The recombinome of IKZF1 deletions in B-ALL

Bruno Lopes 
(

brunolopes@id.uff.br
)
Instituto Nacional de Cancer
https://orcid.org/0000-0003-1072-470X
Claus Meyer 
Johann Wolfgang Goethe-Universität
Heloysa Bouzada 
Instituto Nacional de Câncer
Marius Külp 
Goethe-University of Frankfurt
Ana Luiza Maciel 
Instituto Nacional de Câncer
Patrizia Larghero 
Goethe-University of Frankfurt
Thayana Barbosa 
Instituto Nacional de Cancer
Caroline Poubel 
Instituto Nacional de Câncer
Caroline Blunck 
Instituto Nacional de Cancer (INCA)
Nicola Venn 
Children's Cancer Institute Australia for Medical Research
Luciano Dalla-Pozza 
The Children's Hospital at Westmead
Draga Barbaric 
Sydney Children’s Hospital
Chiara Palmi 
o Ricerca M. Tettamanti, University of Milano-Bicocca
Grazia Fazio 
o Ricerca M. Tettamanti, University of Milano-Bicocca
Claudia Saitta 
Centro Ricerca M. Tettamanti, Pediatrics, University of Milano Bicocca
https://orcid.org/0000-0002-
5842-7774
Thais Aguiar 
Arthur Siqueira Cavalcanti Hematology Institute (HEMORIO)
Mecneide Lins 

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 Instituto de Medicina Integral Prof. Fernando Figueira (IMIP)
Maura Ikoma-Colturato 
Hospital Amaral Carvalho
Marcia Schramm 
Prontobaby Hospital da Criança and Hospital do Câncer I, INCA
Eduardo Chapchap 
Hospital Israelita Albert Einstein
Giovanni Cazzaniga 
Centro Ricerca M. Tettamanti, Università di Milano Bicocca
Rosemary Sutton 
Children's Cancer Institute
https://orcid.org/0000-0002-0188-6005
Rolf Marschalek 
Goethe-University of Frankfurt
https://orcid.org/0000-0003-4870-3445
Mariana Emerenciano 
Instituto Nacional de Câncer
https://orcid.org/0000-0003-2337-8420

Article

Keywords: IKZF1 deletion, acute lymphoblastic leukemia, B-ALL, breakpoints, MLPA, multiplex PCR

Posted Date: March 21st, 2023

DOI: https://doi.org/10.21203/rs.3.rs-2697729/v1

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Abstract
IKZF1 deletions are associated with an increased risk of relapse in B-cell precursor acute lymphoblastic
leukemia (B-ALL), and their accurate detection has great clinical impact. Here, we included four
international cohorts of pediatric and adult patients with B-ALL, and reviewed literature to illustrate the
recombination map of IKZF1 deletions, with a focus at non-recurrent deletions. We provide a substantial
basis for the improvement of diagnostic methods based on MLPA and multiplex PCR for the
identification of IKZF1 deletions, and also demonstrate that rare IKZF1 deletions increase the incidence of
relapse in these patients. Of note, non-recurrent deletions comprised a wide range of alterations, but the
majority were Δ1 and Δ1–3. They were often associated with reciprocal IKZF1 fusions. So far, a total of
23 IKZF1 gene fusions were identified in B-ALL. We also verified the occurrence of the heptamer sequence
(E-value: 9.9 x 10− 9) and an enrichment of GC nucleotides (71% versus 56%; P value = 4.9 x 10− 3)
exclusively within breakpoint clusters, suggesting that RAG recombination and TdT activity may promote
the majority of IKZF1 deletions, although rare types of alterations may be associated with other
molecular mechanism of leukemogenesis, such as microhomology-mediated end joining.

Introduction
The occurrence of IKZF1 deletions increases the risk of relapse in patients with B-cell precursor acute
lymphoblastic leukemia (B-ALL) 1. Therefore, the development of methods for rapid and accurate
detection of this alteration is clinically relevant. Traditionally, the identification of IKZF1 deletions has
been based on multiplex ligation-dependent probe amplification (MLPA), although distinct approaches
are also available, including array-based comparative genomic hybridization (array CGH), optical gene
mapping 2, and whole genome sequencing (WGS). The latter techniques provide a broad panorama of
copy number or structural alterations, but either the validation of results is advisable or the high costs
limit the application in routine diagnosis worldwide. Multiplex (M)-PCR is a feasible and accurate
complementary method for the screening and validation of the majority of IKZF1 deletions, specially
recurrent intragenic ones: Δ2–3, Δ2–7, Δ2–8, Δ4–7, Δ4–8 3,4. In addition, several studies describe the
occurrence of non-recurrent IKZF1 deletions in B-ALL, and proved the importance of these rare alterations
for determining patient outcome 5. For instance, exon 1 deletions and other non-recurrent deletions are
evidenced by MLPA, but lack the possibility of verification by M-PCR. Therefore, here we performed an
international effort to include a large cohort of B-ALL samples, and review the literature to provide the
landscape of IKZF1 deletions in this subtype of leukemia. Our study presents a special focus on non-
recurrent structural alterations encompassing this gene, which was used both for illustrating the genetic
profile of rare lesions and for providing the basis for upgrading current methods to determine IKZF1
status. In addition, we analyze sequence features underlying different types of IKZF1 deletions to explore
possible mechanisms of leukemogenesis.

Materials And Methods

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Patients
This study included pediatric and adult patients with B-ALL. Four patient cohorts were used for analyses:
two from Australia (n = 506 and n = 161), one from Brazil (n = 277), and another from Italy (n = 596). A
total of 1,540 patients were analyzed for the identification of IKZF1 deletions. Participating centers
obtained local ethical committee approval and written informed consent in accordance with the
Declaration of Helsinki. This study was approved by human ethics committees (CAAE
#33709814.7.0000.5274 and SCHN 2019/ETH06161).

Identification of IKZF1 deletions

Copy-number alterations (CNAs) within IKZF1 locus were screened using SALSA MLPA P335. We
considered suspicious deletions when the peak ratio for one MLPA probe was below 0.80. Then, we used
other approaches for confirming these alterations. Suspicious recurrent intragenic deletions of IKZF1
(Δ2–3, Δ2–7, Δ2–8, Δ4–7, Δ4–8) were validated by M-PCR. The remaining samples were further
analyzed with SALSA MLPA P202-C1 or digital MLPA D007 ALL probe mix. Here, deleted loci were
assumed when peak ratios were below 0.65, or when peak ratios were below 0.80 and resided next to at
least one probe peak ratio below 0.65. Biallelic deletions were assumed when peak ratio was below 0.40.
We defined recurrent IKZF1 deletions as those encompassing Δ1–8, Δ2–3, Δ2–7, Δ2–8, Δ4–7, Δ4–8,
while non-recurrent deletions included all the remaining alterations. MLPA analysis was performed with
GeneMarker v2.2.0 (SoftGenetics) and Coffalyser (MRC Holland).

Next-generation sequencing of IKZF1 non-recurrent

deletions
The Illumina DNA Prep with Enrichment was used for sequencing non-recurrent IKZF1 deletions. The
enrichment probes were designed using the DesignStudio Assay Design Tool (Illumina) and included
overlapping probes within IKZF1, as well as breakpoint clusters (BC) outside this gene. The location of
DNA capture probes used in this enrichment protocol is described in Supplementary Table S1. The probes
covered 500 bp up- and downstream each region. The library was prepared according to the
manufacturer’s protocol (Illumina). Briefly, the genomic DNA was fragmented and tagged with adapter
sequences using the Nextera transposon technology. After purification, the tagmented DNA was amplified
and sample-specific indexes were added during the PCR. The tagmented and indexed samples were
pooled in equal amounts, hybridized with biotinylated probes targeting IKZF1 as well as its BC, and
subsequently captured using streptavidin magnetic beads. This step was performed a second time to
increase the specificity. After a final step of amplification and purification, the final library was quantified
using Qubit (Invitrogen, Carlsbad, CA, USA), and the quality was verified using a 4200 TapeStation
(Agilent Technologies, Palo Alto, CA, USA). After denaturation and dilution, the library was loaded onto the
reagent cartridge and sequenced with MiSeq platform. Following the sequencing, Miseq reporter software
generated fastq files containing raw paired-end reads trimmed from Illumina adapters.
Structural variant identification
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The analysis of structural variants and breakpoints was performed in several steps. First, we checked the
quality of the fastq files with FastQC 6. In the second step, we processed paired-end sequence reads with
SpeedSeq 7, which is designed for rapid whole-genome variant detection and interpretation. In this
analysis, we used the module “SpeedSeq-align”, that i) mapped paired-end fastq files with BWA-MEM 8
using the human genome reference GRCh38; ii) marked duplicates, extracted discordant-reads and split-
reads with SAMBLASTER 9; and iii) sorted by position and indexes the BAM files with Sambamba 10. The
following three sorted and indexed BAM files plus their corresponding .bai files were obtained: i) a BAM
file containing the full alignments, ii) a BAM file containing discordant read-pairs and iii) a BAM file
containing split-reads. In addition, we used these three BAM files with LUMPY express version 0.2.13 11 to
identify structural variants. The output is a .vcf file containing the information about possible structural
variations in each sample. All the obtained files were visualized on Integrative Genomics Viewer (IGV) 12
to identify the structural variants and to obtain the breakpoint sequences.

Literature data

We revised previously published data regarding IKZF1 deletions in B-ALL for compiling breakpoint
sequence information of these deletions. We used the term "IKZF1 deletion" AND "acute lymphoblastic
leukemia" for this search on PubMed, which provided 207 articles. Then, we manually inspected all
manuscripts to extract patient and breakpoint sequence data. This database was restricted to breakpoint
sequence information identified at DNA or RNA level, and the respective B-ALL patient data. The
breakpoint sequences retrieved from literature were mapped using BLASTN 13 and the hg38 human
reference genome.

Identification of BC and genetic signatures near the

breakpoints
After retrieving the breakpoint coordinate of every breakpoint in each sample, we identified BC (5'BC or
3'BC) when at least two breakpoints occurred less than 25 bp from each other. For the agnostic motif
search, the sequences of each BC or individual breakpoints plus its 25 pb flanking sequences were
analyzed using MEME (E-value < 10− 4) 14. Next, we identified the former motif inside the breakpoint
region of clusters and individual samples using FIMO (P-value < 10− 2) 15. We also annotated the presence
of filler DNA (additional nucleotides) or microhomologies at the junctions of IKZF1 deletions.

Novel M-PCR for the identification of IKZF1 deletions

The primers were settled to flank the most common BC of rare and recurrent IKZF1 deletions. Some of
them were retrieved from published PCR methods 3,4, and complementary primers were designed at the
vicinity of previously uncovered BC (Supplementary Table S2). The M-PCR was divided into two
independent reactions, covering both recurrent (∆2–7, ∆2–8, ∆4–7 and ∆4–8) and rare (∆1, ∆1–2, ∆1–
3 and ∆2–3) IKZF1 deletions. The reaction was performed using the Platinum™ Taq DNA Polymerase
High Fidelity (Invitrogen) in a volume of 25 uL. A total of 100 ng DNA was added to each reaction, which

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also contained 2.5 mM MgCl2 and 0.20 uM of each primer mix. The amplification was performed with an
initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at
60°C for 30 s, and extension at 72°C for 4 min. The final extension occurred at 72°C for 12 min. Then, M-
PCR products were separated by agarose gel electrophoresis, and stained with GelRed to allow
visualization of amplicons by ultraviolet light.

Chromatin immunoprecipitation (ChIP)

The ChIP was performed using a total of 4x107 NALM-6 cells (ACC 128, DSMZ). The anti-RAG1
monoclonal IgM antibody (sc-377127) or 1 µg isotype control IgM (sc-3881) were used for ChIP. The
quantification was performed by quantitative PCR (qPCR) in triplicates with the StepOnePlus Real Time
PCR system (Applied Biosystems) and ORA qPCR Green ROX H Mix (HighQu QPD020), using the primer
pairs described in Supplementary Table S3. For comparison of enrichment, percent input values were
calculated according to 100 x 2(Cq(adjusted input) - Cq(IP)) displaying the percentage of RAG1 compared to
IgM-bound DNA out of total DNA with adjusted input = Cq(input) − 2. IgM served as an antibody negative
control, while areas covered by control primer pairs (Ctrl01, 02 and 03) were used as negative controls for
RAG1 binding (no RAG1 binding motif identified).

Survival analysis
The B-ALL patients were divided into three comparative groups with i) non-recurrent IKZF1 deletions
versus ii) IKZF1 wild-type or iii) IKZF1 Δ4–7. Reference groups 2 and 3 had twice the number of group 1.
They were matched by leukemia subtype, treatment protocol, age group, WBC group, and sex. The
number of years between diagnosis and death or last follow-up was collected for every patient to
calculate the overall survival (OS). A Kaplan–Meier curve was fitted for each group, which were compared
by means of the log-rank test. Additionally, the time between diagnosis and relapse was calculated for the
analysis of the cumulative incidence of relapse (CIR). The CIR between IKZF1 groups was compared by
means of the Gray test. The analyses were performed using the survminer and tidycmprsk R packages.

Statistical analyses
Data analyses were performed using R studio (version 4.2.1). Categorical variables were compared by
Pearson's Chi-squared test. Continuous data were analyzed by variance test, followed by Student’s t-test
(unpaired and two-tailed). P values < 0.05 were considered significant. Data manipulation and
visualization were performed using tidyverse packages. The majority of plots were generated using
ggplot2 16. In regard to genomic data, the Protein Paint (proteinpaint.stjude.org/FusionEditor) was used
to illustrate IKZF1 gene fusions. Additionally, Bioconductor packages were used for preparing the data
(BSgenome.Hsapiens.UCSC.hg38, version 1.4.4) and for illustrating genomic maps (Gviz, version 1.40.1)
17
.

Reference sequences

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We used the following transcript identification for the description of genes and respective exon/intron
numbering in this study: ABCA13 (NM_152701.5), ADSS1 (NM_152328.5), CDK2 (NM_001798.5), CEP170
(NM_014812.3), COBL (NM_015198.5), DDC (NM_001082971.2), DLG2 (NM_001142699.3), DNAH14
(NM_001367479.1), ENSG00000278996 (NR_146144.1), ETV6 (NM_001987.5), FIGNL1
(NM_001287492.4), GRB10 (NM_001350814.2), IKZF1 (NM_006060.6), LOC105377979 (XR_942936.3),
KMT2A (NM_001197104.2), NUTM1 (NM_001284292.2), PAX5 (NM_016734.3), SETD5
(NM_001080517.3), SPATA48 (NM_001161834.3), STIM2 (NM_020860.4), STK38L (NM_015000.4),
TRPV2 (NM_016113.5), TYW1 (NM_018264.4), ZEB2 (NM_014795.4), and ZPBP (NM_007009.3).

Results
Frequency of IKZF1 alterations
A total of 1,540 B-ALL samples, which represent a combination of four different cohorts, were screened
for IKZF1 deletions: two cohorts from Australia (n = 667; n1 = 506 and n2 = 161), one from Brazil (n = 277)
and another from Italy (n = 596). In this study, the identification of IKZF1 deletions was performed by a
combination of several methodologies. A total of 33 samples initially characterized by MLPA (P335)
presented suspicious non-recurrent IKZF1 deletions. After validation using the MLPA P202 probemix, M-
PCR and/or NGS, 27% demonstrated to be true positive results, while 73% were reclassified as either
IKZF1 wild-type (n = 12) or IKZF1 recurrent deletion (n = 12). We also assessed the peak ratios of MLPA
P335 probes expected to indicate monoallelic deletions (median: 0.63; range: 0.48–1.00) or no alterations
(median: 0.97; range: 0.57–1.74) (Fig. 1a), as well as the peak ratios of MLPA P202 probes expected to
indicate monoallelic deletions (median: 0.64; range: 0.15–1.11) or no alterations (median: 0.98; range:
0.59–1.53) (Fig. 1b). We noticed that a cutoff value at 0.80 allowed for the interpretation of most non-
recurrent IKZF1 deletions, although false positive deletions may be identified. In summary, the overall
frequency of IKZF1 deletion was 17%. Non-recurrent IKZF1 deletions were found in 1.2% of the total
cohort, which represents 7% of all IKZF1 deletions (Fig. 1c; Supplementary Table S4). The clinical
characteristics of these patients are summarized in Supplementary Table S5.

Considering the rarity of some IKZF1 deletions, we reviewed literature to compile information of B-ALL
patients carrying this genetic alteration. We included breakpoint sequences available at DNA level (n = 
478) and RNA level (n = 16). All data from the current work and literature are summarized in
Supplementary Table S6. First, we noticed that non-recurrent IKZF1 deletions comprise miscellaneous
alterations: Δ1, Δ1–2, Δ1–3, Δ1–4, Δ1–5, Δ1–7, Δ2, Δ3, Δ4–6, Δ5, Δ5–7, Δ5–8 and Δ6–8 (Fig. 1d). Most
of them (82%) are associated with the loss of IKZF1 promoter. In addition, a total of 23 IKZF1 gene
fusions were identified with the following partner genes: ABCA13, ADSS1, CDK2, CEP170, COBL, DDC,
DLG2, DNAH14, ENSG00000278996, ETV6, FIGNL1, LOC105377979, KMT2A, NUTM1, PAX5, SETD5,
SPATA48, STIM2, STK38L, TRPV2, TYW1, ZEB2, and ZPBP (Fig. 1e; Supplementary Fig. S1). Seven of
them were exclusively found in this work (Supplementary Table S7). Of note, only 9 out of 36 fusions
were in-frame, while the remaining produced head-to-head (e.g. ZPBP::IKZF1), tail-to-tail (e.g.

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IKZF1::COBL) fusions, or even rearrangements with genes encoding long non-coding RNAs (e.g.
IKZF1::ENSG00000278996), which may not produce functional chimeric IKZF1 proteins.

The sequence data allowed us to investigate BC of IKZF1 deletions. A total of 24 BC were identified, half
of which were associated with 5' and the other half with 3' breaks (Fig. 2a). The detailed genomic
location of each cluster is provided in Supplementary Table S8. The most frequent clusters were 5'BC09,
5'BC12, 3'BC07, and 3'BC10, which are located within IKZF1 intron 1, intron 3, intron 7, and ~ 11.6 kb
downstream this gene, respectively. DNA lesions at these sites promote the most recurrent IKZF1
deletions: Δ2–7 (5'BC09–3'BC07), Δ4–7 (5'BC12–3'BC07), Δ2–8 (5'BC09–3'BC10), and Δ4–8 (5'BC12–
3'BC10). Conversely, the majority of DNA breaks triggering non-recurrent IKZF1 deletions were not located
at BC. Nevertheless, we observed four clusters (5'BC01, 5'BC02, 3'BC01, and 3'BC02) associated with rare
rearrangements. Of note, the majority of ∆1 (9 out of 13) were associated with 3'BC01 (Fig. 2b).

MLPA upgrade for the identification of IKZF1 deletions

As 76% of non-recurrent IKZF1 deletions (29 out of 38 sequences) encompassed exon 1, we aimed at
identifying the best region for positioning MLPA probes for accurate detection of exon 1 deletions. We
observed one commonly deleted region within IKZF1 exon 1 (chr7:50,275,701–50,306,410). Because the
area surrounding its promoter has high GC content (> 50%), we considered a shorter region
(chr7:50,275,701–50,302,000) ideal for designing MLPA probes for detection of exon 1 deletions
(Fig. 3a).

M-PCR upgrade for detection of IKZF1 deletions

To cover the detection of most IKZF1 deletions in a M-PCR assay, we designed primers in the vicinity of
BCs with the highest incidence of DNA breaks in B-ALL. The amplification strategy was based on two
separate reactions: one assay focused on the detection of rare fusions encompassing the first exons of
IKZF1 (∆1, ∆1–2, ∆1–3 and ∆2–3), while the second one aimed at identifying the most recurrent
deletions (∆2–7, ∆2–8, ∆4–7 and ∆4–8). Of note, our novel M-PCR extends the coverage of previous
assays 3,4, as it detected recurrent deletions with breakpoints within 3'BC10, which is often associated
with IKZF1 ∆2–8 and ∆4–8. We selected samples covering all types of recurrent IKZF1 deletions to
standardize and validate this novel assay (Fig. 3b).

Genetic signatures of IKZF1 deletions

We searched for DNA motifs, additional nucleotides and microhomologies associated with IKZF1
deletion. After mapping the genomic coordinates of individual breakpoints and clusters, we retrieved their
respective 25 bp flanking sequences for the identification of breakpoint consensus sequences. Of note,
22 out of 24 BC of IKZF1 deletions had the 5’-CACWGTGKG-3’ sequence (W and K stands for A/T and
G/T, respectively; E-value: 9.9 x 10− 9; Fig. 4a). In addition, recurrent IKZF1 deletions displayed a similar
motif (5’-CACASTG-3’, S means G or C; E-value: 1.1 x 10− 9), which was identified in 58% (529 out of 904)
of these breakpoint sequences (Fig. 4b). On the other hand, non-recurrent IKZF1 deletions presented no
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significant motifs in the vicinity of breakpoints (Supplementary Fig. S2). Since the former DNA motifs are
similar to the canonical heptamer sequence of RAG1-mediated recombination (the first segment of the
recombination signal sequence, RSS), we evaluated the occurrence of cryptic heptamer across BC and
individual breakpoint sequences. An identical heptamer sequence was identified in 7 out of 24 BC
(5'BC02, 5'BC10, 5'BC12, 3'BC02, 3'BC03, 3'BC05, 3'BC09), although most of them presented one or two
bases mismatch (Fig. 4c). Additionally, this DNA motif was identified in 90.6% (874 of 965 sequences) of
IKZF1 deletions: 92.7% (837 of 903) of recurrent, and 59.7% (37 of 62) of non-recurrent deletions. This
result indicates that off-target RAG-mediated recombination may explain most IKZF1 deletions, although
not all these genetic events.

To further explore the role of cryptic RSS on the establishment of IKZF1 deletions, we performed a
quantitative ChIP mapping of RAG1 occupancy within breakpoint regions using the Nalm6 leukemia cell
line (Supplementary Fig. S3). Although we observed an enrichment of RAG1 within 5’BC03 (P value = 
0.001 by t-test), its occupancy levels were similar to control regions (without any breakpoint promoting
IKZF1 deletions) in the remaining breakpoint clusters of recurrent and non-recurrent IKZF1 deletions, as
well as in sporadic breakpoint sites. Our results suggest that a time-dependent RAG1 expression and
activation might have suppressed our ability to demonstrate its role in the establishment of IKZF1
deletions.

One common characteristic of structural alterations is the occurrence of additional nucleotides or

microhomologies at DNA junctions. Therefore, we inspected whether the frequency of those signatures
varies depending on the type of IKZF1 deletion. Recurrent deletions had a divergent spectrum of
signatures compared to non-recurrent IKZF1 alterations (Fig. 4d; P value = 4.99 x 10− 4 by Pearson's Chi-
squared test), and displayed a higher frequency of filler DNA at junctions (93% versus 58%, respectively).
This observation was also true when we compared different deletion ranges (Fig. 4e). Additional DNAs at
breakpoint junctions were common in recurrent intragenic deletions (range: 86–98% of sequences), but
observed in only half of complete IKZF1 deletions. Non-recurrent IKZF1 deletions displayed an
intermediate number of additional nucleotides. Further, we explored whether the occurrence of
breakpoints at clusters or not influence the distribution of genetic signatures at junctions (Fig. 4f; P value 
= 4.99 x 10− 4). In this scenario, the higher the frequency of filler DNA, the greater the number of
breakpoints within clusters (95%, 83%, and 47% for deletions ranging between two clusters, one cluster
and no clusters, respectively).

Although the frequency of filler DNA varied depending on the type of IKZF1 deletion, the size of these
additional sequences was similar between all comparison groups (Fig. 4g–i). The median number of
nucleotides ranged between 4 and 7 base pairs. Conversely, the nucleotide content of filler DNAs was
unbalanced. Although not statistically different, recurrent deletions had the highest frequency of GC
content, while non-recurrent deletions displayed an intermediate frequency (70% versus 64%, respectively;
P value = 0.235 by Pearson's Chi-squared test; Fig. 4j). Remarkably, complete IKZF1 deletions (Δ1–8)
presented the lowest (50%) GC content compared to the majority of the recurrent deletions (Fig. 4k): ∆2–
3 (70%; P value = 4.4 x 10− 2), ∆2–7 (72%; P value = 1.3 x 10− 2), ∆2–8 (71%; P value = 3.1 x 10− 2), ∆4–7
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(72%; P value = 9.5 x 10− 3), ∆4–8 (65%; P value = 0.118). Of note, structural alterations derived from DNA
breaks within clusters at both sides had the highest frequency of GC nucleotides compared to
breakpoints outside any cluster (71% versus 56%; P value = 4.9 x 10− 3; Fig. 3l), whereas deletions derived
from breaks at one cluster had an intermediate GC content (66%; P value = 0.213).

Relevance of non-recurrent IKZF1 deletions to the

prognosis of B-ALL patients
To determine whether non-recurrent IKZF1 deletions also impact patient outcome, we performed Kaplan-
Meier analyses comparing their survival (n = 18) to matched patients with IKZF1 wild-type (n = 36) or ∆4–
7 (n = 36). Patient and clinical information of these comparative groups are detailed in the Supplementary
Table S9. The three IKZF1 status groups had similar OS rates (P value = 0.60 by log-rank test; Fig. 5a).
The 5-year OS rate of patients with non-recurrent IKZF1 deletion was 81.8% (standard error, SE = 11.6%),
compared to 78.9% (SE = 9.0%) and 85.6% (SE = 6.9%) in patients with IKZF1 ∆4–7 or wild-type,
respectively. On the other hand, the 5-year CIR differed between those groups (P value = 0.057 by Gray
test; Fig. 5b), especially between IKZF1 ∆4–7 and wild-type (14.7% vs. 37.7%; P value = 0.027). Despite
the lack of statistical significance, we also observed a higher 5-year CIR rate in patients with non-recurrent
IKZF1 deletion compared to wild-type (14.7% vs. 18.1%; P value = 0.091). Of note, the 10-year CIR was still
14.7% in patients without IKZF1 deletion, but reached 44.6% and 50.1% in the groups with ∆4–7 and non-
recurrent deletion, respectively. Our results highlight that rare and recurrent deletions of IKZF1 have
similar impact on the prognosis of B-ALL patients. Therefore, the identification of these alterations might
contribute to appropriate risk stratification and therapeutic benefits for these patients.

Discussion
IKZF1 deletions are recurrent alterations in B-ALL, and offer relevant information for risk stratification of
these patients, considering their association with a higher relapse risk 1,18. The genetic landscape of
these abnormalities is complex, as they comprise different types of deletions and may co-occur along
with multiple CNA. For instance, B-ALL patients with deletions in CDKN2A, CDKN2B, PAX5, or within the
pseudoautosomal region 1 (PAR1) without ERG deletion have a more adverse outcome, and are defined
as IKZF1plus group 19. Although the first studies addressing the relationship between IKZF1 deletions and
the prognosis of B-ALL had more representation of common deletions (i.e. Δ4–7 and Δ1–8), it has also
been demonstrated that less frequent deletions (i.e. Δ2–7 and Δ2–8) reduce event-free survival of these
patients 5. In this regard, our study adds novel information to those previous results, especially for IKZF1
exon 1 deletions. In summary, we have substantial evidence that IKZF1 deletions confer a dismal
prognosis for B-ALL patients, regardless of the range of deletion.

Traditional methodologies (i.g. MLPA) provide the possibility to detect all IKZF1 deletions in patient
samples whilst a great proportion of clones display this genetic alteration. However, the occurrence of
rare types of deletions may be challenging to confidently interpret, considering that MLPA may provide
false positive results, especially for deletions within exon 1. As previously demonstrated, methodological
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adjustments, including a longer denaturation step in MLPA may overcome this limitation 20. In addition,
the MLPA peak ratios of non-recurrent deletions frequently varied between the threshold of wild-type and
monoallelic deletion, thus indicating that most of them were subclonal. Indeed, we reveal that structural
alterations either within IKZF1 exon 1 or ranging from exon 1 to 3 are the most common non-recurrent
alterations of this gene. This information highlights the importance of considering these rare types of
deletions, although carefully controlling methodological variables to minimize the interpretation of false
positive results. In this regard, we advise the inclusion of an extra MLPA probe within the overlapping
deletion region upstream of IKZF1 promoter.

Previous PCR-based approaches for the detection of IKZF1 deletions have verified a substantial number
of samples escaping MLPA detection (half of B-ALL samples at diagnosis) due to subclonal lesions, low
blast count, or hemodilution 4. As recently discussed, both methods have pros and cons 21. While MLPA
detects a broader range of deletions within IKZF1 and other relevant genes for ALL risk stratification 19,22,
M-PCR is more sensitive and accurate to identify these genetic alterations. Although the prognostic
relevance of subclonal IKZF1 lesions has not been specifically addressed 23, we might assume that the
higher incidence of these genetic alterations at relapse would suffice for proper investigation of IKZF1
status. Therefore, the combination of broader approaches (e.g. MLPA or array techniques) with M-PCR is
ideal. According to the World Health Organization, the astonishing discrepancies in cure rates of
childhood cancer between high-income and low- or middle-income countries include lack of diagnosis,
misdiagnosis or delayed diagnosis 24. These limitations may be partially circumvented by the access to
low cost methodologies for genetic diagnosis of B-ALL. Here, we also improved the M-PCR to cover a
wider spectrum of IKZF1 alterations. Of note, previous PCR methods did not include primers flanking the
3'BC10, which is one of the most recurrent BC leading to Δ2–8 and Δ4–8 deletions. Therefore, we
estimate that our novel M-PCR may detect a higher number of IKZF1 deletions in ALL.

The occurrence of gene fusions is a common feature in acute leukemia. Although they comprise a lower
number of IKZF1 aberrations, here we have identified seven novel partner genes of IKZF1 fusions:
ABCA13, DDC, ENSG00000278996, LOC105377979, PAX5, STK38L, and ZPBP. Among them, only
ABCA13::IKZF1 and IKZF1::DDC were in-frame fusions, and derived from interstitial deletions within
chromosome 7. The ABCA13 belongs to a large family of ATP-binding cassette (ABC) transmembrane
transporters, and contributes to cholesterol internalization 25. It is highly expressed in leukemia cells and
in the bone marrow 26. The predicted chimeric protein lacks both transmembrane domains (TMDs) and
nucleotide-binding domains (NBDs) of ABCA13, thus truncating the normal function of this transporter.
Although all IKZF1 zinc-fingers are conserved, its regulation by the ABCA13 promoter as well as the
overall structure disruption of both proteins may together impact leukemogenesis. The DDC encodes a
protein that catalyzes the decarboxylation of several aromatic amino acids. Its dysfunction results in a
lack of monoamine neurotransmitters, including serotonin and catecholamine 27. Moreover, genetic
variants in this gene were previously linked to childhood ALL 28–30. Of note, previous works have also
identified IKZF1 gene fusions with ADSS131, CDK232, CEP17031, COBL3,33, DLG234, DNAH1435, ETV634,36,
FIGNL137, KMT2A38, NUTM132, SETD532, SPATA4832,36,37, STIM239, TRPV232, TYW140, and ZEB239. As
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the majority of them are reciprocal and out-of-frame IKZF1 fusions, they disrupt its promoter or truncate
its protein to contribute to leukemia promotion.

The process of B-cell maturation includes rearrangements of immunoglobulin genes, which is quite
relevant for the diversification of immune response. This event is orchestrated by recombinase-activating
genes (RAG), which recognizes recombination signal sequences to mediate interstitial deletions at these
genomic loci. Following, the terminal deoxynucleotidyl transferase (TdT) displays template-independent
activity and adds nucleotides to the junctions 41. Here, we demonstrate that most IKZF1 deletions are
associated with genetic signatures of RAG and TdT activity, thus explaining the reason for its recurrence
in B-ALL. Although heptamer sequences for RAG-mediated recombination are expected to occur once per
~ 16 kb, we identified a 74-fold enrichment of these sequences (once per ~ 0.2 kb) within BC. Conversely,
rare types of deletions and rearrangements outside BC lacked those signatures. Of interest, a recent study
identified off-target RAG-mediated rearrangements – which occur outside immunoglobulin genes – in
about 10% of normal lymphocytes 42. In addition, another study including patients at chronic phase of
chronic myeloid leukemia (CML) identified an overexpression of RAG1/2 and DNTT (encoding TdT) prior
to progression to lymphoid blast crisis and acquisition of secondary alterations, such as IKZF1 deletions
43
. This evidence demonstrates that off-target RAG-mediated recombination is a common genetic event
in lymphocytes, and meanwhile promotes secondary alterations in ALL, including the majority of IKZF1
deletions.

Following the cleavage of the DNA double-strand, TdT incorporates random nucleotides in an
untemplated mode. When it reaches the downstream DNA, it becomes template-dependent 44. Although
TdT is able to add several nucleotides to a template strand in vitro, this polymerase is assembled in a
complex of the non-homologous end-joining (NHEJ) machinery in vivo 45, which keeps the upstream and
downstream DNA closer and facilitates the transition between a template-independent to dependent
mode. Therefore, the length of their additional sequences is restricted to 1–20 nucleotides in vivo, and
reaches a maximum at four nucleotides 46. Here, we also observed a similar number of additional
nucleotides at the breakpoint junctions, regardless of the type and range of IKZF1 deletion.

One relevant aspect of TdT is that it requires divalent metal ions as cofactors (Mg2+, Mn2+, Zn2+, and
Co2+), and the incorporation of nucleotides is biased depending on their concentration in the
microenvironment. For instance, purine is more frequently added in the presence of Mg2+, while
pyrimidines are preferred when TdT is chelated to Co2+ 47. In this regard, in vivo studies have shown that
TdT more often incorporates guanine and cytosine nucleotide bases compared to adenine and thymine
48. In this scenario, we observed high GC content in additional nucleotides of the majority of IKZF1
deletions. The most pronounced GC incorporation was observed in sequences derived from
recombination between two BC and intragenic IKZF1 deletions. Conversely, whole-gene deletions had the
lowest frequency of filler DNAs within the breakpoint junction and lacked this nucleotide assimilation
bias. Instead, they presented more often microhomologies between both rearranged strands, thus
indicating that microhomology-mediated end joining (MMEJ) might play a significant role in the

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formation of large structural alterations that deplete the IKZF1 gene. This line of evidence is consistent
with a role of TdT on the establishment of most IKZF1 deletions, though discrete in complete deletions.

In conclusion, this study provides a wide spectrum of structural alterations that affect the IKZF1 gene. It
derives from an international effort to gather information on a large number of B-ALL samples to
illustrate the genetic alterations behind rare types of deletions, and their prognostic relevance for B-ALL
patients. In this regard, we point out methodological adjustments in MLPA and M-PCR to fine-tune the
detection of IKZF1 deletions, once we showed that every patient with either recurrent or non-recurrent
deletions will benefit from accurate risk stratification. Also, we summarize several levels of evidence that
support the idea that RAG and TdT mediate the majority of these alterations, although microhomology-
mediated repair may contribute to the genesis of this secondary alteration in B-ALL to some extent.

Declarations
Acknowledgments

We are grateful to the children and their parents for their participation in research. We also thank
Australian and New Zealand Children’s Haematology and Oncology Group hospital staff and oncologists
and Sydney Children’s Tumour Bank Network for their support and the MRD teams in Sydney and Monza.
We thank Dr. Jinghui Zhang, Dr. Xiaotu Ma, Dr. Qingsong Gao, and Dr. Charles Mullighan for kindly
providing contig sequences of IKZF1 deletions. We also appreciate the contributions made by Luana
Batista as well as the technical assistance provided by Alessandra J. Faro, André F. Duarte and Jodie
Giles.

These results were partially based on data derived from the Therapeutically Applicable Research to
Generate Effective Treatments (TARGET; https://ocg.cancer.gov/programs/target) initiative.

BAL was supported by the Brazilian Ministry of Health and INCA. BAL received a return research grant
(Ref 3.2 - 1193718 - BRA - HFSTCAPES-P) and a fellowship for a short stay in Germany (Ref 3.2 - BRA /
1193718) from the Alexander von Humboldt Foundation. ME is supported by Brazilian National Council
of Technological and Scientific Development – CNPq (PQ-311220/2020-7) and Fundação Carlos Chagas
Filho de Amparo à Pesquisa do Estado do Rio de Janeiro – FAPERJ (E_26/203.214/2017; E-26-
010.101072-2018; and E-26/010.002187//2019) research grants. RM was supported by grants from the
DFG (Ma 1876/12-1) and Wilhelm Sander foundation (2018.070.2). RS, LDP and BD received research
grant funding from NH&MRC Australia APP1128727 and Cancer Australia PdCCRS APP1024232.

Author Contributions

BAL, CM and ME designed the study. ALTM, TCB, CPP, CB, NCV, RS, CP, GF, CS and GC contributed with
MLPA data of individual patients. LDP, DB, CP, GF, TFA, MML, MRVIC, MS, EC, GC and RS provided
samples and patient data. BAL and ALTM performed MLPA validation of suspicious rare deletions. BAL
and ME provided these materials. RM, CM and BAL provided materials for NGS sequencing. BAL and PL

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performed library preparation and NGS sequencing. PL mapped these data. HB carried out M-PCR. MK
performed quantitative ChIP analysis. BAL prepared data, reviewed literature, performed data analyses
and wrote the manuscript. ME reviewed the first draft of the manuscript and provided important insights
to this work. All authors critically reviewed and approved the final version of the manuscript.

Competing Interests

The authors declare no competing financial interests.

Data Availability Statement

All data supporting the findings of this study are available from the corresponding author upon
reasonable request. The TARGET dataset is available at the Genomic Data Commons (GDC) Data Portal
(portal.gdc.cancer.gov/projects).

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Figures

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Figure 1

Genetic landscape of IKZF1 deletions. Individual MLPA peak ratios derived from (a) P335 and (b) P202
were compared between wild-type (WT) or monoallelic deletion loci in samples in which IKZF1 status was
verified. The horizontal line (gray) indicates the peak ratio of 0.8. (c) Frequency of IKZF1 deletions in four
independent cohorts and overall. The gene loss was classified in two groups: recurrent (Δ1–8, Δ2–3, Δ2–
7, Δ2–8, Δ4–7, Δ4–8) and non-recurrent (the remaining deletion ranges). (d) Types of non-recurrent
IKZF1deletions identified in the four cohorts of this study and literature (restricted to DNA and RNA
sequencing data). (e) Spectrum of IKZF1 gene fusions reported so far. They compose in-frame (blue) or
out-of-frame (gray) gene fusions. Bold indicates those found exclusively in this study. *DDCwas a partner
gene in two samples: one had an in-frame fusion, while the other had out-of-frame.

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Figure 2

Breakpoint map of IKZF1 deletions. (a) Genomic location of BC associated with IKZF1 deletions. A total
of 24 BC were identified (horizontal ticks) and located at 5’ (n=12; BC01–12 in blue) and 3’ (n=12; BC01–
12 in red) breakpoints of IKZF1deletions. The counting of all breakpoints is also displayed by genomic
coordinates, where the y-axis illustrates the number of DNA breaks (the maximum value was restricted to
15). (b) Number of IKZF1deletions associated with each combination of BC. The gradient color illustrates
the frequency of non-recurrent IKZF1deletions for every BC combination. The bar plots indicate the
number of sequences for individual 5'BC (right) and 3'BC (above). "No" refers to breakpoints outside BC.

Figure 3

Opportunities to improve methodologies for the determination of IKZF1 status. (a) Genomic map of
IKZF1deletions encompassing exon 1 (n=23; red horizontal bars). The location of commercially available
MLPA probes (P202-C1 assay; orange) and guanine-cytosine content (GC content; gray) are also
Page 19/21
displayed. The common deleted region of IKZF1 promoter is highlighted (light red). After excluding the
region of high GC content, the remaining target area (chr7:50,275,701–50,302,000) may be used for the
design of novel MLPA probes, allowing more accurate definition of exon 1 deletions. (b) M-PCR assay to
determine IKZF1 status. The agarose gel electrophoresis was used to visualize the amplicons of IKZF1
Δ2–3 in the first M-PCR, and Δ2–7, Δ2–8, Δ4–7 in the second reaction. We also included IKZF1wild-type
samples (lanes 3 and 8) as well as negative controls (N).

Figure 4

Genetic signatures of IKZF1 deletions. DNA motifs identified at breakpoints after agnostic search of (a)
BC and (b) individual recurrent IKZF1 deletions. (c) The most significant heptamer-like sequences (5'-
CACAGTG-3') identified at BC. Different types of genetic signatures at the junctions of IKZF1 deletions
were compared based on (d) the frequency of IKZF1 deletions, (e) types of recurrent deletions, and (f)
DNA break at clusters. MH, microhomologies between both breakpoints. The (g–i) size of additional
nucleotides at DNA junctions and the (j–l) frequency of each nucleobase (A, T, G or C) within them was
also compared by the same parameters. Statistical tests were performed between IKZF1 deletion groups:

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Chi-square test (analysis of genetic signatures and frequency of nucleobases) and two-tailed unpaired
Student’s t-test (size of filler DNA).

Figure 5

Outcome of B-ALL patients with non-recurrent IKZF1deletions.Kaplan–Meier curves illustrate the (a) OS
and (b) CIR of non-recurrent IKZF1 deletions versus IKZF1 wild-type or IKZF1 Δ4–7.

Supplementary Files
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SupplementaryTables.pdf

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