Tuomo Mantere

Targeted Next-Generation Sequencing Identifies a Recurrent Mutation in MCPH1 Associating with Hereditary Breast Cancer Susceptibility.

Breast cancer is strongly influenced by hereditary risk factors, a majority of which still remain unknown. Here, we performed a targeted next-generation sequencing of 796 genes implicated in DNA repair in 189 Finnish breast cancer cases with indication of hereditary disease susceptibility and focused the analysis on protein truncating mutations. A recurrent heterozygous mutation (c.904_916del, p.Arg304ValfsTer3) was identified in early DNA damage response gene, MCPH1, significantly associating with breast cancer susceptibility both in familial (5/145, 3.4%, P = 0.003, OR 8.3) and unselected cases (16/1150, 1.4%, P = 0.016, OR 3.3). A total of 21 mutation positive families were identified, of which one-third exhibited also brain tumors and/or sarcomas (P = 0.0007). Mutation carriers exhibited significant increase in genomic instability assessed by cytogenetic analysis for spontaneous chromosomal rearrangements in peripheral blood lymphocytes (P = 0.0007), suggesting an effect for MCPH1 haploinsufficiency on cancer susceptibility. Furthermore, 40% of the mutation carrier tumors exhibited loss of the wild-type allele. These findings collectively provide strong evidence for MCHP1 being a novel breast cancer susceptibility gene, which warrants further investigations in other populations.

Case-control analysis of truncating mutations in DNA damage response genes connects TEX15 and FANCD2 with hereditary breast cancer susceptibility

Several known breast cancer susceptibility genes encode proteins involved in DNA damage response (DDR) and are characterized by rare loss-of-function mutations. However, these explain less than half of the familial cases. To identify novel susceptibility factors, 39 rare truncating mutations, identified in 189 Northern Finnish hereditary breast cancer patients in parallel sequencing of 796 DDR genes, were studied for disease association. Mutation screening was performed for Northern Finnish breast cancer cases (n = 578–1565) and controls (n = 337–1228). Mutations showing potential cancer association were analyzed in additional Finnish cohorts. c.7253dupT in TEX15, encoding a DDR factor important in meiosis, associated with hereditary breast cancer (p = 0.018) and likely represents a Northern Finnish founder mutation. A deleterious c.2715 + 1G > A mutation in the Fanconi anemia gene, FANCD2, was over two times more common in the combined Finnish hereditary cohort compared to controls. A deletion (c.640_644del5) in RNF168, causative for recessive RIDDLE syndrome, had high prevalence in majority of the analyzed cohorts, but did not associate with breast cancer. In conclusion, truncating variants in TEX15 and FANCD2 are potential breast cancer risk factors, warranting further investigations in other populations. Furthermore, high frequency of RNF168 c.640_644del5 indicates the need for its testing in Finnish patients with RIDDLE syndrome symptoms.

FANCM c.5101C > T mutation associates with breast cancer survival and treatment outcome

Breast cancer (BC) is a heterogeneous disease, and different tumor characteristics and genetic variation may affect the clinical outcome. The FANCM c.5101C > T nonsense mutation in the Finnish population associates with increased risk of breast cancer, especially for triple‐negative breast cancer patients. To investigate the association of the mutation with disease prognosis, we studied tumor phenotype, treatment outcome, and patient survival in 3,933 invasive breast cancer patients, including 101 FANCM c.5101C > T mutation carriers and 3,832 non‐carriers. We also examined association of the mutation with nuclear immunohistochemical staining of DNA repair markers in 1,240 breast tumors. The FANCM c.5101C > T mutation associated with poor 10‐year breast cancer‐specific survival (hazard ratio (HR)=1.66, 95% confidence interval (CI) 1.09–2.52, p = 0.018), with a more pronounced survival effect among familial cases (HR = 2.93, 95% CI 1.5–5.76, p = 1.80 × 10−3). Poor disease outcome of the carriers was also found among the estrogen receptor (ER) positive subgroup of patients (HR = 1.8, 95% CI 1.09–2.98, p = 0.021). Reduced survival was seen especially among patients who had not received radiotherapy (HR = 3.43, 95% CI 1.6–7.34, p = 1.50 × 10−3) but not among radiotherapy treated patients (HR = 1.35, 95% CI 0.82–2.23, p = 0.237). Significant interaction was found between the mutation and radiotherapy (p = 0.040). Immunohistochemical analyses show that c.5101C > T carriers have reduced PAR‐activity. Our results suggest that FANCM c.5101C > T nonsense mutation carriers have a reduced breast cancer survival but postoperative radiotherapy may diminish this survival disadvantage.

Rare missense mutations in RECQL and POLG associate with inherited predisposition to breast cancer: Breast cancer risk alleles in RECQL & POLG

Several known breast cancer susceptibility genes with moderate‐to‐high risk alleles encode proteins involved in DNA damage response (DDR). As these explain less than half of the hereditary breast cancer cases, additional predisposing alleles are likely to be discovered. Many of the previous studies utilizing massive parallel sequencing have focused on the protein‐truncating variants, and the role of rare missense mutations has remained poorly addressed. To identify novel susceptibility factors, we have systematically analyzed the data from our parallel sequencing of 796 DDR genes in 189 Northern Finnish hereditary breast cancer patients for rare missense variants, predicted as deleterious. Thirty‐five variants were studied here for the disease association using Finnish breast cancer case (n = 492–2,035) and control (n = 277–1,539) cohorts. As a result, two missense variants in genes involved in DNA replication, RECQL p.I156M and POLG p.L392V, the former involving genomic and the latter mitochondrial DNA replication, showed significant association with risk of breast cancer. Rare RECQL p.I156M allele was observed in breast cancer cases only (6/1,946, 0.3%, p = 0.043), whereas POLG p.L392V was two times more frequent in breast cancer cases (53/2,238, 2.4%) compared to controls (18/1,539, 1.2%, OR = 2.1, 95% CI 1.2–3.5, p = 0.010). Based on the current genetic data, both RECQL p.I156M and POLG p.L392V represent novel breast cancer predisposing alleles.

Primary Myocardial Fibrosis as an Alternative Phenotype Pathway of Inherited Cardiac Structural Disorders

Background: Myocardial fibrosis is a common postmortem finding among young individuals with sudden cardiac death. Because there is no known single cause, we tested the hypothesis that some cases of myocardial fibrosis in the absence of identifiable causes (primary myocardial fibrosis [PMF]) are associated with genetic variants. Methods: Tissue was obtained at autopsy from 4031 consecutive individuals with sudden cardiac death in Northern Finland, among whom PMF was the only structural finding in 145 subjects with sudden cardiac death. We performed targeted next-generation sequencing using a panel of 174 genes associated with myocardial structure and ion channel function when autopsies did not identify a secondary basis for myocardial fibrosis. All variants with an effect on protein and with a minor allele frequency <0.01 were classified as pathogenic or variants of uncertain significance on the basis of American College of Medical Genetics consensus guidelines. Results: Among the 96 specimens with DNA passing quality control (66%), postmortem genetic tests identified 24 variants of known or uncertain significance in 26 subjects (27%). Ten were pathogenic/likely pathogenic variants in 10 subjects (10%), and 14 were variants of uncertain significance in 11 genes among 16 subjects (17%). Five variants were in genes associated with arrhythmogenic right ventricular cardiomyopathy, 6 in hypertrophic cardiomyopathy–associated genes, and 11 in dilated cardiomyopathy–associated genes; 2 were not associated with these disorders. Four unique variants of uncertain significance cosegregated among multiple unrelated subjects with PMF. No pathogenic/likely pathogenic variants were detected in ion channel–encoding genes. Conclusions: A large proportion of subjects with PMF at autopsy had variants in genes associated with arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy, and hypertrophic cardiomyopathy without autopsy findings of those diseases, suggesting that PMF can be an alternative phenotypic expression of structural disease–associated genetic variants or that risk-associated fibrosis was expressing before the primary disease. These findings have clinical implications for postmortem genetic testing and family risk profiling.

Mutation of TP53, translocation analysis and immunohistochemical expression of MYC, BCL-2 and BCL-6 in patients with DLBCL treated with R-CHOP

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma with diverse outcomes. Concurrent translocation of MYC and BCL-2 and/or BCL-6, and concurrent immunohistochemical (IHC) high expression of MYC and BCL-2, have been linked to unfavorable treatment responses. TP53-mutated DLBCL has also been linked to worse outcome. Our aim was to evaluate the aforementioned issues in a cohort of 155 patients uniformly treated with R-CHOP-like therapies. We performed direct sequencing of TP53 exons 5, 6, 7 and 8 as well as fluorescence in-situ hybridization (FISH) of MYC, BCL-2 and BCL-6, and IHC of MYC, BCL-2 and BCL-6. In multivariate analysis, TP53 mutations in L3 and loop-sheet helix (LSH) associated with a risk ratio (RR) of disease-specific survival (DSS) of 8.779 (p = 0.022) and a RR of disease-free survival (DFS) of 10.498 (p = 0.011). In IHC analysis BCL-2 overexpression was associated with inferior DFS (p = 0.002) and DSS (p = 0.002). DLBCL with BCL-2 and MYC overexpression conferred inferior survival in all patients (DSS, p = 0.038 and DFS, p = 0.011) and in patients with non-GC phenotype (DSS (p = 0.013) and DFS (p = 0.010). Our results imply that in DLBCL, the location of TP53 mutations and IHC analysis of BCL-2 and MYC might have a role in the assessment of prognosis.