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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 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 on chromosome 7q encoding a highly conserved 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 increased have 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 , 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 has chromosome been identified 7 in together patients with with the split-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 the submitted identification to DHPLC of a non-sense analysis 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 the paraffin-embedded wild type allele. After breast micro-dissection cancer tissue to of 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). deleterious mutations werein both identified genes. Fourteen in 205 cases additional (22%), cases 119 The entire cohorts of 944 index cases were screened for SHFM1 mutations, irrespective to their BRCA1/BRCA2 mutation status: all patients signed an informed consent to participate in neutralhad rare or variantslikely neutral of unknown (VUS classes biological 1 and 2). significance As expected, while BRCA in the study in accordance to the local ethical committee. SHFM1 mutations89 cases missense were more or frequentintronic variants in ovarian could and be breast/ovarian classified as (Ref Seq Gene NG_009273.1 and NM_006304.1) analysis of coding families (50%), while in families with breast cancer only the sequence and exon-intron boundaries was performed by DHPLC. mutation detection rate did not exceed 29% (overall 13%). Genomic DNA was extracted from whole blood using the SHFM1 Qiagen EZ1 DNA Blood 350 µl Kit on the EZ1 Advanced XL heterozygous nonsense mutation in exon 2, c.169C>T, p (Arg57*) instrument with EZ1 Adv. XL DNA Blood Card, according to (rs199922834),mutation in a BRCA1/BRCA2 screening led wild to the type identification kindred with of four one

JSM Clin Oncol Res 5(2): 1057 (2017) 2/6 Pasini et al. (2017) Email: Central cases of female breast cancer. At 43 years of age, the index case American population; 0.05% in the European-American one), developed a G1 bifocal invasive ductal carcinoma, expressing 8 times in the gnomAD database (0.006%) and is absent in the both oestrogen and progesterone receptors; one out of her 1000Genomes Phase 3 database. three sisters was diagnosed with breast cancer at the age of In order to assess whether loss of heterozygosity (LOH) was 43; the mother and a maternal aunt were also diagnosed with present in the proband’s breast cancer, genomic DNA extracted breast cancer at the age of 77 and 71, respectively; moreover, gastrointestinal cancers were reported in her maternal relatives analysed. Sequencing of SHFM1 showed somatic heterozygosity (Figure 1A and B). SHFM1 analysis on peripheral blood of the atfrom nucleotide formalin-fixed 169 and paraffin-embedded no other somatic pointtumour mutations sections were was affected sister showed the presence of the mutation, suggesting the possible segregation with the disease: no other family members were available for testing. identified.In addition to a known polymorphism (c.15G>A; p.Lys5, MAF: 8.5%), other 5 different SHFM1 This mutation was not found in the remaining 943 analysed all in BRCA-negative index cases: c.89T>C, p.(Leu30Ser) in exon index cases (100 of which were healthy or affected by cancers 2, c.77-9T>C in intron 1 and three sequence nucleotide variants substitutions were identified, in the other than breast /ovarian) or in 548 healthy controls from our 5’UTR of the gene (c.-38C>T; c.-78C>T; c.-119T>C). laboratory. Moreover, it has been reported twice in the ESP6500 database (frequency of 0.03% in the African- and Europen-

The missense variant c.89T>C, p.(Leu30Ser) was identified in

Figure 1 Family with SHFM1 nonsense mutation (c.169C>T, p.Arg57*).

the actual age or age at death (crossed symbols) is indicated; the number following the cancer site indicates the age at diagnosis. Number inside the symbol(A) - Pedigree indicates of thenumber family: of individuals.Head of arrow indicates the index case. For each subject (circle: female; square: male; rhombus: unspecified gender) BrCa: Breast Cancer; GaCa: Gastric Cancer; CUP: Cancer of Unknown Primary; HL: Hodgkin Lymphoma; SalCa: Salivary Glands Cancer; CoCa: Colon Cancer; UtCa: Uterine Cancer; Sen: senectus (B) - Electropherogram of the nonsense mutation of exon 2 of SHFM1 (c.169C>T, p.Arg57*), performed on DNA extracted from a peripheral blood sample.

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Table 1: Phenotype-genotype correlation of 944 index cases analysed for BRCA1, BRCA2 and SHFM1 mutations. N° of affected family Total cases SHFM1 members N° of cases Total BRCA1-2 BRCA1 BRCA2 Phenotype BRCA1-2 mutations Disease status of the analysed mutations (%) mutations mutations WT or VUS [VUS] index case 1 - F BrCa 95 9 (9%) 6 3 86 0 [1] 1 - M BrCa 11 1 (9%) 1 0 10 0 2 195 24 (12%) 13 11 171 0 [2] 3 200 23 (12%) 5 18* 177 0 [1] 4 135 13 (10%) 4 9 122 1 [1] Cases/families 5 to 11 84 24 (29%) 5 19 60 0 with breast cancer Index case with only 612 82 (13%) 32 50 530 1 [4] F BrCa Index case with 33 5 (15%) 1 4 28 0 M BrCa Index case with Other 75 7 (9%) 1 6 68 0 [1] Tumor or healthy Total 720 94 (13%) 34 60 626 1 [5] 1 22 8 (36%) 6 2 14 0 [1] 2 62 20 (32%) 15 5 42 0 3 45 21 (47%) 16 5 24 0 4 40 29 (73%) 25* 4 11 0 [1] Cases/families 5 to 12 39 25 (64%) 18 7 14 0 with breast and Index case with 186 99 (53%) 76 23 87 0 [1] ovarian cancer F BrCa and/or OvCa Index case with 1 0 (0%) 0 0 1 0 M BrCa Index case with Other 21 4 (19%) 4 0 17 0 [1] Tumor or healthy Total 208 103 (50%) 80 23 105 0 [2] 1 3 2 (67%) 2 0 1 0 2 to 4 13 6 (46%) 4 2 7 0 Cases/families with Ovarian cancer Index case with OvCa 12 7 (58%) 6 1 5 0 only Index case with OthTum 4 1 (25%) 0 1 3 0 or healthy Total 16 8 (50%) 6 2 8 0 [0] All phenotypes Total 944 205 (22%) 120 85 739 1 [7] (* Asterisk indicates the two index cases with both a BRCA1 and a BRCA2 deleterious mutation but attributed as mutated case to a single gene, F: Female; M: Male; BrCa: Breast Cancer; OvCa: Ovarian Cancer; OthTum: Other Tumours than Breast or Ovarian; WT: Wild Type, VUS: Variants of

Unknown Biological Significance) a patient with metachronous bilateral breast cancers, diagnosed Taster). Finally, analysis of the nucleotide substitution c.89T>C at 46 and 65 years of age; this variant (rs377420507) is reported only once in the ESP6500 database (frequency of 0.01% effect on SHFM1 mRNA splicing. As this missense variant was considering both African- and European-American populations, withabsent 5 splicing in the twosimulation proband’s tools daughters, failed to predict both diagnosed any significant with 0.02% in the European-American population) and 16 times in breast cancer at the age of 46 years, we could assess it does not the gnomAD database (0,0065%); it is absent in the 1000Genome database Phase 3. This missense variant is located outside the BRCA2-contacting residues [6], and evolutionary analysis through have a significant biological effect. the alignment of human SHFM1 with 16 ortholog proteins from index case of a middle-low risk family (two peri-menopausal cases The intron 1 c.77-9T>C variant was identified in a healthy both closely and distantly related species revealed that Leucine of breast cancer); this variant is absent in both the 1000Genomes 30 is not conserved beyond vertebrates. Most in silico tools Phase 3 database and in the ExAC database. As evaluation with the splicing prediction tools showed no effect on mRNA splicing, Mutation Assessor, Provean; Phyre2). However two software we do not have any element in favour of a possible deleterious predictedclassified the a possible variant deleterious as neutral effect or tolerated (PolyPhen-2, (SIFT; Mutation A-GVGD, effect.

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Two of the 5’UTR variants, c.-38T>C and c.-78C>T, each 6 missense, 2 nonsense and 2 synonymous; of these, 6 were of which was found in two apparently unrelated families, are present in the NCBI database Reference Sequence (rs112474432 mutation, in a stomach carcinoma. Moreover, in the COSMIC and rs138045757, respectively) and have a frequency of 0.1% database,confirmed 153 somatic. CNVs Theare present; p.Arg57* in isparticular, reported 148 once, gain as variants somatic and 5 loss variants. No gene fusion or methylation changes are in one family, as it was present in the index case, diagnosed highlighted. It is interesting to observe that, beside rare loss of within the breast 1000Genome cancer at database. the age of The 30 firstyears, one but did not not in her segregate sister function mutations, SHFM1 overexpression is reported in 842 with breast cancer at 32 years of age. These data show that the samples, whereas underexpression only in 1. As SHFM1 might mimic ssDNA, a possible mechanism in It was not possible to perform any segregation analysis for c.- cancer predisposition could be through a dominant negative 78C>Tvariant nucleotide probably doessubstitution not have and a therefore, significant in biological the absence effect. of of the protein might perturb homologous recombination. mechanism, in which over expression or more affine conformation functional studies, the variant remains of unknown significance. In this context, we can speculate that the nonsense p.Arg57* with lobular breast cancer at 57 years of age and positive family mutation, 14 amino acids up-stream the wild type termination historyThe oflast breast 5’UTR cancer variant, (mother c.-119C>T and sister was identifieddiagnosed in at a50 patient years codon, could escape the nonsense-mediated decay leading to of age). No other family members were available for testing; the a messenger RNA traduced into a truncated protein able to variant is absent in the 1000 Genomes phase 3 database and the interfere with BRCA2 function. This hypothesis is consistent with region is not covered in the ExAC database; functional studies the evidence of no LOH in the cancer tissue of the index case. However, although no LOH was highlighted and no other somatic point mutation was present, another possible mechanism for may be needed to evaluate its biological significance, thus this inactivation of the wild type allele is promoter methylation, 5’UTRCONCLUSION variant remains a variant of unknown significance. which was not tested. To the best of our knowledge, only two studies have been published to date on SHFM1 analysis in breast or ovarian cancer It is however worthy to remember that no LOH, or even loss of the mutant allele, has been described in cancer arisen in patients cancer and the second one [23], on 369 Spanish breast/ovarian with deleterious mutation of well-established breast cancer cancerfamilies: families: the first both one studies [22], on failed 145 to Finnish identify males any with deleterious breast susceptibility genes, such as BRCA1 or BRCA2 [31-33], PALB2 mutation. [34] or CHEK2 [35,36]. In conclusion, although only few cases of familial breast cancer might be due to mutations in SHFM1 gene, In our cohort of 944 index cases, mostly of Italian ancestry, this study increases the number of breast cancer susceptibility SHFM1 nonsense mutation in a familial genes that should be included in massive parallel sequencing breast cancer case negative for BRCA1 and BRCA2 mutations. panels for breast/ovarian cancer families. SHFM1we identified germline the deleterious first mutations seem therefore to be very rare: the prevalence over the whole cohort was 0.1% (944 ACKNOWLEDGEMENTS index cases) and also considering only families without BRCA1 We thank Virna Debenedetti for contribution to BRCA analysis, and BRCA2 mutations (739), without ovarian cancer (626) and Maria Teresa Ricci for help in sample and data collection, Sara with the index case affected by female breast cancer (530), the Albertin for data management and Alfredo Brusco for providing frequency of germline deleterious mutations still remains very control samples. We also thank all patients and their family low (0.19%). members for volunteering to participate in this study and all the As no SHFM1 clinicians who selected patients for genetic counselling. with male breast cancer (58), ovarian cancers (223) or BRCA1/2 mutations (205), mutations we could notor variants assess whether were identified mutations in families in this REFERENCES 1. 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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.

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