Germline Mutations in the CDKN2B Tumor Suppressor Gene Predispose to Renal Cell Carcinoma.

UNLABELLED Familial renal cell carcinoma (RCC) is genetically heterogeneous and may be caused by mutations in multiple genes, including VHL, MET, SDHB, FH, FLCN, PTEN, and BAP1. However, most individuals with inherited RCC do not have a detectable germline mutation. To identify novel inherited RCC genes, we undertook exome resequencing studies in a familial RCC kindred and identified a CDKN2B nonsense mutation that segregated with familial RCC status. Targeted resequencing of CDKN2B in individuals (n = 82) with features of inherited RCC then revealed three candidate CDKN2B missense mutations (p.Pro40Thr, p.Ala23Glu, and p.Asp86Asn). In silico analysis of the three-dimensional structures indicated that each missense substitution was likely pathogenic through reduced stability of the mutant or reduced affinity for cyclin-dependent kinases 4 and 6, and in vitro studies demonstrated that each of the mutations impaired CDKN2B-induced suppression of proliferation in an RCC cell line. These findings identify germline CDKN2B mutations as a novel cause of familial RCC. SIGNIFICANCE Germline loss-of-function CDKN2B mutations were identified in a subset of patients with features of inherited RCC. Detection of germline CDKN2B mutations will have an impact on familial cancer screening and might prove to influence the management of disseminated disease.

[1]  M. Tsai,et al.  Tumor suppressor INK4: comparisons of conformational properties between p16(INK4A) and p18(INK4C). , 1999, Journal of molecular biology.

[2]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[3]  Jan Lubinski,et al.  Mutation analysis of hypoxia-inducible factors HIF1A and HIF2A in renal cell carcinoma. , 2009, Anticancer research.

[4]  P. Bork,et al.  A method and server for predicting damaging missense mutations , 2010, Nature Methods.

[5]  Douglas E. V. Pires,et al.  DUET: a server for predicting effects of mutations on protein stability using an integrated computational approach , 2014, Nucleic Acids Res..

[6]  N. Grishin,et al.  A Novel Germline Mutation in BAP1 Predisposes to Familial Clear-Cell Renal Cell Carcinoma , 2013, Molecular Cancer Research.

[7]  Manuel Serrano,et al.  Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d , 1998, Nature.

[8]  G. Yousef,et al.  Multilevel whole-genome analysis reveals candidate biomarkers in clear cell renal cell carcinoma. , 2012, Cancer research.

[9]  Ming Zhou,et al.  Papillary renal cell carcinoma is associated with PTEN hamartoma tumor syndrome. , 2012, Urology.

[10]  E. Maher,et al.  Population‐based survey of cancer risks in chromosome 3 translocation carriers , 2010, Genes, chromosomes & cancer.

[11]  Matthew P. Banegas,et al.  Renal cell cancer: a shift in approaches for treatment of advanced disease in the United States. , 2014, Journal of the National Comprehensive Cancer Network : JNCCN.

[12]  Aaron R. Quinlan,et al.  Bioinformatics Applications Note Genome Analysis Bedtools: a Flexible Suite of Utilities for Comparing Genomic Features , 2022 .

[13]  Douglas E. V. Pires,et al.  mCSM: predicting the effects of mutations in proteins using graph-based signatures , 2013, Bioinform..

[14]  M. Roussel.  The INK4 family of cell cycle inhibitors in cancer , 1999, Oncogene.

[15]  G. Åkerström,et al.  Evaluation of CDKN2C/p18, CDKN1B/p27 and CDKN2B/p15 mRNA expression, and CpG methylation status in sporadic and MEN1‐associated pancreatic endocrine tumours , 2007, Clinical endocrinology.

[16]  J. Boultwood,et al.  Gene silencing by DNA methylation in haematological malignancies , 2007, British journal of haematology.

[17]  M. Simpson,et al.  Germline FH mutations presenting with pheochromocytoma. , 2014, The Journal of clinical endocrinology and metabolism.

[18]  H. Hakonarson,et al.  ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data , 2010, Nucleic acids research.

[19]  S. Richard,et al.  Familial Non-VHL Clear Cell (Conventional) Renal Cell Carcinoma: Clinical Features, Segregation Analysis, and Mutation Analysis of FLCN , 2008, Clinical Cancer Research.

[20]  A. Arnold,et al.  Germline and Somatic Mutations in Cyclin-Dependent Kinase Inhibitor Genes CDKN1A, CDKN2B, and CDKN2C in Sporadic Parathyroid Adenomas , 2013, Hormones and Cancer.

[21]  C. Sotiriou,et al.  Gene array of VHL mutation and hypoxia shows novel hypoxia-induced genes and that cyclin D1 is a VHL target gene , 2004, British Journal of Cancer.

[22]  S. Cairo,et al.  Abstract 5273: XenTech patient-derived xenograft (PDX) panels: a clinically relevant platform for drug efficacy, target validation and predictive biomarker discovery studies , 2012 .

[23]  Kenny Q. Ye,et al.  An integrated map of genetic variation from 1,092 human genomes , 2012, Nature.

[24]  P. Rustin,et al.  Germline mutations in FH confer predisposition to malignant pheochromocytomas and paragangliomas. , 2014, Human molecular genetics.

[25]  Paolo Vineis,et al.  Genome-wide association study of renal cell carcinoma identifies two susceptibility loci on 2p21 and 11q13.3 , 2011, Nature Genetics.

[26]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[27]  C. Ricketts,et al.  Germline SDHB mutations and familial renal cell carcinoma. , 2008, Journal of the National Cancer Institute.

[28]  M. Simpson,et al.  Acrocallosal syndrome: identification of a novel KIF7 mutation and evidence for oligogenic inheritance. , 2013, European journal of medical genetics.

[29]  S. Marx,et al.  Rare germline mutations in cyclin-dependent kinase inhibitor genes in multiple endocrine neoplasia type 1 and related states. , 2009, The Journal of clinical endocrinology and metabolism.

[30]  E. Cho,et al.  The tumor suppressor, parafibromin, mediates histone H3 K9 methylation for cyclin D1 repression , 2009, Nucleic acids research.

[31]  J. Carpten,et al.  HRPT2, encoding parafibromin, is mutated in hyperparathyroidism–jaw tumor syndrome , 2002, Nature Genetics.

[32]  G. Gyapay,et al.  Germline BAP1 mutations predispose to renal cell carcinomas. , 2013, American journal of human genetics.

[33]  S. Henikoff,et al.  Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm , 2009, Nature Protocols.

[34]  E. Maher.  Genetics of Familial Renal Cancers , 2010, Nephron Experimental Nephrology.

[35]  A. Berns,et al.  p15Ink4b is a critical tumour suppressor in the absence of p16Ink4a , 2007, Nature.