The accurate detection by next-generation sequencing (NGS) of difficult to sequence genes (CALR, CEBPA, FLT3) associated with myeloid disorders using a hybridisation-based enrichment approach

Monday 4 September 2017

Lyudmila Georgieva, Ezam Uddin, Jacqueline Chan and Graham Speight

Presented at CGC 2017, this poster highlights the excellent uniformity of coverage obtained from the hybridisation-based enrichment using the SureSeq myPanel NGS Custom AML Panel.


The application of short read NGS for research into myeloid disorders such as myeloproliferative neoplasms (MPNs) and acute myeloid leukaemia (AML) has been hampered by the difficulty in sequencing certain genes.

Amongst the difficult to sequence genes are calreticulin (CALR), CCAAT/enhancer binding protein alpha (CEBPA) and fms-related tyrosine kinase 3 (FLT3).

The development of robust assays for CEBPA mutation testing is challenging due to the high GC content of the gene (75% in the coding region), the presence of a trinucleotide repeat region, the complexity of the mutations, and the frequent occurrence of mutations in mononucleotide repeats. Genes such as FLT3 are challenging to target because they contain complex repetitive elements that can be long and are generally masked in most panel designs. CALR sequencing is challenging due to the presence of low complexity regions making the detection of insertions and deletions difficult.

Mutations in the CEBPA and FLT3 genes are among the most common molecular alterations in AML. Two mutations on separate alleles of CEBPA with a specific combination of an N-terminal frameshift mutation on one allele and a C-terminal in frame mutation on the other allele are important for prognosis1,2. The prevalence of an internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence and a missense mutation of D835 within the kinase domain of the FLT3 gene occurs in 15-35% and 5-10% of adults with AML, respectively3 . A quarter of patients with essential thrombocythemia or primary myelofibrosis carry a driver mutation of CALR. A 52-bp deletion (type 1) and a 5-bp insertion (type 2 mutation) are the most frequent variants4 .


  1. Behdad A ,et al. A clinical grade sequencing-based assay for CEBPA mutation testing: report of a large series of myeloid neoplasms. J Mol Diagn. 2015 Jan;17(1):76-84. 
  2. Spencer DH, et al. Detection of FLT3 internal tandem duplication in targeted, short-read-length, next-generation sequencing data. J Mol Diagn. 2013 Jan;15(1):81-93. 
  3. Dufour A, et al. Acute myeloid leukemia with biallelic CEBPA gene mutations and normal karyotype represents a distinct genetic entity associated with a favorable clinical outcome. J Clin Oncol. 2010 Feb 1;28(4):570-7. 
  4. Pietra D, et al. Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms. Leukemia. 2016 Feb; 30(2): 431–438.


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