Abstract
Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy in women under 40 years of age1. We sequenced the exomes of six individuals from three families with SCCOHT. After discovering segregating deleterious germline mutations in SMARCA4 in all three families, we tested DNA from a fourth affected family, which also carried a segregating SMARCA4 germline mutation. All the familial tumors sequenced harbored either a somatic mutation or loss of the wild-type allele. Immunohistochemical analysis of these cases and additional familial and non-familial cases showed loss of SMARCA4 (BRG1) protein in 38 of 40 tumors overall. Sequencing of cases with available DNA identified at least one germline or somatic deleterious SMARCA4 mutation in 30 of 32 cases. Additionally, the SCCOHT cell line BIN-67 had biallelic deleterious mutations in SMARCA4. Our findings identify alterations in SMARCA4 as the major cause of SCCOHT, which could lead to improvements in genetic counseling and new treatment approaches.
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Acknowledgements
This work is dedicated to the memory of Georgia Enter and Latosha Durham. We thank K. Hill and the Small Cell Ovarian Cancer Foundation for help in recruitment to this study, B. Vanderhyden (University of Ottawa) for providing the BIN-67 cells, S. Croce and C.S. Choong for aiding in the collection of pathological samples, P.-O. Fiset for help with immunohistochemistry and N. Benlimame, R. Zühlke-Jenisch, C. Kemming, M. Leiße, S. Peetz-Dienhart, L. Raestrup, K. Schmidt and C. Theile for technical assistance. N. Jabado, I. Bah and members of the Foulkes laboratory provided helpful discussions. We also thank the McGill University and Génome Québec Innovation Centre for their cooperation, H. Pierce for her help with contacting family 1 and D. Samelak for her help with family 3. Equipment for immunohistochemistry analysis was made available through Sonderforschungsbereich Transregio (SFB TR) 128 (grant to T. Kuhlmann (Z1)). We received funding from the Jewish General Hospital Foundation and the Jodi Taiger Lazarus Fund (W.D.F.), the Fonds de Recherche du Québec–Santé (L.W.), KinderKrebsInitiative Buchholz/Holm-Seppensen (R.S.) and Interdisziplinären Zentrum für Klinische Forschung (IZKF) Münster (Ha3/016/11) (M.H.).
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L.W. collected samples, performed the experiments and wrote the manuscript. J.C.-Z., S.F. and J.N. carried out bioinformatics analyses and provided text and figures. S.A. and J.A. helped with pathological examination. N.H. oversaw the experiments. E.T., D.G., E.S., C.G., R.G.K., E.A.R., F.R.U., A.M., K.P., S.M.C., J.L., L.M.R., T.M.U., T.A.B., V.G., M.L., A.B., M.T. and C.J.R.S. provided samples and critical input. B.R., E.C. and M.W. aided in experiments. R.S.-S., F.P., A.S. and H.J.M. aided in sample collection. M.A. aided in sample preparation. I.N. and R.S. performed FISH analysis. W.G.M. and B.A.C. provided samples and were the reference pathologists. Y.R. oversaw the preparation of formalin-fixed, paraffin-embedded samples for whole-exome sequencing. M.H. performed all immunohistochemistry analyses. J.M. oversaw all bioinformatics analysis. W.D.F. designed the project, wrote the manuscript and oversaw all aspects of the project.
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Supplementary Figure 1 Pedigrees of four families in which germline SMARCA4 mutations were found.
Pedigrees were not available for familial cases FA5 and FA6. In family 1, members II:2 and III:1 correspond to samples FA1a and FA1b, respectively, in Table 1. In family 2, members II:2 and III:2 correspond to samples FA2a and FA2b, respectively, in Table 1. In family 3, members II:2 and III:1 correspond to samples FA3a and FA3b, respectively, in Table 1. In family 4, members II:2 and III:1 correspond to samples FA4a and FA4b, respectively, in Table 1. SAB, spontaneous abortion; SCCOHT, small cell carcinoma of the ovary, hypercalcemic type; YST, yolk sac tumor; PSU, cancer, primary site unknown; +/+, wild-type for the familial SMARCA4 mutation; +/− , heterozygous for the familial SMARCA4 mutation. A diagonal line through a symbol indicates that the person is deceased. Whole-exome sequencing was carried out using DNA from individuals II:1 and III:1 (family 1); II:2 and III:2 (family 2); and II:1 and III:1 (family 3). The younger age of onset in recent generations in three of the four families is noted; it is likely to be mainly attributable to ascertainment bias. Individual II:4 from family 3 is at high risk for SCCOHT and has consented to a preventive bilateral oophorectomy.
Supplementary Figure 2 LOH analysis on the matched normal-tumor pair from FA1b.
LOH analysis on chromosome 19 for matched normal (FA1b(N)) and tumor (FA1b(T)) DNA from sample FA1b. BAF, B-allele frequency. See the Online Methods for details on LOH analysis. LOH was seen on chromosome 19p, where SMARCA4 is located, and not on 19q (or elsewhere in the genome; Supplementary Figure 4).
Supplementary Figure 3 SMARCA4 results from cases with and without SMARCA4 mutations.
(a) Immunostaining results from familial cases. Chromatograms are shown in Figure 1 where available. (b) Immunostaining results and chromatograms for cases where mutations were found in non-familial cases and cases of unknown family history with SCCOHT, classic type. (c) Immunohistochemistry results and chromatograms of mutations from non-familial cases and cases of unknown family history with SCCOHT, large cell type. (d) Immunohistochemistry results and chromatograms of mutations for the group with unknown family history where the diagnoses were less certain and for non-SCCOHT cases. (e) Immunohistochemistry results for SCCOHT mimics. Note that SMARCA4 immunostaining is retained in all cases.
Supplementary Figure 4 Cycloheximide results for LCLs from family 3 member FA3b.
(a) Agarose gel of the cDNA PCR product amplified across the germline mutation c.2617−3C>G. Lane 1, cDNA from non-treated LCLs. Lane 2, cDNA from LCLs treated with cycloheximide to inhibit translation. Extra (larger) bands appear as parts of the intron are retained. The part of the intron that is retained is subsequently translated and codes for a premature stop codon, making the transcript subject to nonsense-mediated decay in non-treated cells. (b) Chromatogram showing the part of the intron retained as a result of the mutation. An asterisk denotes the mutation. (c) The amino acids encoded at the end of exon 18 are shown in black, and those encoded by the beginning of the retained intron are shown in red. After 12 amino acids, the intron codes for a stop codon, noted by an asterisk. This subjects the allele to nonsense-mediated decay.
Supplementary Figure 5 Genome-wide LOH analysis on SCCOHT samples.
Genome-wide LOH analysis was carried out for samples sequenced by whole-exome sequencing (Online Methods). Recurrent LOH is seen only on chromosome 19p, where SMARCA4 is located (shown in pink). The top three rows show tumor samples with no LOH on chromosome 19p. Rows 4 and 5 show the matched normal and tumor sample seen in Supplementary Figure 1, where row 4 is normal DNA and row 5 is matched tumor DNA with LOH on chromosome 19p. Rows 6−12 show samples with LOH on chromosome 19p, with the region of homozygosity represented in pink.
Supplementary Figure 6 Analysis from two cases that showed abnormal FISH results.
(a) Hematoxylin and eosin staining of UN11 and UN10. (b) SMARCA4 staining. (c) Interphase FISH analysis showing homozygous deletion of the SMARCA4 locus in cases UN11 and UN10 evaluated using Zeiss fluorescence microscopes equipped with appropriate filter sets and documented using ISIS software (MetaSystems, Altlusheim). The left panel for each case shows hybridization with the double-color (red and green) break-apart probe for SMARCA4, and the right panel shows hybridization of the same area with the control probe for the centromere of chromosome 6 (CEP6 labeled in blue, false color display as green to contrast nuclear counterstain in DAPI). Both cases show a significant number of cells lacking SMARCA4 signals but clearly showing CEP6 signals indicating homozygous loss. (d) Chromatograms of the mutations found in both cases.
Supplementary Figure 7 BIN-67 analysis.
(a) Chromatogram of the splice-site mutation in genomic DNA from allele 1. An asterisk denotes the mutation. (b) Chromatogram of the splice-site mutation in genomic DNA from allele 2. An asterisk denotes the mutation. (c) Effect of the mutation on allele 1. Top, chromatograms of cDNA sequence, retaining the beginning of intron 15 and the end of intron 16. Only one allele, allele 1, is being amplified, as the mutation in allele 2 is not present at the end of intron 16 (asterisk). Bottom, diagram of primer location and the expected size of primers. The entire product including both introns was too large to amplify and is not seen on the gel in e. (d) Effect of the mutation on allele 2. Top, chromatogram of cDNA sequence, splicing out exons 15 and 16. Bottom, diagram of primer location and the expected product size. With two exons spliced out, the sequence is in frame with no amino acid changes but does splice out part of the SNF2_N domain, including the DEXDc domain, which is part of the ATPase domain (Figure 1). (e) Gel of BIN-67 results. Lane 1, GAPDH control. Lanes 2 and 3, PCR product from intronic primers 1 and 2 showing that, for allele 2, although the entire product was too large to amplify, the beginning of exon 16 and end of intron 17 are present. Lane 4, BIN-67 cells treated with cycloheximide and non-treated, showing that no larger fragment degraded by nonsense-mediated decay was recovered with cycloheximide. Lane 6, PCR product from control cDNA, showing the correct product size. S, sense; AS, antisense; CHX, cycloheximide; +Ctrl, positive control; Int, intronic primer.
Supplementary Figure 8 Immunoblot analysis of SMARCA4 and SMARCA2 in the BIN-67 cell line.
(a) SMARCA4 and SMARCA2 protein expression in the BIN-67 cell line. HEK 293T and HeLa cell lines were used as controls. No normal SMARCA4 protein was detected in BIN-67 cells as a result of the biallelic mutations (c.2438+1G>A and c.2439−2A>T). SMARCA2 protein expression was substantially decreased in the BIN-67 cell line compared to the HEK 293T and HeLa cell lines. β-actin was used as a loading control. (b) SMARCA2 cDNA expression by PCR seen on an agarose gel. Lane 1, BIN-67; lane 2, positive control; lane 3, negative control. See Supplementary Table 6 for the primers used.
Supplementary Figure 9 Chronology of the study.
SMARCA4 mutations were originally discovered through whole-exome sequencing of samples from three families. As DNA was unavailable for the affected mothers from families 1 and 3, DNA from the unaffected fathers was used instead. We looked for deleterious variants that were present in the affected daughter and were not present in the unaffected father. The rest of the study was carried out as shown.
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Witkowski, L., Carrot-Zhang, J., Albrecht, S. et al. Germline and somatic SMARCA4 mutations characterize small cell carcinoma of the ovary, hypercalcemic type. Nat Genet 46, 438–443 (2014). https://doi.org/10.1038/ng.2931
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DOI: https://doi.org/10.1038/ng.2931
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