Exome sequencing identifies recurrent mutations of the splicing ...

© 2011 Nature America, Inc. All rights reserved.
Here we perform whole-exome sequencing of samples from
05 individuals with chronic lymphocytic leukemia (CLL) ,2 ,
the most frequent leukemia in adults in Western countries.
We found ,246 somatic mutations potentially affecting gene
function and identified 78 genes with predicted functional
alterations in more than one tumor sample. Among these
genes, SF3B1, encoding a subunit of the spliceosomal U2
small nuclear ribonucleoprotein (snRNP), is somatically
mutated in 9.7% of affected individuals. Further analysis
in 279 individuals with CLL showed that SF3B1 mutations
were associated with faster disease progression and poor
overall survival. This work provides the first comprehensive
catalog of somatic mutations in CLL with relevant clinical
correlates and defines a large set of new genes that may drive
the development of this common form of leukemia. The results
reinforce the idea that targeting several well-known genetic
pathways, including mRNA splicing, could be useful in the
treatment of CLL and other malignancies.
Chronic lymphocytic leukemia is a common neoplasia of B lymphocytes
in which these cells progressively accumulate in the bone marrow,
blood and lymphoid tissues 1,2 . The clinical evolution of the disease is
heterogeneous, with the malignancy following an indolent course in
some affected individuals and being characterized by aggressive disease
and short survival times in others. These clinical differences have mainly
been associated with two major molecular subtypes of the disease,
which are defined by the extent to which somatic hypermutations
(SHMs) occur in the variable regions of the immunoglobulin genes 2–4 .
Nature GeNetics ADVANCE ONLINE PUBLICATION
l e t t e r s
Exome sequencing identifies recurrent mutations of the
splicing factor SF3B1 gene in chronic lymphocytic leukemia
Víctor Quesada 1 , Laura Conde 2 , Neus Villamor 2 , Gonzalo R Ordóñez 1 , Pedro Jares 2 , Laia Bassaganyas 3 ,
Andrew J Ramsay 1 , Sílvia Beà 2 , Magda Pinyol 4 , Alejandra Martínez-Trillos 5 , Mónica López-Guerra 2 , Dolors Colomer 2 ,
Alba Navarro 2 , Tycho Baumann 5 , Marta Aymerich 2 , María Rozman 2 , Julio Delgado 5 , Eva Giné 5 ,
Jesús M Hernández 6 , Marcos González-Díaz 6 , Diana A Puente 1 , Gloria Velasco 1 , José M P Freije 1 ,
José M C Tubío 3 , Romina Royo 7 , Josep L Gelpí 7 , Modesto Orozco 7 , David G Pisano 8 , Jorge Zamora 8 ,
Miguel Vázquez 8 , Alfonso Valencia 8 , Heinz Himmelbauer 9 , Mónica Bayés 10 , Simon Heath 10 , Marta Gut 10 ,
Ivo Gut 10 , Xavier Estivill 3 , Armando López-Guillermo 5 , Xose S Puente 1 , Elías Campo 2,11 & Carlos López-Otín 1,11
We have previously used whole­genome sequencing to identify 46
somatic mutations potentially affecting gene function in four individuals
with CLL 5 . Subsequent analysis in additional subjects with CLL
showed that four of the affected genes were recurrently mutated,
thereby showing the usefulness of this approach for the identification
of genes that may drive tumor progression.
To improve our understanding of the genes involved in the pathogenesis
and clinical evolution of CLL, we have performed whole­exome
sequencing of matched tumor and normal samples from 105 individuals
with CLL (cases), comprising 60 subjects with mutated IGHV
regions and 45 individuals in whom these regions are not mutated
(Supplementary Tables 1–3 and Supplementary Note). We identified
a median of 45 somatic mutations per case, which represents about
0.9 mutations per megabase of sequenced DNA, in agreement with
previous reports 6–10 (Supplementary Table 4). To gain insight into the
genes affected by the transformation process in CLL, we focused on the
mutations predicted to result in protein­coding changes, identifying
a total of 1,246 mutations after excluding those occurring in immunoglobulin
loci (Fig. 1a and Supplementary Table 5). The number
of protein­altering mutations per case was higher in IGHV­mutated
CLL than in CLL without mutations in the IGHV region (12.8 ± 0.7
versus 10.6 ± 0.7; P = 0.038) (Fig. 1b). We also confirmed the previous
observation that A>C/T>G transversions are more frequent than other
tranversion types in IGHV­mutated CLL 5 (Fig. 1c).
The distribution of somatic mutations identified by whole­exome
sequencing indicated that a total of 1,100 different genes were
affected in the 105 cases (Supplementary Table 5). We found 60
genes with a somatic mutation rate higher than expected (P < 0.05),
1 Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain. 2 Unidad de Hematopatología,
Servicio de Anatomía Patológica, Hospital Clínic, Universitat de Barcelona, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona,
Spain. 3 Genes and Disease Programme, Center for Genomic Regulation, Pompeu Fabra University (CRG-UPF), Barcelona, Spain. 4 Unidad de Genómica, IDIBAPS,
Barcelona, Spain. 5 Servicio de Hematología, Hospital Clínic, Universidad de Barcelona, Barcelona, Spain. 6 Servicio de Hematología, Hospital Universitario, Centro de
Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain. 7 Programa Conjunto de Biología Computacional, Barcelona Supercomputing Center (BSC),
Institut de Reçerca Biomèdica (IRB), Spanish National Bioinformatics Institute, Universitat de Barcelona, Barcelona, Spain. 8 Structural Biology and Biocomputing
Programme, Spanish National Cancer Research Centre (CNIO), Spanish National Bioinformatics Institute, Madrid, Spain. 9 Ultrasequencing Unit, CRG-UPF,
Barcelona, Spain. 10 Centro Nacional de Análisis Genómico, Parc Científic de Barcelona, Barcelona, Spain. 11 These authors jointly directed this work.
Correspondence should be addressed to C.L.-O. (clo@uniovi.es) or E.C. (ecampo@clinic.ub.es).
Received 12 July; accepted 10 November; published online 11 December 2011; doi:10.1038/ng.1032

© 2011 Nature America, Inc. All rights reserved.
l e t t e r s
a b
761
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117
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091
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064
051
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033
032
019
018
009
007
Chr. 1
Chr. 2
Chr. 3
Chr. 4
Chr. 5
which we defined as class 1 recurrently mutated (RM)­CLL genes.
Furthermore, we identified 18 class 2 RM­CLL genes, which we
defined as those recurrently mutated and present in at least one case
with no mutated class 1 RM­CLL genes, with TP53 mutation representing
a special case (Supplementary Table 6). Of note, about 90%
of the cases in the study had somatic mutations in at least one of the
78 RM­CLL genes, suggesting that we have identified many previously
unrecognized candidate driver genes in CLL. Moreover, an analysis of
the somatic structural variants in ten CLL cases showed that some of
the RM­CLL genes may be affected by large genomic rearrangements
(Supplementary Table 7).
A functional clustering analysis of the mutated genes showed a
substantial enrichment of genes in pathways involved in mRNA splicing
and transport, Toll­like receptor (TLR) signaling and apoptosis,
among others (Supplementary Table 8). The functional and clinical
impact of some of these pathways, including MYD88­associated TLR,
NF­κB and JAK­STAT3 signaling, has already been demonstrated in
our previous whole­genome sequencing study of CLL 5 . The pathway
analysis of the mutated genes also identified notable differences
between the two major CLL subtypes. Mutations in genes of the TLR
Chr. 6
Chr. 7
Chr. 8
Chr. 9
Chr. 10
Chr. 11
Chr. 12
Chr. 13
Chr. 14
Chr. 15
Chr. 16
Chr. 17
Chr. 18
Chr. 19
Chr.20
Chr. 21
Chr. 22
Chr. X
Chr. Y
and pattern recognition pathways were enriched in IGHV­mutated
CLL (Supplementary Fig. 1 and Supplementary Tables 9 and 10),
supporting the idea that different molecular mechanisms might be
implicated in the development of the two major subtypes of CLL.
Other genes recurrently mutated and distinct from those previously
identified in our whole­genome sequencing–based study
of individuals with CLL 5 were those encoding SF3B1, a subunit of
the spliceosomal U2 snRNP 11 ; POT1, a nuclear protein involved in
telomere maintenance 12 ; CHD2, which regulates gene expression
by modification of chromatin structure 13 ; and LRP1B, which has
recently been defined as a tumor suppressor in different malignancies
14 (Table 1). All POT1 somatic mutations appeared in tumors
without IGHV region mutations, whereas CHD2 somatic mutations
exclusively appeared in IGHV­mutated tumors. This finding is consistent
with different mechanisms being involved in disease development
in CLL cases with and without IGHV mutations. Because the
high GC nucleotide content of NOTCH1 hampers sequencing using
exome­capture approaches, we analyzed the coding exons of this
gene by Sanger sequencing of DNA from 260 individuals with CLL 5 .
We found somatic mutations in 25 cases in this series (9.6%; Table 1).
2 ADVANCE ONLINE PUBLICATION Nature GeNetics
RM-CLL
Nonsynonymous mutations
c
Substitution frequency
40
30
20
10
30
25
20
15
10
5
IGHV
mutated
A>C
T>G
A>G
T>C
*
IGHV
unmutated
IGHV mutated
IGHV unmutated
Figure 1 Somatic mutation profiles of 105 CLL exomes. (a) Chromosomal distribution and location of protein-coding mutations (dots) and insertions
and deletions (indels; Xs) identified by exome sequencing of 60 CLL samples with IGHV region mutations (blue) and 45 without IGHV region mutations
(red). RM-CLL genes are highlighted with vertical bars and summarized for each individual with orange dots. (b) Box plot representation comparing
the frequency of coding, nonsynonymous somatic mutations in CLL samples with and without IGHV region mutations. Error bars, range of the data set
(*P = 0.038). (c) Frequency of substitutions in the 105 CLL samples for the six possible mutation classes. Error bars, s.d. (***P = 0.0017).
***
A>T
T>A
C>A
G>T
C>G
G>C
C>T
G>A

© 2011 Nature America, Inc. All rights reserved.
table 1 Most common recurrently mutated genes in Cll
Gene Case ID Exon Encoded alteration Total frequency
Notably, among the class 1 RM­CLL genes, we detected single mutations
in BRAF (encoding p.Asp594Gly or p.Lys601Glu alterations) in
two cases. These mutations differ from that described to encode the
p.Val600Glu variant found in hairy­cell leukemia 15 , but have been
Nature GeNetics ADVANCE ONLINE PUBLICATION
Freq. in IGHV-
mutated cases
l e t t e r s
Freq. in IGHV-
unmutated cases
NOTCH1 415 34 p.Phe2482Phefs*2 12.1% (31/255) 2.8% (2/72) 10.1% (7/69)
457 34 p.Gln2503*
12, 15, 19, 24, 48, 69, 85, 89, 92, 96, 102,
112, 138, 163, 184, 209, 410, 431, 436, 447,
448, 467, 507, 511, 520, 527, 531, 537, 722
34 p.Pro2515Argfs*4
SF3B1 247 14 p.Tyr623Cys 9.7% (27/279) 7.9% (6/76) 20.5% (15/73)
119 14 p.Arg625His
6, 182 14 p.Asn626Tyr
154 14 p.His662Asp
53, 651 14 p.Thr663Ile
102, 116 14 p.Lys666Glu
9, 29, 156, 209, 365, 632, 734, 755, 758 15 p.Lys700Glu
19 15 p.Val701Phe
85 16 p.Lys741Asn
68, 79, 99, 197, 604, 742 16 p.Gly742Asp
83 18 p.Asp894Gly
POT1 44 4_5 Splice site 4.8% (5/105) 0% (0/60) 11.1% (5/45)
184 5 p.Met1Leu
13 6 p.Tyr36Asn
157 7 p.Tyr66*
6 9 p.Tyr223Cys
CHD2 185 16 p.His620Leu 4.8% (5/105) 8.3% (5/60) 0% (0/45)
175 17 p.Leu698Leufs*6
181 27 p.Phe1146Leu
43 30 p.Leu1270Phe
7 35_36 Splice site
LRP1B 5 41 p.Cys2205Phe 4.8% (5/105) 5.0% (3/60) 4.4% (2/45)
a
Human
Fly
Worm
Aspergillus
Rice
Conservation
Human
Fly
Worm
Aspergillus
Rice
Conservation
322 46 p.Cys2567Ser
326 59 p.Val3150Ile
274 62 p.Ser3352Pro
276 86 p.Tyr4436Phe
623 625 626 662 663 666
700 701 741 742 894
Figure 2 Structural impact of SF3B1 alterations. (a) Protein sequence
alignments of the SF3B1 C-terminal domain around the altered residues
(arrows) in evolutionarily diverse species. (b) Schematic representation
of the human SF3B1 protein with the primary structural domains
highlighted. The locations of the different somatic alterations determined
to be encoded in CLL samples (top) and the frequencies of each alteration
(bottom) are shown. (c) Molecular model of the C-terminal portion of
the human SF3B1 protein and detailed view of the altered amino acids
identified in CLL cases.
observed in diffuse large B­cell lymphoma (Catalogue of Somatic Mutations
in Cancer (COSMIC) database; see URLs). A recent study of kinase
genes in CLL also found BRAF mutations in 1.6% of cases, including two
individuals with a p.Lys601Glu alteration 16 . Notably, only one of our
b
Arg625
U2AF2 interaction
SF3B14 interaction
HEAT domain
c
Asn626
Thr663
Lys666
His662
Tyr623
Lys700
Y623C
K700E V701F
R625H H662D
N626Y T663I K666E
K741N
G742D D894G
Val701
Asp894 Gly742
Lys741

© 2011 Nature America, Inc. All rights reserved.
l e t t e r s
a
Coverage
Exons
Q-rich
CLL cases carried a TP53 mutation, which was associated with a 17p
deletion. The low frequency of mutations we observed in this tumor
suppressor gene is probably due to the low­risk CLL characteristics of
our subjects, as our cohort comprises a nonselected, population­based
series (Supplementary Table 1), which carries a lower frequency of
high­risk factors than cases in referral or clinical trial studies 17 .
Among the genes newly found to be recurrently mutated in this
study, we focused on SF3B1, which had somatic point mutations in
~10% of the CLL cases. Moreover, two somatic mutations were present
in more than one case: a mutation encoding a p.Lys700Glu substitution
in four cases and a mutation encoding a p.Asn626Tyr alteration
in two other cases. We confirmed and extended these results by capillary
sequencing of 279 samples from individuals with CLL. We found
somatic mutations in 27 of these subjects (9.7%), including three additional
recurrently mutated residues causing p.Thr663Ile, p.Lys666Glu
and p.Gly742Asp alterations (Fig. 2 and Supplementary Table 11).
These findings make SF3B1 one of the most frequently mutated genes
reported to date in CLL, along with NOTCH1 (refs. 5,18).
SF3B1 encodes a protein involved in the binding of the spliceosomal
U2 snRNP to the branch point close to the 3′ splicing sites 19,20 .
The SF3B1 protein interacts with RNA sequences in the vicinity
of the branch point, as well as with the early 3′­splice­site recognition
factor U2AF65 and the branch point–binding protein SF3B14.
Consistent with such essential roles in gene expression, the amino acid
sequence of SF3B1 shows a high level of phylogenetic conservation,
especially in the regions that are coded for by the CLL somatic mutations
identified in this study (Fig. 2a and Supplementary Fig. 2).
Structurally, the SF3B1 protein has two welldefined
regions: the N­terminal hydrophilic
region, containing several protein­binding
motifs, and the C­terminal region, which
consists of 22 nonidentical HEAT repeats
and is where all somatic alterations identified
in CLL cases are located (Fig. 2b and
Supplementary Fig. 2). To investigate
the impact of the encoded somatic alterations,
we first constructed a model of the
C­terminal domain of the SF3B1 protein. In
this model, most of the alterations occur on
the inner surface of the structure and might
define a binding interface, consistent with
the idea that the helical repeats form the
outer shell of the SF3B complex 21 (Fig. 2c).
The p.Tyr623Cys, p.Arg625His, p.Asn626Tyr,
18
FOXP1w
C2H2-Zf Forkhead
19 19b 20
Control
*
PEST b
p.His662Asp, p.Thr663Ile, p.Lys666Glu, p.Lys700Glu and p.Val701Phe
alterations affect residues that are predicted to be spatially close to
one another and therefore might have a similar functional impact. In
contrast, the p.Lys741Asn and p.Gly742Asp substitutions affect a
predicted external loop, and p.Asp894Gly affects a different area of
the domain, such that these alterations might not result in the same
functional consequences.
Although splicing is a pleiotropic mechanism necessary for cell
function, specific alterations in the splicing of oncogenes and tumor
suppressors have been related to cancer development 22 . Additionally,
splicing factors have been reported to have oncogenic roles in the malignant
transformation of cells 23,24 . Consistent with these observations,
the COSMIC database shows SF3B1 mutations in several additional
tumor types, including breast carcinomas (encoding p.Gln534Pro),
pancreatic cancer (p.Arg568His, p.Gln699His and p.Lys700Glu) and
melanoma (p.Pro718Leu). This pattern of mutations, together with the
findings that some CLL­recurrent alterations, such as p.Lys700Glu, are
also detected in solid tumors and that SF3B1 is the target of antitumor
drugs 25 , reinforces the importance of SF3B1 in cancer development.
In this work, we have also identified somatic mutations in other genes
involved in the splicing machinery, such as SFRS1 (encoding p.Tyr82*
and p.Gly4Glyfs*2), SFRS7 (p.Leu18Gln) and U2AF2 (p.Gln143Leu
and p.Gln190Leu), indicating that alterations in this post­
transcriptional mechanism may be very relevant in CLL pathogenesis.
During the revision of this manuscript, three studies were published
that described associations of somatic mutations affecting SF3B1 and
other components of the splicing machinery with myelodysplastic
4 ADVANCE ONLINE PUBLICATION Nature GeNetics
Log 10 relative quantity
5
4
3
2
1
0
29 156
182 32 152
Mutated SF3B1 Wild-type SF3B1
FOXP1w
Control
Figure 3 Novel alternative splicing of FOXP1 in CLL cases. (a) An expanded view of the protein interval subject to truncation as a result of alternative
splicing is shown. The alternative splicing event that generates the novel transcript encoding this protein, FOXP1w, is shown as a red line, and the
primers used for RT-PCR amplification of FOXP1w and full-length FOXP1 (control) as arrows. Q-rich, glutamine-rich region; C2H2-Zf, Cys 2 His 2 zinc
finger. (b) Quantitative RT-PCR analysis of truncated FOXP1w levels in CLL samples with and without SF3B1 somatic mutations. Error bars, s.d.
a b c
Percentage of cases
WT SF3B1
100 1.0
MUT SF3B1
80
60
40
20
0
*** *
A B
Binet
C
Unmutated
IGHV
High
ZAP-70
Probability of progression
0.8
0.6
0.4
0.2
WT SF3B1
MUT SF3B1
0
0 5.00 10.00 15.00 20.00 25.00
Years
WT SF3B1
MUT SF3B1
Figure 4 Clinical analysis of SF3B1 in CLL. (a) Distribution of disease stage (Binet), IGHV region
mutational status and ZAP-70 expression in individuals with (MUT) or without (WT) mutations in
SF3B1 (***P = 0.004, *P = 0.03). (b) Actuarial probability of disease progression of CLL cases with
mutated or wild-type SF3B1 (P = 0.0001). (c) Actuarial probability of overall survival of CLL cases
with mutated or wild-type SF3B1 (P = 0.002).
Probability of survival
1.0
0.8
0.6
0.4
0.2
0
0 5.00 10.00 15.00 20.00 25.00
Years

© 2011 Nature America, Inc. All rights reserved.
syndromes 26–28 , which reinforces the notion that aberrant splicing
may underlie the development of certain neoplasias. According to
these reports, the only known mutations expected to affect splicing
machinery that are present in both myelodysplasia and CLL are located
in SF3B1. Two other genes, U2AF2 and ZRSR2, are also mutated in
both types of hematological neoplasias, albeit at different positions in
each cancer type. To determine whether SF3B1 could also be mutated
in other lymphoid neoplasias, we sequenced exons 14, 15, 16 and
18 in 156 non­Hodgkin’s lymphomas. No mutations were found in
any of these tumors, suggesting that SF3B1 mutations in lymphoid
neoplasias may be specific for CLL.
Comparative analysis of cases in which SF3B1 is mutated (N = 8) and
not mutated (N = 12) using exon arrays uncovered a set of 184 genes
with exons showing differential inclusion levels between these two subgroups
(false discovery rate (FDR) < 0.005) (Supplementary Table 12).
We analyzed the results of high­throughput sequencing of total RNA
isolated from the tumor cells of 15 cases, including 4 with mutations in
SF3B1. We searched for those splicing junctions that were differentially
expressed between tumors with or without SF3B1 mutations, while
maintaining similar overall gene expression levels (Supplementary
Table 13). All the differential splicing junctions identified with this
method contained a previously described 5′ donor site and a new,
abnormal 3′ acceptor site. Because SF3B1 ensures the fidelity of the
3′ branch point 20 , activation of cryptic 3′ splice sites is the expected
effect of altering SF3B1 function. The low number of candidate splicing
targets obtained with this method suggests that the SF3B1 somatic
mutations found in CLL do not impair the general function of the
protein but rather alter its function in some specific instances.
We confirmed the enhanced expression of truncated mRNAs from
candidate splicing target genes in SF3B1­mutated cases with quantitative
PCR analysis (Fig. 3 and Supplementary Fig. 3). The new
forms include truncated versions of SLC23A2, encoding a vitamin
C transporter 29 , and TCIRG1, one of whose gene products is a
T­cell immune regulator 30 . In addition, one of the novel splicing sites
affects FOXP1, encoding a forkhead transcription factor whose altered
expression has been linked to diffuse large B­cell lymphoma 31 . The
predicted product of the newly identified FOXP1 transcript, which
we call FOXP1w, encodes a protein truncated at its C terminus that
lacks two putative PEST sequences involved in protein degradation
(Fig. 3a). A quantitative RT­PCR analysis showed that the expression
of FOXP1w was three times higher in SF3B1­mutated CLL cases than
in cases without mutation in SF3B1 (Fig. 3b).
Finally, a clinical analysis showed that individuals with SF3B1
somatic mutations present advanced disease at diagnosis and adverse
biological features, such as elevated serum β 2 ­microglobulin and
IGHV loci without mutations, compared to individuals not carrying
these mutations (Fig. 4 and Supplementary Table 14). Furthermore,
individuals with SF3B1 somatic mutations had significantly shorter
time to disease progression (P = 0.0001) and lower 10­year overall
survival rates (P = 0.002). Cox analyses suggested that the mutational
status of SF3B1 has a prognostic value independent of clinical stage
or ZAP­70 or CD38 expression (Supplementary Table 15). However,
when the mutational status of SF3B1 was compared with that of
the IGHV region, only the latter retained prognostic significance.
Collectively, these data suggest that SF3B1 mutations are associated
with aggressive forms of CLL.
In summary, we have performed exome analysis of 105 CLL cases
in the context of the International Cancer Genome Consortium
initiative 32 and have found more than 70 new genes that are recurrently
mutated in CLL, thus illustrating the heterogeneity of the disease
and reinforcing the relevance of genome­wide studies to identify the
l e t t e r s
mutated genes causally involved in each individual with cancer. The
high frequency of mutations in some specific genes, such as SF3B1,
sets the stage for further studies. In addition, the fact that a substantial
fraction of the identified genes participate in well­known genetic
pathways, including gene splicing, suggests that treatment strategies
targeting common pathogenic mechanisms could be used for CLL
and other malignancies. New knowledge provided by these large­scale
efforts may eventually result in the identification of new therapies for
this frequent type of human leukemia.
URLs. COSMIC database, http://www.sanger.ac.uk/genetics/CGP/
cosmic/, European Genotype­phenome Archive (EGA), http://www.
ebi.ac.uk/ega/; Ensembl, http://asia.ensembl.org/index.html; SAMtools,
http://samtools.sourceforge.net/SAM1.pdf; Picard, http://picard.
sourceforge.net/index.shtml/; HHpred, http://toolkit.tuebingen.mpg.
de/hhpred/; Jpred, http://www.compbio.dundee.ac.uk/www­jpred/.
MeTHods
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/naturegenetics/.
Accession numbers. Sequencing, expression and genotyping array
data have been deposited at the European Genome­Phenome
Archive, which is hosted at the European Bioinformatics Institute
(EGAS00000000092).
Note: Supplementary information is available on the Nature Genetics website.
ACkNOwLEDGMENTS
We are grateful to P. Klatt for continuous support, E. Montserrat, J. Valcárcel,
P. Nicolás and C. Romeo­Casabona for helpful comments, S. Guijarro, S. Martín,
C. Capdevila, M. Sánchez and L. Plá for excellent technical assistance, and
N. Villahoz and C. Muro for excellent work in the coordination of the CLL
Spanish Consortium. We are also very grateful to all the individuals with CLL who
participated in this study. This work was funded by the Spanish Ministry of Science
and Innovation (MICINN) through the Instituto de Salud Carlos III (ISCIII)
and Red Temática de Investigación del Cáncer (RTICC) del ISCIII. C.L­O. is an
Investigator of the Botín Foundation.
AUTHOR CONTRIBUTIONS
V.Q., G.R.O., A.J.R., G.V., J.M.P.F. and X.S.P. developed the bioinformatic
algorithms and performed the analysis of sequence data. L.C., P.J., M.P., M.L.­G.,
D.C. and A.N. were responsible for downstream validation analysis and
functional studies. L.B., S.B. and J.M.C.T. studied structural variants. D.A.P.,
H.H., M.B., S.H. and M.G. were responsible for generating libraries, performing
exome capture and running sequencers. M.A. prepared and supervised
the bioethics requirements. N.V., A.M.­T., T.B., J.D., E.G., A.L.­G. and E.C.
performed clinical and biological studies. M.R., M.G.­D., N.V. and J.M.H.
reviewed the pathologic data and confirmed the diagnosis. R.R., J.L.G., M.O.,
D.G.P., J.Z., M.V. and A.V. were in charge of bioinformatics data management.
I.G. coordinated the sequencing efforts and performed primary data analysis.
V.Q., X.S.P., X.E., A. L.­G., E.C. and C.L.­O. directed the research and wrote the
manuscript, which all authors have approved.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturegenetics/.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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oNLINe MeTHods
Samples. The current studies were approved by the institutional review board
(IRB) of Hospital Clinic (Barcelona, Spain). All subjects in the initial screening
gave informed consent for their participation according to the International
Cancer Genome Consortium (ICGC) guidelines, and the subjects in the
mutational screening and clinical validation analysis agreed to IRB­approved
informed consent for genetic studies. Detailed information about the collection
and processing of samples is provided in the Supplementary Note.
Exome enrichment. Genomic DNA (3 µg) from each sample was sheared and
used for the construction of a paired­end sequencing library as described in
the protocol provided by Illumina 33 . Enrichment of exonic sequences was
then performed for each library using the SureSelect Human All Exon 50Mb
kit (Agilent) following the manufacturer’s instructions. Exon­enriched DNA
was precipitated with magnetic beads coated with streptavidin (Invitrogen)
and was washed and eluted. An additional 18 cycles of amplification were
then performed on the captured library. Exon enrichment was validated by
real­time PCR in a 7300 Real­Time PCR System (Applied Biosystems) using a
set of two pairs of primers to amplify exons and one pair to amplify an intron.
Enriched libraries were sequenced in one lane of an Illumina Gene Analyzer
II× sequencer, using the standard protocol.
RNA sequencing. RNA was assessed for quality and was quantified using an
RNA 6000 Nano LabChip kit on a 2100 Bioanalyzer (both from Agilent). The
RNA­Seq libraries were prepared according to the standard Illumina protocol
and the mRNA­Seq Sample Prep and Paired­End Sample Prep kits. cDNA
libraries were checked for quality and quantified using the DNA­1000 kit
(Agilent) on a 2100 Bioanalyzer. Each library was sequenced with the Illumina
Sequencing Kit v4 on one lane of a Gene Analyzer II× sequencer to obtain
76­bp paired­end reads.
Read mapping and processing. For RNA­Seq, reads were aligned to the human
reference genome (GRCh37) with TopHat 34 . For each gene in the Human
Ensembl genes v60, splicing junctions were determined using the CIGAR
string of the mapped reads (SAMtools, see URLs). For SF3B1­mutated samples,
a visual inspection with tview from SAMtools confirmed that the mutant
alleles were expressed at the same levels as the wild­type alleles. For exome
sequencing, reads from each library were mapped to the human reference
genome (GRCh37) using Burrows­Wheeler analysis (BWA) 35 with the sampe
option, and a BAM file was generated using SAMtools 36 . Reads from the same
paired­end libraries were merged, and optical or PCR duplicates were removed
using Picard. Statistics for the number of mapped reads and depth of coverage
for each sample are shown in Supplementary Table 3. For the identification
of somatic substitutions, we used the Sidrón algorithm, which has previously
been described 5 . Because of the robust performance of this algorithm, a single
probability table was used for all the experiments, and we added new cut­off
values for the S parameter (30 for coverage higher than 50 and 50 for coverage
higher than 100). The validation rate of the somatic mutations detected by
Sidrón was higher than 90% as assessed by Sanger sequencing.
Gene and exon expression analysis. Total RNA was extracted with TRIzol
reagent (Invitrogen) following the recommendations of the manufacturer. RNA
integrity was examined with the Agilent 2100 Bioanalyzer, and high­quality
RNA samples were hybridized to Affymetrix GeneChip Human Genome U133
plus 2.0 arrays and Human Exon 1.0 ST arrays according to Affymetrix standard
protocols. The analysis of scanned images for each probe set of the array was
obtained with GeneChip Operating Software (GCOS, Affymetrix). Expression
Console software (Affymetrix) was used to generate summarized expression
values and the detection call for the HU133 Plus 2.0 arrays using the MAS5 algorithm
and the robust multichip average (RMA). Similarly, we used RMA with
the Affymetrix core set to generate summarized signaling and the detection
above background (DABG) score for the exon arrays. In order to retrieve differentially
expressed genes, a supervised analysis with the HU133 Plus 2.0 arrays
was performed with BRB­Array Tools, v.3.6.0 software. Genes were considered
to be expressed in CLL when at least 10% of CLL samples showed a present
detection call. The exon arrays were analyzed with the Partek Genomics Suite
6.5 application to identify transcripts alternatively spliced in cases with SF3B1
doi:10.1038/ng.1032
mutation and control cases. The Affymetrix core probe set was used for splicing
analysis using the ANOVA strategy implemented in the Partek Genomics Suite.
First, we removed the probe sets that showed a DABG score (P > 0.05) in more
than 50% of the samples of at least one group. The probe sets with a low signal
(signal 2 or

© 2011 Nature America, Inc. All rights reserved.
SF3B1 mutations in this case series was assessed with multivariate analyses
including this variable and the main clinical and biological variables that had
prognostic significance in univariate studies (Supplementary Table 15). The
multivariate analysis for survival was performed with the stepwise proportional
hazards model (Cox model).
To test whether a gene was mutated more frequently than expected by
chance, we calculated the basal probability that each gene would acquire a
nonsynonymous mutation (P NS ) as
N NS × l
PNS
=
( NNS + NS ) E
In this equation, N NS is the total number of possible nonsynonymous mutations
and N S is the total number of possible synonymous mutations, with both
of these computed for each gene; l is the length of the open­reading frame
(ORF) of the gene; and E is the total length of the sequenced exome. Thus, the
probability P of finding M or more nonsynonymous mutations in a given gene
from a set of N somatic mutations in all cases is
M −1
⎛ N⎞
j 1−
j
P = 1 − ∑ P P
⎝
⎜ j ⎠
⎟ NS ( 1−
NS)
j = 0
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Nature GeNetics doi:10.1038/ng.1032