Nahuel A Paolini

Martin Attwood

Samuel B Sondalle

Carolina Marques

Santos Vieira

Anita M Van Adrichem

Franca Di Summa

Marie-Françoise O'donohue

Pierre-Emmanuel Gleizes

Swaksha Rachuri

Joseph W Briggs

Franca M Di Summa

Marie- Françoise O'donohue

Roman Fischer

Peter J Ratcliffe

Marcin W Wlodarski

Riekelt H Houtkooper

Marieke Von Lindern

Taco W Kuijpers

Jonathan D Dinman

Susan J Baserga

Matthew E Cockman

Alyson W Macinnes
email: a.w.macinnes@amc.nl

A Ribosomopathy Reveals Decoding Defective Ribosomes Driving Human Dysmorphism

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Ribosomopathies are diseases caused by mutations in genes coding for components of the ribosome, such as ribosomal proteins (RPs), or in one of the many proteins required for ribosome biogenesis and maturation. Classical ribosomopathies have been typically associated with bone marrow failure (BMF). Examples are the inherited BMF Diamond-Blackman anemia (DBA) (OMIM #105650) and acquired 5q-myelodysplastic syndrome (5q-MDS) (OMIM #153550), both of which are linked to RP gene haploinsufficiency and predominantly effect the development of red blood cells 1; 2 . Other inherited BMF syndromes linked to genes important for ribosome biogenesis are dyskeratosis congenita (OMIM #305000) and Shwachman-Diamond syndrome (OMIM #260400), diseases that drive pancytopenia and neutropenia, respectively.

Beyond the hematopoietic phenotype of ribosomopathies are developmental disorders.

Almost 30% of individuals diagnosed with DBA experience physical malformations of the hands (such as abnormal thumbs), face (low nasal bridge, cleft lip/palate), head (microcephaly), heart, or urogenitals [START_REF] Boria | The ribosomal basis of Diamond-Blackfan Anemia: mutation and database update[END_REF] . Treacher-Collins syndrome (OMIM #154500) is a disorder associated with mutations affecting rRNA production that affects development of the facial bones, eyes, and ears. Isolated congenital asplenia (ICA) (OMIM #271400) is linked to extremely rare mutations of the gene coding for ribosomal protein uS2 (RPSA) [START_REF] Bolze | Ribosomal protein SA haploinsufficiency in humans with isolated congenital asplenia[END_REF] . Mutations in the gene coding for ribosomal protein uL16 (RPL10) (OMIM #300847) are found in individuals with intellectual disability (ID), autism spectrum disorder (ASD), and cerebellar hypoplasia.

These individuals also present with microcephaly, anteverted ears, a broad nasal ridge and epicanthus, myopia, urogenital anomalies, and a delay of motor development [START_REF] Brooks | A novel ribosomopathy caused by dysfunction of RPL10 disrupts neurodevelopment and causes X-linked microcephaly in humans[END_REF][START_REF] Thevenon | RPL10 mutation segregating in a family with Xlinked syndromic Intellectual Disability[END_REF][START_REF] Zanni | A Novel Mutation in RPL10 (Ribosomal Protein L10) Causes X-Linked Intellectual Disability, Cerebellar Hypoplasia, and Spondylo-Epiphyseal Dysplasia[END_REF] . While almost all DBA-linked RP gene mutations studied to date are loss of function [START_REF] Vlachos | Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference[END_REF] , the fact that inherited variants of uS2 and uL16 exist in the absence of any hematopoietic phenotype suggests more mechanisms underlying the pathogenicity of these mutations remain to be uncovered. Further support for this comes from the discovery that somatic mutations in genes coding for uL16 and uL18 (RPL5) are associated with the malignancy T-cell acute lymphoblastic leukemia [START_REF] De Keersmaecker | Exome sequencing identifies mutation in CNOT3 and ribosomal genes RPL5 and RPL10 in T-cell acute lymphoblastic leukemia[END_REF] .

Here de novo missense mutations in the RPS23 gene coding for ribosomal protein uS12 were investigated in two unrelated individuals with microcephaly, hearing loss, and overlapping dysmorphic features. uS12 is positioned within the decoding center of the ribosome that serves to maintain translational fidelity by monitoring the complementarity between the mRNA codons being translated and the anti-codons of aminoacyl-tRNAs [START_REF] Stark | Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the codon-recognition complex[END_REF] . Studies in yeast and bacteria have described several amino acid substitutions within two highly conserved loop regions in uS12, PNSA and PGVRY, which disrupt translational fidelity and impair the accuracy of mRNA decoding [START_REF] Sharma | Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome[END_REF][START_REF] Anthony | Alterations in ribosomal protein RPS28 can diversely affect translational accuracy in Saccharomyces cerevisiae[END_REF][START_REF] Synetos | Mutations in yeast ribosomal proteins S28 and S4 affect the accuracy of translation and alter the sensitivity of the ribosomes to paromomycin[END_REF] .

Human uS12 is a known substrate of the 2-oxoglutarate and Fe(II)-dependent oxygenase domain-containing protein 1 (OGFOD1) [START_REF] Singleton | OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation[END_REF] . Acute reductions of OGFOD1 in mammalian cells alter translational fidelity and increase the formation of mRNA-containing stress granules [START_REF] Singleton | OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation[END_REF] .

This study uncovers similar phenotypes in human cells carrying RPS23 mutations that lead to uS12 variants in the PNSA and PGVRY domains. Moreover, the results suggest that the mutations impair OGFOD1 hydroxylation and stabilization of uS12, and that a mechanism exists in cells preventing ribosomes carrying the uS12 variant from entering the translationallyactive polysomal pool.

Materials and Methods

Cell culture. Fibroblasts were obtained by a punch biopsy and maintained in DMEM (Gibco) + 10% FCS and 1% penicillin/streptomycin. LCLs were derived from EBV-immortalization of peripheral mononuclear cells isolated from whole blood using Ficoll (GE Life Sciences) and grown in RPMI (Gibco) + 10% FCS + 1% L-glutamine + 1% penicillin/streptomycin as previously described [START_REF] Hui-Yuen | Establishment of Epstein-Barr virus growth-transformed lymphoblastoid cell lines[END_REF] . The study was approved by the Medical Ethical Committee (METC) of the Amsterdam Medical Center (AMC) and performed in accordance with the ethical standards of the 1964 Declaration of Helsinki. Written informed consent was received from the participants prior to inclusion in this study.

Exome sequencing. Whole exome sequencing was performed with a SOLiDv4 machine from Life TechnologiesTM after enrichment with the Agilent SureSelectXT Human All Exon 50Mb Kit. Data analysis was done with BioScopeTM. The analysis in this indication was focused on identification of de novo variants. Comparing the exome data of the individual with the exome data of both parents identified the de novo variants. Sanger sequencing confirmed the variants in uS12.

Sanger sequencing of fibroblasts. cDNA was synthesized using the QuantiTect reverse transcription kit (Qiagen #205313). The RPS23 gene was amplified by standard PCR using 250 ng of cDNA and with forward primer 5'-TGCGGTGCTTCTCTCTTTCG-3' and reverse primer 5'-CATCTTGGGCCCCTGTAAGA-3'. Unincorporated primers and dNTPs were eliminated with ExoSAP-IT (Fisher Scientific #AF8200200). A BigDye Terminator sequence reaction was performed according to the manufacturer's instructions in a total reaction volume of 10µl using sequencing primer 5'-GTCACCGACGAGACCAGAAG-3'. Sequences were analyzed with 3730XL DNA Analyzer (ThermoFisher Scientific #3730XL) and 4Peaks software.

Ribosome purification. uS12 was isolated from intact ribosomes by sucrose density sedimentation and purified by reverse-phase HPLC using established protocols [START_REF] Singleton | OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation[END_REF] . uS12-containing fractions were lyophilised by vacuum centrifugation prior to solubilization in Laemmli buffer, resolution by SDS-PAGE with Coomassie Blue visualization (Imperial Protein Stain, Thermo Fisher) and in-gel trypsinolysis (Trypsin Singles, Sigma). RNA sequencing. Total RNA was isolated from 1x10 [START_REF] Zanni | A Novel Mutation in RPL10 (Ribosomal Protein L10) Causes X-Linked Intellectual Disability, Cerebellar Hypoplasia, and Spondylo-Epiphyseal Dysplasia[END_REF] of healthy control or uS12 p.Arg67Lys LCLs using TRIzol (Thermo Fisher) and purified by Qiagen RNAeasy columns (Qiagen). RNA-Seq libraries were prepared using poly(A)+ selection of the mRNA. Unstranded RNA-Seq was performed using HiSeq 2000 v4 Paired-End 125bp protocol. The transcripts were pseudoaligned and quantified using Kallisto. Expression levels were expressed as Fragments Per Kilobase of transcript per Million mapped reads (FPKM).

Mass spectrometry.

Liquid chromatography was performed on a Dionex Ultimate 3000 nanoLC system employing a trapping configuration (Pepmap 100, C18, 5um, 100um x 2cm, Thermo Fisher) with 8ul/min flow. Separation was performed with 250 nl/min flowrate on a gradient of 2% to 35% ACN in 0.1% Formic acid and 5% DMSO over 60 minutes on an Easyspray column (Pepmap RSLC, C18, 2um, 75mm x 50cm, Thermo Fisher) with mass spectrometric analysis on an Orbitrap Fusion Lumos mass spectrometer. MS1 scans were acquired between 360 and 1500 m/z with a resolution of 120,000 for up to 50ms and an ion target of 4E5. We acquired an MS1 scan every 3 seconds and used the parallelizable time to maximize MS2 scan events in the linear ion trap (rapid scan, CID with 35% collision energy) for up to 250ms and an ion target of 4E3. Dynamic precursor exclusion was set to 7 seconds and the precursor selection threshold was 5E3 counts after quadrupole isolation (1.2 m/z).

Data processing was performed using PEAKS 7 software (Bioinformatics Solutions) with the following parameters: full tryptic specificity with two missed cleavages; mass tolerance set at 5 ppm for precursor and 0.5 Da for product ions; cysteine carbamidomethylation as a fixed modification; methionine oxidation, hydroxylation, deamidation (Asn/Gln) and Pyro-glu (Gln) were specified as variable modifications; and a maximum of 3 PTMs per peptide. Unspecified PTM searches were enabled via PEAKS PTM algorithm. Data files were searched against human Uniprot database (release 2016_01) supplemented with the uS12 p.Arg67Lys protein sequence. Peptide assignments were filtered to a false discovery rate (FDR) of ≤ 1% and a peptide score (-10 lgP) of ≥ 15. PEAKS searches assigned a number of modifications associated with sample handing to Pro62 containing peptides, including: conversion of Nterminal Gln to pyro-Glu, deamidation of Gln/Asn and carbamylation and carbamidomethylation of Lys. Since detection of these artifactual modifications varied between samples, we measured the most frequently detected peptide variants and aggregated the data in order to derive a robust quantitation (Table S1 for summary of quantitated peptides and counts). Relative peptide quantitation was performed using precursor ion signal intensity. Abundance measurements (peak area integrations of extracted ion chromatograms) were obtained using the Genesis peak detection algorithm in Qual Browser (Xcalibur, Thermo Fisher). Where necessary, manual integrations were performed.

Peptides assigned by MS/MS in at least one sample were used to derive accurate mass and retention time (AMT) signatures of precursor ions for quantitation in all samples. Stringent tolerances for AMT assignment were employed (m/z window +/-5 ppm and 0.5 min retention time deviation) reflecting the high mass accuracy and reproducible chromatography of the nanoLC Lumos platform.

Immunoprecipitations. Endogenous OGFOD1/uS12 protein associations were assayed by OGFOD1 IP/IB (Sigma #HPA003215) and uS12 IB (Abcam # ab57764) using standard immunoprecipitation protocols. Normal rabbit IgG (Cell signaling #2729) served as control antibody for immunoprecipitation. Interactions between ectopic FLAG-OGFOD1 [START_REF] Singleton | OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation[END_REF] and HA-uS12 variants were assayed by transient transfection and immunoprecipitation in HEK293 cells. OGFOD1-and uS12-bound complexes were purified with anti-FLAG (Sigma) and anti-HA (Sigma) agarose affinity gel and immunoblotted with the respective epitope tag specific antibody.

Plasmids and transfections. 3xFLAG-OGFOD1 was constructed as previously described [START_REF] Singleton | OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation[END_REF] .

Wild type and p.Arg67Lys human RPS23 gene fragments (NCBI CCDS47241) with a Cterminal HA tag and 5' BamHI/3' XbaI restriction enzyme sites were purchased from Eurofins Genomics. These fragments were cloned into the pCS2+ vector and transfected into HEK293 Yeast plasmids and strains. A GAL::RPS23A rps23b∆ yeast strain was generated in the parental strain YPH499 (MATa ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1). The endogenous RPS23B locus was knocked out with the HIS3MX6 gene amplified by the polymerase chain reaction (PCR) (oligonucleotides F-deltaB and R-deltaB) with 60 base pair ends homologous to the RPS23B locus from the pFA6a-HIS3MX6 plasmid [START_REF] Longtine | Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae[END_REF] to create an rps23b∆::HIS3MX6 strain in YPH499. The strain was confirmed by PCR from genomic DNA using oligonucleotides F-5'rps23b and R-inhis5 (272 base pair product) as well as oligonucleotides F-inhis5 and R-3'rps23b (334 base pair product) to confirm insertion of the HIS3MX6 gene into the RPS23B locus. The rps23b∆ strain was used to generate a strain where the endogenous RPS23A gene is under the control of a glucose-repressible, galactoseinducible (GAL) promoter by PCR amplification (oligonucleotides F-GAL and R-GAL) of the GAL promoter and the kanMX6 selection marker from pFA6a-kanMX6-PGAL1 [START_REF] Longtine | Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae[END_REF] with 70 base pair ends homologous the upstream region adjacent to the transcription start site of the RPS23A gene. The strain was confirmed by PCR from genomic DNA using oligonucleotides F-inGAL and R-inRPS23A (221 base pair product) to confirm insertion of the GAL promoter upstream of RPS23A.

The RPS23A gene was cloned into a plasmid to heterologously express wild-type and mutated Rps23a protein. RPS23A was amplified by PCR from YPH499 genomic DNA with oligonucleotide primers nested-F and nested-R and PCR amplified subsequently with oligonucleotide primers GWF and GWR, PEG precipitated, and shuttled into the Gateway donor vector pDONR221 using a BP reaction (Life Technologies). To generate Rps23a mutant proteins, a plasmid containing RPS23A was mutated with oligonucleotides MutArg69Lys, MutArg69Glu, MutArg69His, and MutLys62Arg and the Change-IT Multiple Mutation Site Directed Mutagenesis Kit (Affymetrix). The mutated plasmids were confirmed with Sanger sequencing by Genewiz. All genes encoding wild-type and mutated Rps23a were shuttled into the Gateway destination vector p424GPD-CFLAG-gw (TRP1 marker). A stop codon at the end of the genes encoding wild-type and mutated Rps23a prevented expression of the C-terminal FLAG epitope. See Table S2 for oligonucleotide sequences.

Western blotting with yeast. To examine protein levels of wild-type and aberrant Rps23a, the YPH499 GAL::RPS23A rps23b∆ yeast strain was transformed with either empty vector (EV) p424GPD-CFLAG-gw or p424GPD-CFLAG-gw expressing wild-type or mutated Rps23a.

Endogenous Rps23a was depleted by first growing yeast cultures to mid-log phase (OD600 = 0.3-0.8) in synthetic medium containing 2% galactose and 2% raffinose and lacking tryptophan (SG/R-W), and then by shifting yeast cultures to synthetic medium containing 2% dextrose and lacking tryptophan (SD-W) medium for 6 h at 30°C. Protein was then extracted by the alkaline lysis method [START_REF] Kushnirov | Rapid and reliable protein extraction from yeast[END_REF] . Protein extracts (0.25 OD600 equivalents per lane) were separated on a 15% Tris-glycine gel by SDS-PAGE and transferred to a PVDF membrane.

Expression of Rps23a was determined using an anti-RPS23 antibody (AbD Serotec, ca. no.

MCA3433Z; 1:1,000 dilution; anti-mouse-HRP secondary antibody at 1:10,000). An anti-G6PDH antibody (Sigma, ca. no A9521; 1:10,000 dilution; anti-rabbit-HRP secondary antibody at 1:10,000) was used for a loading control.

Yeast growth assays. For analysis of complementation of the growth defect conferred by

Rps23a wild-type and aberrant proteins, the YPH499 GAL::RPS23A rps23b∆ yeast strain was transformed with either empty vector (EV) or plasmids expressing wild-type or mutated Rps23a. For serial dilutions, 0.2 mL of cells at an OD600 of 1 were resuspended in 1 mL water, diluted 1/10 and spotted onto SG/R-W (endogenous and plasmid-borne Rps23a expressed) or SD-W (only plasmid-borne Rps23a expressed). Cells were incubated at 30 °C.

qPCR. RNA was extracted from fibroblasts grown to 70-90% confluency using TRIzol® RNA isolation reagents (ThermoFisher Scientific, #15596018) and cDNA synthesis was performed using the QuantiTect reverse transcription kit (Qiagen, #205313) both according the manufacturer's specifications. qPCR was performed with 5ng of cDNA with 1.25µM of primers in a final volume of 8µl using LightCycler® 480 (Roche, #05015243001) and LightCycler® 480 SYBR Green I Master (Roche, #04707516001). qPCR conditions were 10 min at 95°C, followed by 45 cycles at 95°C for 10s, 60°C for 10s, and 72°C for 15s and then a melting curve at 95°C and cooling to 37°C. Primers used in this assay were: Forward uS12 5'-GGACGCCCTTTTCACTGCTA-3', Reverse uS12 5'-CTGAGGCGCCGGAGATTATT-3', Translational fidelity assays in yeast. Yeast cells yJD 1729 (RPS23A), yJD 1730 (rps23ap.Lys62Arg), and yJD 1731 (rps23a-p.Arg69Lys) were transformed using the alkali cation method [START_REF] Ito | Transformation of intact yeast cells treated with alkali cations[END_REF] . pJD376 (pYDL-LA), pJD1018 (pYDL-EST2), pJD1039 (pYDL-STN1), and pJD1041

(pYDL-EST1) [START_REF] Advani | Yeast telomere maintenance is globally controlled by programmed ribosomal frameshifting and the nonsense-mediated mRNA decay pathway[END_REF] were used to monitor rates of -1 PRF. Rates of +1 PRF were assayed using pJD377 (pYDL-EST3). pJD431 (pYDL-UAA), and pJD432 (pYDL-UAG) were used to assay termination codon readthrough. Suppression of an AGC near-cognate serine codon or a TCT non-cognate serine codon in the firefly luciferase catalytic site was assayed using pJD642

(pYDL-TCT218), and pJD643 (pYDL-AGC218) 20; 21 . pJD375 (pYDL-control) was used as the zero frame dual luciferase reporter. All assays were performed in triplicate a minimum of 3 independent times. The yeast strains were grown overnight in H-tryptophan -uracil liquid media containing galactose and raffinose, and after 24 hrs media was replaced withtryptophan -uracil liquid media containing dextrose. Yeast cells were pelleted by centrifugation, resuspended in 500 μL of lysis buffer (1× PBS pH 7.4, 1 mM PMSF), and lysates were prepared as described previously [START_REF] Harger | An in vivo dual-luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae[END_REF] . Luciferase activities were determined using 20 μL of lysate/sample using the Dual-Luciferase® Assay System and a GloMax®-Multi+ Detection System (Promega). Data were analyzed as described previously [START_REF] Jacobs | Systematic analysis of bicistronic reporter assay data[END_REF] .

Translational fidelity in fibroblast cell lines. Fibroblasts were transfected by electroporation using a Nucleofector TM II apparatus and the Normal Human Dermal Fibroblasts Nucleofector TM kit (Amaxa TM ), per the manufacturer's instructions. For each transfection, 4 -5 x 10 5 cells were mixed with 3 μg of plasmid DNA. pJD2046 (pHDL-fullCMV-UAA) was used to monitor termination codon readthrough. pJD2044 (pHDL-fullCMV-control) was used as the zero frame dual luciferase reporter. To make these constructs, the CMV promoter/enhancer was PCR amplified using pCDNA3.1(+) plasmid template and the following forward and reverse oligonucleotide primers: CMVprom-forward 5'-ACTGTCGGTACCCAGATATACGCGTTGACATTGATTATTGAC-3', CMVprom-reverse 5'-ACTGTCCTGCAGGCCAGTAAGCAGTGGGTTCTCTAG-3'. Primers were designed to have either a KpnI or PstI restriction enzyme digest site so that PCR amplicons could be digested and ligated into similarly digested pJD175f(pHDL-SV40-control), and pJD1643 (pHDL-SV40-UAA) dual luciferase reporter plasmids. All assays were performed in triplicate a minimum of 3 times. Cell lysates were prepared using passive lysis buffer (Promega) and luciferase activities were determined using the Dual-Luciferase® Assay System and a GloMax®-Multi+ Detection System (Promega), per the manufacturer's instructions 24 hours post-transfection.

Data were analyzed as described above for yeast translational fidelity assays.

Structural modeling, alignments, and SNP identification. All structural models of uS12

were generated from a cryo-EM structure of the human 80S ribosome (PDB ID: 4UG0) [START_REF] Khatter | Structure of the human 80S ribosome[END_REF] using the PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC. Identification of polar interactions, distance measurements, and modeling of mutated residues were also performed using PyMol. Multiple protein sequence alignments were performed with ClustalX 2.1 [START_REF] Sievers | Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega[END_REF] . SNPs were collected from the NCBI database and the cBio Cancer Genomics Portal [START_REF] Cerami | The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data[END_REF] .

Growth curve. 50,000 LCLs were plated in triplicate in a 12-well dish with DMEM +10%FCS

and counted daily with a CASY® Cell Counter for five days. Each well was counted in triplicate each day.

De novo protein synthesis measurement. 1x10e5 LCLs were plated in 150µl methioninefree medium (DMEM containing 4,500 mg/L D-glucose, without L-glutamine, sodium pyruvate, L-methionine, and L-cysteine, Invitrogen #21013). Assays performed using the Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay (Thermo Scientific) per the manufacturer's instructions using 2% paraformaldehyde for fixation and 1:1000 dilution of AHA. Fluorescence was measured by flow cytometry using an LSR-II apparatus and analyzed with FACSDiva software (BD Biosciences).

Polysome profiling analysis. 400µg of total protein from freshly lysed cells was loaded onto 17-50% sucrose gradients as previously described [START_REF] Pereboom | A zebrafish model of dyskeratosis congenita reveals hematopoietic stem cell formation failure resulting from ribosomal protein-mediated p53 stabilization[END_REF] . The tubes were centrifuged at 4°C and at 40,000 rpm for 2 hours in a SW41 rotor (Optima L100XP ultracentrifuge; Beckman Coulter).

The gradient fractions were collected at OD254 with a Foxy Jr. gradient collector (Teledyne Isco). 1) 28; 29 . Hypotonia at birth was also noted. No abnormal hematology results were found by routine analysis (Table 2). A case report shows the individual's brittle hair has structural abnormalities compared to family members [START_REF] Alsop | Structural Abnormalities in the Hair of a Patient with a Novel Ribosomopathy[END_REF] .

Confocal analysis.

A variant in yeast uS12 orthologous to p.Arg67Lys affects growth of a unicellular eukaryote. The affected Arg67 lies in a loop region of uS12, PNSA, which is completely conserved in all three domains of life (Figure S1B) [START_REF] Sharma | Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome[END_REF] . The PNSA loop interacts with aminoacyl-tRNAs, 18S rRNA, and the decoding center of the ribosome upon binding of a cognate anticodon-stem-loop [START_REF] Ogle | Recognition of cognate transfer RNA by the 30S ribosomal subunit[END_REF] . Substitution of amino acids within this loop alters translational accuracy, including Lys42 in E. coli and Lys62 in S. cerevisiae (both analogous to Lys60 in humans) [START_REF] Sharma | Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome[END_REF][START_REF] Anthony | Alterations in ribosomal protein RPS28 can diversely affect translational accuracy in Saccharomyces cerevisiae[END_REF][START_REF] Synetos | Mutations in yeast ribosomal proteins S28 and S4 affect the accuracy of translation and alter the sensitivity of the ribosomes to paromomycin[END_REF] . Because of the clear importance of the PNSA loop in ribosome function, the affected human Arg67 was examined at the atomic level. Structural analysis of uS12 from a cryo-EM reconstruction of the human 80S ribosome revealed the position of Arg67 and its predicted polar contacts (hydrogen bonds). Two polar contacts occur between Arg67 and guanosine 617 in the 18S rRNA (Figure 1A). Other polar contacts occur internally with Asp114 in uS12 (Figure 1A) [START_REF] Khatter | Structure of the human 80S ribosome[END_REF] . In addition, Arg67 is closely situated in proximity to a prolyl residue (Pro62) in uS12 that, like Asp114, is completely conserved (Figures 1A andS1A). Substitution of the arginine at position 67 in uS12 with a lysine is predicted by PyMOL to abolish the polar contacts normally maintained by Arg67 with guanosine 617 in the 18S rRNA (Figure 1B).

To investigate the cellular consequences of the uS12 p.Arg67Lys variant, its effects were tested in S. cerevisiae. Haploid yeast cells contain two RPS23 orthologues, presently named RPS23A and RPS23B, which encode identical uS12 proteins. A strain was constructed to test the effects of the mutant form of uS12 as the sole form of uS12 protein in cells. This strain was constructed harboring a complete deletion of RPS23B (rps23b∆::HIS3MX6) in which RPS23A transcription was under the control of the glucose-repressible, galactose-inducible GAL4 promoter (GAL::RPS23A). Episomal high copy vectors were also constructed enabling constitutive heterologous expression of the wild-type and variant forms of uS12 (Figure 1C).

Both the endogenous uS12 and plasmid-borne uS12 are produced in medium containing galactose and lacking glucose, but only plasmid-borne uS12 is produced in media containing glucose and lacking galactose. Depletion of endogenous uS12 was confirmed by western blotting (Figure 1D, lane 2). The levels of uS12 from the plasmid (Figure 1D; lane 4, 6, 8, 10, 12) are at or above the level for undepleted EV (Figure 1D, lane 1), which represents only endogenous levels of uS12 (Rps23) protein. Therefore, all variant uS12 proteins are found near the levels of the endogenous protein in this system.

Site-directed substitutions in the plasmid-expressed uS12 were generated including p.Arg69Lys, p.Arg69Glu, p.Arg69His, and p.Lys62Arg. p.Arg69Lys is orthologous to the human uS12 p.Arg67Lys substitution (see alignment in Figure S1B). p.Arg69Glu and p.Arg69His were employed to test the effect of amino acid charge at that same residue, whilst the p.Lys62Arg substitution (previously shown to negatively affect translational fidelity in yeast) served as a control [START_REF] Anthony | Alterations in ribosomal protein RPS28 can diversely affect translational accuracy in Saccharomyces cerevisiae[END_REF] . Serial dilutions were spotted onto plates containing galactose or glucose of the GAL::RPS23A rps23b∆ strain transformed with the p424GPD plasmids. In galactose (Figure 1E, left panels, 3 and 7 days of growth), no differences among the substitutions can be seen, indicating even spotting of the yeast cultures and no dominant negative effects of the substitutions. In glucose (Figure 1E, right panel), plasmid-borne wildtype uS12 was able to rescue growth defects caused by depletion of endogenous uS12

(Figure 1E, right panel, compare EV vs. WT at 3 days of growth). However, uS12 p.Arg69Lys

did not rescue growth to the same degree as wild-type uS12 (Figure 1E, right panel, 3 days of growth), indicating that p.Arg69Lys is a deleterious variant. The colony size of the yeast producing uS12 p.Arg69Lys is also smaller, indicating impaired growth (Figure 1E, right panel, 3 and 7 days of growth). Interestingly, the known deleterious uS12 p.Arg69Lys variant rescued growth to a comparable degree as uS12 p.Lys62Arg (Figure 1E, right panel, 3 days of growth).

In contrast, replacement of the positively charged arginine at position 69 with a negatively charged glutamate was not able to rescue growth. Moreover, substitution to histidine (a positively charged amino acid like arginine and lysine) at position 69 in uS12 was able to rescue growth of yeast when grown to saturation (Figure 1E, right panel, 7 days of growth), albeit to a lesser degree than the p.Arg69Lys substitution. Taken together, these results highlight the importance of a positively charged amino acid at position 69 in uS12.

Nevertheless, as neither the p.Arg69Lys or p.Arg69His variants were able to rescue growth to the same degree as wild-type uS12, the importance of Arg69 cannot simply lie in its positively charged side chain. These results support the hypothesis that the p.Arg67Lys missense substitution results in a human uS12 that is likely only partially functional, and thus could be a pathogenic hypomorphic allele.

The uS12 p.Arg67Lys variant affects ribosome biogenesis but not cell proliferation, de

novo protein synthesis rates, or the ribosomal stress response. To investigate the functional consequences of uS12 p.Arg67Lys on ribosome biogenesis, EBV-immortalized lymphoblast cell lines (LCLs) were compared to a knockdown of uS12. The knockdown of many RPs, including uS12, results in accumulation of pre-rRNA molecules early in the processing pathway 32; 33 . In recapitulation, uS12 in HeLa cells was knocked down to analyze accumulations of pre-rRNA by RAMP (ratio analysis of multiple precursors) [START_REF] Wang | Two orthogonal cleavages separate subunit RNAs in mouse ribosome biogenesis[END_REF] . Reductions in free 40S subunits by polysome profiling analysis confirmed the efficacy of the knockdown (Figure S2A). An accumulation of 30S pre-rRNA was revealed by northern blotting with ITS1

(internally transcribed spacer-1) probes alongside a reduction of 21S and 18S-E (Figure 2A,B).

Analysis of the effect of the uS12 p.Arg67Lys substitution in the LCL, however, suggested a different and much later defect in pre-rRNA processing. Northern blot analysis revealed an accumulation of 18S-E pre-rRNA in uS12 p.Arg67Lys cells, suggesting one of the final steps of pre-rRNA maturation is impaired (Figures 2A,B). Probes against ITS2 and ITS1-5.8S did not reveal any major differences in cells with knocked down uS12 or carrying the p. Arg67Lys variant (Figure S2B). Included in the analysis is an LCL derived from an individual with DBA carrying a mutation in the RPS24 gene, which codes for eS24 (RPS24). These cells revealed the expected early processing defect evident by 30S pre-rRNA accumulation (Figures 2A,B).

A full schematic of mammalian pre-rRNA processing is shown in Figure S2C and a highlight of the relevant portion of the pathway is shown in Figure 2C 33 .

Polysome profiles of LCL extracts derived from a healthy control individual revealed the expected ratio of small 40S to large 60S ribosomal subunits (Figure 2D,F). In contrast, the number of mature 40S subunits was reduced in cells carrying the uS12 p.Arg67Lys substitution (Figure 2 E,F). Western blot analysis showed that this reduction of 40S subunits was not due to a reduction of total uS12 protein in the cells (Figure S2D,E) and RNA sequencing of polyA-RNAs isolated from LCLs showed there was equal expression of wildtype and mutant alleles transcribing RPS23 mRNAs (Figure 2G).

Further analysis revealed that while LCLs derived from an individual with DBA displayed reduced cell proliferation compared to two unrelated healthy controls, proliferation of the uS12 p.Arg67Lys LCL was unaffected (Figure 2H). Similarly, de novo protein synthesis, as determined by fluorescence-based AHA incorporation, was reduced in the DBA LCL but remained unchanged in the uS12 p.Arg67Lys LCL (Figure 2I).

DBA-linked RP haploinsufficiency causes a number of ribosomal stress responses including autophagy, S6 kinase phosphorylation, and/or stabilization of the p53 tumor suppressor 35; 36 .

Western blotting of cells carrying the uS12 variant did not reveal any changes in the ratio of LC3-I to LC3-II proteins, a measurement indicative of autophagy (Figure S3A) [START_REF] Feng | The machinery of macroautophagy[END_REF] . No increase in S6K phosphorylation or changes in p53 stabilization were observed in uS12 p.Arg67Lys cells compared to healthy control cells (Figure S3B,C). These results together suggest that the ID and dysmorphism in the uS12 individual are due to a ribosomal dysfunction that does not manifest as a reduction of protein synthesis or in the subsequent ribosome stress response.

A second variant linked to dysmorphism destabilizes uS12. Having established that the p.Arg67Lys variant is likely pathogenic but does not cause a DBA-like phenotype, additional evidence was sought to link uS12 to syndromic phenotypes. A 5-year old female was identified with a de novo mutation in RPS23 at Chr5(GRCh37):g.81005179>A; NM_001025.4:c.358T>A (p.[Phe120Ile]). Trio exome sequencing confirmed neither parent carried the mutation. Sanger sequencing confirmed the mutation in fibroblasts (Figure S1A). Queries in BioMuta, DMDM, the NHLBI Exome database, and NCBI did not reveal any previously reported SNPs corresponding to Phe120. The affected phenylalanine lies in another highly conserved loop region of uS12, PGVRY (Figure S1B). This aromatic residue is invariant in Eukarya and strongly conserved in Archaea and Bacteria, where the residue is an aromatic tyrosine (Figure S1B). The PGVRY loop region of uS12, similar to PNSA, makes contact with the decoding center of the ribosome and is also the site of substitutions that affect translational fidelity in E.

coli and reduce growth in S. cerevisiae 10; 11 . The individual with the uS12 p.Phe120Ile variant, similar to the p.Arg67Lys individual, presented with hearing loss and many similar dysmorphisms such as microcephaly, short stature, epicanthic eye folds, low back hairline, low set ears, palmar creases, short fifth digits, and fetal finger pads (Table 1). The p.Phe120Ile

individual also presented with submuccous cleft palate, a bifid uvula, and Raynaud-like phenomena in response to cold. In contrast to the p.Arg67Lys indivivdual, the p.Phe120Ile

individual has no intellectual disability and is communicative (Table 1). The cryo-EM structure of the 80S ribosome revealed that the Phe120 residue is predicted to interact with Phe41 in uS12 through pi-pi stacking (Figure 3A) [START_REF] Khatter | Structure of the human 80S ribosome[END_REF] . Substitution of Phe120 with a non-aromatic isoleucine would abolish pi-pi stacking and potentially result in a structural change that affects the stability of uS12 (Figure 3B).

To determine the extent of the predicted effect of the substitutions on uS12 stability, protein levels of uS12 were measured in human dermal fibroblasts. Untreated wild-type and mutant fibroblasts revealed equivalent levels of uS12 (Figure 3C,D). However, when de novo protein synthesis was blocked with cycloheximide, a reduction of uS12 protein was detected over time in cells carrying the p.Phe120Ile variant but not in healthy control cells (Figures 3C,D). We did not observe significant destabilization of uS12 in p.Arg67Lys fibroblasts treated with cycloheximide (data not shown).

To determine if the substitutions affect the ability of uS12 to assemble into ribosomal complexes, plasmids expressing wild-type and mutant HA-tagged uS12 were transfected into HEK293 cells. In whole cell lysates, a lesser amount of HA-uS12 p.Phe120Ile protein was evident compared to equivalent levels of HA-uS12 wild-type and HA-uS12 p.Arg67Lys protein (Figure 3E,F). Cell lysates were then size-separated by ultracentrifugation on a 32% sucrose cushion. Both HA-uS12 wild-type and HA-uS12 p.Arg67Lys were detected in the high-density pellets that were devoid of tubulin but enriched with uL5 (RPL11) (Figure 3E,F). In contrast, far less HA-tagged protein was detected in the pellets of cells transfected with plasmids encoding HA-uS12 p.Phe120Ile (Figure 3E,F). This suggests the destabilizing effect of the p.Phe120Ile substitution in uS12 may be impairing the involvement of the variant in pre-40S

biogenesis at an earlier point in subunit formation than the p.Arg67Lys substitution.

Polysome profiling of fibroblasts revealed different abnormalities compared to healthy control cells (Figures 3G-I). Compared to the clear reduction of 40S subunits in LCLs with the uS12 p.Arg67Lys variant (Figures 2E,F), the variant in fibroblasts instead has a much more pronounced effect on the 80S monosome (Figures 3H,J). In contrast, the profiles of fibroblasts carrying the p.Phe120Ile substitution do reveal a significant reduction of 40S subunits in addition to fewer 80S monosomes (Figures 3I,J). It is possible that this discrepancy is due to the much faster rate of LCL proliferation compared to fibroblasts. This is evident when comparing the size of the polysome peaks, which are robust in LCLs (Figures 2D,E) and undetectable in fibroblasts (Figures 3G-I). This greater demand for ribosome biogenesis in LCLs may result in an amplification of detectable differences in subunit amounts, and unfortunately LCLs from the individual with the p.Phe120Ile variant were not available to fully test this. However, the polysome profiling of fibroblasts from both individuals reveal reductions of monosomes, and add further evidence that the uS12 p.Phe120Ile variant may have a greater effect on pre-40S subunit formation than the p.Arg67Lys variant.

Quantitative RT-PCR was then used to determine if the expression of uS12 mRNA is compensating for defective uS12 protein in fibroblasts carrying the variant. When expressed relative to βactin mRNA, a striking increase in uS12 mRNA was noted in the variant fibroblasts compared to healthy control cells (Figure 3K). This may reflect an attempt by the cells carrying the variants to ramp up synthesis of wild-type alleles coding for uS12 as a response to the apparent difficulties in generating functional ribosomes for active translation.

p.Arg67Lys impairs uS12 hydroxylation by OGFOD1. The conserved prolyl residue at codon 62 in human uS12 is hydroxylated by the 2-OG oxygenase OGFOD1 [START_REF] Singleton | OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation[END_REF] . OGFOD1 forms discrete associations with uS12 that can be visualized as differentially migrating species in SDS-PAGE [START_REF] Singleton | OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation[END_REF] . Hydroxylation of uS12 leads to the formation of a high-affinity protein complex with OGFOD1 that migrates as an ~100 kDa adduct by SDS-PAGE. This association is separable from a lower-affinity interaction that is hydroxylation-independent and is reducible by denaturing electrophoresis. Using the presence of the 100 kDa adduct as a surrogate for uS12 hydroxylation, parallel OGFOD1 immunoprecipitations were performed in LCLs derived from a healthy control or the individual carrying the uS12 p.Arg67Lys variant. Equivalent capture of uncomplexed OGFOD1 (~75 kDa) was observed in both p.Arg67Lys and control lysates. However, capture of the ~100 kDa hydroxylation-dependent species was significantly reduced in p.Arg67Lys lysate compared to healthy control lysate (Figure 4A; lane 3, 6). This was in the absence of any effect on uS12 binding at its native molecular weight, as determined by uS12 immunoblotting (Figure 4A).

LCLs were not available from the individual carrying the p.Phe120Ile variant. To assay the effect of the p.Phe120Ile substitution on uS12 binding to OGFOD1, plasmids coding for HAtagged uS12 were transiently co-expressed with those coding for FLAG-OGFOD1 in HEK293 cells and anti-HA (uS12) pulldowns were performed. Concordant with endogenous IPs, HA-uS12 p.Arg67Lys co-purified FLAG-OGFOD1 but did not form the 100 kDa adduct (Figure 4B). In contrast, high-affinity HA-uS12 p.Phe120Ile:FLAG-OGFOD1 complexes were detected, albeit to a lesser degree than wild-type uS12 due to lower protein levels of uS12 p.Phe120Ile (Figure 4B). These data suggest that the p.Arg67Lys substitution impairs uS12 hydroxylation by OGFOD1 without affecting protein:protein interaction, and that destabilized uS12 p.Phe120Ile likely results in fewer hydroxylated uS12 proteins.

To directly assay the effect of the p.Arg67Lys substitution on hydroxylation and incorporation of uS12 into the ribosome we isolated endogenous uS12 from polysomes derived from healthy control and p.Arg67Lys LCLs by sucrose density ultracentrifugation and reverse phase chromatography. uS12 was subjected to proteolytic digestion with trypsin and peptides were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The relative abundance of the mutant peptide was monitored in ribosomes derived from the p.Arg67Lys

LCL by measuring peak intensities of equivalent wild-type and p.Arg67Lys precursor ions assigned by MS/MS (Table S1). Ion intensities of wild-type and p.Arg67Lys peptides were summed and expressed as a relative ratio. Quantifying data from 5 independent experiments indicated a marked reduction of p.Arg67Lys peptides in polysomal ribosomes with less than 10% of the total peptide counts carrying the p.Arg67Lys substitution (Figure 4C). As no decrease in polysome size or suppression of mutant allele expression in the LCL was detected (Figure 2C-F), this suggests that a mechanism exists preventing ribosomes carrying the uS12 variant from entering the polysomal pool.

We next considered the effect of the substitution on OGFOD1-dependent catalysis, seeking to corroborate interaction data with direct measurement of hydroxylation by mass spectrometry. Fortuitously, the p.Arg67Lys substitution preserves a trypsin cleavage site and is present on all peptides that contain the target prolyl hydroxylation site. This enabled direct measurement of the effect of the p.Arg67Lys substitution on uS12 hydroxylation at the peptide level in ribosomes isolated from LCLs.

Relative quantitation indicated a deleterious effect of the p.Arg67Lys substitution on Pro62 hydroxylation with <40% of the Lys67-bearing peptides recovered from ribosomes hydroxylated. (Figure 4D, Figure S4 for representative extracted ion chromatograms and MS/MS assignments and Table S1 for Summary). In contrast, wild-type uS12 peptides with Arg67 extracted from the same cells, and from separate healthy control LCLs, were near fully hydroxylated (>95%; Figure 4D). High levels of wild-type uS12 hydroxylation in LCLs carrying p.Arg67Lys uS12 argue against the substitution having a significant dominant negative function in this context. These results show that the substitution of lysine for arginine at amino acid position 67 has detrimental consequences for hydroxylation of uS12 by OGFOD1, and that very few ribosomes carrying the variant protein are incorporated into the polysomal pool.

uS12 variants intensify stress granule formation and affect translational fidelity. Cellular stress such as hypoxia or heat shock induces the formation of cytoplasmic stress granules as a mechanism to suppress the translation of mRNAs not immediately required for the stress response [START_REF] Arimoto | Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways[END_REF] . Stalled pre-initiation complexes that include 40S subunits and small RPs are rapidly sequestered in stress granules upon assembly by the GTPase-activating protein SH3 domain binding protein 1 (G3BP1) [START_REF] Tourriere | The RasGAP-associated endoribonuclease G3BP assembles stress granules[END_REF] . Deregulated stress granule formation is being revealed as an important indicator of several human pathologies, including neuronal dysfunction and degeneration [START_REF] Wolozin | Physiological protein aggregation run amuck: stress granules and the genesis of neurodegenerative disease[END_REF] . Because acute loss of OGFOD1 drives G3BP1+ stress granule formation in U2OS cells [START_REF] Singleton | OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation[END_REF] , their formation in the uS12 variant cells was investigated.

Fibroblasts derived from healthy controls and the individuals carrying uS12 variants were exposed to arsenite to induce oxidative stress. Stress granules in these cells were then stained by immunofluorescence and analyzed by confocal microscopy. In all cell types, G3BP1 localized diffusely throughout the cytoplasm and aggregated in granules of about 100-200 nm in size when treated for 30 minutes with 500 µM arsenite (Figure 5A). However, the percentage of cells revealing stress granules when exposed to increasing concentrations of arsenite was consistently higher in cells carrying either the uS12 p.Arg67Lys or p.Phe120Ile substitution compared to healthy control cells (Figures 5A,B andS5). These data suggest that the uS12 variants affect the dynamics of stress granule formation, and render cells particularly sensitive to oxidative stress.

Increased oxidative stress levels or reduced levels of OGFOD1 change the fidelity of protein translation 13; 41 . This can manifest as changes in suppression of missense mutations, readthrough of stop codons, or in rates of -1 or +1 Programmed Ribosomal Frameshifting (-/+1 PRF) on so-called "slippery sites", and can be measured with dual luciferase reporter plasmids (Figure S6) [START_REF] Jacobs | Systematic analysis of bicistronic reporter assay data[END_REF] .

The uS12 p.Arg69Lys (Arg67 in humans) substitution had a pronounced effect on translational fidelity in yeast. Experiments were performed alongside a strain carrying p.Lys62Arg substitution that has been previously reported to affect translational fidelity 11; 12 . Similar to the uS12 p.Lys62Arg variant, the strain carrying the p.Arg69Lys variant displayed altered rates of -1 PRF in a sequence-dependent manner (Figure 5C). Additionally, uS12 p.Arg69Lys

increased the rate at which ribosomes read through UAA and UAG stop codons (Figure 5D).

The p.Arg218Ser substitution when introduced into the firefly protein renders the luciferase inactive [START_REF] Plant | Differentiating between near-and non-cognate codons in Saccharomyces cerevisiae[END_REF] . However, when this substitution is introduced with a non-cognate DNA mutation (AGA [arginine]->TGT [serine]) yeast cells carrying the uS12 p.Arg69Lys variant were more likely to select the missense arginine residue instead of the serine (Figure 5E). No changes in EST3-directed +1 PRF as a result of uS12 p.Arg69Lys were detected (Figure 5F). +1 PRF is entirely driven by interactions between the peptidyl-tRNA, the ribosomal P-site, and the mRNA. In contrast, -1 PRF involves both the P-and A-sites, while near-and non-cognate tRNA interactions involve the ribosomal A-site [START_REF] Dinman | Mechanisms and implications of programmed translational frameshifting[END_REF] . By process of elimination, it is likely that the uS12 variants are affecting A-site but not P-site function.

Translational fidelity was also assayed in the human fibroblasts. Cells carrying uS12 p.Arg67Lys or p.Phe120Ile revealed a significant increase in the rate of UAA stop codon readthrough compared to healthy control cells (Figure 5G). Together, these results suggest that the human uS12 variants, similar to what was reported previously in E. coli [START_REF] Sharma | Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome[END_REF][START_REF] Anthony | Alterations in ribosomal protein RPS28 can diversely affect translational accuracy in Saccharomyces cerevisiae[END_REF][START_REF] Synetos | Mutations in yeast ribosomal proteins S28 and S4 affect the accuracy of translation and alter the sensitivity of the ribosomes to paromomycin[END_REF] , adversely affect the decoding of mRNA and the fidelity of protein translation.

Discussion

This study suggests that uS12 variants affecting the ability of ribosomes to correctly decode mRNA drive human dysmorphism and in some cases can also impair neurodevelopment.

Comparable developmental defects were presented in unrelated individuals with missense substitutions in two highly conserved loop regions of uS12 that in bacteria and yeast are important for translational accuracy and growth [START_REF] Sharma | Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome[END_REF][START_REF] Anthony | Alterations in ribosomal protein RPS28 can diversely affect translational accuracy in Saccharomyces cerevisiae[END_REF][START_REF] Synetos | Mutations in yeast ribosomal proteins S28 and S4 affect the accuracy of translation and alter the sensitivity of the ribosomes to paromomycin[END_REF] . The p.Phe120Ile substitution destabilized the uS12 protein and the p.Arg67Lys substitution impaired OGFOD1 hydroxylation, an event known to be important for translational fidelity [START_REF] Singleton | OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation[END_REF] . Despite a mild ribosome biogenesis defect in LCLs carrying the p.Arg67Lys substitution, there was no indication of proliferation impairment or changes in the rate of protein synthesis as is commonly observed in LCLs derived from individuals with DBA. However, ribosomes carrying uS12 variants were less able to accurately decode mRNA codons. This is readily apparent in the yeast model, where the only form of uS12 is the variant. Of course this is different in diploid human cells where both wild-type and mutant copies of RPS23 are expressed, thus the human mutations are haploinsufficient as is the case with many other ribosomopathies such as DBA. This underscores the centrality of a functioning ribosome and shows how even small changes can be greatly amplified in complex developmental processes.

The scarcity of uS12 peptides with Lys67 in the polysomal pool of LCLs is particularly interesting in light of the equal expression in these cells of both wild-type and mutant alleles. uS12 p.Arg67Lys does not appear to impair 40S assembly until very late stages (as in LCLs)

if at all (as in fibroblasts), suggesting the existence of a quality control mechanism that identifies 40S subunits with the uS12 variant and largely prevents them from entering the polysomal pool. One possibility is that the uS12 variant is being replaced with a wild-type uS12. This process of ribosome repair by replacing damaged RPs, specifically uS12, has been previously described in E. coli [START_REF] Cukras | Ribosomal proteins S12 and S13 function as control elements for translocation of the mRNA:tRNA complex[END_REF] . A similar mechanism might occur in the human context, where ribosomes carrying an unhydroxylated, unstable, or absent uS12 are held in stalled preinitiation complexes while a repair is made. Since pre-initiation complexes are coordinately recruited to stress granules in response to oxidative stress [START_REF] Kedersha | Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules[END_REF] , this may explain the more rapid formation of these structures in the fibroblasts carrying the uS12 variants upon exposure to arsenite.

These results may also shed light on the question of why RP gene mutations affect such discrete tissue types. All cells in the body express all the RPs, albeit not necessarily equally,

and it has been a puzzle as to why the haploinsufficient loss of specific RP genes results in such tissue-specific effects. Perhaps the answer lies in how much of the wild-type RP gene is expressed and available in a particular cell type or subcellular location for ribosome repair. To use ICA as an example, individuals with RPSA mutations suffer severely impaired development of the spleen but no hematopoietic (or any other major) phenotype. Conversely, asplenia is not reported in individuals with DBA. Tagged uS2 proteins carrying substitutions linked to ICA are, like the uS12 p.Phe120Ile variant reported here, unstable and levels are so low as to be undetectable in some cases [START_REF] Bolze | Ribosomal protein SA haploinsufficiency in humans with isolated congenital asplenia[END_REF] . However these variants, like uS12 p.Arg67Lys, do not seem to adversely affect LCLs in contrast to DBA-linked RP variants [START_REF] Bolze | Ribosomal protein SA haploinsufficiency in humans with isolated congenital asplenia[END_REF] . Thus the ability to compensate for lost or defective RPs seems to depend on which RP is impaired in which cell type. Extending this analysis to DBA, if erythrocyte progenitor cells express less of the wildtype allele to replace the RP variant in comparison to other hematopoietic cell types, this may explain the pure red cell aplasia. If the wild-type RP allele expression in erythrocyte progenitors varies between individuals, this may be why some healthy parents can carry a mutant RP gene (or "silent mutation") that is pathogenic in their offspring. As the number of human disorders linked to RP gene mutation continues to increase, further studies into the reasons underlying tissue-specific defects are definitely warranted.

This study reveals the pathogenicity of a previously undescribed ribosomopathy and suggests that while the hematopoietic phenotypes resulting from RP gene mutations are largely due to reductions in mRNA translation rates, these mutations reported in RPS23 instead drive developmental disorders by coding for uS12 variants that impair the ability of ribosomes to accurately decode mRNA. The implications of this may well go beyond RP gene mutations.

Translational fidelity defects, such as increased rates of frameshifting, have been reported in yeast carrying a mutation in the sister chromatid cohesion 2 gene (SCC2) [START_REF] Zakari | The SMC Loader Scc2 Promotes ncRNA Biogenesis and Translational Fidelity[END_REF] . This mutation impairs rRNA production and ribosome assembly but does not affect protein synthesis rates [START_REF] Zakari | The SMC Loader Scc2 Promotes ncRNA Biogenesis and Translational Fidelity[END_REF] .

Orthologous mutations of this gene, Nipped-B (NIPBL), are found in individuals with Cornelia de Lange syndrome (OMIM #122470). Individuals with this syndrome feature many of the same clinical phenotypes as the individuals reported here, including the ID/ASD, hearing loss, microcephaly, hypotonia at birth and delayed motor development, abnormal hair growth (such as long eyelashes and bushy eyebrows with synophrys), low set ears, the palmar crease, and short fifth digits. Thus translational inaccuracy may be an underlying cause of developmental defects in a much wider spectrum of human disorders than is currently appreciated.

  . Experiments were repeated with three independently grown samples of each cell line. rRNA processing analysis. Lymbloblastoid cell lines and HeLa cells were cultured in RPMI and DMEM, respectively (Gibco). These media were supplemented with 10% fetal bovine serum and 1 mM sodium pyruvate (Sigma). Different 21-mer siRNAs (Eurogentec, Seraing, Belgium), whose efficiency was verified by qPCR, were used to knock down expression of the following human mRNAs in HeLa cells: uS12 (GenBank accession number: NM_001025.siRNA uS12-3) (20819938). Each siRNA solution was added at a final concentration of 500 nM to 200 µl of cell suspension (50 x 10 6 cells/ml diluted in Na phosphate buffer, pH 7.25, containing 250 mM sucrose and 1 mM MgCl2). Electrotransformation was performed at 240 V with a Gene Pulser (Bio-Rad, Hercules, CA)[START_REF] Paganin-Gioanni | Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells[END_REF] . Control HeLa cells were electro-transformed with a scramble siRNA (siRNA-negative control duplex; Eurogentec). After 10 min incubation at ambient temperature, cells were plated and grown at 37°C for 48 hours.

RNA extraction and analysis

  by northern blot. Total RNAs were extracted with Trizol from cell pellets containing 20-30 x 10 6 cells. The aqueous phase was further extracted with phenolchloroform-isoamylic alcohol (25:24:1; Sigma), then with chloroform. Total RNAs were recovered after precipitation with 2-propanol. For Northern blot analyses, RNAs were dissolved in formamide, denatured for 10 min at 70°C and separated on a 1.2% agarose gel containing 1.2% formaldehyde and 1X Tri/Tri buffer (30 mM triethanolamine, 30 mM tricine, pH 7.9) (3 µg RNAs/lane). RNAs were transferred to a Hybond N + nylon membrane (GE Healthcare, Orsay, France) by passive transfer and cross-linked under UV light. Prehybridization was performed for 1 hour at 45°C in 6X SSC, 5X Denhardt's solution, 0.5% SDS, 0.9 g/ml tRNA. The 5'-radiolabeled oligonucleotide probe was incubated overnight. The sequences of the probes were: 5'-ITS1 (5'-CCTCGCCCTCCGGGCTCCGTTAATGATC-3'), ). Membranes were washed twice for 10 min in 2X SSC, 0.1% SDS and once in 1X SSC, 0.1% SDS, and then exposed. Signals were acquired with a Typhoon Trio PhosphorImager (GE Healthcare) and quantified using the MultiGauge software.

Figure 1 .

 1 Figure 1. Substitution of Arg69 in yeast uS12, orthologous to Arg67 in human uS12, results in growth defects. (A) Arg67 in wild-type uS12 is shown to be in close proximity with

Figure 2 .

 2 Figure 2. uS12 p.Arg67Lys affects ribosome biogenesis but not proliferation or de novo protein synthesis (A) Northern blot analysis of siRNA-treated HeLa cells or LCLs with 5'ITS1

Figure 3 .

 3 Figure 3. p.Phe120Ile affects stabilization of uS12 and 40S subunit levels while both

Figure 4 .

 4 Figure 4. Substitutions perturb OGFOD1 binding and uS12 Pro62 hydroxylation. (A)

Figure 5 .

 5 Figure 5. uS12 substitutions increase stress granules formation and impair

  cells by calcium phosphate precipitation. The p.Phe120Ile substitution was introduced into the

	HA-tagged	wild-type	uS12	pCS2+	plasmid	with	forward	primer	5'-
	CTGGAGTCCGCATTAAGGTTGTC-3'	and	reverse	primer	5'-
	GACAACCTTAATGCGGACTCCAG-3' using Quickchange II Site Directed Mutagenesis kit
	(Agilent).

is identified in RPS23 at c.200G>A in an individual with ID, ASD,

  RPS23 gene of a 10-year old male that was absent in both parents. Sanger sequencing confirmed the mutation in fibroblasts (FigureS1A). Queries in BioMuta, DMDM, the NHLBI Exome database, and NCBI did not reveal any previously reported SNPs corresponding to Arg67. The individual presents with a mild ID, ASD, and hearing loss. Additionally the individual has a broad range of dysmorphic features often associated with ID/ASD such as simian palmar creases, epicanthic folds in the eyes, fetal finger pads, extra front teeth, low back hairline, facial asymmetry, and high palate (Table

	Results
	Cells were then permeabilized for 5 minutes at RT with 0.1% Triton X-100 (Sigma #T8787) in Sucrose cushion experiment. Transfected HEK293 cells were lysed in polysomal lysis buffer
	PBS and washed 3x with PBS. Cells were then blocked for 30 minutes at RT with 1% BSA (supplemented with 110mM potassium acetate, 20mM magnesium acetate, 10mM HEPES, A missense mutation and hearing loss. Trio exome sequencing identified a de novo
	(Sigma #A2153) in PBS and incubated with G3BP1 antibodies (BD Transduction Laboratories 100mM potassium chloride, 10mM magnesium chloride, 0.1% NP-40, 2mM DTT, 40U/mL
	Chr5(GRCh37):g.81572302C>T; NM_001025.4:c.200G>A (p.[Arg67Lys]) mutation in the
	#611126) at a 1:100 dilution in blocking buffer at RT for 30 minutes. Cells were then incubated RNase inhibitor [Thermo Fisher #10777019), 100ug/ml cycloheximide [Sigma #C104450] and
	at RT for 30 minutes with anti-mouse immunoglobulin conjugated to biotin diluted at 1:200 in 1X Protease inhibitor Cocktail V [Calbiochem]). Lysates were treated with 1500 U RNAse1
	blocking buffer (Dako #E0433), followed by 2x washes with PBS and a final incubation in the (Ambion) for 30 min at 21° C and loaded onto 6 ml polysome lysis buffer layered over 4 ml
	dark for 30 minutes at RT with streptavidin conjugated to FITC at a 1:200 dilution in blocking 32% sucrose cushions. Tubes were centrifuged in a SW41 rotor for 2.5h in a Beckman Coulter
	buffer. Cells were then washed 3x with PBS and mounted on slides with Prolong® Gold L-100 XP ultracentrifuge. The supernatants were removed and pellets were dissolved in 6x
	SDS loading buffer for western blot analysis.
	tubulin (Sigma #T6199), and LC3 (Abcam #ab51520). 10% acrylamide gels were run for
	detection of p53 (Santa Cruz, #sc-6243) and phospho-S6 kinase (Cell Signaling #92345). 4-
	20% acrylamide gels were used for the cycloheximide experiments. Primary antibodies were
	diluted 1:1000 in 5% milk in TBST and incubated overnight at 4°C. For detection of p53, pS6k,
	and LC3HRP-conjugated secondary antibodies were diluted 1:10,000 at RT for one hour and

Fibroblasts were plated to 50% confluence on glass coverslips in a 24well plate. The following day cells were treated for 30 minutes with varying concentrations of arsenite (Sigma #S7400) dissolved in DMEM + 10% FCS at 37°C. Cells were fixed for 10 minutes at RT with 2% paraformaldehyde (Sigma #P6148) in PBS and washed 2x with PBS.

Antifade Mountant (ThermoFisher #P36935) and left to dry overnight in the dark at RT. Images were acquired with a Leica SP-8 confocal microscope. For quantifications, biological triplicates of each condition were analyzed by generating 3x3 tiles at 40x objective three times from each slide.

Western blot analysis. Bafilomycin A (Sigma, #B1793) treatment was at 20nM for 6 hours.

Cycloheximide (Sigma #C104450) treatment was at 200µg/ml and cells lysed at 6, 12, or 24 hours after addition. For p53 stabilization, cells were exposed to 25 Gy ionizing radiation and lysed 4 hours later. SDS/PAGE gels were run under reducing conditions and immunoblotted on nitrocellulose membranes. 25µg of protein was run on 12% acrylamide gels for detection of HA (Sigma #H3663), uS12 (Santa Cruz #sc-100837), uL5 (RPL11) (Abcam #ab79352), detected with ECL reagents (Amersham) and exposed to film. For detection of uS12, HA, uL5, and tubulin the Odyssey detection kit (LI-COR) was used. Quantifications were performed with ImageJ software.

Acknowledgements

Very special thanks go to all the individuals in this study and their families. We thank Prof.

Hans Brunner of the

Radboud University Nijmegen Medical Centre and Prof. Nine Knoers of the University Medical Center Utrecht for performing the exam sequencing as well as Daisy Picavet and the AMC Cellular Imaging Core Facility for assistance with the confocal microscopy. Mass spectrometry analysis was performed in the TDI MS laboratory led by Benedict M. Kessler. AM, AA, MW, PEG, and MOD are supported under the frame of E-Rare-2, ERA-Net for Research on Rare Diseases (ZonMW #113301205 and #40-44000-98-1008 in the Netherlands, #BMBF 01GM1301 in Germany, #ANR-15-RAR3-0007-04 in France). The Landsteiner Foundation supported NP and FS for Blood Transfusion Research (#1239). CV was supported by the National Institutes of Health (NIH) / National Heart Lung Blood Institute (R01HL119439 to JD). SB was supported by NIH / General Medical Sciences (R01GM115710). SS was supported by National Institutes of Health (NIH) / Diabetes and Digestive and Kidney Diseases / General Medical Sciences / Child Health and Human Development (F30DK109582, T32GM07205, and T32HD007149 to SS). The Ludwig Institute of Cancer Research supported MA, MC and PR. PG and MO were supported by the Agence National de la Recherche, (project DBA-Multigenes).


Taina T Nieminen

Marie-Françoise O'donohue

Yunpeng Wu

Hannes H Lohi

Stephen W Scherer

Andrew D Paterson

Pekka Ellonen

Wael M Abdel-Rahman

Satu Valo

Jukka-Pekka Mecklin

Heikki J Järvinen

Pierre-Emmanuel Gleizes

Päivi Peltomäki

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

Keywords: Colon Cancer, Hereditary Nonpolyposis Colorectal Cancer, Ribosome, Exome Sequencing nonpolyposis colorectal cancer type X, rRNA, ribosomal RNA

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H ereditary nonpolyposis colorectal cancer as defined by the Amsterdam criteria 1,2 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. 3 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. 4 We recently discovered germline mutations in the gene for bone morphogenetic protein receptor type IA in 2 Amsterdampositive families of 18 FCCX families investigated (11%). 5 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. 6 Tumors from mutation carriers showed no loss of the wild-type allele (Supplementary Figure 2B), arguing against 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 (þ, 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 k light polypeptide gene enhancer in B cells, kinase b (IKBKB) SNV alteration was unlikely to explain the colorectal cancer susceptibility of F56 and was excluded from further consideration. U, noncarrier.

Knudson's 2-hit mechanism for tumor-suppressor genes. 7 The absence of loss of heterozygosity complies with observations from zebrafish showing that ribosomal protein genes act as haploinsufficient suppressors of tumorigenesis. 8 RPS20 is required during the late steps of 18S ribosomal RNA (rRNA) formation. 9 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. 9 The ribosomal protein gene family comprises 80 genes, 8 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. 12 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. 13 RPS20 was proposed to be primarily of the detector type, with reduction perturbing ribosomal biogenesis 9 (Figure 2 and Supplementary Figure 3), leading to stabilization of p53 13 (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 Materials and Methods

Patients and Samples

This study was based on 26 FCCX families from Finland, 15 of which fulfilled the Amsterdam criteria 1,2 and 11 fulfilled the Bethesda criteria. 3 The families had no DNA mismatch repair defects in tumor tissue or in the germline 4 ; furthermore, germline mutations in bone morphogenetic protein receptor type IA were excluded. 5 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 cancers 6 (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.

Genome-Wide Linkage Analysis

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) 7 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), 8 using the following parameters: autosomal-dominant mode of inheritance, gene frequency of 0.001, 4 age-dependent liability classes, 9 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 and Data Analysis

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 pipeline 10 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 algorithm 10 developed at the Institute for Molecular Medicine Finland was used for single-nucleotide variant calling, and Pindel 11 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.

Exclusion of Inhibitor of k Light Polypeptide Gene Enhancer in B Cells Kinase b as a Likely Susceptibility Gene for F56

The c.1933C>T (RefSeq NM_001556.2) alteration in inhibitor of k light polypeptide gene enhancer in B cells kinase b 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 k light polypeptide gene enhancer in B cells. Inhibitor of k light polypeptide gene enhancer in B cells kinase b 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 k light polypeptide gene enhancer in B cells kinase b has an established disease connection, with homozygous germline inactivation underlying severe combined immunodeficiency. 12 Sanger Sequencing of RPS20 RPS20 was screened for point mutations by exonspecific sequencing with intronic primers from Sjoblom et al 13 and for large rearrangements by long-range genomic polymerase chain reaction with primers 5'-ATTTTTGGTCCG CACGCTCCT-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'-CTCGTTAATGTTA GTGTAGAAGGTG-3', reverse primer: 5'-GAACCTGAATTT AGTCAACATC-3').

Analysis of Loss of Heterozygosity in Tumor Tissue

Fluorescent fragment analysis was performed as described, 14 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'-CAG-CATTTTTCCTCAGAGCAG-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'.

Analysis of the Effects of RPS20 Mutation on Pre-rRNA Processing by Northern Blot Total RNAs were extracted by the TRIzol (Grand Island, NY) method (modified from Chomczynski 15 ). Northern blot analysis of pre-rRNA species with probes hybridizing to the internal-transcribed spacer 1 or 2 was performed as described. 16 HeLa cells treated for 48 hours with a scramble small interfering RNA or a small interfering RNA targeting RPS20 messenger RNA (5'-GGUGGCAAUUCACCGAAUUdTdT-3') (Eurogentec, Liege, Belgium) and lymphoblastoid cells of individuals with or without the RPS20 c.147dupA mutation were included in the analysis. Precursor rRNAs were detected with a 5' internal-transcribed spacer 1 probe, which is complementary to the junction of the 18S and the internal-transcribed spacer 1. Mature rRNAs were shown with 18S and 28S probes. Each pre-rRNA species was measured by phosphorimaging, quantified using MultiGauge (Tokyo, Japan) software, and normalized to 28S rRNA. For each species, the value of the mean of the 3 control samples arbitrarily was set to 1. RNA analyses were performed 3 times and led to similar results.

Figure 1 .

 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 (þ, mutation carrier, -, noncarrier). Arrow denotes the index person. Clinical diagnoses are specified in Supplementary Table1. 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 k light polypeptide gene enhancer in B cells, kinase b (IKBKB) SNV alteration was unlikely to explain the colorectal cancer susceptibility of F56 and was excluded from further consideration. U, noncarrier.

Figure 2 .

 2 Figure 2. (A) Northern blot analysis of total RNAs from HeLa cells treated for 48 hours with a scramble smallinterfering 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. 9 (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.

Acknowledgments

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.

Conflicts of interest

The authors disclose no conflicts.

Funding

Supported by grants from the Paulo Foundation (T.T.N.), Suomen Akatemia (257795 to P.P.), The Finnish Cancer Organizations (P.P., J.-P.M.), the Sigrid Juselius Foundation (P.P., H.L., J.-P.M.), the Päivikki and Sakari Sohlberg Foundation (P.P.), Nordic Cancer Union (P.P.), Biocentrum Helsinki (P.P.), European Research Council (FP7-ERC-232635 to P.P.), Kuopio University Hospital (B13-104 and B14-01 to J.-P.M.), Agence Nationale pour la Recherche (ANR-10-BLAN-1115-1 to P.-E.G.), Association pour la Recherche sur le Cancer (PJA20131200432 to M.-F.O.'D.), and Genome Canada through the Ontario Genomics Institute (S.W.S. and A.D.P.). The Centre for Applied Genomics provided infrastructure support.

Supplementary Material

Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at http://dx.doi.org/10.1053/ j.gastro.2014.06.009.

Author names in bold designate shared co-first authorship.

Received April 15, 2014. Accepted June 4, 2014.

Reprint requests

Address requests for reprints to: Taina Nieminen, PhD, Department of Medical Genetics, Haartman Institute, PO Box 63, Haartmaninkatu 8, FI-00014 University of Helsinki, Finland. e-mail: Taina.Nieminen@Helsinki.Fi; fax: (358) 294125105.

Analysis of Ribosomes by Sucrose Density Gradient Centrifugation

Polysome analysis was conducted by sucrose density gradient centrifugation of cytoplasmic fractions of cycloheximide-treated cells as described in detail previously. 16

Western Blot Analysis of p53 Protein

Total proteins were extracted from lymphoblastoid cells, separated by electrophoresis on Novex (Carlsbad, CA) NuPAGE 4%-12% Bis-Tris Gels (1.0 mm), blotted on nitrocellulose membranes, hybridized with the primary and secondary antibodies, visualized with the Amersham (Buckinghamshire, UK) ECL Prime Western Blotting Detection reagents, and scanned with the CCE camera. P53 rabbit polyclonal antibody (9282; Cell Signaling Technology, Danvers, MA) with 1:1000 dilution was used as the primary antibody to detect p53 protein. The housekeeping protein glyceraldehyde-3-phosphate dehydrogenase served as a loading control and was detected with rabbit polyclonal antibody (1:1000 dilution, FL-335; Santa Cruz Biotechnology, Dallas, TX).

URL Addresses for Web Resources Used

The following online resources were used: Online Mendelian Inheritance in Man (OMIM), available at: http:// www. Supplementary Figure 1. Results from genetic linkage analysis for chromosome 8 in F56, based on multipoint analysis with the program MERLIN v1.0-alpha (Supplementary Materials and Methods). The area between positions 61.133 and 79.265 on the Haldane marker map (interpolated from DeCode genetic map provided by Affymetrix) assuming no interference yielded lod scores over 1 (note that linkage analysis was based on a more concise pedigree compared to that shown in Fig. 1A, reflecting sample availability of the time).