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.
*Corresponding authorBarbara Pasini, Medical Genetics Unit, AOU Città della Salute e della Scienza di Torino, Turin, Italy, Tel: 39-11-6336681; Fax: 39-11-6335181; Email:
A Truncating SHFM1DSS1 Germline Mutation in a Familial Breast Cancer Case: the List of Breast Cancer Susceptibility Genes is Getting LongerFrancesca Vignolo Lutati1, Cecilia Bracco1,2,3, Anna Allavena1, Paola Ogliara1, Guido C. Casalis Cavalchini1,3, Giorgia Mandrile1, Daniela F. Giachino3, and Barbara Pasini1,2,3*1Medical Genetics Unit, San Luigi University Hospital, Italy2Department of Medical Sciences, University of Turin, Italy3Fondazione del Piemonte per l’Oncologia-IRCCS, Italy
ABBREVIATIONSHR: Homologous Recombination
INTRODUCTIONBreast cancer is one of the most frequent malignancies,
affecting 8 to 10 percent of women in Western Countries. About 20% of patients have a positive family history for breast cancer but only a small percentage of familial cases are due to germline mutations in BRCA1 or BRCA2, the genes responsible for the autosomal dominant Hereditary Breast and Ovarian Cancer (HBOC). Indeed germline mutations in BRCA1 and BRCA2 are found in approximately 50% of breast and ovarian cancer kindreds but in only 10-20% of breast cancer families [1], personal data). Assessing cancer risk in BRCA-negative families represents therefore a relevant clinical challenge.
It is well known that mutations in other genes responsible for cancer prone hereditary diseases such as TP53 (Li Fraumeni syndrome), CDH1 (hereditary diffuse gastric cancer), PTEN (Cowden syndrome), STK11 (Peutz-Jeghers syndrome) and NF1
(Neurofibromatosis type 1) confer an increased risk for breast cancer. Moreover, although no other major high penetrance breast cancer susceptibility genes have been discovered, emerging evidences highlighted the presence of a number of “moderate penetrance” genes conferring a 2-3 fold increased risk of developing breast cancer, such as ATM, CHEK2, PALB2, BRIP1, SLX4, FANCM, BARD1, MRE11A, RAD50, NBN [2], on the other hand, RAD51C and RAD51D seem to increase the risk mainly for ovarian cancer. However mutations in these genes are extremely rare, overall accounting for less than 5-10% of familial breast cancers [3].
BRCA1 and BRCA2 play an essential role in DNA repair through homologous recombination (HR) and most of the breast cancer susceptibility genes are involved in pathways whose role is to preserve genome integrity [4]. Therefore it is worthy to study other genes belonging to DNA repair and the HR network in mutation-negative breast cancer families. Direct interaction with BRCA1 and BRCA2 has been demonstrated for many different proteins, among which BARD1, BRIP1, PALB2 and the structurally and functionally highly conserved SHFM1 [5-7].
Keywords•Breast cancer•SHFM1•DSS1•BRCA1•BRCA2
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.
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SHFM1 is a 70 amino acids highly acidic protein (with 40% of residues being either Aspartate or Glutamate) encoded by the SHFM1 gene (also known as DSS1, SEM1, SHFD1, SHSF1). SHFM1 has been identified together with the homeobox genes DLX5 and DLX6 in the minimal deleted region on the long arm of chromosome 7 in patients with split-hand/split-foot malformation type 1, a limb developmental disorder [8]. However, knock-out experiments on mouse models have suggested that combined haploin sufficiency of both DLX5 and DLX6 seems the main cause of the reduction in cell proliferation underlining the limb developmental defect [9-11].
SHFM1 binds to BRCA2 between residues 2472-2957, which is the longest conserved region of the protein [5]. It has been shown that the majority of endogenous BRCA2 in mammalian cells resides in complex with SHFM1, which may mimic ssDNA [6]. The stability of the BRCA2 protein in mammalian cells depends on the presence of SHFM1 and depletion of SHFM1 in human cell lines dramatically increases BRCA2 degradation leading to protein loss [12]. Moreover, deletion or inactivation of Shfm1 in Ustilago maydis leads to HR deficiency and genomic instability [13]. These findings support the hypothesis that loss of function mutations of SHFM1 or suppression of its expression could be a potential causative mechanism for human breast and/or ovarian cancer development.
We therefore assessed for germline SHFM1 mutations 944 index cases referred for BRCA1 and BRCA2 testing in order to verify whether SHFM1 mutations are responsible for a proportion of high risk BRCA-negative families or could have an effect in modifying cancer risk in BRCA mutation carriers.
MATERIALS AND METHODS944 families were selected for BRCA1 and BRCA2 testing at
three cancer family clinics of Piedmont Region (Italy) according to the following criteria: a) at least one case of early onset (<36 years) or triple negative (1 - F BrCa in Table 1) or male breast 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 patient; c) at least two first degree relatives with premenopausal (< 50 years) or bilateral breast cancer (any age) or ovarian cancer; d) at least three first degree relatives diagnosed with breast or ovarian cancer at any age. In most families, the index case was affected by breast and/or ovarian cancers (844 cases, 810 females, 34 males) while in 100 families the available index case was healthy (88 cases) or affected by another cancer (12 cases). BRCA1 and BRCA2 genes were tested for germline mutations along the entire coding sequence by DHPLC analysis (exon 11 of both BRCA1 and BRCA2 was analysed by PTT in 784 cases) followed by MLPA analysis in wild type cases (SALSA MLPA probemix P002-B and P045-B2).
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 the study in accordance to the local ethical committee. SHFM1 (Ref Seq Gene NG_009273.1 and NM_006304.1) analysis of coding sequence and exon-intron boundaries was performed by DHPLC.
Genomic DNA was extracted from whole blood using the Qiagen EZ1 DNA Blood 350 µl Kit on the EZ1 Advanced XL instrument with EZ1 Adv. XL DNA Blood Card, according to
the manufacturer’s instructions. Polymerase chain reactions (PCRs) were performed in a final volume of 25 µl containing 3 µl template DNA (about 25-50 ng), 12.5 pmol of each primer, 100 µM dNTPs, standard PCR buffer (1.5 mM MgCl2), and 0.5U of thermostable DNA polymerase (GoTaq Hot Start Polymerase, Promega) for exons 2 and 3 and standard PCR buffer (2.5 mM MgCl2), and 0.5U of thermostable DNA polymerase (AmpliTaq Gold polymerase, Perkin-Elmer) for exon 1. PCR amplifications were carried out using a touchdown protocol in order to reduce miss-primed products (annealing temperature: 69°C, -1°C per 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 60°C for 35 cycles for exon 3). DHPLC analysis was performed on a Wave 3500HT DNA Fragment Analysis System (ADS Biotech). Primer sequences and DHPLC analysis conditions are available on request. Fragments showing heterozygous elution profiles were analysed by direct sequencing to determine the exact nucleotide variation. Sequencing was performed using BigDye Terminator v1.1 Cycle Sequencing Kit on a 3130xl Genetic analyzer (Applied Biosystems).
Following the identification of a non-sense SHFM1 mutation in a familial female breast cancer case, 548 cancer-free unrelated controls were submitted to DHPLC analysis of SHFM1 exon 2. Sections from the paraffin-embedded breast cancer tissue of the SHFM1 mutation positive patient were obtained to assess the somatic loss of the wild type allele. After micro-dissection to enrich for cancer cells, tumour DNA was extracted using Qiagen EZ1 Tissue Kit on the EZ1 Advanced XL instrument with EZ1 Adv. XL DNA Paraffin Section Card, according to the manufacturer’s instructions.
In silico evolutionary protein sequence evaluation of the missense variant was performed with seven different tools: SIFT [14], A-GVGD [15,16], Mutation Assessor [17], Provean [18], Phyre2 [19], Mutation Taster [20], and PolyPhen-2 [21].
In silico splicing evaluation was performed with five different splicing tools: http://www.fruitfly.org/seq_tools/splice.html; http://deepc2.psi.iastate.edu/cgi-bin/sp.cgi;
RESULTS AND DISCUSSIONA cohort of 944 index cases was analysed for BRCA1, BRCA2
and SHFM1 mutations: personal and familial histories of cancer with respect to mutation status are described in Table 1. BRCA1 and BRCA2 mutations were identified in 205 cases (22%), 119 with BRCA1 mutations, 84 with BRCA2 mutations and 2 with deleterious mutations in both genes. Fourteen additional cases had rare variants of unknown biological significance while in 89 cases missense or intronic variants could be classified as neutral or likely neutral (VUS classes 1 and 2). As expected, BRCA mutations were more frequent in ovarian and breast/ovarian families (50%), while in families with breast cancer only the mutation detection rate did not exceed 29% (overall 13%).
SHFM1 mutation screening led to the identification of one heterozygous nonsense mutation in exon 2, c.169C>T, p (Arg57*) (rs199922834), in a BRCA1/BRCA2 wild type kindred with four
cases of female breast cancer. At 43 years of age, the index case developed a G1 bifocal invasive ductal carcinoma, expressing both oestrogen and progesterone receptors; one out of her three sisters was diagnosed with breast cancer at the age of 43; the mother and a maternal aunt were also diagnosed with breast cancer at the age of 77 and 71, respectively; moreover, gastrointestinal cancers were reported in her maternal relatives (Figure 1A and B). SHFM1 analysis on peripheral blood of the affected sister showed the presence of the mutation, suggesting the possible segregation with the disease: no other family members were available for testing.
This mutation was not found in the remaining 943 analysed index cases (100 of which were healthy or affected by cancers other than breast /ovarian) or in 548 healthy controls from our laboratory. Moreover, it has been reported twice in the ESP6500 database (frequency of 0.03% in the African- and Europen-
American population; 0.05% in the European-American one), 8 times in the gnomAD database (0.006%) and is absent in the 1000Genomes Phase 3 database.
In order to assess whether loss of heterozygosity (LOH) was present in the proband’s breast cancer, genomic DNA extracted from formalin-fixed paraffin-embedded tumour sections was analysed. Sequencing of SHFM1 showed somatic heterozygosity at nucleotide 169 and no other somatic point mutations were identified.
In addition to a known polymorphism (c.15G>A; p.Lys5, MAF: 8.5%), other 5 different SHFM1 sequence variants were identified, all in BRCA-negative index cases: c.89T>C, p.(Leu30Ser) in exon 2, c.77-9T>C in intron 1 and three nucleotide substitutions in the 5’UTR of the gene (c.-38C>T; c.-78C>T; c.-119T>C).
The missense variant c.89T>C, p.(Leu30Ser) was identified in
Figure 1 Family with SHFM1 nonsense mutation (c.169C>T, p.Arg57*).(A) - Pedigree of the family: Head of arrow indicates the index case. For each subject (circle: female; square: male; rhombus: unspecified gender) 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 indicates number of individuals.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.
Phenotype
N° of affected family members N° of cases
analysedTotal BRCA1-2 mutations (%)
BRCA1 mutations
BRCA2 mutations
Total casesBRCA1-2
WT or VUS
SHFM1mutations
[VUS]Disease status of the index case
Cases/familieswith breast cancer
only
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]
5 to 11 84 24 (29%) 5 19 60 0Index case with
F BrCa 612 82 (13%) 32 50 530 1 [4]
Index case withM BrCa 33 5 (15%) 1 4 28 0
Index case with Other Tumor or healthy 75 7 (9%) 1 6 68 0 [1]
Total 720 94 (13%) 34 60 626 1 [5]
Cases/familieswith breast and ovarian cancer
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]
5 to 12 39 25 (64%) 18 7 14 0Index case with
F BrCa and/or OvCa 186 99 (53%) 76 23 87 0 [1]
Index case withM BrCa 1 0 (0%) 0 0 1 0
Index case with Other Tumor or healthy 21 4 (19%) 4 0 17 0 [1]
Total 208 103 (50%) 80 23 105 0 [2]
Cases/familieswith Ovarian cancer
only
1 3 2 (67%) 2 0 1 0
2 to 4 13 6 (46%) 4 2 7 0
Index case with OvCa 12 7 (58%) 6 1 5 0Index case with OthTum
or healthy 4 1 (25%) 0 1 3 0
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 at 46 and 65 years of age; this variant (rs377420507) is reported only once in the ESP6500 database (frequency of 0.01% considering both African- and European-American populations, 0.02% in the European-American population) and 16 times in 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 the alignment of human SHFM1 with 16 ortholog proteins from both closely and distantly related species revealed that Leucine 30 is not conserved beyond vertebrates. Most in silico tools classified the variant as neutral or tolerated (SIFT; A-GVGD, Mutation Assessor, Provean; Phyre2). However two software predicted a possible deleterious effect (PolyPhen-2, Mutation
Taster). Finally, analysis of the nucleotide substitution c.89T>C with 5 splicing simulation tools failed to predict any significant effect on SHFM1 mRNA splicing. As this missense variant was absent in the two proband’s daughters, both diagnosed with breast cancer at the age of 46 years, we could assess it does not have a significant biological effect.
The intron 1 c.77-9T>C variant was identified in a healthy index case of a middle-low risk family (two peri-menopausal cases of breast cancer); this variant is absent in both the 1000Genomes Phase 3 database and in the ExAC database. As evaluation with the splicing prediction tools showed no effect on mRNA splicing, we do not have any element in favour of a possible deleterious effect.
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Two of the 5’UTR variants, c.-38T>C and c.-78C>T, each of which was found in two apparently unrelated families, are present in the NCBI database Reference Sequence (rs112474432 and rs138045757, respectively) and have a frequency of 0.1% in the 1000Genome database. The first one did not segregate in one family, as it was present in the index case, diagnosed with breast cancer at the age of 30 years, but not in her sister with breast cancer at 32 years of age. These data show that the variant probably does not have a significant biological effect. It was not possible to perform any segregation analysis for c.-78C>T nucleotide substitution and therefore, in the absence of functional studies, the variant remains of unknown significance.
The last 5’UTR variant, c.-119C>T was identified in a patient with lobular breast cancer at 57 years of age and positive family history of breast cancer (mother and sister diagnosed at 50 years of age). No other family members were available for testing; the variant is absent in the 1000 Genomes phase 3 database and the region is not covered in the ExAC database; functional studies may be needed to evaluate its biological significance, thus this 5’UTR variant remains a variant of unknown significance.
CONCLUSIONTo the best of our knowledge, only two studies have been
published to date on SHFM1 analysis in breast or ovarian cancer families: the first one [22], on 145 Finnish males with breast cancer and the second one [23], on 369 Spanish breast/ovarian cancer families: both studies failed to identify any deleterious mutation.
In our cohort of 944 index cases, mostly of Italian ancestry, we identified the first SHFM1 nonsense mutation in a familial breast cancer case negative for BRCA1 and BRCA2 mutations. SHFM1 germline deleterious mutations seem therefore to be very rare: the prevalence over the whole cohort was 0.1% (944 index cases) and also considering only families without BRCA1 and BRCA2 mutations (739), without ovarian cancer (626) and with the index case affected by female breast cancer (530), the frequency of germline deleterious mutations still remains very low (0.19%).
As no SHFM1 mutations or variants were identified in families with male breast cancer (58), ovarian cancers (223) or BRCA1/2 mutations (205), we could not assess whether mutations in this gene influence the risk for cancers other than female breast or modify the penetrance of BRCA1/2 mutations.
Data from recent studies show that multigene panel testing on breast and/or ovarian cancer patients highlight the presence of mutations in two different susceptibility genes in less than 1% (range 0.1-0.8%) of subjects [24-30]. In the family with the deleterious SHFM1 mutation, no BRCA1/2 mutation was identified but we cannot exclude the presence of other genetic susceptibility factors, such as mutations in moderate penetrance genes or unfavourable allele combinations at low penetrance loci. Beside the nonsense mutation, only one missense variant of uncertain significance (p.Leu30Ser), one intronic variant and three 5’UTR variants were identified in 8 patients: this led the rate of SHFM1 variants to 0.8%.
SHFM1 is a small gene with an observed mutation rate lower than the expected (25 mutations identified in the ExAC database despite 40 expected). In the COSMIC database, out of 23.027 samples tested, 10 unique mutations are present, of which
6 missense, 2 nonsense and 2 synonymous; of these, 6 were confirmed somatic. The p.Arg57* is reported once, as somatic mutation, in a stomach carcinoma. Moreover, in the COSMIC database, 153 CNVs are present; in particular, 148 gain variants and 5 loss variants. No gene fusion or methylation changes are highlighted. It is interesting to observe that, beside rare loss of function mutations, SHFM1 overexpression is reported in 842 samples, whereas underexpression only in 1.
As SHFM1 might mimic ssDNA, a possible mechanism in cancer predisposition could be through a dominant negative mechanism, in which over expression or more affine conformation of the protein might perturb homologous recombination.
In this context, we can speculate that the nonsense p.Arg57* mutation, 14 amino acids up-stream the wild type termination codon, could escape the nonsense-mediated decay leading to a messenger RNA traduced into a truncated protein able to interfere with BRCA2 function. This hypothesis is consistent with 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 inactivation of the wild type allele is promoter methylation, which was not tested.
It is however worthy to remember that no LOH, or even loss of the mutant allele, has been described in cancer arisen in patients with deleterious mutation of well-established breast cancer susceptibility genes, such as BRCA1 or BRCA2 [31-33], PALB2 [34] or CHEK2 [35,36]. In conclusion, although only few cases of familial breast cancer might be due to mutations in SHFM1 gene, this study increases the number of breast cancer susceptibility genes that should be included in massive parallel sequencing panels for breast/ovarian cancer families.
ACKNOWLEDGEMENTSWe thank Virna Debenedetti for contribution to BRCA analysis,
Maria Teresa Ricci for help in sample and data collection, Sara Albertin for data management and Alfredo Brusco for providing control samples. We also thank all patients and their family members for volunteering to participate in this study and all the clinicians who selected patients for genetic counselling.
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