HUMARA assay
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HUMARA assay is one of the most widely used methods to determine the clonal origin of a tumor.[1][2] The method is based on X chromosome inactivation and it takes advantage of the different methylation status of the gene HUMARA (short for human androgen receptor) located on the X chromosome. Considering the fact that once one X chromosome is inactivated in a cell,[3] all other cells derived from it will have the same X chromosome inactivated, this approach becomes a tool to differentiate a monoclonal population from a polyclonal one in a female tissue. The HUMARA gene, in particular, has three important features that make it highly convenient for the purpose:
- The gene is located on the X chromosome and it goes through inactivation by methylation in normal embryogenesis of a female infant.[3] Because most genes on the X chromosome undergo inactivation, this feature is important.
- Human androgen receptor gene alleles have varying numbers of CAG repeats.[4] Thus, when DNA from a healthy female tissue is amplified by polymerase chain reaction (PCR) for a specific region of the gene, two separated bands can be seen on the gel.
- The region that is amplified by PCR also has certain base orders that make it susceptible to be digested by HpaII (or HhaI) enzyme when it is not methylated.[4] This detail gives the opportunity to researchers to differentiate a methylated allele from the unmethylated allele.
Due to these qualities of the HUMARA gene, clonal origin of any tissue from a female mammalian organism can be determined.