title: Clonal hematopoiesis as determined by the HUMARA assay is a marker for acquired mutations in epigenetic regulators in older women.
abstract: Recent large cohort studies revealed that healthy older individuals harbor somatic mutations that increase their risk for hematologic malignancy and all-cause cardiovascular deaths. The majority of these mutations are in chromatin and epigenetic regulatory genes (CERGs). CERGs play a key role in regulation of DNA methylation (DNMT3A and TET2) and histone function (ASXL1) and in clonal proliferation of hematopoietic stem cells. We hypothesize that older women manifesting clonal hematopoiesis, defined here as a functional phenomenon in which a hematopoietic stem cell has acquired a survival and proliferative advantage, harbor a higher frequency of somatic mutations in CERGs. The human androgen receptor gene (HUMARA) assay was used in our study to detect the presence of nonrandom X inactivation in women, a marker for clonal hematopoiesis. In our pilot study, we tested 127 blood samples from women ≥65 years old without a history of invasive cancer or hematologic malignancies. Applying stringent qualitative criteria, we found that 26% displayed clonal hematopoiesis; 52.8% displayed polyclonal hematopoiesis; and 21.3% had indeterminate patterns (too close to call by qualitative assessment). Using Illumina MiSeq next-generation sequencing, we identified somatic mutations in CERGs in 15.2% of subjects displaying clonal hematopoiesis (three ASXL1 and two DNMT3A mutations with an average variant allele frequency of 15.7%, range: 6.3%-23.3%). In a more limited sequencing analysis, we evaluated the frequency of ASXL1 mutations by Sanger sequencing and found mutations in 9.7% of the clonal samples and 0% of the polyclonal samples. By comparing several recent studies (with some caveats as described), we determined the fold enrichment of detecting CERG mutations by using the HUMARA assay as a functional screen for clonal hematopoiesis. We conclude that a functional assay of clonal hematopoiesis is enriching for older women with somatic mutations in CERGs, particularly for ASXL1 and TET2 mutations and less so for DNMT3A mutations.
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health & Science University, Portland, OR, USA.
authors.forename: Julia Erin
authors.initials: JE
authors.lastname: Wiedmeier
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health & Science University, Portland, OR, USA.
authors.forename: Catherine
authors.initials: C
authors.lastname: Kato
authors.affiliation: School of Public Health, Oregon Health & Science University, Portland, OR, USA.
authors.forename: Zhenzhen
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health & Science University, Portland, OR, USA.
authors.forename: Hyunjung
authors.initials: H
authors.lastname: Lee
authors.affiliation: Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA; Department of Pathology, Oregon Health & Science University, Portland, OR, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Dunlap
authors.affiliation: Department of Pathology, Oregon Health & Science University, Portland, OR, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Nutt
authors.affiliation: Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA; Department of Pathology, Oregon Health & Science University, Portland, OR, USA.
authors.forename: Rogan
authors.initials: R
authors.lastname: Rattray
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health & Science University, Portland, OR, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: McKay
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health & Science University, Portland, OR, USA; Howard Hughes Medical Institute, Portland, OR, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Eide
authors.affiliation: Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA; Department of Pathology, Oregon Health & Science University, Portland, OR, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Press
authors.affiliation: School of Public Health, Oregon Health & Science University, Portland, OR, USA; Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA.
authors.forename: Motomi
authors.initials: M
authors.lastname: Mori
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health & Science University, Portland, OR, USA; Howard Hughes Medical Institute, Portland, OR, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Druker
authors.affiliation: Knight Cancer Institute, Hematology and Medical Oncology, Oregon Health & Science University, Portland, OR, USA. Electronic address: daok@ohsu.edu.
authors.forename: Kim-Hien
authors.initials: KH
authors.lastname: Dao
mesh_terms: D000367:Age Factors; D000368:Aged; D000369:Aged, 80 and over; D060965:Clonal Evolution / Q000235:genetics*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D000090205:DNA Methyltransferase 3A; D044127:Epigenesis, Genetic; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006410:Hematopoiesis / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D011944:Receptors, Androgen / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics
chemical_list: C473949:ASXL1 protein, human; C000717967:DNMT3A protein, human; D011944:Receptors, Androgen; D012097:Repressor Proteins; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A