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WGS in MBL and ultra-stable CLL
in HC-MBL_2, which also harbored the FBXW7 mutation. Two somatic non-synonymous variants were identified in 2 different PMN samples: an ATM variant (p.R337C; VAF 20%) detected in an LC-MBL case, frequently reported and probably with a low functional impact, and a TP53 mutation (p.G245A, VAF 3%) found in a HC-MBL case, previously reported in several human cancers and lym- phomas40 (Online Supplementary Table S8).
Non-coding variants in MBL and ultra-stable CLL target genes in pathways relevant to CLL pathogenesis
Coding and non-coding regions enriched for mutations were detected using Fishhook29 in 10 kilobase windows across the genome and with compensation for replication timing. In line with previous findings,9 the analysis revealed highest mutational enrichment in the IG loci and within sites known to be recurrently affected by off-target somatic hypermutation (e.g. BTG2, BCL6 and TCL1A) (Online Supplementary Figure S1).
Funseq2,41 a bioinformatics tool investigating the link- age between NCVs and target genes using integrated bisulfite sequencing, ChIP-Sequencing, and RNA-sequenc- ing data from the Roadmap Epigenomics Project, was used for the examination of the NCVs. This analysis revealed a total of 1517 variants in the MBL/CLL samples and 39 in the PMN samples. After stringent filtering, 106 NCVs of potential relevance to MBL and CLL were iden- tified (Online Supplementary Table S9): 29 in LC-MBL (aver- age 4.8), 45 in HC-MBL (average 9), and 32 in CLL samples (average 6.4), respectively; only 4 NCVs were found in 2 PMN samples. Since we intentionally selected for NCVs in transcription factor (TF) highly occupied regions (see Online Supplementary Appendix), not unexpectedly most variants were located in gene promoter sites (Figure 4A). Twenty-nine variants (26.4%) concerned 16 cancer-associ- ated genes and were evenly distributed amongst the 3 entities: 9 in LC-MBL samples, 11 in HC-MBL, and 9 in ultra-stable CLL samples. Three of these cancer-associated genes were recurrently targeted: 9 variants concerned the BTG2 gene in 4 samples (2 CLL, 1 HC-MBL and 1 LC- MBL), 5 variants involved the BCL6 gene in 2 samples (1 HC-MBL and 1 LC-MBL), and 2 variants targeted the BIRC3 gene in 2 samples (1 CLL and 1 HC-MBL). We also identified 6 variants concerning the ST6GAL1 gene in 3 CLL samples and the same NKIRAS1-related variant in 2 CLL samples (Figure 4B). Moreover, pathway analysis with Enrichr31 showed that 30 of 110 (27.3%) of the vari- ants targeted genes were implicated in key CLL pathways and cellular processes, such as the PI3K-AKT pathway (TCL1A, CCND1, BCL2, PKN1, DDIT4 and SGK3) (P<0.05), the NF-κB pathway (BIRC3, BCL2 and PLAU) (P<0.05) and the spliceosome machinery (DDX46 and HSPA2). In most of these cases (22 of 30, 73.3%), the vari- ants were located at promoter sites. Comparison to the series by Puente et al.9 identified 4 common gene targets: BTG2, BCL6, BACH2 and TCL1A; none of our samples carried variants affecting either the 3’ UTR of the NOTCH1 gene or the PAX5 gene enhancer.
We also analyzed the predicted impact of the NCVs on TF binding and found that 72 of 110 (65.5%) of the vari- ants could result in a motif-breaking event (LC-MBL: n=21, HC-MBL: n=33, ultra-stable CLL: n=18) (Online Supplementary Table S10). We subsequently investigated genes and gene pathways that may be affected by such TF motif breaks. In 55 of 72 (76.4%) cases, variants disrupted
a DNase I hypersensitive site, while enrichment analysis of the implicated target genes using Enrichr31 led to the identification of genes participating in pathways relevant to CLL pathogenesis, such as the MAPK, WNT and AP-1 pathways (P<0.0005) (Online Supplementary Table S11).
Moreover, we examined the potential relation of the NCVs that affect TF binding to AID activity by checking if they occurred in the known hotspots (WRCY, RGYW, WA, TW). According to our findings, 21 of 72 (29.2%) NCVs were located at AID hotspots. Gene enrichment analysis of the remaining 51 target genes revealed similar pathways as in the original analysis (namely AP-1 and DNA damage response pathways).
Shared mutations between CLL and polymorphonuclear cells indicate that somatic variants can arise before CLL onset
Shared mutations between MBL/CLL samples and their respective PMN samples were identified in all samples irrespective of origin. Regarding exonic mutations, the same synonymous GSE1 mutation was found in an HC- MBL case and its paired PMN sample with comparable VAF (28% vs. 26%). In addition, a LC-MBL sample and its paired PMN sample carried an identical mutation within the ncRNA gene LOC339874, though with different VAF (16% vs. 31%). In the case of non-exonic mutations, 179 shared NCVs were identified between MBL/CLL and PMN samples (Online Supplementary Table S12); the aver- age number per sample was 15.8 for LC-MBL, 8.2 for HC- MBL, and 9 for ultra-stable CLL (range: 2-34), respectively. Most of these mutations were intergenic (128 of 179, 71.5%) (Figure 4C). Interestingly, 6 NCVs were recurrent- ly found in more than one MBL/CLL-PMN sample pair: 3 were intergenic and the other 3 were intronic (Online Supplementary Table S13). Finally, we also examined the mutational signatures for shared mutations between MBL/CLL and PMN samples but did not observe clear cor- relations with any signature (data not shown).
In order to exclude the possibility of contamination of the PMN cell fraction by MBL/CLL DNA, we designed allele-specific primers (Online Supplementary Table S14) and performed PCR amplification of the clonotypic IGH gene rearrangement in both the MBL/CLL and the respective PMN samples in 11 of 16 cases with available material. We identified the clonotypic rearrangement in all 11 MBL/CLL samples but in none of the corresponding PMN samples examined, effectively ruling out the possibility that the observed results were due to contamination.
Somatic copy-number analysis
sCNA analysis was performed in 3 samples from each entity and their respective PMN control samples, as well as in 4 additional PMN samples from 1 HC-MBL and 3 LC-MBL cases. In total, 16 sCNAs were identified in the MBL/CLL samples (average: 1.8, range: 1-6): 7 in LC-MBL, 4 in HC-MBL, and 5 in the ultra-stable CLL samples, all but one concerning deletion events (Online Supplementary Table S15). Of the recurrent cytogenetic aberrations included in the Döhner hierarchical model,42 del(17p), del(11q) and trisomy 12 were not identified in any of the samples, whereas del(13q) was detected in 7 of 9 MBL/CLL cases (2 LC-MBL, 3 HC-MBL and 2 CLL cases). FISH analysis gave concordant results in 5 of 7 cases where data from both techniques were available; in the remaining 2 cases del(13q) was detected with a single
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