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.

See Covering the Cover synopsis on page 547; see editorial on page 554.

Hereditary nonpolyposis colorectal cancer as defined by the Amsterdam criteria1, 2 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. 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.

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%). 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).

Figure 1

(A) Pedigree of FCCX family 56. Numbers below the symbols are patient identifiers; key members also are marked with a letter code A1–A8 for affected and U1 for unaffected. Carrier status for the c.147dupA in RPS20 is shown (+, mutation carrier, -, noncarrier). Arrow denotes the index person. Clinical diagnoses are specified in Supplementary Table 1. Nonessential pedigree features were omitted or modified to protect confidentiality. (B) Exomic sequencing of blood DNAs from individuals A2, A3, A5, and A8 (see Supplementary Materials and Methods for details). The stepwise reduction in the number of insertions or deletions and single-nucleotide variants (SNV) remaining for consideration is shown, ultimately resulting in 2 exonic alterations shared by the 4 affected members. The RPS20 insertion or deletion (indel) alteration fulfilled the prerequisites of a predisposing mutation and was characterized fully in this investigation whereas the available evidence (incomplete co-segregation, occurrence in healthy controls, equivocal pathogenicity by predictions, as well as other data detailed in the Supplementary Materials and Methods) suggested that the inhibitor of κ light polypeptide gene enhancer in B cells, kinase β (IKBKB) SNV alteration was unlikely to explain the colorectal cancer susceptibility of F56 and was excluded from further consideration. U, noncarrier.

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.

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. The absence of loss of heterozygosity complies with observations from zebrafish showing that ribosomal protein genes act as haploinsufficient suppressors of tumorigenesis.

RPS20 is required during the late steps of 18S ribosomal RNA (rRNA) formation. 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.

Figure 2

(A) Northern blot analysis of total RNAs from HeLa cells treated for 48 hours with a scramble small interfering RNA (siRNA) or a small interfering RNA targeting RPS20 messenger RNA, and lymphoblastoid RNAs from controls (C1–C3), a noncarrier (U), and affected mutation carriers (A1–A4). Precursor rRNAs were detected with a 5’ internal-transcribed spacer 1 probe. (B) Mature rRNAs detected with 18S and 28S probes. (C) Quantification of pre-rRNA species by phosphorimaging after normalization to 28S rRNA. For each species, the value of the mean of the 3 control samples arbitrarily was set to 1.

All RPSs are essential in human cells, except RPS25. The ribosomal protein gene family comprises 80 genes, 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.10, 11 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. 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. RPS20 was proposed to be primarily of the detector type, with reduction perturbing ribosomal biogenesis (Figure 2 and Supplementary Figure 3), leading to stabilization of p53 (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.

Supplementary Table 1

Clinical Diagnoses of Members From F56 Tested for RPS20 c. 147dupA

Individual ID	Mutation carrier status	Tumor diagnosis (age at diagnosis in years)
II:2	Carrier	Carcinoma of sigmoid colon (75)
III:1	Carrier	Carcinoma of ascending colon (24), carcinoma of transverse colon (60)
III:2	Obligatory carrier	Carcinoma of transverse colon (52)
III:4	Carrier	Carcinoma of ascending colon (64)
III:7	Noncarrier	Carcinoma of breast (55)
III:8	Carrier	Carcinoma of cecum (50), carcinoma of rectum (59)
III:10	Carrier	Carcinoma of sigmoid colon (43), carcinoma of rectum (45)
III:12	Noncarrier	–
III:15	Noncarrier	–
III:17	Noncarrier	Hyperplastic polyp of cecum (47), tubular adenoma of ascending colon (53)
III:18	Carrier	Carcinoma of descending colon (54)
III:20	Noncarrier	–
IV:1	Carrier	Carcinoid tumor of rectum (33)