poland (795 cases and controls). Results. Beyond individual effects, the analysis revealed statistically significant linear cumulative effects for these 6 markers for all samples except of the Latvian one (corrected P value = 0.018 for the Estonian, corrected P value = 0.0034 for the Lithuanian, and corrected P value = 0.0076 for the Polish sample). Conclusions. The significant linear cumulative effects demonstrated here support the idea of using sets of low-risk markers for delimiting new groups with high-risk of CRC in clinical practice that are not carriers of the usual CRC high-risk markers.

Lynch syndrome mutations shared by the Baltic States and Poland.

To the Editor : The definite diagnosis of Lynch syndrome (LS) is based on the identification of a mutation within one of the DNA mismatch repair (MMR) genes: MLH1 , MSH2 , MSH6 and PMS2 . To date, hundreds of pathogenic and suspected-pathogenic DNA variants in the MMR genes have been described worldwide (1). Large rearrangements account for about 10% of all MMR changes and can be easily detected by multiplex ligation-dependent probe amplification (MLPA), whereas substitutions, small deletions and insertions account for about 90% (2) and only full screening using either denaturing high-performance liquid chromatography (DHPLC), high resolution melting (HRM) or direct DNA sequencing seems to be the option for genetic analysis. Despite significant progress in the detection of gene mutations full screening remains expensive and time-consuming. According to the previous studies, majority of Polish families are affected by recurrent mutations found at least twice in our own series (2). That tendency provided an opportunity to design a common inexpensive assay for recurrent mutations, an approach that has been successfully applied as prescreening method (3). Founder mutations have been reported from a number of countries and geographical regions and are thought to be a result of population bottlenecks occurring throughout history. It is well recognized that populations from neighboring countries can share genetic changes and as such the probability of finding common mutation is greater. With respect to the regions bounded by Estonia, Latvia, Lithuania and Poland there are reports of common mutations occurring in these populations (4, 5).

Apparently "BRCA-related" breast and ovarian cancer patient with germline TP53 mutation.

Abstract:  Germline TP53 gene mutations are associated with complex cancer predisposition syndrome, the Li–Fraumeni syndrome, and are not as rare as were previously thought. Currently, the identification of Li–Fraumeni syndrome is mostly based on a conformance to descriptive criteria, which recently were amended to include wider spectrum of malignancies. The presence of very young age‐onset breast cancers in TP53 mutations families is a feature that overlaps with hereditary breast/ovarian cancer families with BRCA1/2 genes mutations. Peri‐diagnostic germline TP53 testing results in breast cancer patients can significantly affect surgical and adjuvant radiotherapy choices. The aim of this case report is to emphasize the importance of peri‐diagnostic germline TP53 molecular testing in patients with early‐onset breast cancer and its effect on the management and outcome of the disease. We present the apparent BRCA1‐related, although mutation negative, breast and ovarian cancer patient who subsequently was confirmed to be TP53 c.817C>T (p.R273C) mutation carrier and discuss the importance of peri‐diagnostic oncogenetic TP53 testing in early breast cancer cases. Histopathology and genetic modifiers (MDM2 SNP309G; TP53 R72P, PIN3) data are also addressed.

Extremely low risk of pheochromocytomas in complete VHL gene deletion cases.

We read with great interest article by Franke et al. [2009] on the extensive characterization of large deletion breakpoints and delineation of Alu-mediated recombination mechanism in the gross deletions of von Hippel-Lindau (VHL) syndrome. Franke et al. confirmed previous observations [Cascon et al., 2007; Maranchie et al., 2004] and our data on the role of HSPC300 gene retention for increased risk of renal cell carcinoma (RCC) and also observed increased risk for retinal angiomas [Franke et al., 2009]. However, misleading interpretation of the pheochromocytoma risk needs particular caution because of the significant clinical implication. Franke et al. [2009] declared that their results did not support previous observations on reduced risk of pheochromocytomas in VHL deletion families, which were found in 18% (7/39) of gross VHL deletion informative families (from the 54 studied overall). It should be stressed, however, that this estimation was presented by pooling informative complete VHL gene deletion families (n 5 14) with partial VHL deletion families (n 5 25). Hes et al. [2000] and Cybulski et al. [2002] were the first to discriminate these two different entities, which previously were interchangeably grouped into gross rearrangement group, and showed the phenotypic differences between complete and partial VHL deletion cases (in respect of reduced RCC and pheochromocytoma frequency observed in patients with complete VHL gene deletions). Franke et al. [2009] did not discriminate these two types of families, and used them interchangeably for the calculations. Indeed, Franke et al. [2009] found only one pheochromocytoma case (family 48) in the complete VHL gene deletion informative family (1/14), although number of analyzed patients with complete VHL deletion is unknown. This is actually in agreement with previous observations of low pheochromocytomas risk in complete VHL deletion families [Cascon et al., 2007; Cybulski et al., 2002; Hes et al., 2000, 2007; Huang et al., 2007; Maranchie et al., 2004; Ong et al., 2007]. To our knowledge, there were at least 71 complete VHL gene deletion cases reported in the literature with available clinical data (including three of our patients), which are summarized in Table 1. There were no pheochromocytoma observed in any of these cases and even after the inclusion of Franke et al. [2009] data, overall pheochromocytomas risk should comprise less than 1% (1/85) in whole VHL deletion cases, what is extremely low. It would be also worthwhile to substantiate the first reported case of pheochromocytoma in more detail (age at onset, activity, symptoms, etc.). Conversely, pheochromocytoma phenotype is expressed in up to 14% (2/14) of partial VHL gene deletion cases [Cascon et al., 2007], with pooled average of 5% (9/195) (Table 1). Franke et al. [2009] observed pheochromocytomas in 17% (6/36) of informaOFFICIAL JOURNAL

Abstract LB-044: Germline mutations in patients with hereditary breast and ovarian cancer establish ERCC2 as a cancer susceptibility gene

Introduction: Breast and ovarian cancer (BC/OC) predisposition is associated with a number of high- and low-penetrance susceptibility genes. Despite comprehensive testing there is still a large portion of high risk cases without mutation in any of the known susceptibility loci. Therefore novel candidate genes need to be identified. Here we report on the results of testing 94 genes in 717 BC/OC patients from Germany and Lithuania. Method: Inclusion criteria for the patients in this study were defined by the German Consortium for Breast and Ovarian Cancer. Next generation sequencing (NGS) was performed on an Illumina MiSeq sequencer, with 150 bp paired end sequencing chemistry and an average base coverage of 300 fold. Target enrichment was performed with the Illumina TruSight cancer panel, which includes 94 genes associated with both common (e.g., breast, colorectal) and rare cancers. Results: In 19.7% of the patients, BRCA1 or BRCA2 variations have been found. These were either clearly pathogenic loss-of-function mutations (43%) or very rare, unclassified missense variations with high probability of a deleterious effect (57%). In 17.9% of the patients we found null-mutations and rare, unclassified missense variants in the acknowledged BC/OC susceptibility genes ATM, CDH1, CHEK2, NBN, PALB2, RAD51C/D and TP53. Analysis of the non-BC/OC genes on the NGS panel identified the “excision repair cross-complementing rodent repair deficiency, complementation group 2” gene (ERCC2 or XPD) as a promising BC/OC predisposition candidate: we found 3 frame-shift mutations and 1 splice-site mutation in four independent BC/OC families. Additionally we found 20 rare, unclassified sequence variations in ERCC2. These variants have a cumulative allele frequency of 2.9% in our BC/OC cohort, which is 14.5-fold overrepresentation compared to the “exome aggregation consortium” (ExAC) cohort (61486 exomes). In all affected families tested so far, the ERCC2 mutations co-segregate with the occurrence of BC and/or OC. Functional assays testing the ERCC2 variants have been initiated. First results show that at least some of the missense variants (e.g. NM_000400.3:p.Val536Met) have lost their DNA repair ability. Conclusion: Deleterious mutations and probably pathogenic missense variations in ERCC2, which are significantly overrepresented in our BC/OC families and co-segregate with the affected individuals, define ERCC2 clearly as a susceptibility gene for BC/OC predisposition. Functional assays will be continued in order to identify missense variations that diminish the DNA-repair capacity of ERCC2. Affected individuals with excluded mutations in the known BC/OC predisposition genes should be tested for mutations in ERCC2. Citation Format: Evelin Schrock, Anna Benet-Pages, Steffen Schubert, Ramūnas Janavicius, Karl Hackmann, Elitza Betcheva-Krajcir, Luisa Mackenroth, Janin Lehmann, AM Nissen, Janine Altmueller, Holger Thiele, Nataliya Di Donato, Barbara Klink, Jan D. Kuhlmann, Andreas Tzschach, Karin Kast, Pauline Wimberger, Elke Holinski-Feder, Alfons Meindl, Steffen Emmert, Andreas Rump. Germline mutations in patients with hereditary breast and ovarian cancer establish ERCC2 as a cancer susceptibility gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-044. doi:10.1158/1538-7445.AM2015-LB-044

Multiple endocrine neoplasia type 1: a case report and review of the literature

Multiple endocrine neoplasia syndrome, type 1 (MEN1) is an underdiagnosed autosomal dominant inherited cancer predisposition syndrome with inter- and intrafamilial variability without a known genotype-phenotype correlation. Disease is caused by mutations in the MEN1 gene located on chromosome 11, but other genes (CDKN1B, AIP) and mechanisms might be involved too. We performed retrospective case series study of MEN1 syndrome patient and his family and present our experience of management of genetically confirmed 9 MEN1 syndrome large family members from Lithuania with novel MEN1 gene mutation (MEN1 exon 6 — c. 879delT (p.Pro293Profs*76)) and delineate its clinical phenotype. At present the diagnosis of MEN1 syndrome must be established by direct mutation testing. MEN1 syndrome patients, their relatives and patients suspected of MEN1 are eligible for mutation testing. Patients with MEN1 have a shorter life expectancy than the general population. MEN1 patients and mutation carriers should be subjected to periodic screening in order to detect manifestations in an early stage. Early genetic diagnosis and subsequent periodic screening is associated with less morbidity and mortality at follow-up. Our study confirmed the absence of genotype-phenotype correlation and showed high intrafamilial clinical expression variability of the MEN1 syndrome.