Dr. Pawel Mroz

Lab Medicine & Pathology
Medical School
Twin Cities
Project Title: 
Haplotype Phase of CEPBA Mutations in Acute Myeloid Leukemia

The CCAAT enhancer binding protein alpha (CEBPA) gene is a 1077 base pair long, intronless gene that maps to chromosome 19q13.1 and encodes a transcription factor. Mutations in the CEBPA gene contribute to leukemic transformation of myeloid progenitors and occur in approximately 10-15% of cases of cytogenetically normal acute myeloid leukemia (CN-AML). The presence of two CEBPA mutations is a favorable prognostic indicator in AML in CN-AML and constitutes a separate diagnostic AML entity in WHO2017 classification. Clinically significant mutations are predicted to cause loss of protein function and tend to cluster in the N-terminal and C-terminal regions of the gene. Regardless of the location, CEBPA mutations show three typical patterns in AML: single mutated, single mutated with loss of heterozygosity, and double mutated. Double CEBPA mutations are assumed to be biallelic because the haplotypic phase of double CEBPA mutations has not been thoroughly investigated. This is primarily due to the limitations of standard short-read sequencing technologies, namely, Sanger sequencing and NGS. Recently, long-read sequencing technologies, such as Pacific Bioscience’s Single Molecule Real-Time (PacBio’s SMRT) sequencing platform, have been developed to overcome some of the shortcomings of Sanger and NGS. While the maximum read length obtained by NGS is approximately 500 bp, PacBio SMRT sequencing yields average read lengths of 10-14 Kb, enabling direct determination of the haplotypic phase of variants.

To address the shortcomings of the current sequencing platforms these researchers are evaluating PacBio’s SMRT sequencing platform applicability to determine the phase of the CEPBA variants in cases of CN-AML, hypothesizing that both cis and trans configurations of double (“biallelic”) CEBPA mutations may occur. There are two aims:

  • To evaluate if the PacBio’s SMRT sequencing platform can be effectively applied to detect single and double CEBPA mutations in CN-AML patients
  • To determine the haplotypic phase of CEBPA mutations in cases of CN-AML with double CEBPA mutations

The results of this study will contribute to understanding of the biology of CEBPA double mutation-positive AML and will pave the way for work exploring the impact of CEBPA variant phase on patient outcomes.

The pilot study developed and validated a PacBio’s SMRT sequencing platform for detection and phasing of CEBPA mutations. The "training set" of samples included a combination of negative samples, a single CEBPA mutation positive samples and samples with single mutations and common polymophisms. The researchers established that PacBio’s SMRT as an effective method for sequencing and phasing of CEBPA variants. They are in the process of sequencing additional ~80 samples with single and double CEBPA pathogenic variants and anticipate that direct haplotype phasing will reveal whether double CEBPA mutations occur in cis and/or trans configuration. They will subsequently explore any associations between haplotype phase of double CEBPA mutations with event-free survival (EFS) and overall survival (OS) in patients treated with induction chemotherapy.

The researchers have established that PacBio SMRT is a viable option to phase CEBPA double variants in CN-AML cases. If cis and trans configurations of double CEBPA mutations do occur and have different clinical outcomes this may lead to major change in WHO AML classification. To the researchers' knowledge this is a first time a SMRT sequencing was applied to clinical CEBPA mutation testing and the first time that haplotype phase of CEBPA double mutations is elucidated.

Project Investigators

Todd Knutson
Ryan Martinez
Dr. Pawel Mroz
Dr. Sarah Munro
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