A noncoding regulatory variant in IKZF1 increases acute lymphoblastic leukemia risk in Hispanic/Latino children

Abstract

Hispanic/Latino children have the highest risk of acute lymphoblastic leukemia (ALL) in the US compared to other racial/ethnic groups, yet the basis of this remains incompletely understood. Through genetic fine-mapping analyses, we identified a new independent childhood ALL risk signal near IKZF1 in self-reported Hispanic/Latino individuals, but not in non-Hispanic White individuals, with an effect size of ∼1.44 (95% confidence interval = 1.33-1.55) and a risk allele frequency of ∼18% in Hispanic/Latino populations and <0.5% in European populations. This risk allele was positively associated with Indigenous American ancestry, showed evidence of selection in human history, and was associated with reduced IKZF1 expression. We identified a putative causal variant in a downstream enhancer that is most active in pro-B cells and interacts with the IKZF1 promoter. This variant disrupts IKZF1 autoregulation at this enhancer and results in reduced enhancer activity in B cell progenitors. Our study reveals a genetic basis for the increased ALL risk in Hispanic/Latino children.

Keywords: B-ALL; GWAS; IKZF1; Indigenous American; acute lymphoblastic leukemia; cancer disparity; cancer predisposition; childhood leukemia; fine-mapping; hematopoiesis.

Graphical abstract

Figure 1

Novel childhood ALL risk locus at IKZF1 is associated with Indigenous American ancestry and demonstrates ancient origins and positive selection in Hispanic/Latino populations (A–C) LocusZoom plots showing an approximately 600 Mb region at chromosome 7p12.2 centered on the IKZF1 gene region (±250 kb) from (A) signal 1, the unconditional GWAS of childhood ALL in Hispanic/Latino individuals; (B) signal 2, results conditioned on the lead SNP (rs4917017) in signal 1; and (C) signal 3, results conditioned on lead SNPs in signals 1 (rs4917017) and 2 (rs10272724), identifying the novel ALL risk locus tagged by lead SNP rs76880433. Diamond symbols in purple indicate the lead SNP in each locus. Color of remaining SNPs is based on linkage disequilibrium (LD) in the admixed American superpopulation in the 1KG Project (which includes Mexican ancestry in Los Angeles [MXL], Peruvian in Lima, Peru [PEL], Colombian, and Puerto Rican populations) as measured by r² with the lead SNP in each signal. All coordinates are in genome build hg38. (D) The rs76880433 risk allele T is positively associated with Indigenous American ancestry among Hispanic/Latino cases and controls in the California Cancer Record Linkage Project ALL GWAS. Error bars correspond to standard errors. (E) Frequency of the haplotype derived from Indigenous American ancestry at IKZF1 signal 3 is significantly higher in childhood ALL cases than controls among Hispanic/Latino individuals. (F) Mapping the origins of the putative causal rs1451367 SNP with shotgun sequencing data of ancient DNA samples suggests the mutation arose at least 12,700 years ago. (G) Reconstructed genealogies in 1KG populations provide evidence for positive selection at the haplotype containing signal 3 lead SNP rs76880433 only in Hispanic/Latino populations (MXL and PEL) but not in European (IBS) or East Asian (CHS) populations.

Figure 2

Two independent childhood ALL risk alleles in Hispanic/Latino individuals reside in an enhancer for IKZF1 that is most active in pro-B cells (A) Top: putative causal variants underlying Hispanic/Latino ALL GWAS signal 2 and signal 3, rs17133807 and rs1451367, respectively, lie 26 bp apart in a region with strong interaction with IKZF1 promoter in promoter capture Hi-C data. Bottom: single-cell assay for transposase-accessible chromatin using sequencing (ATAC sequencing) data showed strong accessibility in B cells and precursors, especially pro-B cells, but minimal or no accessibility in T cells or myeloid cells obtained from healthy donors (see Figure S10 for details). (B) ATAC sequencing coverage in 156 patients with B cell ALL displayed by genotype for putative causal variants underlying GWAS signals 1–3. Genotypes are ordered by non-risk allele homozygotes, non-risk/risk allele heterozygotes, and risk allele homozygotes (rs11765436: T/T, T/A, A/A; rs1451367: C/C, C/T, T/T; rs17133807: G/G, G/A, A/A). For each SNP, the patients with B cell ALL were categorized into three groups based on a read coverage cutoff of 5: non-risk/non-risk, where non-risk allele reads are >5 and risk allele reads are <5; non-risk/risk, where both risk and non-risk allele reads are >5; and risk/risk, where risk allele reads are >5 and non-risk allele reads are <5. ATAC sequencing read counts normalized by reads per million are shown (see Table S11 for details), and the p values were calculated using one-tailed Wilcoxon tests.

Figure 3

Functional impact of IKZF1 variants on enhancer activity, IKZF1 regulation, and implications for B cell development (A) IKZF1 putative causal variants were found to reduce enhancer activity in lentiviral reporter assays. Lentiviral reporters were introduced at the same multiplicity of infection as assessed by quantitative PCR of the WPRE element into (top) REH, (middle) HepG2, and (bottom) HEK293T cells. Geometric mean fluorescence intensity of GFP was measured by flow cytometry. Two-sided p values of 0.05 or less were considered to indicate statistical significance. All data are presented as the mean ± the standard deviation (n = 3; ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (B) rs1451367 overlaps IKZF1 binding peak in human B lymphoblastoid cell chromatin immunoprecipitation sequencing (ChIP-seq) data. (C) rs1451367 risk allele disrupts an IKZF1 binding motif based on global ChIP-seq data analysis. (D) IKZF1 expression changes along the B cell developmental trajectory. Single-cell RNA sequencing revealing increasing IKZF1 expression from early hematopoietic progenitors to mature B cells is shown as a pseudo-time trajectory. (E) Reduced IKZF1 mRNA expression in B cell progenitors derived from differentiation of human HSPCs by qPCR after ribonucleoprotein-mediated introduction of microdeletions involving the IKZF1 transcription factor binding motif within the enhancer region. All data are presented as the mean ± the standard deviation (n = 3; ∗p < 0.05). (F) Genome editing of IKZF1 results in an accumulation of CD10⁺CD19⁻ B cell progenitors and impaired differentiation of CD19⁺CD10⁺ maturing B precursors after 3 weeks of coculture of human hematopoietic stem and progenitor cells on MS-5 stromal cells.