SUPPLEMENTARY APPENDIX

Supplementary Methods

Supplementary Tables

Table S1. Summary of case and control cohorts

Table S2. Summary of patient characteristics (n=646)

Table S3. Recurrent germline variants in CDK1 and ATM (Discovery Cohort: n = 516 CLL cases versus 8,920 controls)

Table S4. RNASeq confirmation of ATM variant calls in carriers and non-carriers

Table S5. Significant hits in extension analysis (n=646 CLL cases versus n=8,920 controls)

Table S6. All ATM germline variants identified in the extension study, sorted by frequency in CLL cases (n=646 CLL cases versus n=8,920 controls)

Table S7. Characteristics of patients with ATM germline variants (n=646 CLL cases versus n=8,920 controls)

Table S8. All CDK1 germline variants identified in the extension study, sorted by frequency in CLL cases (n=646 CLL cases versus n=8,920 controls)

Table S9. Characteristics of patients with CDK1 germline variants (n=646 CLL cases versus n=8,920 controls)

Table S10. Characteristics of patients analyzed for progression-free and overall survival (CLL8 Cohort)

Table S11. Progression-free survival in patients with germline variants and del(11q), including treatment arm in the multivariate analysis

Table S12. Progression-free survival in patients with germline variants and del(11q), including treatment arm, del(17p) and unmutated IGHV in the multivariate analysis

Table S13. Overall survival in patients with germline variants and del(11q), including treatment arm in the multivariate analysis

Table S14. Summary of sample-level quality control

Table S15. Summary of genotype- and variant-level quality control

Table S16. Genes with significant somatic mutations in CLL extension cohort (n=609)

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Supplementary Figures

Figure S1. Overview of quality control and analysis workflow

Figure S2. Gene-based association test is well calibrated after controlling for residual population stratification

Figure S3. Rare germline variants in CDK1 are enriched in the CLL discovery cohort.

Figure S4. PolyPhen2 HDIV scores for rare ATM missense mutations in cases versus controls

Figure S5. Association of del(11q) status, but not ATM germline variants, with progression-free survival (PFS)

Figure S6. No association of ATM germline variants or del(11q) status with overall survival (OS)

Figure S7. Top 25 genes with significant somatic mutations in CLL extension cohort (n=609)

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SUPPLEMENTARY METHODS

Patient cohort and sample collection

A summary of our entire workflow from sample collection to association analysis is shown in Supplementary Figure S1. A total of 646 CLL patient samples were included in this study: 516 patients were used in the discovery phase, and an additional 130 patients were included in the extension phase. Cohorts are summarized in Supplementary Table S1 and patient characteristics for the entire cohort are summarized in Supplementary Table S2. Among the 516 patients in the discovery cohort, 235 were CLL patients enrolled in a prospective cohort natural history study at the Dana-Farber Cancer Institute. The study was approved by the Dana-Farber Institutional Review Board, and all subjects provided their written informed consent. Of these, 128 samples had been previously described in our earlier study of subclonal somatic mutations in CLL1, while the remaining 107 are a previously unpublished subset. CLL cells were isolated from peripheral blood via density gradient centrifugation using Ficoll-PaqueTM Plus (GE Healthcare). B-cell fractions were enriched using Rosette Sep (Stemcell Technologies) and were used as the source of tumor DNA. Saliva samples were used as the source for germline DNA.

Additionally, 281 of the 516 patient samples in this study were from our recent study of somatic drivers and clonal evolution in CLL2. For these samples, heparinized blood was obtained from patients enrolled in the prospective, randomized, open label CLL8 trial before the first cycle of treatment3, 4. Immuno-magnetic enrichment for CD-19 positive cells was performed, achieving > 95% purity. CD-19 negative fractions were used as the source of germline DNA. For some of these samples, the source of germline DNA was PBMC collected after chemoimmunotherapy, when the samples were evaluated by flow cytometry as minimal residual disease-negative (Kiel Laboratory, Germany). Patient characteristics for these samples have been previously published and are included in Supplementary Table S2 and S102.

For the 130 additional patients included in the extension cohort, 24 were from our previously published whole-genome sequencing (WGS) study in CLL5, while 106 came from the ICGC’s published WES data6.

Controls representing the general population were collected from 3 major sources: 2,520 from the 1000 Genomes Project7; 6,852 from the Exome Sequencing Project8; and 7,611 from a large study of genetic risk for myocardial infarction conducted at the Broad Institute9. After applying quality-control filters, ethnicity matching, and duplicate removal, a total of 8,920 control samples were included in the association analysis (see Quality Control below). The final cohort of control samples consisted of 463 samples from the 1000 Genomes Project, 2,995 samples from the ESP, and 5,462 samples from the Broad myocardial infarction study.

Whole-exome sequencing

For the CLL case cohort, genomic DNA was isolated from tumor samples using either the Qiagen All-Prep Kit or the QIAamp Blood DNA Kit (Qiagen Inc). DNA from normal (saliva) samples was isolated using the Oragene DNA kit (Oragene). Libraries for WES were constructed using standard Broad Institute protocols and quality control procedures as previously described2, and were sequenced on either an Illumina HiSeq 2000 or GA-IIX, using 76 bp paired-end reads. WES sequences for the normal population control cohort were collected from previously sequenced sample cohorts, and details on their respective sequencing procedures have been previously described. Once the binary sequence alignment map (BAM)

3

files were generated or retrieved (for cases and controls, respectively), a germline call set was generated from case and control exomes using the Broad Genome Analysis Tool Kit (GATK v3.2.2).10 Germline variants were called, combined, jointly genotyped, and filtered according to current best practices (https://software.broadinstitute.org/gatk/ best-practices/bp_3step.php? case=GermShortWGS).

Sample quality control

We examined a number of sample-level quality control metrics, including the overall call rate (i.e., the percentage of all possible variant sites with a called genotype in a given sample), mean sequencing read depth, mean genotype quality, ratio of transitions to transversions called, and the ratio of heterozygous to homozygous alternate genotypes called. Samples which featured at least two outlying metrics (e.g., with values greater or less than 3 standard deviations from the mean calculated over all samples of the same ethnicity) were removed (see Supplementary Table S14 for summary).

Genotype and variant quality control

All genotype calls with a genotype quality score less than 20 (the phred-scaled confidence in the genotype call) were removed from the call set, along with multi-allelic sites, sites violating Hardy-Weinberg equilibrium (p<.001), poorly covered sites (missing >6% of genotypes across all samples), and variants with frequencies of >1% in the total cohort (Supplementary Table S15). Indel sites with a QD (quality-by-depth) value less than 5 were removed. We applied a final variant filter by removing variants that were flagged as not passing the GATK Variant Quality Score Recalibration workflow in the Exome Aggregation Consortium dataset8 (http://exac.broadinstitute.org/), a set of over 60,000 jointly called normal population exomes that was processed using the same calling workflow as described above.

Principal Components Analysis

We also inferred the ethnicity of all samples in the germline call set using principal components analysis (PCA) over a set of 5,848 common autosomal variants11. Variants were chosen based on work by Purcell and others11 such that they were (i) on autosomal chromosomes; (ii) polymorphic across multiple ethnic populations; (iii) present in the targeted coding regions of most exome capture platforms; (iv) in approximate linkage equilibrium; and (v) in Hardy-Weinberg equilibrium11. Using EIGENSTRAT12, we obtained 10 principal component vectors and using known (self-reported) ethnicity annotations for a subset of the controls, we inferred the ethnicity of samples of unknown ancestry based on their projection onto the first two principal components (PCs). These two PCs were empirically found to give the lowest error rate for predicting ethnicity (when splitting the cases with self-reported ethnicity to training sets of 40%, 50%, 60%, 70%, 80%, and 90%, and test sets of the remaining 60%, 50%, 40%, 30%, 20%, and 10%, respectively). For each of the ethnic groups we calculated the center in the two-principal-component space and assigned samples with unknown ethnicity based on the closest centroid (using Euclidean distance). Our largest ethnic cohort was of European ancestry, which included all CLL cases, and controls were matched by ethnicity to the CLL cases. A total of 4,331 control samples of non-European ancestry were removed.

After determining the final set of samples to be included in the study (see below, “Identification and removal of duplicate and cryptically related samples”), we performed a second round of PCA exclusively on the included samples in order to obtain principal components for use as covariates in the case-control association. These principal components

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were used to control for any population stratification within the European cohort that remained after case-control matching. PCA analysis was conducted in the same manner on the extension cohort.

Because CLL predominantly affects individuals of European descent, we chose to focus our study on patients of European ethnicity, both for the purpose of maximizing the study size and therefore increasing our statistical power to detect meaningful associations, and also for the reason that any findings from the study would have potentially widespread clinical relevance to the patient population at greatest risk for CLL. We expect, however, that further studies examining patients of different ethnic backgrounds may uncover additional germline risk genes not detectable in the European study cohort. Indeed, the presence of residual population substructure within the cohort of European subjects in this study suggests to us that there may be additional germline predisposition genes affecting different European sub-populations that we have not yet identified. Future studies concentrating on rare germline variation in more homogeneous, specific sub-populations (of both European and non-European descent) will likely yield new and significant associations.

Identification and removal of duplicate and cryptically related samples

We examined cryptic relatedness within the European cohort by running KING13 on the same set of 5,848 sites curated by Purcell and others to discover duplicates and first- or second-degree relatives in the cohort. This removed an additional 378 such samples from the call set.

Rare-variant case-control association

After applying all quality control filters, we used the Variant Effect Predictor (VEP v75: http://www.ensembl.org/info/docs/tools/vep/index.html), with the Loss-of-Function Transcript Effect Estimator (LOFTEE: https://github.com/konradjk/loftee) plugin, to annotate all variant sites for their predicted functional impact. For the case-control association, we included all bi-allelic coding variants as defined by VEP, except synonymous variants and low confidence loss-of-function variants based on LOFTEE. We tested independently, for each of the 15,868 genes with at least one rare germline variant, whether the burden of rare, non-synonymous coding variants in the CLL cases was significantly different from that of the controls. Burden testing was done using EPACTS v3.3 with the “b.collapse” test. The EPACTS collapse test scores the presence or absence of any rare variant in a given gene and a given individual as an independent variable and performs a logistic regression of the case-control phenotype against this variable. A Wald test is then performed on the fitted regression coefficient, producing a p-value for that gene. We used the first three principal components from the PCA on the final, matched European-ancestry cohort as covariates in the association in order to account for subtle allele frequency differences across European sub-populations. This gene-based test yielded a properly calibrated quantile-quantile (Q-Q) plot that deviated from the null expectation only at the extreme end of the observed p-value distribution (Supplementary Figure S2). We corrected for multiple hypothesis testing using the Benjamini-Hochberg procedure and considered genes with q≤0.05 as significantly associated with CLL.

RNASeq

For samples with available RNASeq data, expression of the alternate germline allele in variants of interest was confirmed by direct visualization of the aligned BAM file in the Integrated Genomics Viewer (IGV). We also checked that the alternate allele fraction in RNA transcripts

5

was comparable to the germline alternate allele fraction in the genomic DNA (Supplementary Table S4). All germline variants had approximately 50 or 100% allele frequency and thus are highly unlikely to result from somatic contamination.

Somatic mutation calling and LOH identification

We applied the standard MuTect workflow (v1.1.6)14 to matching tumor exome data for each of our germline samples, followed by an additional panel-of-normals filtering step to catch residual common germline variants and other sequencing artifacts. A final filtering step was performed on somatic mutation calls via local realignment of sequencing reads and re-evaluation of the mutation likelihoods at candidate sites. We used ABSOLUTE v1.3.4.015 to estimate the absolute local copy number across the exome and to identify regions of loss-of-heterozygosity in tumor samples, including both copy-number-altered and copy-neutral LOH. We also applied MutSig2CV16 across the entire CLL cohort to identify CLL drivers and ensure that drivers in our cohort were consistent with previously studied CLL cohorts (Table S16, Figure S7).

Statistical analyses of clinical data

Statistical analyses of clinical features from the CLL8 clinical trial3, 4 were performed on an intention-to-treat basis, including all eligible subjects as randomized who had available samples. This included fit and previously untreated CLL patients who were randomized to first line therapy with fludarabine cyclophosphamide (FC) or FC with rituximab (FCR).3, 4

Progression-free survival (PFS) was defined as the time from randomization to disease progression or death, whichever occurred first. PFS was estimated using the Kaplan-Meier method and the difference was compared using the log-rank test. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated using Cox regression adjusting for the treatment arm. All p-values were two-sided and considered significant at the 0.05 level. Statistical analyses were performed with SPSS version 23.

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TABLES

Table S1. Summary of case and control cohorts

Sample CohortCohort Type

Cohort Size

Germline Sample Source

Sequencing Method Analyses

DFCI CLL prospective study (unpublished)

Case(discovery) 107 Saliva

Illumina HiSeq 2000 or GA-IIX

Association test, somatic second hit analysis

DFCI CLL prospective study1

Case(discovery) 128 Saliva

Illumina HiSeq 2000 or GA-IIX

Association test, somatic second hit analysis

GCLLSG CLL8 trial2-4

Case(discovery) 281

CD19-negative fraction from peripheral blood mononuclear cells (PBMC)

Illumina HiSeq 2000 or Illumina HiSeq 2500

Association test, somatic second hit analysis

Whole-genome CLL5

Case(extension) 24 Saliva

Illumina HiSeq 2000

Association test, somatic second hit analysis

International Cancer Genome Consortium CLL6

Case(extension) 106

CD19-negative fraction from peripheral blood mononuclear cells (PBMC)

Illumina GA-IIX

Association test, somatic second hit analysis

1000 Genomes Project7 Control 463

Immortalized lymphoblastoid cell lines

454 GS FLX/Titanium or Illumina GA-II

Association test

Exome Sequencing Project8 Control 2995 Blood

Illumina GA-IIX or HiSeq 2000

Association test

Early Onset Myocardial Infarction Study9 Control 5462 Blood

Illumina GA-IIX or HiSeq 2000

Association test

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Table S2. Summary of patient characteristics (N=646)Characteristic n %Sex (N= 644) Male 436 67.7 Female 208 32.3Age at Diagnosis (N=538) Median (Range) 59 (31 – 86)Family History (N=258) Familial 80 31.0 Sporadic 178 69.0Binet Stage (N=383) A 73 19.1 B 202 52.7 C 108 28.2Rai Stage (N=356) 0 159 44.7 I-II 140 39.3 III-IV 57 16.0β2-Microglobulin (N=236) Median (Range) 2.7 (1.1 – 16.5)Treated at Sampling (N=643) No 482 75.0 Yes 161 25.0Total Number of Therapies in Patients Treated at Sampling (N=161) Median (Range) 4 (1-12)IGHV Status (N=614) Unmutated 328 53.4 Mutated 286 46.6ZAP70 Positive (N=471) No 270 57.3 Yes 201 42.7Del17p by FISH (N=620) No 555 89.5 Yes 65 10.5Dell11q by FISH (N=620) No 493 79.5 Yes 127 20.5Trisomy 12 by FISH (N=619) No 532 85.9 Yes 87 14.0Normal Cytogenetics by FISH (N=619) No 498 80.4 Yes 121 19.5Del13q by FISH (N=619) No 269 43.5 Yes 350 56.5* Cytogenetics data represent presence of abnormalities unclassified by Dohner hierarchy.

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Table S3. Recurrent germline variants in CDK1 and ATM (Discovery Cohort)Gene Chromosomal

Locus

Reference

Allele

Alternate

Allele

Protein Change PolyPhen2

Category**

# of Cases with

Rare Variants (%)

# of

Controls

with Rare

Variants (%)

OR (95% CI)

CDK1 10:62544600 C T p.R59C D 5 (1.0 %) 10 (0.1%) 8.74 (3.0-25.7)

ATM 11:108138003 T C p.F858L P 13 (2.5%) 186 (2.1%) 1.21 (0.7-2.1)

11:108196896 C T p.L2307F D 12 (2.3%) 21 (0.2%) 10.11 (4.9-20.7)

11:108123551 C T p.P604S P 9 (1.7%) 62 (0.7%) 2.56 (1.3-5.2)

11:108170506 A C p.S1691R B 8 (1.6%) 29 (0.3%) 4.83 (2.2-10.6)

11:108119654 T G N/A (Splice-site) N/A 6 (1.2%) 32 (0.4%) 3.29 (1.4-7.9)

11:108175463 A T p.D1853V P 6 (1.2%) 107 (1.2%) 0.97 (0.4-2.2)

11:108180917 T C p.L73P*** N/A 6 (1.2%) 103 (1.2%) 1.02 (0.4-2.3)

11:108160480 T G p.F1463C D 5 (1.0%) 29 (0.3%) 3.01 (1.2-7.8)

11:108114727 G C p.V182L B 3 (0.6%) 19 (0.2%) 2.76 (0.8-9.4)

11:108117787 C T p.S333F P 3 (0.6%) 42 (0.5%) 1.24 (0.4-4.0)

11:108160416 T C p.Y1442H D 3 (0.6%) 4 (0.04%) 13.05 (2.9-58.5)

11:108164137 T C p.V1570A B 3 (0.6%) 23 (0.3%) 2.26 (0.7-7.6)

11:108202772 G T N/A (Splice-site) N/A 3 0.6%) 42 (0.5%) 1.24 (0.4-4.0)

11:108100014 A G p.S99G D 2 (0.4%) 2 (0.02%) 17.4 (2.4-123.8)

11:108137907 A C p.I826L B 2 (0.4%) 2 (0.02%) 17.35 (2.4-123.4)

11:108155007 AG A p.E1267fs N/A 2 (0.4%) 0 (0%) N/A

11:108201108 T G p.L2492R D 2 (0.4%) 2 (0.02%) 17.35 (2.4-123.4)

* Recurrent variants are defined as occurring ≥2 times in cases. ** PolyPhen2 categories are as follows: D, probably damaging; P, possibly damaging; B, benign. *** Annotation based on a shorter alternative transcript.

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Table S4. RNASeq confirmation of ATM variant calls in carriers and non-carriers

Hugo Symbol

Patient ID

Variant Carrier Status

Protein Change Locus

Ref Allele

Var Allele

Reads with

reference base

Reads with alternate

base

ATM 27 carrier p.E26Q11:108098506 G C 29 28

ATM 26 carrier p.S49C11:108098576 C G 43 32

ATM 13 carrier p.S49C11:108098576 C G 58 69

ATM 23 carrier p.S99G11:108100014 A G 13 16

ATM 8 carrier p.S99G11:108100014 A G 26 33

ATM 20 carrier p.V182L11:108114727 G C 38 31

ATM 14 carrier p.V182L11:108114727 G C 51 72

ATM 21 carrier p.P604S11:108123551 C T 24 9

ATM 29 carrier p.E649K11:108124587 G A 37 33

ATM 1 carrier p.F763L11:108128246 T A 13 12

ATM 10 carrier p.F858L11:108138003 T C 18 12

ATM 24 carrierp.H1136R

11:108151726 A G 15 17

ATM 19 carrier* p.Y1252F11:108154962 A T 23 0

ATM 18 carrier**p.E1267fs

11:108155007 AG A 1 1

ATM 25 carrier p.Y1442H11:108160416 T C 44 52

ATM 28 carrier p.K1454R11:108160453 A G 61 47

ATM 16 carrier p.V1570A11:108164137 T C 72 47

ATM 25 carrier p.D1853V11:108175463 A T 78 68

ATM 22 carrier p.L73P11:108180917 T C 40 35

ATM 22 carrier p.A1931A11:108180917 T C 40 35

ATM 17 carrierp.G2023R

11:108186610 G A 33 20

ATM 15 carrier p.V2079I11:108188136 G A 61 65

ATM 2 carrier p.L2307F11:108196896 C T 13 68

ATM 12 carrier p.L2307F11:108196896 C T 121 115

ATM 9 carrier p.L2492R11:108201108 T G 6 108

10

ATM 14 carrierp.R2854C

11:108216611 C T 156 148

ATM 11 carrierp.Q2989E

11:108235923 C G 285 280

ATM 3 no p.P604S11:108123551 C T 114 0

ATM 4 no p.P604S11:108123551 C T 8 0

ATM 5 no p.P604S11:108123551 C T 92 0

ATM 6 no p.P604S11:108123551 C T 74 0

ATM 7 no p.P604S11:108123551 C T 77 0

ATM 3 no p.S1691R11:108170506 A C 184 0

ATM 4 no p.S1691R11:108170506 A C 22 0

ATM 5 no p.S1691R11:108170506 A C 163 0

ATM 6 no p.S1691R11:108170506 A C 153 0

ATM 7 no p.S1691R11:108170506 A C 145 0

ATM 3 no p.L2307F11:108196896 C T 251 0

ATM 4 no p.L2307F11:108196896 C T 56 0

ATM 7 no p.L2307F11:108196896 C T 197 0

ATM 5 no p.L2307F11:108196896 C T 204 1

ATM 6 no p.L2307F11:108196896 C T 170 1

* Patient 19 carried a nonsense germline variant (p.Y1252F) that showed no alternate reads in the RNA, which is likely the result of nonsense-mediated decay.1, 5

**Patient 18 had very low RNASeq coverage at the p.E1267fs variant site.

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Table S5. Significant hits in extension analysis (n=646 CLL cases versus n=8,920 controls) Gene p-value FDR

(q-value)# of

Cases with Rare Variants

(%)

# of Controls with Rare Variants

(%)

OR(95% CI)

Fisher OR(95% CI)

ATM 1.034x10-8 1.67x10-4 170 (26.32%)

1483(16.55%)

1.8(1.5-2.17)

1.8(1.49-2.17)

SDK2 4.31x10-6 0.035 36(5.57%)

222(2.48%)

2.33(1.62-3.34)

2.33(1.57-3.35)

KRTAP21-2 6.56x10-6 0.035 11(1.7%)

31(0.35%)

4.99(2.5-9.98)

4.99(2.25-10.27)

DCAF11 1.16x10-5 0.047 28(4.33%)

162(1.81%)

2.46(1.64-3.71)

2.46(1.57-3.73)

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Table S6. All ATM germline variants identified in the extension study, sorted by frequency in CLL cases

Locus Ref Allele

Alt Allele

Protein Change Hetero-

zygous

Cases

Hetero-zygous Control

s

Homo-zygous Alt

Cases

Homo-zygous

Alt Control

s

Homo-zygous

Ref Cases

Homo-zygous

Ref Control

s11:108098576 C G p.S49C 22 207 0 1 623 871211:108138003 T C p.F858L 22 184 0 2 624 873411:108196896 C T p.L2307F 12 21 0 0 634 889911:108123551 C T p.P604S 9 62 1 0 632 885011:108170506 A C p.S1691R 9 29 0 0 637 889111:108119654 T G Splice Site 7 32 0 0 632 881211:108175463 A T p.D1853V 7 107 0 0 639 881111:108180917 T C p.A1931A 7 103 0 0 633 881711:108114727 G C p.V182L 4 20 0 0 633 882111:108117787 C T p.S333F 4 42 0 0 641 887811:108119823 T C p.V410A 4 61 0 1 642 885811:108160416 T C p.Y1442H 4 4 0 0 641 891111:108160480 T G p.F1463C 4 29 1 0 640 889111:108164137 T C p.V1570A 3 23 0 0 643 889011:108202772 G T Splice Site 3 42 0 0 643 887411:108100014 A G p.S99G 2 2 0 0 642 891111:108137907 A C p.I826L 2 2 0 0 644 891611:108155007 AG A p.E1267fs 2 0 0 0 644 892011:108199845 C G p.T2396S 2 6 0 0 644 891411:108201108 T G p.L2492R 2 2 0 0 644 891811:108098506 G C p.E26Q 1 0 0 0 645 892011:108106435 A G p.I124V 1 6 0 0 644 891411:108114793 G A p.G204R 1 6 0 0 644 891411:108114830 C CTAT

p.217_218insI 1 0 0 0 645 8920

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11:108115601 G A p.R250Q 1 3 0 0 644 891711:108117798 C T p.R337C 1 1 0 0 644 891811:108121787 G A p.C532Y 1 4 0 0 645 891411:108122683 T C p.I576T 1 0 0 0 643 892011:108122700 T C p.F582L 1 44 0 0 644 887611:108124587 G A p.E649K 1 0 0 0 645 892011:108124740 C T p.Q700* 1 0 0 0 645 892011:108126966 C T p.R717W 1 1 0 0 641 891611:108128207 G C Splice Site 1 0 0 0 643 891811:108128246 T A p.F763L 1 10 0 0 645 890911:108129722 A C p.N796H 1 2 0 0 645 891811:108129785 G A p.D817N 1 0 0 0 645 892011:108141789 A G Splice Site 1 0 0 0 645 891811:108141988 T C p.S978P 1 23 0 0 643 889711:108143552 G A p.R1086H 1 0 0 0 645 892011:108151726 A G p.H1136R 1 0 0 0 644 891811:108154962 A T p.Y1252F 1 0 0 0 638 879211:108155132 G A p.A1309T 1 18 0 0 644 890011:108155171 C A p.L1322I 1 1 0 0 644 891911:108160453 A G p.K1454R 1 0 0 0 645 891911:108160454 A C p.K1454N 1 12 0 0 645 890711:108160516 A G p.Y1475C 1 13 0 0 638 889611:108170480 A G p.D1682G 1 0 0 0 645 892011:108170524 A G p.T1697A 1 2 0 0 645 891811:108172386 G A p.R1730Q 1 4 0 0 644 891611:108172516 G A p.K1773K 1 0 0 0 640 891811:108175517 C T p.T1871I 1 0 0 0 645 891911:10817870 G C p.R1918T 1 3 0 0 641 8915

14

211:108180900 A *,G p.T1926A 1 2 0 0 642 891711:108183194 A C,* p.K1992T 1 12 0 0 645 890711:108186610 G A p.G2023R 1 55 0 0 645 886511:108186787 T G p.Y2049D 1 0 0 0 645 892011:108188131 T C p.L2077P 1 0 0 0 645 892011:108188136 G A p.V2079I 1 10 0 0 645 891011:108188224 A G p.Q2108R 1 0 0 0 644 892011:108188244 G A p.V2115I 1 0 0 0 644 892011:108196222 A C p.K2253T 1 0 0 0 644 8920

11:108198391

TTACA

CTACA,T

p.2332_2332LT>PTY,p.LT2332fs 1 4 0 0 645 8915

11:108202273 G A p.V2540I 1 0 0 0 638 891911:108202604 A C Splice Site 1 0 0 0 645 892011:108203488 G A Splice Site 1 0 0 0 642 890611:108203516 A G p.I2606V 1 1 0 0 643 891811:108203575 T G p.D2625E 1 0 0 0 641 892011:108203576 G C p.A2626P 1 0 0 0 641 891911:108216611 C T p.R2854C 1 3 0 0 645 891711:108224555 A G p.R2912G 1 6 0 0 645 891411:108235923 C G p.Q2989E 1 0 0 0 645 892011:108098392 A G p.Q14R 0 1 0 0 646 891911:108098509 G A p.V27I 0 1 0 0 646 891911:108098524 C T p.R32C 0 1 0 0 646 891811:108098525 G T p.R32L 0 1 0 0 646 891811:108098545 A G p.T39A 0 1 0 0 646 891911:108098555 A G p.H42R 0 1 0 0 646 891911:108098563 C T p.R45W 0 1 0 0 646 891911:10809862 T C Splice Site 0 1 0 0 645 8870

15

211:108099898 C T Splice Site 0 2 0 0 627 872811:108099921 A G p.I68V 0 1 0 0 640 888011:108106483 G C p.D140H 0 1 0 0 646 891911:108106543 T C p.S160P 0 1 0 0 645 891911:108106565 T C Splice Site 0 8 0 0 646 891211:108114701 G T p.R173M 0 2 0 0 623 838011:108114838 T C p.C219R 0 1 0 0 646 891911:108114842 C T p.A220V 0 1 0 0 646 891811:108115522 A G p.K224E 0 1 0 0 645 891911:108115654 C T p.Q268* 0 1 0 0 646 891911:108117691 G A p.G301D 0 2 0 0 645 890211:108117735 T C p.Y316H 0 2 0 0 645 891711:108117799 G A p.R337H 0 2 0 0 646 891711:108117838 C T p.A350V 0 1 0 0 646 891911:108117844 T C p.I352T 0 1 0 0 646 891911:108119667 A G p.N358S 0 1 0 0 644 891111:108119732 T A p.Y380N 0 1 0 0 645 891911:108119749 A T p.K385N 0 1 0 0 646 891911:108119769 G T p.G392V 0 1 0 0 646 891911:108121463 C A p.P424H 0 1 0 0 646 891011:108121543 C T p.R451C 0 1 0 0 646 891911:108121593 CAA C p.K468fs 0 1 0 0 646 891911:108121636 A C p.K482Q 0 3 0 0 646 891711:108121656 G T p.W488C 0 1 0 0 646 891911:108121658 G T p.C489F 0 1 0 0 646 891911:108121660 A G p.I490V 0 1 0 0 646 891911:108121708 G T p.G506C 0 5 0 0 646 8915

16

11:108121752 CAG C p.R521fs 0 2 0 0 646 891811:108122604 A G p.I550V 0 1 0 0 646 891911:108122746 C T p.P597L 0 2 0 0 644 891711:108122766 A G Splice Site 0 1 0 0 644 891311:108123575 C A p.L612I 0 1 0 0 645 891511:108123578 G T p.V613L 0 1 0 0 644 891511:108123596 A C p.N619H 0 1 0 0 644 891711:108123621 T G p.F627C 0 2 0 0 645 891611:108123629 G A p.V630M 0 1 0 0 642 890911:108124585 T C p.V648A 0 1 0 0 646 891911:108124602 C A p.Q654K 0 2 0 0 645 891811:108124663 A G p.H674R 0 1 0 0 644 891911:108124716 C T p.R692C 0 1 0 0 646 891911:108124729 G A p.G696E 0 1 0 0 646 891911:108124759 C T p.S706L 0 1 0 0 645 891211:108124764 G A p.E708K 0 1 0 0 645 890311:108127067 G A p.K750K 0 1 0 0 646 891011:108127069

TAGGAG

A

T Splice Site 0 1 0 0 646 8900

11:108128217 C A p.Q754K 0 1 0 0 645 891911:108128227 G A p.G757E 0 1 0 0 646 891911:108128232 A G p.S759G 0 4 0 0 646 891611:108128319 A C p.S788R 0 1 0 0 646 891911:108129732 C T p.A799V 0 3 0 0 646 891611:108129749 C T p.R805* 0 2 0 0 646 891611:108129750 G A p.R805Q 0 1 0 0 646 891811:108129778 C A p.D814E 0 3 0 0 646 891711:108137889 TC T Splice Site 0 1 0 0 643 8918

17

11:108137925 C T p.R832C 0 8 0 0 646 891111:108137926 G A p.R832H 0 1 0 0 646 891811:108138039 A G p.N870D 0 1 0 0 646 891911:108138071 T C Splice Site 0 1 0 0 646 891911:108139142 A G p.I882V 0 1 0 0 646 891911:108139187 T A p.F897I 0 1 0 0 646 891911:108139196 A G p.M900V 0 1 0 0 646 891911:108139203 A C p.K902T 0 1 0 0 646 891911:108139239 A G p.N914S 0 1 0 0 646 891911:108139268 C T p.R924W 0 4 0 0 645 891611:108139326 A C p.H943P 0 1 0 0 646 891911:108141825 A G p.E958G 0 1 0 0 646 891911:108141867 C T p.P972L 0 1 0 0 646 891911:108141871 A G p.L973L 0 1 0 0 646 891911:108141873 C T p.S974F 0 1 0 0 646 891911:108141874 G A Splice Site 0 2 0 0 646 891811:108142000 C T p.R982C 0 1 0 0 645 891911:108142001 G A p.R982H 0 1 0 0 645 891811:108142057 C G p.L1001V 0 2 0 0 646 891811:108142070 A G p.N1005S 0 6 0 0 646 891411:108142072 A G p.M1006V 0 1 0 0 646 891911:108143445 G A Splice Site 0 11 0 0 646 890611:108143470 G A p.A1059T 0 1 0 0 646 891911:108143537 A G p.N1081S 0 2 0 0 646 891811:108143551 C T p.R1086C 0 1 0 0 646 891911:108150261 G A p.A1110T 0 1 0 0 629 891311:108150289 C T p.A1119V 0 1 0 0 643 891911:10815031 A G p.Q1128R 0 1 0 0 645 8917

18

611:108151719 A C Splice Site 0 1 0 0 645 891011:108151786 C T p.T1156M 0 1 0 0 646 891911:108151793 AG A p.A1159fs 0 1 0 0 645 891911:108151824 G A p.E1169K 0 1 0 0 646 891911:108151882 A C p.H1188P 0 1 0 0 646 891911:108151884 C T p.L1189F 0 1 0 0 645 891911:108151902 A G Splice Site 0 1 0 0 645 891911:108153437 G A p.V1193I 0 1 0 0 646 891011:108153473 C T p.R1205C 0 1 0 0 646 890411:108153536 G C p.D1226H 0 1 0 0 643 891511:108153555 C G p.S1232C 0 1 0 0 643 888311:108153603 A G p.Y1248C 0 1 0 0 642 878411:108154975 T G p.I1256M 0 1 0 0 644 889611:108155048 A T p.S1281C 0 1 0 0 646 891911:108158393 C A p.P1354T 0 15 0 0 646 890411:108158399 A G p.N1356D 0 2 0 0 646 891811:108158412 G A p.S1360N 0 1 0 0 646 891911:108158420 A G p.T1363A 0 1 0 0 646 891911:108160339 A G p.Q1416R 0 1 0 0 644 889811:108160396 A G p.K1435R 0 2 0 0 645 891311:108160398 C T p.H1436Y 0 5 0 0 644 890811:108160421 C A p.H1443Q 0 1 0 0 645 891711:108160462 T C p.L1457S 0 1 0 0 646 891911:108160467 G A p.G1459R 0 2 0 0 646 891811:108160471 C T p.A1460V 0 1 0 0 646 891911:108160488 C G p.R1466G 0 1 0 0 645 891911:108163339 A G Splice Site 0 1 0 0 646 8919

19

11:108163354 G A p.C1482Y 0 1 0 0 646 891911:108163374 C T p.R1489C 0 1 0 0 646 891911:108163410 G A p.V1501I 0 1 0 0 646 891911:108163467 A C p.I1520L 0 1 0 0 646 891911:108164094 T C p.Y1556H 0 1 0 0 645 891511:108164101 C T p.T1558M 0 1 0 0 645 891711:108164131 A G p.H1568R 0 3 0 0 646 891211:108164188 C A p.P1587H 0 1 0 0 646 891411:108164196 C T p.L1590F 0 2 0 0 646 890711:108165661 A G p.N1595S 0 1 0 0 646 891911:108165702 A G p.T1609A 0 1 0 0 646 891911:108165730 G A p.R1618Q 0 2 0 0 646 891811:108165733 G A p.R1619K 0 1 0 0 646 891911:108165748 A G p.H1624R 0 4 0 0 646 891611:108165789 G A Splice Site 0 2 0 0 646 891811:108168020 C T p.P1639L 0 1 0 0 645 891811:108168115 CTA C Splice Site 0 2 0 0 641 891711:108168116 T C,* Splice Site 0 2 0 0 641 891511:108170444 C T p.A1670V 0 1 0 0 646 891911:108170491 A G p.I1686V 0 2 0 0 646 891811:108170515 G A p.A1694T 0 2 0 0 646 891811:108170536 A G p.K1701E 0 1 0 0 646 891911:108170582 C A p.T1716N 0 1 0 0 646 891911:108170611 T G p.C1726G 0 1 0 0 646 891911:108172382 G C p.V1729L 0 2 0 0 643 891811:108172416 T G p.I1740S 0 1 0 0 646 891911:108172424 A G p.T1743A 0 1 0 0 646 891911:10817251 C A Splice Site 0 1 0 0 642 8918

20

911:108173597 A T p.R1779S 0 1 0 0 646 891811:108173678 A G p.I1806M 0 1 0 0 645 891911:108173712 G A p.G1818S 0 1 0 0 642 891911:108175459 C A p.Q1852K 0 1 0 0 646 891711:108175528 C T p.R1875* 0 1 0 0 645 890311:108175538 C T p.S1878L 0 1 0 0 645 884911:108178655 T TA p.K1903fs 0 1 0 0 643 891811:108178675 T C p.M1909T 0 1 0 0 645 891911:108178699 G C p.R1917T 0 1 0 0 644 8919

11:108180899

AACAATTTTTAATGA

T Ap.TIFND1926del 0 1 0 0 644 8919

11:108180945 G C p.V1941L 0 4 0 0 642 891611:108181006 A G p.Y1961C 0 2 0 0 645 891811:108181014 A G p.K1964E 0 4 0 0 645 891611:108183193

AAAAGT A p.KS1992fs 0 1 0 0 646 8918

11:108186568 T C p.Y2009H 0 2 0 0 646 891811:108186631 A G p.I2030V 0 1 0 0 643 891911:108186735 T C Splice Site 0 1 0 0 644 891811:108188127 A G p.I2076V 0 1 0 0 646 891911:108188156 T A p.D2085E 0 1 0 0 646 891911:108188194 T C p.L2098P 0 1 0 0 646 891911:108188222 G A p.M2107I 0 1 0 0 645 891911:108188254 A C Splice Site 0 1 0 0 645 891911:108190771 T A p.S2146R 0 1 0 0 642 891411:108192075 A G p.Y2167C 0 1 0 0 646 891911:108192118 G T p.E2181D 0 1 0 0 646 8919

21

11:108192126 G C p.S2184T 0 2 0 0 646 891811:108192151 T C Splice Site 0 2 0 0 646 891811:108196135 T C p.M2224T 0 1 0 0 645 891911:108196167 A G p.M2235V 0 1 0 0 643 891911:108196797 G A p.A2274T 0 5 0 0 643 891511:108196800 A G p.I2275V 0 1 0 0 644 891911:108196848 T C p.W2291R 0 1 0 0 645 891711:108196908 A G p.I2311V 0 1 0 0 646 891911:108196951 C T p.A2325V 0 1 0 0 645 891911:108196959 T A Splice Site 0 1 0 0 645 891911:108198376 A T p.N2327I 0 1 0 0 646 891811:108198384 C G p.L2330V 0 1 0 0 646 891711:108198394 C A,* p.T2333K 0 1 0 0 646 891811:108198484 A T p.K2363M 0 2 0 0 644 891311:108199780 A C p.E2374D 0 1 0 0 646 891911:108199923 A G p.E2422G 0 1 0 0 645 891811:108199929 T G p.V2424G 0 1 0 0 645 890911:108200953 G C p.K2440N 0 1 0 0 643 891911:108200987 C G p.L2452V 0 2 0 0 645 891711:108200991 G A p.R2453H 0 2 0 0 645 891711:108201008 C T p.R2459C 0 2 0 0 644 891711:108201023 T C p.C2464R 0 6 0 0 645 891411:108201125 T C p.S2498P 0 1 0 0 646 891911:108201135 A G p.N2501S 0 1 0 0 646 891911:108202246 A G p.M2531V 0 1 0 0 644 891911:108202247 T C p.M2531T 0 3 0 0 643 891711:108202716 A C p.R2580S 0 1 0 0 645 891911:10820275 A G p.Q2593R 0 2 0 0 646 8917

22

411:108202764 G A p.E2596E 0 1 0 0 646 891911:108203535 G A p.R2612K 0 1 0 0 644 891811:108203571 G C p.C2624S 0 2 0 0 642 891811:108203619 C T p.T2640I 0 4 0 0 644 890811:108203632 G GT Splice Site 0 8 0 0 638 887911:108204681 A G p.T2666A 0 2 0 0 646 891811:108205690 T G Splice Site 0 1 0 0 644 891911:108205700 A C p.D2672A 0 1 0 0 645 891911:108205751 A G p.E2689G 0 1 0 0 646 891911:108205756 C T p.R2691C 0 1 0 0 645 891811:108205785 A C p.K2700N 0 1 0 0 646 891911:108205798 G T p.V2705L 0 1 0 0 646 891911:108205832 T C p.V2716A 0 1 0 0 646 891911:108206564 T G Splice Site 0 1 0 0 645 891311:108206566 C T Splice Site 0 2 0 0 644 891011:108206576 G A p.R2719H 0 4 0 0 644 891111:108206607 A C p.Q2729H 0 1 0 0 645 891511:108206609 A G p.Q2730R 0 1 0 0 646 891711:108206648 C T p.T2743M 0 1 0 0 645 891911:108206663 G C p.R2748T 0 1 0 0 646 891811:108206686 A *,T p.K2756* 0 1 0 0 645 891511:108206694 T A Splice Site 0 1 0 0 645 891211:108213955 C G p.P2759A 0 1 0 0 646 891911:108213957

CCTCT C p.LS2760fs 0 1 0 0 646 8919

11:108214033 G A p.D2785N 0 1 0 0 646 891911:108216479 A C p.K2810Q 0 3 0 0 646 891611:108216546 G A p.R2832H 0 1 0 0 645 8919

23

11:108216548 T C p.Y2833H 0 1 0 0 646 891911:108216581 A G p.I2844V 0 1 0 0 646 891911:108216609 C T p.T2853M 0 3 0 0 646 891711:108216616 T A p.S2855R 0 1 0 0 646 891911:108216617 G A p.V2856I 0 1 0 0 646 891911:108216638 A G Splice Site 0 1 0 0 646 891911:108218017 C G p.L2866V 0 1 0 0 644 891711:108218026 G A p.G2869S 0 1 0 0 646 891511:108218038 G A p.V2873I 0 1 0 0 646 891811:108218045 A G p.N2875S 0 1 0 0 645 891911:108224558 G C p.D2913H 0 1 0 0 646 891911:108224561 A T p.I2914F 0 1 0 0 646 891911:108224583 C T p.T2921M 0 1 0 0 646 8919

11:108225534

CTAGATGCT

G C p.IDA2929del 0 1 0 0 646 891911:108225605 A C Splice Site 0 1 0 0 646 891711:108235801 C T Splice Site 0 1 0 0 646 891311:108235818 T C p.Y2954H 0 1 0 0 646 891811:108235879 C T p.P2974L 0 2 0 0 646 891811:108235926 G A p.E2990K 0 1 0 0 646 891911:108236086 C T p.R3008C 0 1 0 0 646 891911:108236087 G A p.R3008H 0 1 0 0 646 891911:108236134 A C p.T3024P 0 1 0 0 646 891911:108236143 A G p.S3027G 0 1 0 0 646 891911:108236150 G A p.G3029D 0 5 0 0 646 891511:108236183 T C p.I3040T 0 1 0 0 646 891811:108236230 G T p.V3056L 0 1 0 0 644 8903

24

* For synonymous variants, please see full germline sequencing data in dbGAP under accession number phs000879.v1.p1. One of the additional recurrent variants identified in the extension analysis, p.S49C, was present at an allele frequency of 0.01 in the discovery cohort and thus did not meet the allele-frequency threshold for inclusion in the discovery analysis. This variant was subsequently included in the extension analysis after the allele frequency dropped to 0.0095 in the second call set. The p.S49C variant was enriched in CLL cases (OR 1.48, CI .95-2.31) in the extension set and therefore contributed to the increase in statistical significance of ATM variation. In the entire extension cohort (n=646), we found 44 variants observed in both cases and controls, with 36 enriched in cases. Twenty-seven variants were unique to the CLL cases, and all but one were singletons.

The majority of recurrent variants we observed in ATM were non-synonymous missense variants. This is in contrast to the nonsense or frameshift ATM mutations observed in ataxia telangiectasia syndrome17, a hereditary disorder associated with greater than 100-fold increased risk of leukemias and lymphomas18. This is consistent with the fact that the CLL phenotype is not as severe as that of ataxia telangiectasia.

25

Table S7. Characteristics of patients with ATM germline variantsCharacteristic Presence of ATM Germline Variant

No (n=476) Yes (n=170)Sex N % N % Male 282 66.8 86 73.5 Female 140 33.2 31 26.5Age at Diagnosis* Median (Range) 59 (31 – 86) 57 (34 – 83)Family History Familial 62 30.8 18 31.6 Sporadic 139 69.1 39 68.4Binet Stage A 13 5.9 2 3.3 B 135 61.1 38 63.3 C 73 33.0 20 33.3Rai Stage 0 92 46.7 27 47.4 I-II 75 38.1 21 36.8 III-IV 30 15.2 9 15.8β2-Microglobulin Median (Range) 2.7 (1.1 – 13) 2.7 (1.3 – 16.5)Treated at Sampling No 296 70.3 81 69.2 Yes 125 29.7 36 30.8IGHV Status Unmutated 221 55.1 62 57.4 Mutated 180 44.9 46 42.6ZAP70 Positive No 155 63.5 46 57.5 Yes 139 57.0 34 42.5Del17p by FISH No 350 87.1 102 89.5 Yes 52 12.9 12 10.5Del11q by FISH No 321 79.8 82 71.9 Yes 81 20.1 32 28.1Trisomy 12 by FISH No 337 84.0 101 88.6 Yes 64 16.0 13 11.4Normal Cytogenetics No 323 80.5 94 82.5 Yes 78 19.4 20 17.5Del13q by FISH No 169 42.1 46 40.3 Yes 232 57.8 68 59.6* In our cohorts, ATM status was not significantly associated with any clinical parameters including age at diagnosis.

26

Table S8. All CDK1 germline variants identified in the extension study, sorted by frequency in CLL cases

Locus Ref Allele

AltAllele

Protein Change

Hetero-zygous Cases

Hetero-zygous

Controls

Homo-zygous

Alt Cases

Homo-zygous

Alt Controls

Homo-zygous

RefCases

Homo-zygous

Ref Controls

10:62544600 C T p.R59C 5 10 0 0 641 891010:62539896 T C Splice Site 1 4 0 0 642 891210:62544511 T C p.V29A 1 0 0 0 645 892010:62552001 C T p.A250V 1 0 0 0 643 892010:62539967 G A Splice Site 0 1 0 0 640 888810:62544508 A G p.Q28R 0 1 0 0 646 891910:62545435 C T p.L70F 0 1 0 0 645 891910:62551694 C G p.A179G 0 1 0 0 643 890910:62551735 A G p.I193V 0 1 0 0 643 891510:62551953 T C p.L234S 0 1 0 0 641 891910:62552047 G A p.S265S 0 2 0 0 641 891610:62553630 C T Splice Site 0 3 0 0 639 889210:62553666 T C p.I276T 0 1 0 0 643 8914* For synonymous variants, please see full germline sequencing data in dbGAP under accession number phs000879.v1.p1.

27

Table S9. Characteristics of patients with CDK1 germline variantsCharacteristic Presence of CDK1 Germline Variant

No (n=638) Yes (n=8)Sex N=531 % N=8 % Male 364 68.5 4 50 Female 167 31.4 4 50Age at Diagnosis N=532 N=8 Median (Range) 58 (31-86) 61 (42-66)Family History N=256 % N=2 % Familial 79 30.9 1 50 Sporadic 177 69.1 1 50Binet Stage N=275 % N=6 % A 15 5.4 0 0 B 169 61.4 4 66.7 C 91 33.1 2 33.3Rai Stage N=252 % N=2 % 0 118 56.8 1 50 I-II 95 37.7 1 50 III-IV 39 15.5 0 0β2-Microglobulin N=256 N=2 Median (Range) 2.7 (1.1 – 16.5) 2.5 (1.3 3.8)Treated at Sampling N=530 % N=8 % No 371 70 6 75 Yes 159 30 2 25IGHV Status N=501 % N=8 % Unmutated 280 55.9 3 37.5 Mutated 221 44.1 5 62.5ZAP70 Positive N=370 % N=4 % No 198 53.5 3 75 Yes 172 46.5 1 25Del17p by FISH N=508 % N=8 % No 444 87.4 8 100 Yes 64 12.6 0 0Del11q by FISH N=508 % N=8 % No 395 77.8 8 100 Yes 113 22.2 0 0Trisomy 12 by FISH N=507 % N=8 % No 431 85.0 7 87.5 Yes 76 14.9 1 12.5Normal Cytogenetics N=507 % N=8 % No 411 81.1 6 75 Yes 96 18.9 2 25Del13q by FISH N=507 % N=8 % No 212 41.8 3 37.5 Yes 295 58.2 5 62.5* In our cohorts, CDK1 status was not significantly associated with any clinical parameters including age at diagnosis.

28

Table S10. Characteristics of patients analyzed for progression-free and overall survival (CLL8 Cohort)

Characteristic Sample not available,

N (%)

Sample available,

N (%)

P value*

Total

N (%)All patients on CLL8 , N 536 281 817Treatment arm

FC 263 (49.1) 146 (52.0) 409

FCR 273 (50.9) 135 (48.0) 408

Age at diagnosis (years) 534 280 814Median (range) 58 (20-78) 57 (30-81) 0.106 58 (20-81)

SexMale 397 (74.1) 210 (74.7) 0.836 607 (74.3)

Binet stage 533 281 817A 25 (4.7) 15 (5.3) 0.442 40 (4.9)

B 349 (65.5) 173 (61.6) 522 (63.9)

C 159 (29.8) 93 (33.1) 252 (31.0)

Presence of B symptoms 533 280 813Yes 236 (44.3) 128 (45.7) 0.695 364 (44.8)

β2-Microglobulin (mg/L) 342 249 591Median (range) 2.9

(0.7-10.2)2.8(0.9-8.0)

0.219 2.9(0.7-10.2)

IGHV mutational status 354 268 622Unmutated 234 (66.1) 158 (59.0) 0.068 392 (63.0)

Mutated 120 (33.9) 110 (41.0) 230 (37.0)

Genetic classification according to hierarchical model by FISH

350 266 616

Del(17p) 41 (11.7) 10 (3.8) 0.003 51 (8.3)

Del(11q) 76 (21.7) 66 (24.8) 142 (23.1)

Trisomy 12 27 (7.7) 34 (12.8) 61 (9.9)

No abnormalities 76 (21.7) 62 (23.3) 138 (22.4)

Del(13q) 130 (37.1) 94 (35.3) 224 (36.4)

Response to treatment 491 268 759Response (CR/PR) 442 (90.0) 255 (95.1) 0.014 697 (91.8)

CR 151 (30.8) 101 (37.7) 0.053 252 (33.2)* PFS analyses were limited to the subgroup analyzed for germline variants, i.e. those with samples available.

29

Table S11. Progression-free survival in patients with germline variants and del(11q), including treatment arm in the multivariate analysis

COX regressionPFS

Univariate comparison

Hazard ratio[HR]

95% Confidence Interval

p value

Lower UpperGermline variant and del(11q)

Germline variant/ del(11q)

vs. No germline variant / del(11q) 1.186 0.633 2.222 NS

No germlinevariant/ del(11q)

vs. No germline variant / No

del(11q)1.701 1.176 2.460 0.005

Germline variant/ No del(11q)

vs. No germline variant / No

del(11q)1.221 0.795 1.875 NS

Germline variant/ del(11q)

vs. No germline variant / No

del(11q)2.018 1.120 3.634 0.019

Treatment armFCR vs. FC 0.582 0.430 0.788 < 0.001

30

Table S12. Progression-free survival in patients with germline variants and del(11q), including treatment arm, del(17p) and unmutated IGHV in the multivariate analysisCOX regressionPFS

Univariate comparison

Hazard ratio[HR]

95% Confidence Interval

p value

Lower UpperGermline variant and deletion 11q

Germline variant/ del (11q)

vs. No germline variant / del(11q) 1.131 0.598 2.137 0.705

No germlinevariant/ del (11q)

vs. No germline variant / No

del(11q)1.383 0.940 2.035 0.100

Germline variant/ No del (11q)

vs. No germline variant / No

del(11q)1.128 0.726 1.754 0.591

Germline variant/ del (11q)

vs. No germline variant / No

del(11q)1.564 0.862 2.838 0.141

Treatment armFCR vs. FC 0.622 0.459 0.843 0.002

IGHV mutational statusUnmutated vs. mutated 1.943 1.373 2.750 < 0.001

del(17p)Present vs. absent 17.711 7.557 41.505 < 0.001

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Table S13. Overall survival in patients with germline variants and del(11q), including treatment arm in the multivariate analysisCOX regressionOS

Univariate comparison

Hazard ratio[HR]

95% Confidence Interval

p value

Lower UpperGermline variant and deletion 11q

Germline variant/ del (11q)

vs. No germline variant / del(11q) 0.647 0.220 1.903 0.429

No germlinevariant/ del (11q)

vs. No germline variant / No

del(11q)1.421 0.831 2.429 0.349

Germline variant/ No del (11q)

vs. No germline variant / No

del(11q)1.154 0.612 2.174 0.658

Germline variant/ del (11q)

vs. No germline variant / No

del(11q)0.920 0.331 2.557 0.872

Treatment armFCR vs. FC 0.533 0.342 0.831 0.005

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Table S14. Summary of sample-level quality controlCohort Sample Size

Discovery CohortOriginal cohort 17,711After removal of non-European samples 13,380After removal of duplicate individuals, samples with restricted use, tumors, and samples from 1st- and 2nd-degree relatives 11,428After removal of samples failing on ≥ 2 quality control metrics 10,251After removal of samples with low call rates (missing ≥ 6% variants) 9,436Extension CohortDiscovery cohort controls + additional extension samples 9,573After removal of samples failing on ≥ 2 quality control metrics 9,571After removal of related samples 9,566

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Table S15. Summary of genotype- and variant-level quality control Genotype Quality Control

Discovery ExtensionAverage number of genotyped sites* per individual 4,510,641 4,177,077Average number of genotyped sites per individual (GQ>=20) 4,241,601 94.04% 3,871,618 92.69%

Variant Quality ControlDiscovery Extension

Overall number of variants 4,755,662 4,834,173Number of variants (missing data <= 6%) 2,948,243 61.99% 3,025,106 62.58%Number of variants (QD > 5) 2,918,754 90.00% 3,002,463 99.25%Number of variants (passing VQSR) 2,697,204 92.41% 2,819,975 93.92%Number of variants (HWE p > .001) 2,693,164 99.85% 2,815,580 99.84%Overall % variants kept 56.63% 58.24%

*A genotyped site is a site in a given individual where at least one non-reference allele exists in the cohort across all patients, and where there is sufficient coverage for a genotype call to be made. Variant-level coverage is the percent of non-missing genotype calls across all individuals in the call set. GQ is the genotype quality score; QD is the quality-by-depth score filter that was applied to indels; VQSR is the Genome Analysis Tool Kit (GATK) variant quality score recalibration procedure for scoring the quality of variants called by the GATK HaplotypeCaller. Variants passing the VQSR filter were kept if they were assigned “PASS” status in the Exome Aggregation Consortium database or, if they were unique to our call set, were assigned “PASS” status by VQSR in our call set. HWE is the Hardy-Weinberg equilibrium test, which was run only on controls.

Percentages show the portion of genotypes or fractions kept from the previous filtering step. We applied a conservative coverage filter (along with other genotype- and variant-level filters) to ensure the quality of variants included in the call set and to avoid potential false positive results from calling artifacts. We found that after independent quality control of the discovery and extension call sets that the variants we observed in ATM and CDK1 were consistent across call sets.

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Table S16. Genes with significant somatic mutations in CLL extension cohort (n=609)* rank gene %pat nmis nstp nspl nind nnon npat nsite p q

1 SF3B1 20.85 111 0 30 3 144 127 35 1.00E-16 1.66E-132 ATM 13.14 64 13 15 30 122 80 111 1.00E-16 1.66E-133 TP53 12.64 65 8 12 2 87 77 65 1.00E-16 1.66E-134 NOTCH1 7.06 2 6 0 36 44 43 13 1.00E-16 1.66E-135 POT1 5.75 29 5 5 1 40 35 32 1.00E-16 1.66E-136 XPO1 4.6 28 0 0 0 28 28 4 1.00E-16 1.66E-137 EGR2 4.11 25 0 0 0 25 25 3 1.00E-16 1.66E-138 BRAF 3.78 21 0 3 0 24 23 14 1.00E-16 1.66E-139 KRAS 2.96 18 0 0 0 18 18 10 1.00E-16 1.66E-13

10 BIRC3 2.79 4 1 0 16 21 17 17 1.00E-16 1.66E-1311 MYD88 2.63 3 13 0 0 16 16 3 1.00E-16 1.66E-1312 NRAS 1.97 12 0 0 0 12 12 4 1.78E-15 2.70E-1213 NXF1 1.64 1 5 1 3 10 10 10 5.88E-15 8.26E-1214 CHD2 4.27 13 3 4 6 26 26 26 1.45E-14 1.90E-1115 IGLL5 2.79 6 0 11 1 18 17 8 3.84E-14 4.67E-1116 MAP2K1 2.13 15 0 0 0 15 13 11 5.23E-14 5.97E-1117 FUBP1 1.64 0 4 2 4 10 10 10 5.63E-14 6.04E-1118 BAZ2A 1.64 0 2 2 6 10 10 10 2.42E-12 2.46E-0919 ZMYM3 1.97 3 3 2 4 12 12 12 1.03E-11 9.90E-0920 RPS15 4.76 28 1 0 0 29 29 12 1.62E-11 1.48E-0821 DDX3X 2.13 9 1 1 2 13 13 13 1.20E-09 1.05E-0622 DYRK1A 1.48 6 0 0 3 9 9 5 1.78E-09 1.48E-0623 ZC3H18 0.99 1 3 0 2 6 6 6 3.22E-09 2.56E-0624 BCOR 1.81 2 2 2 5 11 11 11 7.37E-09 5.61E-0625 SAMHD1 2.3 13 1 4 0 18 14 18 9.29E-09 6.78E-0626 EWSR1 0.82 4 0 0 1 5 5 3 3.70E-08 2.60E-0527 MED12 1.48 9 0 0 0 9 9 5 5.36E-08 3.62E-0528 GPS2 0.66 0 1 0 3 4 4 4 1.01E-07 6.55E-0529 PTPN11 1.15 7 0 0 0 7 7 6 1.22E-07 7.71E-0530 FAM50A 0.66 5 0 0 0 5 4 4 2.02E-07 1.23E-0431 MGA 2.96 5 3 0 10 18 18 17 3.65E-07 2.14E-0432 SPEN 1.31 0 4 0 5 9 8 9 3.75E-07 2.14E-0433 HIST1H1E 1.48 8 1 0 1 10 9 10 5.95E-07 3.29E-0434 CNOT3 0.99 6 0 0 0 6 6 5 8.73E-07 4.69E-0435 IKBKB 0.66 4 0 0 0 4 4 3 9.25E-07 4.82E-0436 ASXL1 0.99 0 4 0 2 6 6 6 1.05E-06 5.33E-0437 ELF4 0.66 3 1 0 0 4 4 4 2.37E-06 1.17E-0338 GNB1 0.82 5 0 0 0 5 5 2 2.46E-06 1.18E-0339 GNB2L1 0.66 5 0 1 0 6 4 6 5.22E-06 2.44E-0340 GLI2 1.31 6 3 0 0 9 8 9 5.92E-06 2.70E-0341 TRAF2 0.66 2 2 0 1 5 4 5 1.55E-05 6.90E-0342 SETD2 0.99 1 3 0 3 7 6 7 2.04E-05 8.87E-0343 IRF4 1.97 13 0 0 0 13 12 4 2.94E-05 1.23E-0244 FBXW7 1.97 13 3 0 0 16 12 14 2.98E-05 1.23E-0245 KIAA1755 0.66 3 1 0 0 4 4 4 3.04E-05 1.23E-0246 C11orf41 1.15 5 2 0 0 7 7 6 4.19E-05 1.66E-0247 IKZF3 2.13 13 0 0 0 13 13 1 4.63E-05 1.80E-0248 KLHL6 1.31 10 1 0 0 11 8 10 5.61E-05 2.09E-0249 ARID2 0.99 1 0 1 4 6 6 6 5.62E-05 2.09E-0250 COL4A4 0.33 0 1 1 0 2 2 2 6.78E-05 2.47E-0251 CYP8B1 0.49 2 1 0 0 3 3 3 9.53E-05 3.41E-02

35

52 BRCC3 1.15 3 0 1 3 7 7 7 1.55E-04 5.45E-0253 ZNF296 0.33 0 2 0 0 2 2 2 1.79E-04 6.17E-0254 TFCP2 0.66 4 0 0 0 4 4 4 2.15E-04 7.27E-0255 ETV1 0.99 6 0 0 0 6 6 5 2.32E-04 7.71E-0256 CHEK2 0.66 3 0 0 1 4 4 4 2.38E-04 7.75E-0257 DNAH7 1.64 6 1 2 1 10 10 10 3.06E-04 9.66E-0258 INO80 0.33 1 0 1 1 3 2 3 3.07E-04 9.66E-02*MutSig2CV16 analysis of somatic mutations in 609 patients in the validation cohort identified 58 significantly mutated somatic driver genes (q<0.1). Patients not included in the MutSig2CV analysis were missing somatic data (n=28), were extreme outliers for somatic mutation rate (n=6), or were excluded for quality control reasons (n=3). “Nsil” indicates the number of silent mutations, “nmis” indicates the number of missense mutations, “nstp” indicates the number of stop mutations, “nspl” indicates the number of splice site mutations, “nind” indicates number of indels, “nnon” indicates the total number of non-synonymous mutations (sum of missense, stop, splice, and indel mutations), “npat” indicates the number of unique patients with mutations, “%pat” indicates the percentage of patients in the cohort with non-synonymous mutations, and “nsite” indicates the number of unique mutated sites found in each gene.

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FIGURES

37

Figure S1. Overview of quality control and analysis workflow. Workflow shown illustrates the steps taken to create and analyze the final call set, consisting of the discovery case cohort (blue figures), extension case cohort (purple figures), and control cohort (grey figures). Individuals carrying a germline variant in a gene of interest are shown with a red dot. The presence of a variant is also indicated by the red bar on the grey DNA strand. An example of a fully genotyped region from a gene of interest is shown for four patients, with the reference sequence indicated. Bases in red indicate germline variants. After inferring the ethnicity of all samples in the call set using principal components analysis, non-European samples were removed, along with samples identified as duplicates or as cryptically related individuals. Samples with at least two outlier QC metrics were also removed. After all sample removal (figures with broken lines), the final call set consisted of 646 CLL cases (516 cases in the discovery case cohort and 130 in the extension cohort) and 8,920 controls. Variants that did not pass the GATK Variant Quality Score Recalibration workflow were removed, along with variants violating Hardy-Weinberg equilibrium, variants with poor coverage, and common variants with minor allele frequency > 1%. These variants were deemed likely artifacts (or outside the scope of this study as in the case of common variants). All observed genotypes in all samples corresponding to these variants were removed (removal of an entire variant from the call set is represented by light grey bases shown within the boxed column). Additionally, individual genotype calls for specific variants in specific samples were filtered when their genotype quality scores were < 20 (i.e., when confidence in the genotype call was low). Only high-confidence genotype calls, whether reference or non-reference, were included to improve association testing. Individually removed genotypes are also shown as grey bases within individually boxed regions, shown here for two representative cases. Variant sites were annotated for their predicted functional impact. All bi-allelic coding variants, except synonymous variants and low confidence loss-of-function variants, were then included in the association testing. For each gene with at least one rare germline variant, we independently tested the burden of rare, non-synonymous coding variants in the CLL cases against the burden in controls. This was performed both for the discovery case cohort (left panel under association testing) and, as validation, for the discovery case cohort combined with the extension cohort (right panel). Burden testing was conducted using EPACTS with the “b.collapse” test, producing a p-value for each gene. We corrected for multiple hypothesis testing using the Benjamini-Hochberg procedure and considered genes with q≤0.05 as significantly associated with CLL.

38

A.

B.

Figure S2. The gene-based association test is well calibrated after controlling for residual population stratification. A. Principal components (PCs) computed on cases and controls show residual population structure within the ethnically matched European sample set. To eliminate potential spurious associations due to population substructure, we used the first three PCs (plotted in pairs in the left and right panels) as covariates in the association testing. B. Quantile-quantile plot of the p-values obtained from the exome-wide, gene-based association test for rare, non-synonymous coding region variants.

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Figure S3. Rare germline variants in CDK1 are enriched in the CLL discovery cohort. A. Rare CDK1 germline variants observed in CLL lie in the protein kinase domain of CDK1. Variants found in CDK1 are displayed with the total number of cases (above the protein track) and controls (below), along with the corresponding percentages in their respective cohorts. B. 3D rendering of the CDK1 protein highlights the recurrent p.R59C variant (purple) and singletons (blue) in CLL cases. Pink residues indicate published somatic mutations in other cancers.

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Figure S4. PolyPhen2 HDIV scores for rare ATM missense mutations in cases versus controls. Compared to scores from the control cohort, PolyPhen2 scores in cases were significantly shifted towards the deleterious end of the scale (0 indicating minimal effect on protein function, and 1 indicating substantial effect on protein function).

41

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Figure S5. Association of del(11q) status, but not ATM germline variants, with progression-free survival (PFS).

43

Figure S6. No association of ATM germline variants or del(11q) status with overall survival (OS).

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Figure S7. Top 25 genes with significant somatic mutations in CLL extension cohort (n=609). Somatic mutation information is shown for the top 25 significantly mutated genes (q<0.1) analyzed through MutSig2CV. Genes are ranked by significance from top to bottom. For each patient sample (arranged along the x-axis), the presence of a mutation is marked by a colored bar (mutation type indicated in legend). Grey indicates the absence of a somatic mutation in a given gene. Top panel shows the mutation rate per Mb for each sample, with synonymous and non-synonymous mutation status indicated by green and orange coloring, respectively. Left panel shows the absolute number of somatic mutations observed in a given gene, along with the percentage of the cohort affected. Bottom panel summarizes the mutation contexts of all the mutations found in each patient.

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