Germline Mutation of RPS20, Encoding a Ribosomal Protein, Causes Predisposition to Hereditary Nonpolyposis Colorectal Carcinoma Without DNA Mismatch Repair Deficiency

Little is known about the genetic factors that contribute to familial colorectal cancer type X (FCCX), characterized by hereditary nonpolyposis colorectal carcinoma with no mismatch repair defects. Genetic linkage analysis, exome sequencing, tumor studies, and functional investigations of 4 generations of a FCCX family led to the identification of a truncating germline mutation in RPS20, which encodes a component (S20) of the small ribosomal subunit and is a new colon cancer predisposition gene. The mutation was associated with a defect in pre–ribosomal RNA maturation. Our findings show that mutations in a gene encoding a ribosomal protein can predispose individuals to microsatellite-stable colon cancer. Evaluation of additional FCCX families for mutations in RPS20 and other ribosome-associated genes is warranted. Little is known about the genetic factors that contribute to familial colorectal cancer type X (FCCX), characterized by hereditary nonpolyposis colorectal carcinoma with no mismatch repair defects. Genetic linkage analysis, exome sequencing, tumor studies, and functional investigations of 4 generations of a FCCX family led to the identification of a truncating germline mutation in RPS20, which encodes a component (S20) of the small ribosomal subunit and is a new colon cancer predisposition gene. The mutation was associated with a defect in pre–ribosomal RNA maturation. Our findings show that mutations in a gene encoding a ribosomal protein can predispose individuals to microsatellite-stable colon cancer. Evaluation of additional FCCX families for mutations in RPS20 and other ribosome-associated genes is warranted. See Covering the Cover synopsis on page 547; see editorial on page 554. See Covering the Cover synopsis on page 547; see editorial on page 554. Hereditary nonpolyposis colorectal cancer as defined by the Amsterdam criteria1Vasen H.F. et al.Dis Colon Rectum. 1991; 34: 424-425Crossref PubMed Google Scholar, 2Vasen H.F. et al.Gastroenterology. 1999; 116: 1453-1456Abstract Full Text Full Text PDF PubMed Scopus (1996) Google Scholar includes 2 distinct entities with roughly comparable shares. Families with germline mutations in DNA mismatch repair genes represent Lynch syndrome (MIM 120435-6), with some 3000 unique predisposing mutations known.3Thompson B.A. et al.Nat Genet. 2014; 46: 107-115Crossref PubMed Scopus (365) Google Scholar Familial colorectal cancer type X (FCCX) is a collective designation for families with no evidence of DNA mismatch repair deficiency, wherein type X refers to the as yet unknown nature of predisposition.4Lindor N.M. Surg Oncol Clin N Am. 2009; 18: 637-645Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar We recently discovered germline mutations in the gene for bone morphogenetic protein receptor type IA in 2 Amsterdam-positive families of 18 FCCX families investigated (11%).5Nieminen T.T. et al.Gastroenterology. 2011; 141: e23-e26Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Among families with no bone morphogenetic protein receptor type IA mutations, family F56 fulfilling the Amsterdam criteria (Figure 1A) was chosen for closer scrutiny by genetic linkage analysis, exome sequencing, and tumor investigations. The mean age at colorectal cancer diagnosis was 52.3 years, with a 6–8 ratio of proximal to distal cancers. Genome-wide linkage analysis of the core pedigree resulted in the highest multipoint lod score (1.6) for D8S507 (Genethon) and D8S1115 (Marshfield), both of which reside in the area of linkage between D8S255 and D8S1718 on chromosome 8p11-8q12 (Supplementary Materials and Methods and Supplementary Figure 1). However, because a few other chromosomal regions also showed lod scores greater than 1, we opted for exome sequencing and chose 4 siblings with colorectal cancer from F56 to be included in the analysis (Figure 1B). A single truncating alteration of RPS20 (c.147dupA, RefSeq NM_001023.3) (Supplementary Figure 2A), a ribosomal protein gene, turned out to be shared by all 4 affected members investigated. It leads to frameshift and premature truncation (p.Val50SerfsX23). RPS20 is located on 8q12.1 in the region identified by genetic linkage analysis. The alteration showed a full co-segregation with microsatellite-stable colorectal cancer in F56 (Figure 1A), yielding a lod score of 3.0 for segregation. The sequence change was absent in healthy controls (allele count 0 of 584); moreover, it has not been reported in 4300 European Americans and 2203 African Americans (Exome Variant Server; available: http://evs.gs.washington.edu/EVS/; date accessed: April 1, 2014). We subsequently screened RPS20 for mutations in blood DNA from 25 other FCCX families from Finland and in tumor DNA from 61 primary colorectal cancers and cancer cell lines (Supplementary Materials and Methods); no RPS20 mutations were detected. Based on COSMIC (http://cancer.sanger.ac.uk) and TCGA (http://cancergenome.nih.gov/) databases (date accessed: May 28, 2014), at least 11 unique missense variants of RPS20 in cancer are known (mutation frequency of up to 2.6% depending on tumor type), with pathogenicity varying from benign to deleterious by in silico predictions. At least one colon cancer case with a somatic missense change (R79C) is included.6Cancer Genome Atlas NetworkNature. 2012; 487: 330-337Crossref PubMed Scopus (6300) Google Scholar Tumors from mutation carriers showed no loss of the wild-type allele (Supplementary Figure 2B), arguing against Knudson’s 2-hit mechanism for tumor-suppressor genes.7Knudson Jr., A.G. Proc Natl Acad Sci U S A. 1971; 68: 820-823Crossref PubMed Scopus (5881) Google Scholar The absence of loss of heterozygosity complies with observations from zebrafish showing that ribosomal protein genes act as haploinsufficient suppressors of tumorigenesis.8Amsterdam A. et al.PLoS Biol. 2004; 2: E139Crossref PubMed Scopus (359) Google Scholar RPS20 is required during the late steps of 18S ribosomal RNA (rRNA) formation.9O'Donohue M.F. Choesmel V. et al.J Cell Biol. 2010; 190: 853-866Crossref PubMed Scopus (131) Google Scholar Indeed, Northern blot analysis showed that small interfering RNA depletion of RPS20 in HeLa cells led to a significant increase of 21S pre-rRNAs (which are distributed in 2 close bands in this cell type), as well as an accumulation of 18S-E pre-rRNAs (Figure 2A). This was accompanied by a strong decrease of the 18S/28S ratio (Figure 2B). Patients carrying the RPS20 c.147dupA mutation (A1–A4) showed a marked increase of 21S pre-rRNAs compared with healthy unrelated controls (C1–C3), while the 18S-E pre-rRNA level was in the same range in control, noncarrier, and patient samples (Figure 2C). The 18S/28S ratios were unchanged in patient cells compared with controls and a noncarrier. Altogether, these results show a late pre-rRNA processing defect in mutation carrier cells consistent with RPS20 haploinsufficiency. Polysome analysis showed a slight increase in the 60S peak relative to the 40S peak in mutation carriers compared with a noncarrier and a healthy unrelated control (Supplementary Figure 3). Collectively, RNA results suggest that the RPS20 mutation disturbs ribosome biogenesis by affecting the equilibrium between the different pre-rRNA species and the formation of mature 18S rRNA. All RPSs are essential in human cells, except RPS25.9O'Donohue M.F. Choesmel V. et al.J Cell Biol. 2010; 190: 853-866Crossref PubMed Scopus (131) Google Scholar The ribosomal protein gene family comprises 80 genes,8Amsterdam A. et al.PLoS Biol. 2004; 2: E139Crossref PubMed Scopus (359) Google Scholar at least 11 of which are known to be mutated in Diamond–Blackfan anemia, a dominantly inherited form of pure red cell aplasia, growth retardation, and congenital anomalies.10Gazda H.T. et al.Hum Mutat. 2012; 33: 1037-1044Crossref PubMed Scopus (122) Google Scholar, 11Landowski M. et al.Hum Genet. 2013; 132: 1265-1274Crossref PubMed Scopus (88) Google Scholar No such features were present in colon cancer patients from F56. Why is the RPS20 mutation associated with colorectal cancer susceptibility, while mutations in 11 other ribosomal protein genes cause predisposition to Diamond–Blackfan anemia? Haploinsufficiency for RPS19 or RPS20 in mice was shown to stabilize p53, which in turn had different effects in different cell types.12McGowan K.A. et al.Nat Genet. 2008; 40: 963-970Crossref PubMed Scopus (300) Google Scholar Mouse findings make it tempting to speculate that cell type–specific effects of RPS20 haploinsufficiency might play a role in RPS20-associated colon tumorigenesis in human beings, with disturbed ribosome biogenesis, altered p53 dosage, or various downstream events as possible mediators. Among ribosomal proteins, “detector” and “effector” types have been distinguished based on contribution to p53 stress response.13Daftuar L. et al.PLoS One. 2013; 8: e68667Crossref PubMed Scopus (117) Google Scholar RPS20 was proposed to be primarily of the detector type, with reduction perturbing ribosomal biogenesis9O'Donohue M.F. Choesmel V. et al.J Cell Biol. 2010; 190: 853-866Crossref PubMed Scopus (131) Google Scholar (Figure 2 and Supplementary Figure 3), leading to stabilization of p5313Daftuar L. et al.PLoS One. 2013; 8: e68667Crossref PubMed Scopus (117) Google Scholar (Supplementary Figure 4). Conversely, the constant activation of p53 consecutive to ribosomal stress induced by RPS20 mutation could favor, in the long run, the selection of cells that escape regulation by p53. In summary, we show that inactivating germline mutation of RPS20 is associated with a dominant predisposition to colorectal cancer. This report links germline mutation of RPS20 to human disease. Future investigations are necessary to establish the prevalence of RPS20 mutations in FCCX families worldwide as well as the exact tumorigenic mechanisms and the basis of apparent tumor-type specificity. Finally, our study encourages investigations into the possible involvement of other ribosomal protein genes in colon cancer susceptibility. The authors thank Saila Saarinen for expert technical assistance and Tuula Lehtinen and Kirsi Pylvänäinen for help in collecting clinical data. The authors also thank Dr Hanna Gazda for helpful discussions. This study was based on 26 FCCX families from Finland, 15 of which fulfilled the Amsterdam criteria1Vasen H.F. et al.Dis Colon Rectum. 1991; 34: 424-425Crossref PubMed Scopus (1778) Google Scholar, 2Vasen H.F. et al.Gastroenterology. 1999; 116: 1453-1456Abstract Full Text Full Text PDF PubMed Scopus (2146) Google Scholar and 11 fulfilled the Bethesda criteria.3Umar A. et al.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2609) Google Scholar The families had no DNA mismatch repair defects in tumor tissue or in the germline4Renkonen E. et al.J Clin Oncol. 2003; 21: 3629-3637Crossref PubMed Scopus (86) Google Scholar; furthermore, germline mutations in bone morphogenetic protein receptor type IA were excluded.5Nieminen T.T. et al.Gastroenterology. 2011; 141: e23-e26Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar In addition, 11 colorectal cancer cell lines including 7 with microsatellite instability (HCA7, HCT115, HCT116, KM12, LIM1215, LoVo, and RKO) and 4 microsatellite-stable (T84, SW480, SW837, and CACO2), 50 primary colorectal cancers6Kuismanen S.A. Holmberg M.T. et al.Am J Pathol. 2000; 156: 1773-1779Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar (18 with microsatellite instability and 32 microsatellite stable), as well as 197 healthy blood donors from Finland and 95 from the United Kingdom were screened for RPS20 mutations. The Institutional Review Board of the Helsinki University Central Hospital (Helsinki, Finland) approved this study. Genetic linkage analysis of the core pedigree of F56 was conducted at Ohio State University, taking advantage of microsatellite markers from the Genethon collection (ABI PRISM Linkage Mapping Set Version 2, Grand Island, NY)7Huang J. et al.Cancer Res. 2001; 61: 1619-1623PubMed Google Scholar and at the University of Toronto using single-nucleotide polymorphisms and microsatellite markers from the Marshfield collection (Human Mapping 10K array V2; Affymetrix, Santa Clara, CA) (Supplementary Figure 1). Parametric multipoint analysis was performed with MERLIN v1.0-alpha (Ann Arbor, MI),8Abecasis G.R. et al.Nat Genet. 2002; 30: 97-101Crossref PubMed Scopus (2805) Google Scholar using the following parameters: autosomal-dominant mode of inheritance, gene frequency of 0.001, 4 age-dependent liability classes,9Peltomäki P. Aaltonen L.A. et al.Science. 1993; 260: 810-812Crossref PubMed Scopus (824) Google Scholar and 4 age-dependent phenocopy rates (0.00, 0.01, 0.03, and 0.05 corresponding to liability classes 1–4, respectively). Family members with a colorectal polyp as the only tumor were treated in 2 alternative ways, considering them either to have an unknown status or to be affected. Exome sequencing was conducted at the Institute for Molecular Medicine Finland (Helsinki, Finland). Exome sequencing was performed on an Illumina HiSeq 2000 platform (San Diego, CA) with Roche NimbleGen SeqCap EZ Exome Library 2.0 (Roche, Basel, Switzerland). The probes cover a total size of 44.1 Mb, comprising the whole human exome and more than 20,000 genes. A variant calling pipeline10Sulonen A.M. et al.Genome Biol. 2011; 12: R94Crossref PubMed Scopus (217) Google Scholar version 2.0 for quality control, short read alignment, variant identification, and annotation were used for the primary analysis of sequence data. Paired-end reads were aligned to the GRCh37/Hg19 human genome build. An algorithm10Sulonen A.M. et al.Genome Biol. 2011; 12: R94Crossref PubMed Scopus (217) Google Scholar developed at the Institute for Molecular Medicine Finland was used for single-nucleotide variant calling, and Pindel11Ye K. et al.Bioinformatics. 2009; 25: 2865-2871Crossref PubMed Scopus (1481) Google Scholar was used for insertion or deletion calling. The minimum acceptable read depth was 7, and the required quality score was 20 for single-nucleotide variants and 50 for insertions or deletions. The coverage depth was 20. After filtering out low-quality variations as well as common and homozygous variants, the individual Excel (Microsoft, Redmond, WA) files were analyzed by Microsoft Access software to detect variants shared by all 4 family members. As an alternative to the scheme shown in Figure 1B with all 4 colorectal cancer patients expected to be carriers of the same mutation, we also analyzed the data allowing for any one of the individuals to be a possible noncarrier (phenocopy). The latter method showed no additional noteworthy candidates to be considered as susceptibility genes. The c.1933C>T (RefSeq NM_001556.2) alteration in inhibitor of κ light polypeptide gene enhancer in B cells kinase β that was shared by all 4 affected family members besides the RPS20 mutation (Figure 1B) constitutes a missense change in a gene encoding an inhibitor of the κ light polypeptide gene enhancer in B cells. Inhibitor of κ light polypeptide gene enhancer in B cells kinase β is located on 8p11.2 in an interval flanking the linked region, close to marker D8S255. The alteration results in an arginine to tryptophan substitution (p.R645W) and is suggested to be damaging by SIFT (Rockville, MD) and a polymorphism by MutationTaster (Berlin, Germany). Segregation analysis in the entire family 56 showed that the variant was absent in 1 member affected with a colorectal tumor (Figure 1A, IV:1) and present in the remaining colorectal cancer patients; in addition, it was present in 1 clinically unaffected member (Figure 1A, III:12) and absent in the remaining individuals not affected with colorectal cancer. The variant occurred in healthy controls with an allele count of 0.2% (1 of 450). Finally, inhibitor of κ light polypeptide gene enhancer in B cells kinase β has an established disease connection, with homozygous germline inactivation underlying severe combined immunodeficiency.12Pannicke U. Baumann B. Fuchs S. Henneke P. et al.N Engl J Med. 2013; 369: 2504-2514Crossref PubMed Scopus (144) Google Scholar RPS20 was screened for point mutations by exon-specific sequencing with intronic primers from Sjoblom et al13Sjoblom T. Jones S. Wood L. Parsons D.W. et al.Science. 2006; 314: 268-274Crossref PubMed Scopus (2908) Google Scholar and for large rearrangements by long-range genomic polymerase chain reaction with primers 5’-ATTTTTGGTCCGCACGCTCCT-3’ (from the 5’ untranslated region) and 5’-CACTCTAAGATACCCATATATTCCACC-3’ (from the 3’ untranslated region). The c.147dupA mutation in exon 3 (Supplementary Figure 2A) was detected with primers from flanking introns (forward primer: 5’-CTCGTTAATGTTAGTGTAGAAGGTG-3’, reverse primer: 5’-GAACCTGAATTTAGTCAACATC-3’). Fluorescent fragment analysis was performed as described,14Abdel-Rahman W.M. et al.Oncogene. 2005; 24: 1542-1551Crossref PubMed Scopus (77) Google Scholar taking advantage of 7 microsatellite markers used for haplotype and linkage analysis (from pter to qter: D8S255, D8S1828, D8S507, D8S260, D8S543, D8S1805, and D8S270). Microsatellite marker D8S507 (Supplementary Figure 2B) was investigated with forward primer 5’- CCCCTATTCCTTCTGCCTTT-3’, and reverse primer 5’-CAGCATTTTTCCTCAGAGCAG-3’. Loss of heterozygosity analysis with the germline mutation (RPS20 c.147dupA) as an intragenic marker (Supplementary Figure 2B) used forward primer 5’-GCAGAAGTGAAGGCCTTAA-3’ and reverse primer 5’-GAACCTGAATTTATGCAACATC-3’. Total RNAs were extracted by the TRIzol (Grand Island, NY) method (modified from Chomczynski15Chomczynski P. Biotechniques. 1993; 15 (536–537): 532-534PubMed Google Scholar). Northern blot analysis of pre-rRNA species with probes hybridizing to the internal-transcribed spacer 1 or 2 was performed as described.16O'Donohue M.F. Choesmel V. et al.J Cell Biol. 2010; 190: 853-866Crossref PubMed Scopus (155) Google Scholar HeLa cells treated for with a small interfering RNA or a small interfering RNA RPS20 RNA and cells of individuals with or the RPS20 c.147dupA mutation were included in the analysis. were detected with a 5’ internal-transcribed spacer 1 which is to the of the 18S and the internal-transcribed spacer were shown with 18S and pre-rRNA species was by using and to rRNA. the of the mean of the 3 control samples was to RNA were performed 3 and led to Polysome analysis was conducted by of of cells as in M.F. 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