Inherited genetic susceptibility to acute lymphoblastic leukemia in Down syndrome

Abstract

Children with Down syndrome (DS) have a 20-fold increased risk of acute lymphoblastic leukemia (ALL) and distinct somatic features, including CRLF2 rearrangement in ∼50% of cases; however, the role of inherited genetic variation in DS-ALL susceptibility is unknown. We report the first genome-wide association study of DS-ALL, comprising a meta-analysis of 4 independent studies, with 542 DS-ALL cases and 1192 DS controls. We identified 4 susceptibility loci at genome-wide significance: rs58923657 near IKZF1 (odds ratio [OR], 2.02; Pmeta = 5.32 × 10-15), rs3731249 in CDKN2A (OR, 3.63; Pmeta = 3.91 × 10-10), rs7090445 in ARID5B (OR, 1.60; Pmeta = 8.44 × 10-9), and rs3781093 in GATA3 (OR, 1.73; Pmeta = 2.89 × 10-8). We performed DS-ALL vs non-DS ALL case-case analyses, comparing risk allele frequencies at these and other established susceptibility loci (BMI1, PIP4K2A, and CEBPE) and found significant association with DS status for CDKN2A (OR, 1.58; Pmeta = 4.1 × 10-4). This association was maintained in separate regression models, both adjusting for and stratifying on CRLF2 overexpression and other molecular subgroups, indicating an increased penetrance of CDKN2A risk alleles in children with DS. Finally, we investigated functional significance of the IKZF1 risk locus, and demonstrated mapping to a B-cell super-enhancer, and risk allele association with decreased enhancer activity and differential protein binding. IKZF1 knockdown resulted in significantly higher proliferation in DS than non-DS lymphoblastoid cell lines. Our findings demonstrate a higher penetrance of the CDKN2A risk locus in DS and serve as a basis for further biological insights into DS-ALL etiology.

Graphical abstract

Figure 1.

Flowchart of study design and analytic approach. GSA, Global Screening Array; MiDSALL, Michigan-based DS-ALL study.

Figure 2.

Manhattan plot of DS-ALL GWAS meta-analysis results. Genome-wide −log10(P) values from meta-analysis of 7 separate DS-ALL GWA studies, including 4 European (in COG/NDSP study 1 and 2, Michigan-based DS-ALL study, and IS-DSAL), 2 Hispanic (in COG/NDSP study 2 and IS-DSAL), and 1 African American ancestry (COG/NDSP study 1) case-control sets. Analysis included 6 758 624 autosomal and non–chromosome 21 SNPs (trisomic genotypes analyzed separately), and results are reported for SNPs successfully imputed in at least 6 out of 7 studies. Red horizontal line represents the genome-wide significance threshold of P = 5 × 10⁻⁸.

Figure 3.

Functional characterization of the IKZF1 susceptibility locus. (A) Epigenetic profile of the IKZF1 SNP rs58923657 susceptibility locus. Tracks show positions of rs58923657 and SNPs in LD (r² ≥ 0.6); IKZF1; DNase I hypersensitive site clusters; GM12878 chromatin state (ChromHMM) corresponding to transcribed regions (green), candidate weak enhancers (yellow), or candidate strong enhancers (orange); and ChIP-seq data for H3K4Me1, H3K4Me3, H3K27ac and transcription factors from ENCODE. Data are displayed in the UCSC genome browser (http://genome.ucsc.edu/) and the WashU Epigenome Browser (http://epigenomegateway.wustl.edu/). (B) Chromatin spatial organization of chromosome 7 from 50.0-50.6 MB. Tracks show a Hi-C heatmap of chromatin contact frequencies in GM12878; chromatin contact domain determined by the Arrowhead algorithm; CD19⁺ B-cell superenhancers; canonical transcripts; CTCF ChIP-seq data in GM12878 from ENCODE; and CTCF ChIA-PET mapping data in GM12878. Black rectangle indicates the rs58923657 locus. (C) Risk and nonrisk allele-specific enhancers encompassing rs58923657 were cloned into the pGL3-promoter vector (Promega) and transfected into 20 LCLs (10 DS and 10 non-DS). Enhancer reporter assay demonstrates relative luciferase activity measured 24 hours later for the nonrisk and risk alleles relative to the empty vector. Bars show the mean ± SEM from transfections performed in triplicate in the 20 LCLs. (D) EMSAs were performed using nuclear extracts from 6 patient-derived LCLs (3 DS and 3 non-DS) reacted with double-stranded DNA probes encompassing the indicated IKZF1 SNPs. Bars show mean ratios ± SEM of risk to nonrisk allele protein binding. (E) IKZF1 knockdown results in increased cellular proliferation with a greater effect in DS LCLs. Serial cell counts demonstrated significantly greater cellular proliferation for 4 DS LCLs expressing IKZF1-shRNA compared with NT-shRNA (P = .026) and 4 non-DS LCLs expressing IKZF1-shRNA compared with NT-shRNA (P = .017), and the effect of IKZF1-shRNA was significantly greater in the DS LCLs than in the non-DS LCLs (P = .047). Data shown are means of experiments performed in triplicate. P values in panels C-E were determined by a Student 2-tailed t test. *P < .05; **P < .001; ***P < .0001.