Clonality assay of the HUMARA gene. (A) HUMARA locus on X chromosome. The first exon of the human androgen receptor gene contains a highly polymorphic CAG repeat with more than 20 different alleles and a heterozygosity frequency of 90%. One hundred base pairs 5′ to the (CAG)_n lies a site of differential methylation between Xa (X active) and Xi (X inactive). The HUMARA assay has reliable methylation patterns that have been documented using an androgen receptor expression assay at the same locus. Within the differential methylation site are 2 restriction sites for the methylation sensitive restriction endonuclease Hpa II. Hpa II will cleave unmethylated, active X alleles, precluding amplification of these alleles by PCR primers 1 and 2. (B) The HUMARA clonality assay. DNA is digested with Hpa II and amplified by PCR using ³²P end-labeled primers. In a polyclonal population of cells (b), both the maternal (M) and paternal (P) X alleles (X inactive) will be amplified and discriminated as two bands of different molecular weight on a denaturing polyacrylamide gel by the variable CAG repeat. In contrast, for a clonal population of cells (a and c), in which all cells are derived from a single common progenitor, only the maternal or paternal allele (X inactive) will be amplified, giving a single band. In accordance with published literature, patients are considered to have clonal hematopoiesis if the corrected allelic ratio (Cr) is greater than 3:1. Ratios between 1:1 and 3:1 (ie, between 1 and 3) are considered to represent polyclonal hematopoiesis ([▪] paternal allele; [□] maternal allele; large open box denotes longer CAG expansion associated with paternal allele; small hatched box denotes shorter CAG expansion associated with maternal allele; slash mark indicates active unmethylated allele cleaved by Hpa II).