Published Ahead of Print on April 20, 2017, as doi:10.3324/haematol.2016.162198.
Copyright 2017 Ferrata Storti Foundation.
Low frequency mutations in ribosomal proteins RPL10 and RPL5
in multiple myeloma
by Isabel J.F. Hofman, Stephanie Patchett, Mark van Duin, Ellen Geerdens, Jelle Verbeeck,
Lucienne Michaux, Michel Delforge, Pieter Sonneveld, Arlen W. Johnson,
and Kim De Keersmaecker
Haematologica 2017 [Epub ahead of print]
Citation: Hofman IJF, Patchett S, van Duin M, Geerdens E, Verbeeck J, Michaux L, Delforge M,
Sonneveld P, Johnson AW, and De Keersmaecker K. Low frequency mutations in ribosomal proteins RPL10
and RPL5 in multiple myeloma. Haematologica. 2017; 102:xxx
doi:10.3324/haematol.2016.162198
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LETTER TO THE EDITOR
Low frequency mutations in ribosomal proteins RPL10 and RPL5 in multiple
myeloma
Isabel JF Hofman1, Stephanie Patchett2, Mark van Duin3, Ellen Geerdens 4,5, Jelle Verbeeck1, Lucienne
6
7
3
2
Michaux , Michel Delforge , Pieter Sonneveld , Arlen W. Johnson , Kim De Keersmaecker
1
1
Department of Oncology, KU Leuven - University of Leuven, LKI - Leuven Cancer Institute, Leuven,
Belgium
2
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA
3
Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, The Netherlands
4
Center for Cancer Biology, VIB, Leuven, Belgium
5
Center for Human Genetics, KU Leuven - University of Leuven, LKI - Leuven Cancer Institute,
Leuven, Belgium
6
7
Center for Human Genetics, KU Leuven - University Hospitals Leuven, Leuven, Belgium
LKI - Leuven Cancer Institute, Department of Development and Regeneration, KU Leuven -
University Hospitals Leuven, Leuven, Belgium
Word count (max 1500) = 1496
Tables: 1
Figures: 1
Low frequency mutations in ribosomal proteins RPL10 and RPL5 in multiple myeloma
Genomic screening studies recently revealed that mutations in ribosomal protein (RP) genes represent
a novel class of defects in cancer. In T-cell acute lymphoblastic leukemia (T-ALL), 20% of children
harbor acquired mutations and deletions in RPL10 (uL16 in the new nomenclature1), RPL5 (uL18) and
RPL22 (eL22), 3 proteins of the large 60S ribosomal subunit.
2,3
Strikingly, 7.9% of pediatric T-ALL
2
patients carried the same RPL10 R98S missense mutation. Somatic mutations in RPs are not
confined to T-ALL. RPL5 is mutated in 11-34% of glioblastoma, melanoma and breast cancer samples
and 10-20% of chronic lymphocytic leukemia samples have RPS15 mutations.4,5,6 The plasma cell
malignancy multiple myeloma (MM) is an attractive candidate for harboring RP mutations: initial
genome sequencing revealed that half of the patients carry mutations in genes that may be
functionally linked to protein translation and we recently described that RPL5 is in a 58 kb minimal
deleted region on 1p22 that is deleted in ≥20% of MM cases.7,8 In this study, integration of published
sequencing data, targeted resequencing of all 81 RP genes in a cohort of 37 MM cases and Sanger
sequencing of RPL10 in 141 MM cases revealed rare somatic defects in RPL5 and RPL10.
Interestingly, the RPL10 mutations clustered in a different region as compared to the described
mutational hotspot in T-ALL.2
Initiating events of MM consist of translocations involving the IgG locus or hyperdiploidy of uneven
chromosomes. Further malignant progression is driven by NFkB and MAPK/ERK signaling.
9,10
Until a
few years ago, the mutational landscape for MM was largely unknown. The first whole genome
sequencing study uncovered only ten significantly mutated genes, two of which were the previously
identified NRAS and KRAS. One of the novel findings in that study was that nearly half of the patients
carry mutations in genes with a function that may be linked to protein translation. Of interest are the
mutations in FAM46C in 13% of MM cases. FAM46C expression correlates with that of ribosomal
proteins and translational initiation and elongation factors.7
The supplements of this first MM sequencing paper also described RPL10 mutations (E66G and I70L)
7
in 2/38 patients analyzed (Figure 1A and Table 1, Chapman cohort). The same group later expanded
this cohort and identified 3 additional RPL10 mutations (I70L, I70M and I33V) out of 165 new patients
(Figure 1A and Table 1, Lohr cohort). Intriguingly, 2 out of 3 mutations again affected residue 70 of
RPL10. The supplements of this paper also included rare mutations in several other RPs (Table 1).11
Given these observations, we explored the spectrum of mutations in ribosomal protein genes in MM in
more detail. We started by Sanger sequencing of the entire RPL10 coding region in 141 MM samples.
This uncovered two RPL10 mutations (I70M and I33V), which were expressed on RNA level and
absent in the germline DNA of the patients, confirming their somatic nature (Figure 1A-B; UZ LeuvenErasmus MC cohort). Putting these results together with those in the published genome sequencing
studies,7,11 there are 7 mutations in RPL10 in 344 patients, or a mutation frequency of 2%. On a linear
view of the RPL10 protein, three variants lie close to one another, with I33V located more towards the
N-terminus of the protein (Figure 1A). Interestingly, in a 3D conformational view of the protein, the
mutations cluster in a region that is distinct from the mutational hotspot described in T-ALL (Figure
1C).2 The mutated residues are conserved (Figure 1D), and SIFT predictions for all these mutations
are deleterious, suggesting a damaging effect on protein function (Table 1). Polyphen scores are more
conservative, with possibly/probably damaging predictions for mutants E66G, I70L and I70M but a
benign prediction for mutant I33V. To further test whether the identified RPL10 mutations could alter
RPL10 function, we engineered yeast cells expressing wild type (wt) Rpl10 or the identified Rpl10
mutants as the sole copy of Rpl10, similar to the experiments we previously did for the T-ALL
associated R98S mutation.2 In yeast proliferation assays, the I33V mutant did not show any difference
from wt Rpl10 expressing yeast, whereas the remaining three Rpl10 mutants showed a decrease in
proliferation, which was most pronounced in the I70L mutant (Figure 1E). To investigate the effect of
the mutants on ribosome biogenesis, polysome profiling was used to measure the relative abundance
of the 60S and 40S subunits, mature 80S ribosomes, and actively translating ribosomes associated
with mRNA (polysomes). Only the I70L mutant showed a pronounced phenotype, with an increase in
60S subunit abundance and absence of 40S subunit signal (Figure 1F). While further research is
needed to clarify the effect of these mutants in the cell and their role in carcinogenesis, it is
conceivable that mutants in RPL10, which reaches into the catalytic center of the ribosome, could
differentially alter the translation of certain transcripts. Further studies with these mutants in human
MM cell lines would be required to validate this hypothesis.
The ribosome is composed of 81 ribosomal proteins. We suspected that defects in other ribosomal
proteins besides RPL10 might also occur in MM. To explore this, we ran a custom-designed Haloplex
targeted capture assay covering all exonic regions of the 81 ribosomal genes followed by nextgeneration sequencing on 37 UZ Leuven MM samples. We identified 6 variants targeting 5 different
RP genes in 5 MM patients (Table 1). All variants for which Sanger sequencing could be performed
were confirmed in diagnostic material, and when available, the somatic nature of the variant was
tested by Sanger sequencing of germline material. One somatic variant (in RPLP0) has previously
never been reported in SNP databases or in disease-associated databases such as COSMIC. Two
other somatic variants (in RPL5 and RPL3L (uL3)) have been described before as very rare SNPs
(Multiple Allele Frequency (MAF) ≤ 0.001). The variant in RPL5 is interesting as deletion of this gene is
recurrent in MM and because the same variant has also been described in the ribosomopathy
Diamond-Blackfan anemia (DBA), a congenital disease caused by mutations in RP genes such as
RPL5.8,12 Additionally, two mutations were found that could not be tested by Sanger sequencing (both
in RPL29 (eL29)) and one mutation turned out not to be somatic (in RPL15 (eL15)). One of the RPL29
variants (R150G) is described both as a rare SNP (MAF < 0.001) and as a mutation identified in lung
cancer (COSM340672), while the other is a novel variant (T155K). The supplements of the extended
sequencing study (Lohr cohort) included another 12 variants in RP genes (Table 1). Interestingly, one
of these variants again targets RPL5, while all others affect distinct RPs from those picked up in our
Haloplex assay. It thus seems that RPL5 and RPL10 are the only RP genes recurrently mutated in
MM.
RPL10 is mutated at a low frequency at what might be a MM-specific hotspot. Although the mutations
did not show a significant ribosome biogenesis defect in yeast, their modest growth defect suggests an
impact on Rpl10 function. Moreover, the somatic nature of the mutations, conservation of affected
residues, and clustering in a mutational hotspot argue against them being passengers. We can only
speculate why the MM hotspot is different from the one in T-ALL. The R98 residue mutated in T-ALL is
close to the catalytic center of the ribosome, while the identified mutations in MM occur in a distinct
region that could differentially impact ligand binding to the ribosome.
Mutation analysis of all other ribosomal proteins did not uncover any other strikingly recurrent defects.
However, RPL5 stays an interesting candidate in MM because it is deleted in 20-40% of MM cases
and it seems the only other recurrently mutated ribosomal protein gene in MM, besides RPL10, when
putting together multiple sequencing studies.8,11 It is worth pointing out that also another group
reported one missense and one splice site mutation in RPL5.13 Overall, our data point to a low
frequency of mutations in ribosomal proteins in MM, fitting with the observation of few recurrent
mutations in the disease in general.11,13,14 Other mechanisms besides deletions and mutations might
influence expression of RPs in MM. For RPL5, we previously showed that some patients show a
8
lowered expression in the absence of mutation or deletion. Interestingly, Table 1 shows that 1 patient
can carry multiple RP defects (MM14 and MM0571).
Although we failed to identify any RPs recurrently mutated at a high frequency, our results do support
that the ribosome in general, and RPL10 and RPL5 in particular, are targets of mutation in MM.
Together with the recurrent deletion of RPL5 in ≥20% of MM and the observation that half of MM
patients carry mutations in genes linked to translation,
7,8
it seems that defects in the ribosome and in
translation in general are a significant class of defects in MM. In light of our recent finding that
deletions in RPL5 are associated with a better response to clinically used proteasome inhibitors such
8
as bortezomib in MM, it will be of interest to determine whether this is also the case for these other
lesions in the translation machinery.
ACKNOWLEGDEMENTS
IH was paid by a fellowship of the Flemish Agency for Innovation through Science and Technology
(Agentschap voor Innovatie door Wetenschap en Technologie; IWT). This research was funded by an
ERC starting grant (n°334946), FWO funding (1505913N and G084013N) and a Stichting Tegen
Kanker grant (grant n° 2012-176) to KDK and an NIH grant (GM53655) to AJ.
REFERENCES
1.
Ban N, Beckmann R, Cate JHD, et al. A new system for naming ribosomal proteins. Curr Opin
Struct Biol. 2014;24:165–169.
2.
De Keersmaecker K, Atak ZK, Li N, et al. Exome sequencing identifies mutation in CNOT3 and
ribosomal genes RPL5 and RPL10 in T-cell acute lymphoblastic leukemia. Nat Genet.
2013;45(2):186–190.
3.
Rao S, Lee S-Y, Gutierrez A, et al. Inactivation of ribosomal protein L22 promotes
transformation by induction of the stemness factor, Lin28B. Blood. 2012;120(18):3764–3773.
4.
Fancello L, Kampen KR, Hofman IJ, Verbeeck J, De Keersmaecker K. The ribosomal protein
gene RPL5 is a haploinsufficient tumor suppressor in multiple cancer types. Oncotarget.
2017;8(9):14462-14478.
5.
Ljungstrom V, Cortese D, Young E, et al. Whole-exome sequencing in relapsing chronic
lymphocytic leukemia: clinical impact of recurrent RPS15 mutations. Blood. 2015;127(8):1007.
6.
Landau DA, Tausch E, Taylor-Weiner AN, et al. Mutations driving CLL and their evolution in
progression and relapse. Nature. 2015;526(7574):525.
7.
Chapman MA, Lawrence MS, Keats JJ, et al. Initial genome sequencing and analysis of
multiple myeloma. Nature. 2011;471(7339):467–472.
8.
Hofman IJF, van Duin M, De Bruyne E, et al. RPL5 on 1p22.1 is recurrently deleted in multiple
myeloma and its expression is linked to bortezomib response. Leukemia. 2017 Jan 3. [Epub
ahead of print]
9.
Morgan GJ, Walker BA, Davies FE. The genetic architecture of multiple myeloma. Nat Rev
Cancer. 2012;12(5):335–348.
10.
Kuehl WM, Bergsagel PL. Molecular pathogenesis of multiple myeloma and its premalignant
precursor. J Clin Invest. 2012;122(10):3456–3463.
11.
Lohr JG, Stojanov P, Carter SL, et al. Widespread Genetic Heterogeneity in Multiple Myeloma:
Implications for Targeted Therapy. Cancer Cell. 2014;25(1):91–101.
12.
Quarello P, Garelli E, Carando A, et al. Diamond-Blackfan anemia: genotype-phenotype
correlations in Italian patients with RPL5 and RPL11 mutations. Haematologica.
2010;95(2):206–213.
13.
Bolli N, Avet-Loiseau H, Wedge DC, et al. Heterogeneity of genomic evolution and mutational
profiles in multiple myeloma. Nat Commun. 2014;5:2997.
14.
Walker BA, Boyle EM, Wardell CP, et al. Mutational Spectrum, Copy Number Changes, and
Outcome: Results of a Sequencing Study of Patients With Newly Diagnosed Myeloma. J Clin
Oncol. 2015;33(33):3911–3920.
Table 1 RP mutations across different cohorts
Gene
Sample
Genomic mutation
AA
change
Sanger
Somatic
VAF
Known variant
SIFT score
Polyphen score
Patient
status
RPL10 mutants
UZ Leuven cohort
RPL10
MM10
g.chrX:153628163T>G
p.I70M
Confirmed
Yes
20%
No
deleterious (0)
probably damaging (0.97)
Diagnostic
RPL10
T44
g.chrX:153627842A>G
p.I33V
Confirmed
Yes
NA
No
deleterious (0.05)
benign (0.02)
Diagnostic
Chapman cohort
RPL10
MM-0282
g.chrX:153628150A>G
p.E66G
NA
Yes
76%
No
deleterious (0)
possibly damaging (0.8)
Treated
RPL10
MM-0347
g.chrX:153628161A>C
p.I70L
NA
Yes
93%
COSM3034228
deleterious (0)
possibly damaging (0.5)
Diagnostic
RPL10
MM-0191
g.chrX:153628161A>C
p.I70L
NA
Yes
3%
COSM3034228
deleterious (0)
possibly damaging (0.5)
Treated
RPL10
MM-0516
g.chrX:153628163T>G
p.I70M
NA
Yes
7%
No
deleterious (0)
probably damaging (0.97)
Treated
RPL10
MM-0524
g.chrX:153627842A>G
p.I33V
NA
Yes
9%
No
deleterious (0.05)
benign (0.02)
Treated
Lohr cohort
Other RP mutants
UZ Leuven cohort
RPLP0
MM07
g.chr12:120637269G>A
p.P25L
Confirmed
Yes
53%
No
tolerated (0.8)
possibly damaging (0.9)
Diagnostic
RPL5
MM09
g.chr1:93297677G>C
Splice Site
Confirmed
Yes
48%
rs200628272, CS100830
NA
NA
Diagnostic
RPL3L
MM14
g.chr16:1995913C>T
p.G324R
Confirmed
Yes
90%
rs375754739
deleterious (0)
probably damaging (0.9)
Diagnostic
RPL29
MM14
g.chr3:52027797G>C
p.R150G
NA
NA
20%
rs754268159, COSM340672
tolerated (1)
benign (0)
Diagnostic
RPL29
MM15
g.chr3:52027781G>T
p.T155K
NA
NA
23%
No
tolerated (0.06)
possibly damaging (0.8)
Diagnostic
RPL5
MM-0465
g.chr1:93298955A>G
p.K5E
NA
Yes
41%
COSM2153192,COSM3493393
deleterious (0)
benign (0.3)
Diagnostic
RPL26L1
MM-0512
g.chr5:172395544G>A
p.R84Q
NA
Yes
15%
rs375645667
tolerated (0.05)
benign (0.02)
NA
RPL27
MM-0624
g.chr17:41151975G>A
p.R36H
NA
Yes
39%
rs776186138,COSM979717
tolerated (0.3)
benign (0.003)
Treated
RPL36
MM-0571
g.chr19:5691445T>G
p.L70R
NA
Yes
29%
No
tolerated (0.2)
probably damaging (0.98)
Diagnostic
RPL36AL
MM-0329
g.chr14:50085527C>T
p.R99K
NA
Yes
5%
No
tolerated (0.3)
benign (0.006)
Treated
RPL3L
MM-0423
g.chr16:2002974A>C
p.V89G
NA
Yes
5%
No
damaging (0)
probably damaging (0.98)
Treated
RPL4
MM-0533
g.chr15:66791990G>T
p.H347N
NA
Yes
72%
No
tolerated (0.5)
benign (0.006)
Diagnostic
RPL6
MM-0528
g.chr12:112844637_112844638insT
p.K131fs
NA
Yes
25%
No
NA
NA
Diagnostic
RPS11
MM-0499
g.chr19:50000463delT
p.Y10fs
NA
Yes
36%
No
NA
NA
Treated
RPS12
MM-0508
g.chr6:133137642G>T
p.E58D
NA
Yes
8%
rs767922042
deleterious (0.02)
benign (0.01)
Treated
RPS16
MM-0485
g.chr19:39924389C>T
p.V55I
NA
Yes
11%
No
tolerated (0.7)
benign (0)
Treated
RPS24
MM-0571
g.chr10:79795145G>A
p.M13I
NA
Yes
40%
No
tolerated (0.2)
benign (0.2)
Diagnostic
RPSA
MM-0637
g.chr3:39452456G>A
p.R155H
NA
Yes
32%
No
tolerated (0.3)
benign (0.007)
Diagnostic
Lohr cohort
Abbreviations: AA change: amino acid change; VAF: variant allele frequency.
FIGURE LEGEND
Figure 1: RPL10 mutations in MM
(A) Linear view of the RPL10 protein with mutations from different cohorts indicated. (B)
Chromatograms of mutations found by Sanger sequencing. Germline DNA control, tumor DNA,
and tumor cDNA are shown. (C) 3D model of the 60S subunit of the ribosome with RPL10 in blue.
In the blow-up, the mutant residues I33, E66 and I70 colored red, green and orange respectively.
(D) Alignment of the RPL10 protein sequence in different species with mutant residues indicated
by red stars. (E-F) Functional studies in yeast testing the effect of the different mutants in Rpl10 on
proliferation (e) and polysome profiles (f).
A
UZ Leuven - Erasmus MC cohort (n = 141)
Lohr cohort (n = 165)
Chapman cohort (n = 38)
I33V
RPL10 1
B
T44
c.97A>G
p.I33V
E66G I70M (2)
I70L (2)
Germline DNA
Tumor DNA
TT CGCATTT T T
TT CGCA TTT T T
G
TT CGCATTT T T
G
CGAATTTGTGC
G
CGAATTTGTGC
G
p.I33
MM10
c.T>G
p.I70M
214
cDNA
p.I33
p.I33V
CGA ATTT GTGC
p.I70
p.I33
p.I33V
p.I70
p.I70M
C
p.I70
p.I70M
D
I33
E66
20
I70
40
30
human S R F C R GV P DAK I
mouse S R F C R GV P DAK I
zebrafish S R F C R GV P DP K I
S R F C R GV P DP K I
fruitfly
S R Y NR AV P DS K I
yeast
50
RI
RI
RI
RI
RI
60
F DL GR K K AK V DE F P L C
F DL GR K K AK V DE F P L C
F DL GR K K AK V DE F P L C
F DL GR K K AT V E DF P L C
Y DL GK K K AT V DE F P L C
70
human GHMV S DE Y E QL S S E AL E AAR I
mouse GHMV S DE Y E QL S S E AL E AAR I
zebrafish AHMV S DE Y E QL S S E AL E AAR I
V HL V S DE Y E QL S S E AL E AGR I
fruitfly
V HL V S NE L E QL S S E AL E AAR I
yeast
F
E
Rpl10 wt
vector
Rpl10 I33V
C ANK Y MV K S
C ANK Y MV K S
C ANK Y MV K T
C C NK Y L V K Y
C ANK Y MT T V
80S
60S
40S
polysomes
Rpl10 wt
Rpl10 I33V
80S
Rpl10 E66G
Rpl10 I70M
60S
Rpl10 I70L
polysomes
40S
Rpl10 I70M
Rpl10 I70L
Rpl10 E66G
SUPPLEMENTARY MATERIAL
LETTER TO THE EDITOR
Low frequency mutations in ribosomal proteins RPL10 and RPL5 in multiple
myeloma
Isabel JF Hofman1, Stephanie Patchett2, Mark van Duin3, Ellen Geerdens4,5, Jelle Verbeeck1, Lucienne
Michaux6, Michel Delforge7, Pieter Sonneveld3 , Arlen W. Johnson2, Kim De Keersmaecker1
1
Department of Oncology, KU Leuven - University of Leuven, LKI - Leuven Cancer Institute, Leuven,
Belgium
2
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA
3
Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, The Netherlands
4
Center for Cancer Biology, VIB, Leuven, Belgium
5 Center
for Human Genetics, KU Leuven - University of Leuven, LKI - Leuven Cancer Institute,
Leuven, Belgium
6
Center for Human Genetics, KU Leuven - University Hospitals Leuven, Leuven, Belgium
7 LKI
- Leuven Cancer Institute, Department of Development and Regeneration, KU Leuven - University
Hospitals Leuven, Leuven, Belgium
SUPPLEMENTAL MATERIALS AND METHODS
Patient samples
We studied 75 diagnostic MM bone marrow samples from UZ Leuven (Leuven, Belgium) and 66
diagnostic MM bone marrow samples from the Erasmus Medical Center (Erasmus MC) (Rotterdam, The
Netherlands). All analyzed samples contained at least 70% plasma cells (UZ Leuven range: 70-100%,
median 79%; EMC Rotterdam range: 78-100%, median 92%; Supplementary table S1). For the UZ
Leuven cohort, no sample purification was done and purity was determined by morphological
assessment of a bone marrow aspirate. Diagnostic samples from the EMC Rotterdam cohort were
purified using CD138 magnetic microbeads (Miltenyi Biotec). Purity was analyzed by performing flow
cytometry for the CD138 marker (CD138-PE; Beckman Coulter). Percentages of CD138 cells in
analyzed samples are reported in supplementary Table 1. This study was approved by the ethics
committees of the institutes involved and informed consent was obtained from the participants. Samples
and clinical data were stored in accordance with the declaration of Helsinki. The public cohorts we
included in our analysis (“Chapman cohort”1 and “Lohr cohort”2) were a mix of diagnostic and treated
patients.
1
Haloplex resequencing
We analyzed 37 cases using a custom-designed Haloplex (Agilent technologies) sequence enrichment
assay that captures the coding sequences of the 81 ribosomal genes as well as a set of leukemia
implicated genes followed by Illumina massive parallel sequencing of the library (Supplementary
table S2). Sequencing data are available at EGA (accession number EGAS00001002405). Data
analysis was performed using NextGENe software (v2.2.1, Softgenetics, State College, PA, USA),
performing the following steps: (i) the fastQ output file was converted into a FASTA file to eliminate
reads that were not “paired” and that did not meet the criteria of the default settings; (ii) reads from the
converted unique FASTA file were aligned to the reference genome (Human_v37.2). After alignment a
*.pjt file was created and opened in the NextGENe Viewer; (iii) a mutation report was created using the
coordinates from the targeted enrichment kit as a *.bed file to enable calling of single nucleotide variants
and small insertion/deletions (indels) in the regions of interest. For variant calling, we required a
minimum read depth of 20 and an allele frequency of at least 3%. Detected variants were confirmed by
Sanger sequencing on diagnostic material and were tested for their somatic origin on germline DNA if
available.
Sanger sequencing
The entire coding sequence of the RPL10 gene was PCR amplified and Sanger sequenced in diagnostic
material from all 75 UZ Leuven cases and on whole genome amplified material from the 66 cases from
Erasmus MC. Analysis of Sanger chromatograms was performed using CLC Main Workbench (CLC
Bio, Aarhus, Denmark). Detected variants were confirmed on original, non-amplified material and were
tested for their somatic origin on germline DNA if available. Primers used for Sanger sequencing are
listed in Supplementary table S3.
Yeast experiments
Appropriate codons of yeast (Saccharomyces cerevisiae) Rpl10 in the centromeric LEU2 vector
pAJ2522 were changed by site-specific mutagenesis. Wild-type and mutant were then introduced into
the rpl10 deletion strain AJY1437 (MATα rpl10::KanMX lysΔ0 met15Δ0 his3Δ0 leu2Δ0 ura3Δ0 pAJ392
- RPL10 URA3 CEN) by plasmid shuffle and assayed for growth by plating ten-fold serial dilutions onto
selective medium. Polysome profiles were analyzed as described. (Klinge, Science, 2011) Wild-type
and mutant RPL10 were introduced into AJY1837, containing a glucose repressible RPL10 gene (GALRPL10), the leptomycin-B-sensitive allele of CRM1-T539C and NMD3-GFP, and AJY2766, containing
GAL-RPL10 and TIF6-GFP. Cultures were grown in selective medium containing galactose. Glucose
was added to repress expression of wild-type genomic RPL10 for 2 hours. Images were captured using
a Nikon E800 microscope fitted with a 100X Plan Apo objective and a Photometrics CoolSNAP ES
camera controlled by NIS-Elements AR 2.10 software. Images were prepared using Adobe Photoshop
7.0.
2
Table S1 Patient information
UZ Leuven cohort (no CD138 purification)
MM
Date
Plasma
cells in BM
(%)
Translocation
Hyperdiploidy
MM01
29/02/2012
70
t(11;14)
No
MM02
26/08/2011
70
No
No
MM03
4/01/2012
90
t(11;14)
No
MM04
22/04/2011
77
t(11;14)
No
MM05
8/11/2011
80
No
MM06
21/09/2011
75
MM07
30/03/2011
75
t(11;14)
Anomaly at
14q32 but no
confirmed
translocation
No
Yes
MM08
11/03/2011
80
No
Yes
MM09
3/02/2011
74
t(11;14)
No
MM10
22/02/2011
83
t(11;14)
No
MM11
25/05/2011
84
No
Yes
MM12
27/05/2011
81
No
Yes
MM13
1/06/2011
70
t(11;14)
No
MM14
15/06/2011
77
t(11;14)
Yes
MM15
19/10/2011
76
No
No
MM16
31/10/2011
71
t(14;20)
No
MM17
21/02/2012
86
No
Yes
MM18
15/03/2012
91
t(6;14)
No
MM19
9/02/2012
70
No
Yes
MM20
9/02/2012
79
t(11;14)
No
MM21
7/12/2011
72
No
MM22
10/11/2011
72
MM23
12/10/2010
95
No
t(14q32) IGH
translocation
with unknown
partner
t(14;16)
MM24
15/01/2010
80
No
Yes
MM26
8/01/2010
79
t(14;16)
No
MM27
18/11/2009
99
No
Yes
MM28
8/12/2011
100
No
Yes
MM29
22/04/2010
85
t(4;14)
No
MM30
22/12/2009
78
No
Yes
MM31
24/06/2010
82
t(11;14)
No
MM32
4/01/2010
87
t(11;14)
No
MM33
23/11/2010
81
No
Yes
MM34
10/11/2009
80
No
Yes
MM35
23/12/2011
78
No
Yes
MM36
16/11/2010
78
No
Yes
MM37
11/06/2010
70
No
Yes
MM38
19/08/2010
75
t(11;14)
No
No
No
No
3
UZ Leuven cohort (no CD138 purification)
Minimal plasma cell % in BM
70
Maximal plasma cell % in BM
100
Median plasma cell % in BM
79
EMC Rotterdam (CD138 purified)
Minimal plasma cell % in purified sample
78
Maximal plasma cell % in purified sample
100
Median plasma cell % in purified sample
92
4
TableS2 Haloplexdesign
Targetregions
Totalsize
Coverage
404921bp
99,10%
Gene
Source
ABL1
AKAP6
AKT1
ARPP21
BCL11B
BMS1
BRAF
CDKN2A
CDKN2B
CNOT3
CT47B1
CTCF
DCLRE1C
DNM2
DNMT3A
DRG1
DUSP12
ECT2L
EED
EFTUD1
EIF2A
EIF6
EP300
EPDR1
ETV6
EZH2
FAT1
FAT2
FAT3
FAT4
FAU
FBXW7
FLT3
GATA3
GRID2
GTPBP4
HIST1H1B
HMCN1
HNRNPA1
HNRNPR
IDH1
CCDS35165.1,CCDS35166.1
CCDS9644.1
CCDS9994.1
NM_001025068,NM_001025069,NM_016300,NM_198399
CCDS9949.1,CCDS9950.1
CCDS7199.1
CCDS5863.1
CCDS6510.1,CCDS6511.1,CCDS34998.1
CCDS6512.1,CCDS6513.1
CCDS12880.1
CCDS48161.1
CCDS10841.1
CCDS31149.1,CCDS7105.1,CCDS31150.1
CCDS32908.1,CCDS45968.1,CCDS32907.1,CCDS45969.1
CCDS1718.2,CCDS33157.1,CCDS46232.1
CCDS13897.1
CCDS1234.1
CCDS43508.1
CCDS8274.1,CCDS8273.1
CCDS42070.1,CCDS42071.1
CCDS46935.1
CCDS13250.1,CCDS13249.1
CCDS14010.1
CCDS5454.1
CCDS8643.1
CCDS5892.1,CCDS5891.1
CCDS47177.1
CCDS4317.1
CCDS44706.1
CCDS3732.3
CCDS8095.1
CCDS3778.1,CCDS34078.1,CCDS3777.1
CCDS31953.1
CCDS7083.1,CCDS31143.1
CCDS3637.1
CCDS31132.1
CCDS4635.1
CCDS30956.1
CCDS44909.1,CCDS41793.1
CCDS232.1,CCDS44085.1
CCDS2381.1
1976
5
IDH2
IGF1R
IKZF1
IL7R
JAK1
JAK2
JAK3
JAKMIP2
KDM6A
KRAS
LCK
LEF1
LPHN2
LSG1
MAGEC3
MLH3
MRTO4
MTMR8
MYB
NIP7
NMD3
NOTCH1
NPM1
NRAS
ODZ2
PAX5
PHF6
PIK3CA
PKHD1L1
PTCH1
PTEN
PTPN11
PTPN2
PTPRC
RB1
RELN
RPL10
RPL10A
RPL10L
RPL11
RPL12
RPL13
RPL13A
RPL14
RPL15
RPL17
RPL18
RPL18A
CCDS10359.1
CCDS10378.1
NM_006060
CCDS3911.1
CCDS41346.1
CCDS6457.1
CCDS12366.1
CCDS4285.1
CCDS14265.1
CCDS8702.1,CCDS8703.1
CCDS359.1
CCDS47122.1,CCDS3679.1,CCDS47123.1
CCDS689.1
CCDS33922.1
CCDS14676.1,CCDS14677.1
CCDS32123.1,CCDS9837.1
CCDS191.1
CCDS14379.1
CCDS5174.1,CCDS47482.1,CCDS47481.1
CCDS10877.1
CCDS3194.1
CCDS43905.1
CCDS4376.1,CCDS43399.1,CCDS4377.1
CCDS877.1
NM_001122679
CCDS6607.1
CCDS14639.1,CCDS14640.1
CCDS43171.1
CCDS47911.1
CCDS47995.1,CCDS43851.1,CCDS47996.1,CCDS6714.1
NM_000314
CCDS9163.1
CCDS11864.1,CCDS11863.1,CCDS11865.1
CCDS1397.1,CCDS1399.1,CCDS1398.1,CCDS44291.1
CCDS31973.1
CCDS47680.1,CCDS34722.1
CCDS14746.1
CCDS4806.1
CCDS32071.1
CCDS238.1
CCDS6872.1
CCDS10979.1
CCDS12768.1
CCDS33739.1,CCDS43070.1
CCDS2640.1
CCDS45865.1
CCDS12726.1
CCDS12367.1
6
RPL19
RPL21
RPL22
RPL23
RPL23A
RPL24
RPL26
RPL26L1
RPL27
RPL27A
RPL28
RPL29
RPL3
RPL30
RPL31
RPL32
RPL34
RPL35
RPL35A
RPL36
RPL36A
RPL36AL
RPL37
RPL37A
RPL38
RPL39
RPL39L
RPL3L
RPL4
RPL41
RPL5
RPL6
RPL7
RPL7A
RPL7L1
RPL8
RPL9
RPLP0
RPLP1
RPLP2
RPS10
RPS11
RPS12
RPS13
RPS14
RPS15
RPS15A
RPS16
CCDS42312.1
CCDS9320.1
CCDS58.1
CCDS11330.1
CCDS11241.1
CCDS33809.1
CCDS11142.1
CCDS4382.1
CCDS11449.1
CCDS7790.1
CCDS46189.1,CCDS46190.1,CCDS46192.1,CCDS46191.1,CCDS12924.1
CCDS2845.1
CCDS13988.1
CCDS34928.1
CCDS46374.1,CCDS2049.1,CCDS46373.1
CCDS2614.1
CCDS3680.1
CCDS6858.1
CCDS33930.1
CCDS12147.1
CCDS14483.1
CCDS9689.1
CCDS3934.1
CCDS2404.1
CCDS11696.1
CCDS14586.1
CCDS3286.1
CCDS10450.1
CCDS10218.1
CCDS44919.1
CCDS741.1
CCDS9162.1
CCDS6212.1
CCDS6965.1
CCDS4873.1
CCDS6433.1
CCDS3452.1
CCDS9193.1
CCDS10234.1,CCDS10233.1
CCDS7717.1
CCDS4792.1
CCDS12769.1
CCDS5164.1
CCDS7823.1
CCDS4307.1
CCDS12067.1
CCDS10571.1
CCDS12535.1
7
RPS17
RPS18
RPS19
RPS2
RPS20
RPS21
RPS23
RPS24
RPS25
RPS26
RPS27
RPS27A
RPS27L
RPS28
RPS29
RPS3
RPS3A
RPS4X
RPS4Y1
RPS4Y2
RPS5
RPS6
RPS7
RPS8
RPS9
RPSA
RSL24D1
RUNX1
SBDS
SETD2
SH2B3
SUZ12
TBP
TDRD6
TET1
TET2
TET3
TLR1
TP53
TSR1
UBA52
UNC5D
USP9X
WT1
CCDS10320.1
CCDS4771.1
CCDS12588.1
CCDS10452.1
CCDS6163.1
CCDS13497.1
CCDS47241.1
CCDS7355.1,CCDS44443.1,CCDS7356.1
CCDS8406.1
CCDS31832.1
CCDS1059.1
CCDS33202.1
CCDS42048.1
CCDS45953.1
CCDS32072.1,CCDS9685.1
CCDS8236.1
CCDS3775.1
CCDS14418.1
CCDS14773.1
CCDS44028.1
CCDS12978.1
CCDS6492.1
CCDS1648.1
CCDS513.1
CCDS12884.1
CCDS2686.1
CCDS10152.1
CCDS13639.1,CCDS42922.1,CCDS46646.1
CCDS5537.1
CCDS2749.2
CCDS9153.1
CCDS11270.1
CCDS5315.1
CCDS34470.1
CCDS7281.1
CCDS47120.1,CCDS3666.1
CCDS46339.1
CCDS33973.1
CCDS11118.1,CCDS45605.1,CCDS45606.1
CCDS32525.1
CCDS12382.1
CCDS6093.2
CCDS43930.1
CCDS7877.2,CCDS7878.2,CCDS44562.1,CCDS44561.1
8
Table S3 Sanger sequencing primers
Gene
Exon
Forward
Reverse
RPL10
Exon 1
TTGCTGGTTCTCACACCTTTT
ATTGTTTTCAGCGGCCATAG
Exon 2-3
AAAGTGCCTGTTGGGCTTT
AGTGTATGTGGGTGGGGTTG
Exon 4
ACTCAGCCAACACAGTTCCC
CAGACCAAGCTCACCTGTCA
Exon 5-6
AGTGTACGCAGCCTGTTGGT
GGGGCTGCAGCACTACATAC
RPLP0
Exon 7
CAGGAGGGTGGTGGGTAATA
AAGTTGGTTGCTTTTTGGTGA
RPL5
Exon 1
GACTTGGTCGAGGTGCAGT
CCGCACTCAGGCTGTCTAC
RPL3L
Exon 8
GGATCCCCACACTTGATGTT
AGCTCCAGCTCCCTACTCG
Supplementary references
1. Chapman MA, Lawrence MS, Keats JJ, Cibulskis K, Sougnez C, Schinzel AC, et al. Initial
genome sequencing and analysis of multiple myeloma. Nature. 2011;471(7339):467–72.
2. Lohr JG, Stojanov P, Carter SL, Cruz-Gordillo P, Lawrence MS, Auclair D, et al. Widespread
Genetic Heterogeneity in Multiple Myeloma: Implications for Targeted Therapy. Cancer Cell.
2014;25(1):91–101.
9