JMG Online First, published on April 24, 2015 as 10.1136/jmedgenet-2014-102912 Cancer genetics
SHORT REPORT
A germline mutation in PBRM1 predisposes to renal cell carcinoma Patrick R Benusiglio,1,2 Sophie Couvé,3,4,5 Brigitte Gilbert-Dussardier,1,6 Sophie Deveaux,1 Hélène Le Jeune,3,4,5 Mélanie Da Costa,3,4,5 Gaëlle Fromont,7 Françoise Memeteau,8 Mokrane Yacoub,9 Isabelle Coupier,1,10,11 Dominique Leroux,1,12 Arnaud Méjean,1,13,14 Bernard Escudier,15 Sophie Giraud,1,16 Anne-Paule Gimenez-Roqueplo,17 Christophe Blondel,18 Eric Frouin,19 Bin T Teh,20,21,22 Sophie Ferlicot,1,23 Brigitte Bressac-de Paillerets,18 Stéphane Richard,1,3,4,5 Sophie Gad3,4,5 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jmedgenet-2014-102912). For numbered affiliations see end of article. Correspondence to Dr Sophie Gad, Laboratoire de Génétique Oncologique EPHE, INSERM U753, Gustave Roussy Cancer Campus, 114 rue Edouard Vaillant, Villejuif 94800, France; [email protected] Received 20 November 2014 Revised 10 March 2015 Accepted 4 April 2015
To cite: Benusiglio PR, Couvé S, GilbertDussardier B, et al. J Med Genet Published Online First: [please include Day Month Year] doi:10.1136/ jmedgenet-2014-102912
ABSTRACT Background Many cases of familial renal cell carcinoma (RCC) remain unexplained by mutations in the known predisposing genes or shared environmental factors. There are therefore additional, still unidentified genes involved in familial RCC. PBRM1 is a tumour suppressor gene and somatic mutations are found in 30–45% of sporadic clear cell (cc) RCC. Methods We selected 35 unrelated patients with unexplained personal history of ccRCC and at least one affected first-degree relative, and sequenced the PBRM1 gene. Results A germline frameshift mutation (c.3998_4005del [ p.Asp1333Glyfs]) was found in one patient. The patient’s mother, his sister and one niece also had ccRCC. The mutation co-segregated with the disease as the three affected relatives were carriers, while an unaffected sister was not, according with autosomal-dominant transmission. Somatic studies supported these findings, as we observed both loss of heterozygosity for the mutation and loss of protein expression in renal tumours. Conclusions We show for the first time that an inherited mutation in PBRM1 predisposes to RCC. International studies are necessary to estimate the contribution of PBRM1 to RCC susceptibility, estimate penetrance and then integrate the gene into routine clinical practice.
About 3% of renal cell carcinomas (RCC) are familial, and inherited genetic abnormalities likely explain most of these cases (reviewed in ref. 1). The main RCC predisposing genes are VHL, MET, FH, FLCN and SDHs associated respectively with von Hippel–Lindau disease, the hereditary papillary RCC, hereditary leiomyomatosis and papillary RCC, Birt–Hogg–Dubé syndrome and succinate dehydrogenase RCC. Mutations in these different genes predispose to different types of RCC; VHL and SDHs predispose to clear cell (cc) RCC (the most common histological type), FLCN to benign oncocytoma, chromophobe, hybrid chromophobeoncocytoma and ccRCC, MET to papillary type 1 RCC and FH to papillary type 2 RCC. Additionally, chromosome 3 translocations are a
rare cause of familial ccRCC, and karyotyping is mandatory in this context. In 2011, we and others identified a rare missense mutation in the MITF oncogene, associated with the development of melanoma and, in some cases, RCC.2 3 Other still unidentified genes are involved in susceptibility to RCC as many familial cases remain unexplained by mutations in known genes or by shared environmental factors.4 Genes that are somatically mutated in sporadic RCC play a role in the majority of familial RCC, the VHL tumour suppressor gene being the prime example as it is inactivated in 80% of ccRCC, and causes von Hippel–Lindau disease when mutated in the germline.5 VHL loss of function is central to renal carcinogenesis through the activation of the hypoxia-inducible factor (HIF) pathway.6 In the past few years, high-throughput sequencing technologies have enabled the discovery of two other genes with frequent somatic mutations in ccRCC: PBRM1 (Poly bromo-1) and BAP1 (BRCA1-associated protein-1).7–9 Both are tumour suppressors located close to VHL on chromosome 3p. Somatic mutations in PBRM1, encoding BAF180, a subunit of ATP-dependent chromatinremodelling complex, and BAP1, encoding a ubiquitin carboxy-terminal hydrolase, are found in 30–45% and 10–15% of ccRCC, respectively. BAP1 has only recently been associated with familial RCC as germline mutations were identified in families with multiple cases of ccRCC and other tumours.10 11 To search for novel RCC predisposing genes, we performed a candidate gene study in a series of ccRCC families and, for the first time, discovered PBRM1 to be mutated at the germline level in a family. We selected for study 35 unrelated French patients with familial ccRCC, that is, individuals with a personal history of ccRCC and at least one first-degree relative with RCC of cc or undocumented histology. All patients had been seen in the clinic by a cancer geneticist between 1997 and 2011, were negative for VHL germline mutations and had given informed consent for the exploratory analysis of candidate genes other than VHL. Blood samples were provided by the French NCI
Benusiglio PR, et al. J Med Genet 2015;0:1–5. doi:10.1136/jmedgenet-2014-102912
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.
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Cancer genetics
Figure 1 Pedigree for the family carrying a PBRM1 germline mutation. The mutation segregates with diagnoses of renal cell carcinoma. Arrow, index case. Dg, age at diagnosis of renal cell carcinoma; Dcd, deceased (age of death); Mut, mutation carrier; Wt, wild-type.
(INCa) Centre Expert National Cancers Rares PREDIR in le Kremlin-Bicêtre near Paris, Hôpital Edouard Herriot in Lyon, CHRU—Hôpital Arnaud de Villeneuve in Montpellier, and the Centre Hospitalier Universitaire in Poitiers. PBRM1 was analysed by screening the 30 exons and their flanking regions (gene accession number ENST00000296302, isoform 1 of 1689 amino acids, corresponding to the full-length protein) by Sanger
sequencing on germline DNA, with the ABI3730 analyser (Applied Biosystems), and inspection of the sequences using Sequencher V.4.10.1 software (Gene Codes Corporation). Detailed protocols are available on request. Among the 35 patients included, mean age at diagnosis of ccRCC (age at first RCC in case of multiple tumours) was 50 (age range 30–72). Only three patients had multiple ccRCC, and in these cases both kidneys were affected. Twenty-eight, six and one patients had respectively one, two and three relatives with a history of RCC, with a mean age at RCC diagnosis in relatives of 58 (age range 34–84). A germline mutation located in exon 24 of PBRM1 was found in the index case of the family with four cases of ccRCC. This patient (II.2, figure 1) was a previously healthy Caucasian male who presented with unexplained abdominal pain age 38. Ultrasonography and abdominal CT scan revealed a 25 mm tumour of the right kidney, and he underwent partial nephrectomy. The pathologist described a 20 mm grade 3 ccRCC. Family history was highly relevant as his mother (I.2), a sister (II.4) and a niece (III.1, daughter of the affected sister) had ccRCC respectively ages 64/70 (bilateral), 42 (unilateral) and 36 (unilateral). Both his mother and sister had died of the disease. Family history was otherwise unremarkable. Four other siblings were unaffected, and there were no other cancers or nononcological manifestations in the patient or his relatives, suggesting a known genetic susceptibility to RCC, or to cancer in general. In addition to VHL, we had excluded germline mutations in the other genes associated with familial ccRCC (FLCN, SDHB and BAP1) as well as karyotype abnormalities. The
Figure 2 Sequencing chromatograms for PBRM1 exon 24. A heterozygous frameshift mutation (arrow) was detected in the germline DNA of the index case (II.2) and his affected niece (III.1), but not in an unaffected sister (II.6). There is loss of heterozygosity in the niece’s renal tumour (III.1). The reference sequence (commercial DNA) is located at the top of the figure.
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Benusiglio PR, et al. J Med Genet 2015;0:1–5. doi:10.1136/jmedgenet-2014-102912
Cancer genetics proband was found to carry a heterozygous germline deletion in PBRM1, c.3998_4005del ( p.Asp1333Glyfs) (figure 2). This is a frameshift, truncating mutation that creates a stop codon three amino acids downstream codon 1333. This deletion is novel compared with PBRM1 somatic mutations identified so far in sporadic RCC.6 8 Furthermore, no PBRM1 truncating mutation was reported in 6503 Caucasian and Afro-American individuals included in the NHLBI Exome Sequencing Project (http://evs.gs. washington.edu/EVS). We confirmed through co-segregation and somatic studies that PBRM1 c.3998_4005del ( p.Asp1333Glyfs) was associated with ccRCC in the family. We extracted DNA from the affected niece’s circulating white blood cells and frozen renal tissue (III.1). Both were heterozygote for the frameshift mutation, while the renal tumour was almost homozygote, demonstrating therefore loss of heterozygosity (LOH) (figure 2). On the contrary, the mutation was not found in the germline DNA of another sister with no history of cancer age 61 (II.6, figure 2). We then analysed DNA extracted from paraffin-embedded normal and tumour renal tissue in the affected mother (I.2) and sister (II.4), using specific primers designed for a smaller amplicon given the degraded state of the DNA. The frameshift mutation was present in all samples (see online supplementary figure S1). Finally, we carried out BAF180 (PBRM1) immunohistochemistry studies on the niece’s tissue blocks. We observed loss of nuclear protein expression in the tumour tissue, whereas expression was conserved in normal tissue and in inflammatory cells (figure 3). In the current era of exome sequencing, our study demonstrates that the simpler and cheaper candidate gene approach remains useful when the genes are selected through relevant clues. Indeed, by combining somatic observations and biological plausibility, we selected for study PBRM1 and showed for the first time that it was an RCC predisposing gene. We identified a germline mutation in this gene in a family with four RCC-affected individuals among three generations. The frameshift c.3998_4005del ( p.Asp1333Glyfs) is expected to be a loss-of-function mutation as it results either in a truncated protein lacking the key, highly conserved High Mobility Group-box DNA binding domain of BAF180 (Uniprot Q86U86) or in a null allele through nonsense mRNA-mediated decay, a cellular mechanism eliminating mRNAs containing premature termination codons and thus helping to limit the synthesis of abnormal proteins.12 Somatic studies supported our germline findings, as we observed LOH in renal tumours. As with other tumour suppressor genes, tumours associated with PBRM1 arise when both gene copies are inactivated.7 9 We also demonstrated loss of BAF180 (PBRM1) protein expression in tumoural tissue, illustrating the fact that the frameshift mutation prevented the expression of a functional protein. Finally, the germline mutation clearly co-segregated with ccRCC, as it was not found in a first-degree relative with no history of cancer, and was transmitted in an autosomal-dominant fashion. Only two families with a germline mutation in the BAP1 gene have been reported so far (out of 115 studied).10 11 Likewise, PBRM1 germline mutations are rare in familial RCC as only one out of 35 families (2.9%) carried a pathogenic mutation in our study. However, we might have underestimated the frequency of mutations in inherited, familial RCC as some cases included in our study had a weak, late-onset phenotype. Indeed, we did not set an age limit for inclusion as long as family history was positive. For example, we analysed one case who had ccRCC age 72, his sister had the same diagnosis age 79, and the advanced ages at diagnosis in this family could suggest a non-genetic aetiology. Furthermore, while germline VHL analysis was done for Benusiglio PR, et al. J Med Genet 2015;0:1–5. doi:10.1136/jmedgenet-2014-102912
Figure 3 Immunohistochemistry for the BAF180 (PBRM1) protein. Immunohistochemical analysis was performed on deparaffinised sections of renal tissue for the niece (III.1), using polyclonal rabbit anti-BAF180 antibody (dilution, 1:50; cat. no. A301-591A; Bethyl Laboratories, Montgomery, Texas, USA). According to the Bond Polymer Refine detection kit (Menarini diagnostics), sections were treated with a solution of peroxidase-labelled streptavidin and colour reaction was developed by incubation with 3, 30 -diaminobenzidine. Then, nuclear counterstaining with haematoxylin was performed. (A) Normal renal tissue. Strong nuclear staining is visible in a glomerulus and in atrophic tubules (×10). (B) There is loss of BAF180 (PBRM1) expression in tumour tissue (×20). The rare cells showing nuclear staining are inflammatory cells. all included patients, testing for the other ccRCC predisposing genes like FLCN and SDHB, and karyotyping were not systematic, and disease-causing mutations or chromosomal translocations could therefore have been missed. Finally, we could only perform Sanger sequencing of the coding regions and intron– exon junctions, and did not look for large genomic rearrangements. International collaborative studies including if possible hundreds of families are needed to determine more precisely the contribution of PBRM1 to genetic susceptibility to RCC, estimate RCC penetrance and then integrate the gene into the 3
Cancer genetics routine clinical practice of cancer genetics. In addition to RCC, somatic PBRM1 mutations have also been described in intrahepatic cholangiocarcinoma (with an enrichment in nonsense and frameshift mutations), gallbladder carcinoma and in epithelial tumours of the thymus.13 14 A frameshift somatic mutation was even observed in a malignant mesothelioma cell line.15 The implication of PBRM1 in predisposition to cancers other than RCC is therefore a real possibility, and large-scale studies will hopefully help determine the whole spectrum of clinical manifestations associated with inherited abnormalities. There are direct clinical implications to the discovery of a new cancer susceptibility gene, and PBRM1 should be no exception. Our index case had RCC in his thirties and the three other known carriers in the family also developed the disease, two of them at a young age, suggesting that the penetrance associated with mutations is high. At this stage and until data based on more families are published, we would therefore recommend annual renal imaging in carriers starting age 18–20, ideally with MRI, a surveillance protocol inspired by the recommendations made to patients with VHL disease.16 Finally, and from a biological point of view, PBRM1 encodes a SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodelling complex protein.7 9 This complex is notably involved in the normal cellular response to hypoxia-induced cell cycle arrest.17 The main RCC predisposing genes reported so far are involved in oxygen, iron, energy or nutrient sensing pathways.1 The identification in this context of PBRM1 as a new RCC predisposing gene is compelling. Author affiliations 1 Centre Expert National Cancers Rares PREDIR AP-HP/INCa, Hôpital Bicêtre, Le Kremlin Bicêtre, France 2 Département de Médecine Oncologique, Consultation d’Oncogénétique, Gustave Roussy Cancer Campus, Villejuif, France 3 Ecole Pratique des Hautes Etudes, Paris, France 4 Laboratoire de Génétique Oncologique EPHE, INSERM U753, Villejuif, France 5 Faculté de Médecine Université Paris-Sud, Le Kremlin-Bicêtre, France 6 Service de Génétique Médicale, CHU de Poitiers, Poitiers, France 7 Service d’Anatomie et Cytologie Pathologiques, INSERM UMR1069, CHRU de Tours, Tours, France 8 Département d’Anatomie et de Cytologie Pathologique, Centre Hospitalier de Niort, Niort, France 9 Service de Pathologie, CHU Pellegrin de Bordeaux, Bordeaux, France 10 Unité d’oncogénétique, CRLC Val-d’Aurelle, Montpellier, France 11 Service de Génétique Médicale, Unité d’Oncogénétique, CHU de Montpellier, Montpellier, France 12 Département d’Hématologie, Oncogénétique et Immunologie, CHU de Grenoble site Nord—Institut de biologie et de pathologie, Boulevard de la Chantourne, La Tronche, France 13 Service d’Urologie, Hôpital Européen Georges-Pompidou, AP-HP, Paris, France 14 Université Paris-Descartes, 12 Rue de l’École de Médecine, Paris, France 15 Département de Médecine Oncologique, Consultation des tumeurs rénales, et INSERM U753, Villejuif, France 16 Génétique Moléculaire et Clinique, Hôpital Edouard Herriot, Lyon, France 17 Département de Génétique Moléculaire, Hôpital Européen Georges Pompidou, AP-HP, Paris, France 18 Département de Biopathologie, Service de Génétique, Gustave Roussy Cancer Campus, Villejuif, France 19 Service d’Anatomie et de Cytologie Pathologiques, CHU de Poitiers, Poitiers, France 20 NCCS-VARI Translational Research Laboratory, Division of Medical Sciences, National Cancer Centre, Singapore, Singapore 21 Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, Singapore 22 Cancer Science Institute of Singapore, National University of Singapore, Centre for Life Sciences, Singapore, Singapore 23 Service d’Anatomie Pathologique, Hôpital Bicêtre, AP-HP, Le Kremlin Bicêtre, France Acknowledgements We thank the families for their participation. We are grateful to Christine Bombled, Johny Bombled, Odile Cabaret, Sébastien Forget, Marine 4
Guillaud-Bataille and Giovanni Stevanin for their technical and scientific contribution, and Betty Gardie and Charles-Henry Gattolliat for critical reading of the manuscript. Finally, we would like to thank the NHLBI GO Exome Sequencing Project and its ongoing studies that produced and provided exome variant calls for comparison. Contributors PRB and SC contributed equally. SR and SG are joint senior authors. HLJ and MDC performed PBRM1 sequencing. PRB and SC analysed the data and wrote the manuscript. BG-D, SD, IC, DL, AM, BE, SGi, A-PG-R, CB and SR were in charge of the patients, either in the clinic or in the laboratory (analysis of known RCC predisposing genes). GF, FM, MY, EF and SF provided pathological expertise and SF performed IHC. BB-dP and BTT revised the manuscript. SR and SGa conceived the study and co-wrote the manuscript. All authors read and approved the final version of the manuscript. Funding Ligue nationale contre le Cancer (Comités de l’Indre et du Cher), the French National Cancer Institute ‘PNES Rein’ project and the Fondation Gustave Roussy. Competing interests None declared. Patient consent Obtained. Ethics approval CCPPRB (Ethical Committee), Le Kremlin-Bicêtre, France. Provenance and peer review Not commissioned; externally peer reviewed.
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SHORT REPORT
A germline mutation in PBRM1 predisposes to renal cell carcinoma Patrick R Benusiglio,1,2 Sophie Couvé,3,4,5 Brigitte Gilbert-Dussardier,1,6 Sophie Deveaux,1 Hélène Le Jeune,3,4,5 Mélanie Da Costa,3,4,5 Gaëlle Fromont,7 Françoise Memeteau,8 Mokrane Yacoub,9 Isabelle Coupier,1,10,11 Dominique Leroux,1,12 Arnaud Méjean,1,13,14 Bernard Escudier,15 Sophie Giraud,1,16 Anne-Paule Gimenez-Roqueplo,17 Christophe Blondel,18 Eric Frouin,19 Bin T Teh,20,21,22 Sophie Ferlicot,1,23 Brigitte Bressac-de Paillerets,18 Stéphane Richard,1,3,4,5 Sophie Gad3,4,5 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jmedgenet-2014-102912). For numbered affiliations see end of article. Correspondence to Dr Sophie Gad, Laboratoire de Génétique Oncologique EPHE, INSERM U753, Gustave Roussy Cancer Campus, 114 rue Edouard Vaillant, Villejuif 94800, France; [email protected] Received 20 November 2014 Revised 10 March 2015 Accepted 4 April 2015
To cite: Benusiglio PR, Couvé S, GilbertDussardier B, et al. J Med Genet Published Online First: [please include Day Month Year] doi:10.1136/ jmedgenet-2014-102912
ABSTRACT Background Many cases of familial renal cell carcinoma (RCC) remain unexplained by mutations in the known predisposing genes or shared environmental factors. There are therefore additional, still unidentified genes involved in familial RCC. PBRM1 is a tumour suppressor gene and somatic mutations are found in 30–45% of sporadic clear cell (cc) RCC. Methods We selected 35 unrelated patients with unexplained personal history of ccRCC and at least one affected first-degree relative, and sequenced the PBRM1 gene. Results A germline frameshift mutation (c.3998_4005del [ p.Asp1333Glyfs]) was found in one patient. The patient’s mother, his sister and one niece also had ccRCC. The mutation co-segregated with the disease as the three affected relatives were carriers, while an unaffected sister was not, according with autosomal-dominant transmission. Somatic studies supported these findings, as we observed both loss of heterozygosity for the mutation and loss of protein expression in renal tumours. Conclusions We show for the first time that an inherited mutation in PBRM1 predisposes to RCC. International studies are necessary to estimate the contribution of PBRM1 to RCC susceptibility, estimate penetrance and then integrate the gene into routine clinical practice.
About 3% of renal cell carcinomas (RCC) are familial, and inherited genetic abnormalities likely explain most of these cases (reviewed in ref. 1). The main RCC predisposing genes are VHL, MET, FH, FLCN and SDHs associated respectively with von Hippel–Lindau disease, the hereditary papillary RCC, hereditary leiomyomatosis and papillary RCC, Birt–Hogg–Dubé syndrome and succinate dehydrogenase RCC. Mutations in these different genes predispose to different types of RCC; VHL and SDHs predispose to clear cell (cc) RCC (the most common histological type), FLCN to benign oncocytoma, chromophobe, hybrid chromophobeoncocytoma and ccRCC, MET to papillary type 1 RCC and FH to papillary type 2 RCC. Additionally, chromosome 3 translocations are a
rare cause of familial ccRCC, and karyotyping is mandatory in this context. In 2011, we and others identified a rare missense mutation in the MITF oncogene, associated with the development of melanoma and, in some cases, RCC.2 3 Other still unidentified genes are involved in susceptibility to RCC as many familial cases remain unexplained by mutations in known genes or by shared environmental factors.4 Genes that are somatically mutated in sporadic RCC play a role in the majority of familial RCC, the VHL tumour suppressor gene being the prime example as it is inactivated in 80% of ccRCC, and causes von Hippel–Lindau disease when mutated in the germline.5 VHL loss of function is central to renal carcinogenesis through the activation of the hypoxia-inducible factor (HIF) pathway.6 In the past few years, high-throughput sequencing technologies have enabled the discovery of two other genes with frequent somatic mutations in ccRCC: PBRM1 (Poly bromo-1) and BAP1 (BRCA1-associated protein-1).7–9 Both are tumour suppressors located close to VHL on chromosome 3p. Somatic mutations in PBRM1, encoding BAF180, a subunit of ATP-dependent chromatinremodelling complex, and BAP1, encoding a ubiquitin carboxy-terminal hydrolase, are found in 30–45% and 10–15% of ccRCC, respectively. BAP1 has only recently been associated with familial RCC as germline mutations were identified in families with multiple cases of ccRCC and other tumours.10 11 To search for novel RCC predisposing genes, we performed a candidate gene study in a series of ccRCC families and, for the first time, discovered PBRM1 to be mutated at the germline level in a family. We selected for study 35 unrelated French patients with familial ccRCC, that is, individuals with a personal history of ccRCC and at least one first-degree relative with RCC of cc or undocumented histology. All patients had been seen in the clinic by a cancer geneticist between 1997 and 2011, were negative for VHL germline mutations and had given informed consent for the exploratory analysis of candidate genes other than VHL. Blood samples were provided by the French NCI
Benusiglio PR, et al. J Med Genet 2015;0:1–5. doi:10.1136/jmedgenet-2014-102912
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.
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Cancer genetics
Figure 1 Pedigree for the family carrying a PBRM1 germline mutation. The mutation segregates with diagnoses of renal cell carcinoma. Arrow, index case. Dg, age at diagnosis of renal cell carcinoma; Dcd, deceased (age of death); Mut, mutation carrier; Wt, wild-type.
(INCa) Centre Expert National Cancers Rares PREDIR in le Kremlin-Bicêtre near Paris, Hôpital Edouard Herriot in Lyon, CHRU—Hôpital Arnaud de Villeneuve in Montpellier, and the Centre Hospitalier Universitaire in Poitiers. PBRM1 was analysed by screening the 30 exons and their flanking regions (gene accession number ENST00000296302, isoform 1 of 1689 amino acids, corresponding to the full-length protein) by Sanger
sequencing on germline DNA, with the ABI3730 analyser (Applied Biosystems), and inspection of the sequences using Sequencher V.4.10.1 software (Gene Codes Corporation). Detailed protocols are available on request. Among the 35 patients included, mean age at diagnosis of ccRCC (age at first RCC in case of multiple tumours) was 50 (age range 30–72). Only three patients had multiple ccRCC, and in these cases both kidneys were affected. Twenty-eight, six and one patients had respectively one, two and three relatives with a history of RCC, with a mean age at RCC diagnosis in relatives of 58 (age range 34–84). A germline mutation located in exon 24 of PBRM1 was found in the index case of the family with four cases of ccRCC. This patient (II.2, figure 1) was a previously healthy Caucasian male who presented with unexplained abdominal pain age 38. Ultrasonography and abdominal CT scan revealed a 25 mm tumour of the right kidney, and he underwent partial nephrectomy. The pathologist described a 20 mm grade 3 ccRCC. Family history was highly relevant as his mother (I.2), a sister (II.4) and a niece (III.1, daughter of the affected sister) had ccRCC respectively ages 64/70 (bilateral), 42 (unilateral) and 36 (unilateral). Both his mother and sister had died of the disease. Family history was otherwise unremarkable. Four other siblings were unaffected, and there were no other cancers or nononcological manifestations in the patient or his relatives, suggesting a known genetic susceptibility to RCC, or to cancer in general. In addition to VHL, we had excluded germline mutations in the other genes associated with familial ccRCC (FLCN, SDHB and BAP1) as well as karyotype abnormalities. The
Figure 2 Sequencing chromatograms for PBRM1 exon 24. A heterozygous frameshift mutation (arrow) was detected in the germline DNA of the index case (II.2) and his affected niece (III.1), but not in an unaffected sister (II.6). There is loss of heterozygosity in the niece’s renal tumour (III.1). The reference sequence (commercial DNA) is located at the top of the figure.
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Benusiglio PR, et al. J Med Genet 2015;0:1–5. doi:10.1136/jmedgenet-2014-102912
Cancer genetics proband was found to carry a heterozygous germline deletion in PBRM1, c.3998_4005del ( p.Asp1333Glyfs) (figure 2). This is a frameshift, truncating mutation that creates a stop codon three amino acids downstream codon 1333. This deletion is novel compared with PBRM1 somatic mutations identified so far in sporadic RCC.6 8 Furthermore, no PBRM1 truncating mutation was reported in 6503 Caucasian and Afro-American individuals included in the NHLBI Exome Sequencing Project (http://evs.gs. washington.edu/EVS). We confirmed through co-segregation and somatic studies that PBRM1 c.3998_4005del ( p.Asp1333Glyfs) was associated with ccRCC in the family. We extracted DNA from the affected niece’s circulating white blood cells and frozen renal tissue (III.1). Both were heterozygote for the frameshift mutation, while the renal tumour was almost homozygote, demonstrating therefore loss of heterozygosity (LOH) (figure 2). On the contrary, the mutation was not found in the germline DNA of another sister with no history of cancer age 61 (II.6, figure 2). We then analysed DNA extracted from paraffin-embedded normal and tumour renal tissue in the affected mother (I.2) and sister (II.4), using specific primers designed for a smaller amplicon given the degraded state of the DNA. The frameshift mutation was present in all samples (see online supplementary figure S1). Finally, we carried out BAF180 (PBRM1) immunohistochemistry studies on the niece’s tissue blocks. We observed loss of nuclear protein expression in the tumour tissue, whereas expression was conserved in normal tissue and in inflammatory cells (figure 3). In the current era of exome sequencing, our study demonstrates that the simpler and cheaper candidate gene approach remains useful when the genes are selected through relevant clues. Indeed, by combining somatic observations and biological plausibility, we selected for study PBRM1 and showed for the first time that it was an RCC predisposing gene. We identified a germline mutation in this gene in a family with four RCC-affected individuals among three generations. The frameshift c.3998_4005del ( p.Asp1333Glyfs) is expected to be a loss-of-function mutation as it results either in a truncated protein lacking the key, highly conserved High Mobility Group-box DNA binding domain of BAF180 (Uniprot Q86U86) or in a null allele through nonsense mRNA-mediated decay, a cellular mechanism eliminating mRNAs containing premature termination codons and thus helping to limit the synthesis of abnormal proteins.12 Somatic studies supported our germline findings, as we observed LOH in renal tumours. As with other tumour suppressor genes, tumours associated with PBRM1 arise when both gene copies are inactivated.7 9 We also demonstrated loss of BAF180 (PBRM1) protein expression in tumoural tissue, illustrating the fact that the frameshift mutation prevented the expression of a functional protein. Finally, the germline mutation clearly co-segregated with ccRCC, as it was not found in a first-degree relative with no history of cancer, and was transmitted in an autosomal-dominant fashion. Only two families with a germline mutation in the BAP1 gene have been reported so far (out of 115 studied).10 11 Likewise, PBRM1 germline mutations are rare in familial RCC as only one out of 35 families (2.9%) carried a pathogenic mutation in our study. However, we might have underestimated the frequency of mutations in inherited, familial RCC as some cases included in our study had a weak, late-onset phenotype. Indeed, we did not set an age limit for inclusion as long as family history was positive. For example, we analysed one case who had ccRCC age 72, his sister had the same diagnosis age 79, and the advanced ages at diagnosis in this family could suggest a non-genetic aetiology. Furthermore, while germline VHL analysis was done for Benusiglio PR, et al. J Med Genet 2015;0:1–5. doi:10.1136/jmedgenet-2014-102912
Figure 3 Immunohistochemistry for the BAF180 (PBRM1) protein. Immunohistochemical analysis was performed on deparaffinised sections of renal tissue for the niece (III.1), using polyclonal rabbit anti-BAF180 antibody (dilution, 1:50; cat. no. A301-591A; Bethyl Laboratories, Montgomery, Texas, USA). According to the Bond Polymer Refine detection kit (Menarini diagnostics), sections were treated with a solution of peroxidase-labelled streptavidin and colour reaction was developed by incubation with 3, 30 -diaminobenzidine. Then, nuclear counterstaining with haematoxylin was performed. (A) Normal renal tissue. Strong nuclear staining is visible in a glomerulus and in atrophic tubules (×10). (B) There is loss of BAF180 (PBRM1) expression in tumour tissue (×20). The rare cells showing nuclear staining are inflammatory cells. all included patients, testing for the other ccRCC predisposing genes like FLCN and SDHB, and karyotyping were not systematic, and disease-causing mutations or chromosomal translocations could therefore have been missed. Finally, we could only perform Sanger sequencing of the coding regions and intron– exon junctions, and did not look for large genomic rearrangements. International collaborative studies including if possible hundreds of families are needed to determine more precisely the contribution of PBRM1 to genetic susceptibility to RCC, estimate RCC penetrance and then integrate the gene into the 3
Cancer genetics routine clinical practice of cancer genetics. In addition to RCC, somatic PBRM1 mutations have also been described in intrahepatic cholangiocarcinoma (with an enrichment in nonsense and frameshift mutations), gallbladder carcinoma and in epithelial tumours of the thymus.13 14 A frameshift somatic mutation was even observed in a malignant mesothelioma cell line.15 The implication of PBRM1 in predisposition to cancers other than RCC is therefore a real possibility, and large-scale studies will hopefully help determine the whole spectrum of clinical manifestations associated with inherited abnormalities. There are direct clinical implications to the discovery of a new cancer susceptibility gene, and PBRM1 should be no exception. Our index case had RCC in his thirties and the three other known carriers in the family also developed the disease, two of them at a young age, suggesting that the penetrance associated with mutations is high. At this stage and until data based on more families are published, we would therefore recommend annual renal imaging in carriers starting age 18–20, ideally with MRI, a surveillance protocol inspired by the recommendations made to patients with VHL disease.16 Finally, and from a biological point of view, PBRM1 encodes a SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodelling complex protein.7 9 This complex is notably involved in the normal cellular response to hypoxia-induced cell cycle arrest.17 The main RCC predisposing genes reported so far are involved in oxygen, iron, energy or nutrient sensing pathways.1 The identification in this context of PBRM1 as a new RCC predisposing gene is compelling. Author affiliations 1 Centre Expert National Cancers Rares PREDIR AP-HP/INCa, Hôpital Bicêtre, Le Kremlin Bicêtre, France 2 Département de Médecine Oncologique, Consultation d’Oncogénétique, Gustave Roussy Cancer Campus, Villejuif, France 3 Ecole Pratique des Hautes Etudes, Paris, France 4 Laboratoire de Génétique Oncologique EPHE, INSERM U753, Villejuif, France 5 Faculté de Médecine Université Paris-Sud, Le Kremlin-Bicêtre, France 6 Service de Génétique Médicale, CHU de Poitiers, Poitiers, France 7 Service d’Anatomie et Cytologie Pathologiques, INSERM UMR1069, CHRU de Tours, Tours, France 8 Département d’Anatomie et de Cytologie Pathologique, Centre Hospitalier de Niort, Niort, France 9 Service de Pathologie, CHU Pellegrin de Bordeaux, Bordeaux, France 10 Unité d’oncogénétique, CRLC Val-d’Aurelle, Montpellier, France 11 Service de Génétique Médicale, Unité d’Oncogénétique, CHU de Montpellier, Montpellier, France 12 Département d’Hématologie, Oncogénétique et Immunologie, CHU de Grenoble site Nord—Institut de biologie et de pathologie, Boulevard de la Chantourne, La Tronche, France 13 Service d’Urologie, Hôpital Européen Georges-Pompidou, AP-HP, Paris, France 14 Université Paris-Descartes, 12 Rue de l’École de Médecine, Paris, France 15 Département de Médecine Oncologique, Consultation des tumeurs rénales, et INSERM U753, Villejuif, France 16 Génétique Moléculaire et Clinique, Hôpital Edouard Herriot, Lyon, France 17 Département de Génétique Moléculaire, Hôpital Européen Georges Pompidou, AP-HP, Paris, France 18 Département de Biopathologie, Service de Génétique, Gustave Roussy Cancer Campus, Villejuif, France 19 Service d’Anatomie et de Cytologie Pathologiques, CHU de Poitiers, Poitiers, France 20 NCCS-VARI Translational Research Laboratory, Division of Medical Sciences, National Cancer Centre, Singapore, Singapore 21 Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, Singapore 22 Cancer Science Institute of Singapore, National University of Singapore, Centre for Life Sciences, Singapore, Singapore 23 Service d’Anatomie Pathologique, Hôpital Bicêtre, AP-HP, Le Kremlin Bicêtre, France Acknowledgements We thank the families for their participation. We are grateful to Christine Bombled, Johny Bombled, Odile Cabaret, Sébastien Forget, Marine 4
Guillaud-Bataille and Giovanni Stevanin for their technical and scientific contribution, and Betty Gardie and Charles-Henry Gattolliat for critical reading of the manuscript. Finally, we would like to thank the NHLBI GO Exome Sequencing Project and its ongoing studies that produced and provided exome variant calls for comparison. Contributors PRB and SC contributed equally. SR and SG are joint senior authors. HLJ and MDC performed PBRM1 sequencing. PRB and SC analysed the data and wrote the manuscript. BG-D, SD, IC, DL, AM, BE, SGi, A-PG-R, CB and SR were in charge of the patients, either in the clinic or in the laboratory (analysis of known RCC predisposing genes). GF, FM, MY, EF and SF provided pathological expertise and SF performed IHC. BB-dP and BTT revised the manuscript. SR and SGa conceived the study and co-wrote the manuscript. All authors read and approved the final version of the manuscript. Funding Ligue nationale contre le Cancer (Comités de l’Indre et du Cher), the French National Cancer Institute ‘PNES Rein’ project and the Fondation Gustave Roussy. Competing interests None declared. Patient consent Obtained. Ethics approval CCPPRB (Ethical Committee), Le Kremlin-Bicêtre, France. Provenance and peer review Not commissioned; externally peer reviewed.
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