Beyond clonal hematopoiesis of indeterminate potential: Understanding how the trisomy 21 context promotes TET2 mutations Free

Clonal hematopoiesis of indeterminate potential (CHIP) is the observation in the peripheral blood of a somatic mutation in a gene associated with hematologic malignancy at a variant allele frequency (VAF) at or above 2%. CHIP has been used to uncover associations with numerous disorders including cardiovascular disease and leukemia. Despite this progress, the guidelines of CHIP may prevent additional discovery and opportunities to understand hematologic somatic evolution. Notably, the threshold of 2% only highlights clones in later stages of development and prevents us from understanding how the hematopoietic microenvironment is influencing the mutational landscape to enable that clonal growth. Furthermore, due to leukemia presenting most frequently in the elderly population, the detection of CHIP is largely centered around age. This overlooks other factors that influence clonal growth and hematopoietic dysregulation such as congenital disorders, which impact millions of people globally. Down syndrome (DS) - the result of trisomy in chromosome 21 – is associated with chronic inflammation, autoimmunity, and increased risk of childhood leukemia, making it a condition worth study for clonal hematopoiesis (CH). In this project, we use a rare-mutation detection technique to characterize the mutation landscape of the blood from people with DS to understand how their hematopoietic microenvironment influences CH.

To detect rare mutations and characterize the mutation landscape of people with DS, we use a technique called DuplexSeq. This targeted-sequence technique covers ~50 kB spanning 37 genes that are implicated in CHIP and leukemia in people with DS. Through collaborations in the Linda Crnic Institute for Down Syndrome, we procured DNA from the leukocytes of the peripheral blood of ~150 people with DS and age-matched euploid controls in their Human Trisome Project. To get appropriate depth to characterize the mutation landscape, we used 400ng of input DNA for each sample, enabling detection of clones of at least 0.01% VAF.

Using several bioinformatics analyses, we uncover several genes that are differentially mutated and with larger clonal expansions in people with DS. When stratifying for specific mutations that have been previously seen in cancer, we observe a disproportionate prevalence of mutations in JAK2 and KRAS, among others. However, TET2 shows the greatest and most consistent prevalence in people with DS relative to those without DS. It is worth noting that there were no discernible differences observed in DNMT3A – the most prevalently mutated gene in CHIP – and only 2 participants had somatic mutations with VAFs high enough to be considered CHIP.In comparative RNAseq analysis of the blood of people with DS, heme metabolism is upregulated and pathways involved in DNA repair and cell proliferation are downregulated in those harboring notable TET2 mutations. These results lead us to speculate that disrupted iron metabolism and/or restoration of cell cycle are aspects of the DS microenvironmental context that may promote selection for TET2 mutations. To further investigate TET2 mutations in the DS context, we recapitulated the mutations observed with DuplexSeq in mouse cells using CRISPR technology. Given that TET2 is a tumor suppressor gene, an effective knockout in these cells can mimic the indels and premature stop codons we observe in people with DS. In in vitro experiments, we observe greater expansions of Tet2 mutant hematopoietic stem and progenitor cells from Dp16 mice – a mouse model of DS – than from their control littermates. In vivo experiments are underway to elucidate whether these Tet2 expansions are more attributable to cell-intrinsic or cell-extrinsic factors of the DS context.

In conclusion, these experiments will help us understand how the DS context can promote disruptive clonal expansions. In the future, we will conduct epigenetic experiments to determine how TET2 loss is impacting DNA methylation and subsequent gene expression. Furthermore, this methodology of characterizing the mutational landscape can be applied to other congenital hematological disorders such as sickle cell disease and Fanconi anemia to provide mechanistic insight on features that contribute to hematopoietic disorder and creating avenues for potential interventions to enhance the quality of life for millions of people with predispositions to hematologic malignancies.