A Truncating SHFM1DSS1 Germline Mutation in a Familial Breast Cancer Case: the List of Breast Cancer Susceptibility Genes Is Getting Longer
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Central JSM Clinical Oncology and Research Research Article *Corresponding author Barbara Pasini, Medical Genetics Unit, AOU Città della Salute e della Scienza di Torino, Turin, Italy, Tel: 39-11- DSS1 6336681; Fax: 39-11-6335181; Email: A Truncating SHFM1 Germline Submitted: 28 July 2017 Mutation in a Familial Breast Accepted: 09 August 2017 Published: 11 August 2017 Cancer Case: the List of Breast Copyright © 2017 Pasini et al. Cancer Susceptibility Genes is OPEN ACCESS Keywords • Breast cancer Getting Longer • SHFM1 Francesca Vignolo Lutati1, Cecilia Bracco1,2,3, Anna Allavena1, • DSS1 • BRCA1 1 1,3 1 Paola Ogliara , Guido C. Casalis Cavalchini , Giorgia Mandrile , • BRCA2 Daniela F. Giachino3, and Barbara Pasini1,2,3* 1Medical Genetics Unit, San Luigi University Hospital, Italy 2Department of Medical Sciences, University of Turin, Italy 3Fondazione del Piemonte per l’Oncologia-IRCCS, Italy Abstract Although approximately 20% of breast cancer cases have a positive family history for the disease, less than 25% of familial cases carry an identified germline mutation in the “high risk” susceptibility genes, BRCA1, BRCA2 and TP53, or in the so called “moderate penetrance” susceptibility genes such as ATM, CHEK2, PALB2, SLX4, BRIP1, BARD1, MRE11A, RAD50 and NBN. These genes are involved in pathways related to DNA repair thus suggesting that a failure in maintaining genome integrity can increase breast cancer risk. Moreover, tumours with impaired DNA repair through homologous recombination as those occurring in BRCA1 or BRCA2 mutation carriers seem particularly sensitive to PARP inhibitors thus underlining the need of a better knowledge of the mechanisms promoting cancer development. With the aim to identify additional breast/ovarian cancer susceptibility genes belonging to the homologous recombination pathway, we focus our attention on SHFM1DSS1, a three exons gene on chromosome 7q encoding a highly conserved protein interacting with the longest region of evolutionary conservation of BRCA2. SHFM1DSS1 analysis in a consecutive series of 944 cases previously screened for BRCA1 and BRCA2 mutations led to the identification of a non-sense germline mutation in a family with four cases of female breast cancer diagnosed between 43 and 77 years. The mutation was absent in 548 healthy controls. Although rare in the Italian population, SHFM1DSS1 germline mutations can potentially increase the risk of breast cancer thus extending the list of cancer susceptibility genes to be considered for testing. ABBREVIATIONS cancer. Moreover, although no other major high penetrance HR: Homologous Recombination breast(Neurofibromatosis cancer susceptibility type 1) confer genes an increasedhave been risk discovered, for breast INTRODUCTION emerging evidences highlighted the presence of a number of “moderate penetrance” genes conferring a 2-3 fold increased risk Breast cancer is one of the most frequent malignancies, of developing breast cancer, such as ATM, CHEK2, PALB2, BRIP1, affecting 8 to 10 percent of women in Western Countries. SLX4, FANCM, BARD1, MRE11A, RAD50, NBN [2], on the other About 20% of patients have a positive family history for breast hand, RAD51C and RAD51D seem to increase the risk mainly for cancer but only a small percentage of familial cases are due to ovarian cancer. However mutations in these genes are extremely germline mutations in BRCA1 or BRCA2, the genes responsible rare, overall accounting for less than 5-10% of familial breast for the autosomal dominant Hereditary Breast and Ovarian cancers [3]. Cancer (HBOC). Indeed germline mutations in BRCA1 and BRCA2 are found in approximately 50% of breast and ovarian cancer BRCA1 and BRCA2 play an essential role in DNA repair kindreds but in only 10-20% of breast cancer families [1], through homologous recombination (HR) and most of the breast personal data). Assessing cancer risk in BRCA-negative families cancer susceptibility genes are involved in pathways whose role represents therefore a relevant clinical challenge. is to preserve genome integrity [4]. Therefore it is worthy to study other genes belonging to DNA repair and the HR network in It is well known that mutations in other genes responsible mutation-negative breast cancer families. Direct interaction with for cancer prone hereditary diseases such as TP53 (Li Fraumeni BRCA1 and BRCA2 has been demonstrated for many different syndrome), CDH1 (hereditary diffuse gastric cancer), PTEN proteins, among which BARD1, BRIP1, PALB2 and the structurally (Cowden syndrome), (Peutz-Jeghers syndrome) and STK11 NF1 and functionally highly conserved SHFM1 [5-7]. Cite this article: Lutati FV, Bracco C, Allavena A, Ogliara P, Casalis Cavalchini GC, et al. (2017) A Truncating SHFM1DSS1 Germline Mutation in a Familial Breast Cancer Case: the List of Breast Cancer Susceptibility Genes is Getting Longer. JSM Clin Oncol Res 5(2): 1057. Pasini et al. (2017) Email: Central SHFM1 is a 70 amino acids highly acidic protein (with 40% the manufacturer’s instructions. Polymerase chain reactions of residues being either Aspartate or Glutamate) encoded by the SHFM1 gene (also known as DSS1, SEM1, SHFD1, SHSF1). µl template DNA (about 25-50 ng), 12.5 pmol of each primer, (PCRs) were performed in a final volume of 25 µl containing 3 SHFM1 100 µM dNTPs, standard PCR buffer (1.5 mM MgCl2), and 0.5U DLX5 and DLX6 in the minimal deleted region on the long of thermostable DNA polymerase (GoTaq Hot Start Polymerase, arm of haschromosome been identified 7 in togetherpatients withwith thesplit-hand/split-foot homeobox genes Promega) for exons 2 and 3 and standard PCR buffer (2.5 mM malformation type 1, a limb developmental disorder [8]. However, MgCl2), and 0.5U of thermostable DNA polymerase (AmpliTaq knock-out experiments on mouse models have suggested that DLX5 and DLX6 seems the were carried out using a touchdown protocol in order to reduce main cause of the reduction in cell proliferation underlining the miss-primedGold polymerase, products Perkin-Elmer) (annealing for temperature: exon 1. PCR 69amplifications°C, -1°C per combinedlimb developmental haploin sufficiency defect [9-11]. of both cycle for 7 cycles and then 62°C for 35 cycles for exons 1 and 2; annealing temperature: 67°C, -1°C per cycle for 7 cycles and then SHFM1 binds to BRCA2 between residues 2472-2957, which is 60°C for 35 cycles for exon 3). DHPLC analysis was performed on the longest conserved region of the protein [5]. It has been shown a Wave 3500HT DNA Fragment Analysis System (ADS Biotech). that the majority of endogenous BRCA2 in mammalian cells Primer sequences and DHPLC analysis conditions are available on resides in complex with SHFM1, which may mimic ssDNA [6]. The stability of the BRCA2 protein in mammalian cells depends on the analysed by direct sequencing to determine the exact nucleotide presence of SHFM1 and depletion of SHFM1 in human cell lines variation.request. Fragments Sequencing showing was performed heterozygous using elution BigDye profiles Terminator were dramatically increases BRCA2 degradation leading to protein v1.1 Cycle Sequencing Kit on a 3130xl Genetic analyzer (Applied loss [12]. Moreover, deletion or inactivation of Shfm1 in Ustilago Biosystems). maydis SHFM1 mutation of SHFM1leads or tosuppression HR deficiency of its and expression genomic instability could be [13].a potential These in a familial female breast cancer case, 548 cancer-free unrelated causativefindings support mechanism the hypothesis for human that breast loss and/or of function ovarian mutations cancer controlsFollowing were thesubmitted identification to DHPLC of a non-senseanalysis of SHFM1 exon 2. development. the SHFM1 mutation positive patient were obtained to assess We therefore assessed for germline SHFM1 mutations 944 theSections somatic from loss the of theparaffin-embedded wild type allele. Afterbreast micro-dissection cancer tissue toof index cases referred for BRCA1 and BRCA2 testing in order to enrich for cancer cells, tumour DNA was extracted using Qiagen verify whether SHFM1 mutations are responsible for a proportion EZ1 Tissue Kit on the EZ1 Advanced XL instrument with EZ1 Adv. of high risk BRCA-negative families or could have an effect in modifying cancer risk in BRCA mutation carriers. instructions. MATERIALS AND METHODS XL DNA Paraffin Section Card, according to the manufacturer’s In silico evolutionary protein sequence evaluation of the 944 families were selected for BRCA1 and BRCA2 testing at missense variant was performed with seven different tools: SIFT three cancer family clinics of Piedmont Region (Italy) according [14], A-GVGD [15,16], Mutation Assessor [17], Provean [18], to the following criteria: a) at least one case of early onset (<36 Phyre2 [19], Mutation Taster [20], and PolyPhen-2 [21]. years) or triple negative (1 - F BrCa in Table 1) or male breast In silico cancer (1 - M BrCa in Table 1); b) at least one case of epithelial ovarian cancer or both breast and ovarian cancer in the same http://deepc2.psi.iastate.edu/cgi-bin/sp.cgi splicing evaluation was performed; with five different splicing tools: http://www.fruitfly.org/seq_tools/splice.html; (< 50 years) or bilateral breast cancer (any age) or ovarian http://www.tigr.org/tdb/GeneSplicer/gene_spl.html; patient; c) at least two first degree relatives with premenopausal http://www.cbs.dtu.dk/services/NetGene2; breast or ovarian cancer at any age. In most families, the index http://genes.mit.edu/GENSCAN.html). casecancer; was d) affected at least by three breast first and/or degree ovarian relatives cancers diagnosed (844 cases, with 810 females, 34 males) while in 100 families the available RESULTS AND DISCUSSION index case was healthy (88 cases) or affected by another cancer A cohort of 944 index cases was analysed for BRCA1, BRCA2 (12 cases). BRCA1 and BRCA2 genes were tested for germline and SHFM1 mutations: personal and familial histories of cancer mutations along the entire coding sequence by DHPLC analysis with respect to mutation status are described in Table 1. BRCA1 (exon 11 of both BRCA1 and BRCA2 was analysed by PTT in 784 and BRCA2 cases) followed by MLPA analysis in wild type cases (SALSA with BRCA1 mutations, 84 with BRCA2 mutations and 2 with MLPA probemix P002-B and P045-B2).