Molly A. Morgan, MFS, Thomas J. Parsons, Ph.D., and Mitchell M. Holland, Ph.D.
DoD DNA Registry, Armed Forces Institute of Pathology and American Registry of Pathology, Rockville, MD 20850-3125

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Nuclear pseudogenes homologous to mitochondrial DNA (mtDNA) sequences have recently been detected in many organisms (e.g. Collura and Stewart 1995, Zischler et al., 1995). These elements, apparently caused by rare events of migration and integration of mtDNA sequences to the nucleus, create the potential for complications in human identity testing based on mtDNA sequence analysis. To our current knowledge, only a single nuclear pseudogene corresponding to human control region sequences has been detected. The insert has been mapped to chromosome 11 and its frequency has been characterized in various populations (Zischler et al., 1995). The element apparently reflects an ancient genetic transposition from the mitochondrion to the nucleus and differs from typical mtDNA sequences by approximately 7.5%.

Through our extensive experience with mtDNA casework, we have encountered no evidence that nuclear pseudogenes are generally amplified in "routine" applications. The probable reason for this is that an individual's authentic mtDNA sequences will greatly predominate in copy number over any nuclear pseudogenes. The control region pseudogene that has been identified so far is a single copy nuclear element.

However, we have observed amplification of the nuclear pseudogene in two specific instances. Interestingly, in both cases, identifiable conditions existed that caused the preferential amplification of the pseudogene. The first instance involved a point mutation in the authentic mtDNA sequence at the 3'-most primer position that prevented amplification of authentic mtDNA, and permitted amplification of the nuclear pseudogene (easily recognizable by unique, unusual polymorphisms). Other primer pairs were capable of amplifying authentic DNA from these same extracts. The second instance involved an attempt to amplify mtDNA from a sperm head fraction of vaginal swabs. DNA preparations from the sperm head fraction are known to be highly deficient in mtDNA, and amplification of the pseudogene in this case can be traced to a higher representation of nuclear than mtDNA templates.

Our results indicate that forensic scientists should be aware of the potential for amplification of the nuclear pseudogene. Additionally, studies in our laboratory are underway to search for additional nuclear pseudogenes that correspond to control region sequences and could possibly complicate mtDNA identity testing. An initial population screen is being performed to demonstrate that the mtDNA primer pairs used in testing do not have potential nuclear pseudogene targets.

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The opinions and assertions expressed herein are solely those of the authors and are not to be construed as official or as the views of the United States Department of Defense or the United States Department of the Army.