The search for trans-acting factors affecting somatic mutation

Abstract

Somatic hypermutation of immunoglobulin genes is an elusive process that causes point mutations to occur at an exceptionally high rate of 10 -4--10-3-per base pair per generation and is vital to providing immunity. We have undertaken three approaches aimed at determining possible mechanisms for this unique process. Since nothing was known about the mechanism of mutation, it has been tempting to evaluate any genetic defect that results in genetic instability and is associated with an immunodeficiency. Our first project undertook the examination of the Bloom's Syndrome (BS) using a similar approach to that which was used to examine another inheritable syndrome, xeroderma pigmentosum. The gene for this disorder, BLM, has recently been identified as a DNA helicase. If this gene were to play a role in immunoglobulin mutations, then patients with BS may lack normally mutated antibodies implicating BLM in immunoglobulin hypermutation. Sequences of immunoglobulin variable (V) regions were analyzed from small-unsorted blood samples obtained from BS individuals and compared with germ-line sequences. BS V regions displayed the normal distribution of mutations indicating that the defect in BS is not related to the mechanism of somatic mutation. These data strongly argues against BLM being involved in this process.;A second approach to unraveling this mysterious mutation process is to directly challenge hypotheses dealing with a proposed mechanism. One mechanism that was proposed over 10 years ago by Steele and Pollard was that an intrinsic reverse transcriptase (RT) copies the nascent pre-mRNA creating the large number of observed point mutations due to its high error rate. A cDNA copy of the mutated V region would then replace the endogenous DNA through a gene conversion like event, thus integrating these point mutations into the genome. Using AZT and ddC to inhibit endogenous RTs, we have assayed for somatic mutation using in vitro and in vivo models. Somatic mutation occurred at the same rate with or without treatment suggesting that mechanisms other than standard reverse transcription must be responsible for antibody V region hypermutation. (Abstract shortened by UMI.).