CHEK2 mutations as predisposition alleles for inherited hematopoietic malignancies

The overall goal of this award is to test the hypothesis that inherited mutations of CHEK2 confer risk for the blood and bone marrow cancers.  We have been working on experiments that test this hypothesis, organized in two Aims:

(1) To determine if additional genetic alterations exist in patients with familial CHEK2 mutations who have bone marrow-derived cancers; and

(2) To generate a mouse model that mimics the CHEK2 mutation seen most frequently in these families.

 Progress to date:

            Aim 1: To determine if additional genetic alterations exist in patients with familial CHEK2 mutations who have bone marrow-derived cancers.

Eight samples of patients with hematopoietic malignancies and the CHEK2 I157T variant have undergone whole genome sequencing (WGS).  We have established a bioinformatic pipeline to call variants in these samples.  In two families, we identified actionable mutations in two genes (one each in the two independent families) that have clinical implications.  Each individual chose to receive this information on a research basis, and we provided information on how to obtain clinical validation of these variants.

We are currently determining the genetic haplotypes of the individuals through examination of microsatellite markers.  This will allow us to group the individuals by genotype, which will help us to interpret the genomic variants that have been called.  Recently, a similar study has been performed for a modifier of a TP53 allele present in the Brazilian population, and we are closely following this approach to test if a genetic modifier of CHEK2 I157T allele exists.

Aim 2: To generate a mouse model that mimics the CHEK2 mutation seen most frequently in these families. 

The University of Chicago Transgenic Mouse and Embryonic Stem Cell Facility generated a targeting construct that directs the expression of the homologous Chek2 murine amino acid, I161T.  We confirmed the correct sequence of the construct, and germline knock-in animals were generated from two distinct ES clones.  These founder animals were bred into two distinct lines of mice, and these are being followed for phenotypes.  We have confirmed that the mice express the engineered I161T allele.  The mice are currently being employed in three experiments: (i) We are testing if the I161T allele has an impact in the mouse embryo.  Matings to generate homozygous mice have been established, and we expect 25% of the progeny to be homozygous for the I161T allele.  However, we only observe the homozygous animals at a frequency of about 15%, suggesting that the I161T allele confers embryonic lethality.  This is a very important observation, since the Chek2 knock-out animal is completely viable and fertile.  This implies that the I161T allele has a dominant activity and is not a simple null allele.  This result supports our overall hypothesis.  Our current work is aimed at obtaining enough animals to reach statistical significance, and following that, we will performed timed matings to identify the stage at which the embryos are dying and by what mechanism.  (ii) We are examining the peripheral blood cell counts of the animals expressing the I161T allele over time to see if there is a phenotype in the blood of these animals.  With time, we will also perform necropsies at designated time intervals to see if there is any organ pathology.  (iii) We have treated the Chek2 knock-in mice with radiation and are following them over time to look at recovery from radiation (which largely appears normal) and subsequent bone marrow function.