Whole exome sequencing reveals a C-terminal germline variant in CEBPA-associated acute myeloid leukemia: 45-year follow up of a large family

Abstract

Familial acute myeloid leukemia is rare and linked to germline mutations in RUNX1, GATA2 or CCAAT/enhancer binding protein-α (CEBPA). We re-evaluated a large family with acute myeloid leukemia originally seen at NIH in 1969. We used whole exome sequencing to study this family, and conducted in silico bioinformatics analysis, protein structural modeling and laboratory experiments to assess the impact of the identified CEBPA Q311P mutation. Unlike most previously identified germline mutations in CEBPA, which were N-terminal frameshift mutations, we identified a novel Q311P variant that was located in the C-terminal bZip domain of C/EBPα. Protein structural modeling suggested that the Q311P mutation alters the ability of the CEBPA dimer to bind DNA. Electrophoretic mobility shift assays showed that the Q311P mu-tant had attenuated binding to DNA, as predicted by the protein modeling. Consistent with these findings, we found that the Q311P mutation has reduced transactivation, consistent with a loss-of-function mutation. From 45 years of follow up, we observed incomplete penetrance (46%) of CEBPA Q311P. This study of a large multi-generational pedigree reveals that a germline mutation in the C-terminal bZip domain can alter the ability of C/EBP-α to bind DNA and reduces transactivation, leading to acute myeloid leukemia.

Copyright© Ferrata Storti Foundation.

Figures

Figure 1.

Pedigree of NCI AML family. CEBPA Q311P genotyping was performed on all individuals with available DNA. Filled symbols: AML cases; symbol with a dot in the center: obligate carrier. Boxed symbols: exome-sequenced samples; Q311 is the wild-type allele and Q311P is the variant at this position. Arrow: proband.

Figure 2.

Comparison of the wild-type C/EBP-α -DNA fragment (PDB: 1NWQ; left) complex to the final conformation from the simulated double Q311P mutant (right). Residue 311 is shown in “ball-and-stick style”. The mutant C/EBPα is predicted to bind to DNA with less avidity than the wild-type, affecting transcriptional regulation.

Figure 3.

Transactivation potential of CEBPA Q311P versus wild-type. (A) CEBP promoter transactivation assays in MOLM16 cells for pcDNA, CEBPA Q311 (WT) and CEBPA Q311P (and all combinations), show a loss-of-function of CEBPA Q311P (experiments were performed in triplicate). P-value paired t-test values: pcDNA/Q311 vs. pcDNA/Q311P, P=0.0002; pcDNA/Q311 vs. Q311/Q311P, P=0.0011; Q311/Q311 vs. Q311P/Q311P, P= 0.0021; Q311/Q311 vs. Q311/Q311P, P=0.0343; Q311P/Q311P vs. Q311/Q311P, P=0.0013. (B) anti-CEBPA and anti-GAPDH Western blot of total extracts of transiently transfected MOLM16 cells.

Figure 4.

DNA binding potential of CEBPA Q311P versus CEBPA Q311 (wild-type). Electrophoretic mobility shift assay performed with nuclear extracts of H1299 cells transiently transfected with equal amounts of pcDNA, CEBPA Q311 (wild-type) and CEBPA Q311P expression plasmids. (A) Binding to high affinity site and (B) binding to low affinity site.