Immunoglobulin Heavy Chain (IgH) Gene Somatic Hypermutation Status Using Homoduplex-Separated RT-PCR Products.

Assessment of IgH somatic hypermutation status has been shown to be a valuable indicator for judging the prognosis of patients with chronic lymphocytic leukemia (CLL). Our laboratory has developed a streamlined method to improve the rate of successful evaluation of IgH mutation status. Six individual PCR reactions are first performed using random hexamer-generated cDNA as template. These reactions have identical reaction parameters, use a common JH reverse primer and one of six VH class-specific forward primers within Framework 1. PCR products are separated on acrylamide or MetaPhor® agarose gels following formamide denaturation. In almost all cases, a single homoduplex band is resolved indicating a class-specific clonal product. The homoduplex band is excised from the gel, eluted and directly sequenced. Mutation analysis is performed using the NCBI Ig BLAST program with percent identity determined for the region from the beginning of CDR1 to the end of Framework 3. To date, less than 10% of cases analyzed have not yielded a clearcut clonal PCR product using this approach