Detection of radiation and cyclophosphamide-induced mutations in individual mouse sperm at a human expanded trinucleotide repeat locus transgene

A method to measure the germline mutations induced by cancer treatment in humans is needed. To establish such a method we used a transgenic mouse model consisting of a human DNA repeat locus that has a high spontaneous mutation frequency as a biomarker. Alterations in repeat number were measured in individual sperm from mice hemizygous for an expanded (CTG)(162) human myotonic dystrophy type 1 (DM1) microsatellite repeat using single genome-equivalent (g.e.) PCR and detection by a DNA fragment analyzer. Mutation frequencies were measured in DNA from sperm from controls and sperm derived from stem spermatogonia, differentiating spermatogonia, and spermatocytes exposed to radiation and from spermatocytes of mice treated with cyclophosphamide. There was no increase above control levels in mutations, scored as greater than 1 repeat changes, in any of the treated groups. However, moderately large deletion mutants (between 9 and 20 repeat changes) were observed at frequencies of 2.2% when spermatocytes were treated with cyclophosphamide and, 1.8 and 2.5% when spermatocytes and stem cells, respectively, were treated with radiation, which were significantly higher than the frequency of 0.3% in controls. Thus, radiation and cyclophosphamide induced deletions in the expanded DM1 trinucleotide repeat. PCR artifacts were characterized in sperm DNA from controls and from mice treated with radiation; all artifacts involved losses of more than 20 DM1 repeats, and surprisingly the artifact frequency was higher in treated sperm than in control sperm. The radiation-induced increase in the frequency of PCR artifacts might reflect alterations in sperm DNA that destabilize the genome not only during PCR amplification but also during early embryonic development

Age and insertion site dependence of repeat number instability of a human <i>DM1</i> transgene in individual mouse sperm

Precise measurement of germline repeat number mutations is important for understanding the molecular etiology of expanded trinucleotide repeat diseases. We used single genome-equivalent PCR of sperm DNA to measure the mutation frequencies in two lines of Dmt transgenic mice containing an expanded CTG·CAG tract on an identical genetic background. Single genome-equivalent PCR indicated that apparent mutational spectra derived in other investigations from PCR of bulk sperm DNA were largely the consequence of PCR stutter and not mutations. Here we show that sperm from 8-week-old &lt;i&gt;Dmt&lt;/i&gt;-D mice had a significantly higher mutation frequency (change of &#62;1 repeat) (14.2%) than those of &lt;i&gt;Dmt&lt;/i&gt;-E mice of the same age (5.5%), in agreement with pedigree analysis. Furthermore, the mutation frequency in sperm of &lt;i&gt;Dmt&lt;/i&gt;-D mice increased significantly with age (28.0% at 17 weeks). The age dependence of the degree of expansion implies that mutations accumulate with time in spermatogenic stem cells. Similar rates of expansion per spermatogenic cycle in man would yield the large expansions observed in human diseases such as myotonic dystrophy type 1. Pedigree data showed a significant age-dependent bias toward repeat contraction in female transmissions and a trend towards expansion with age in male transmissions. Thus, direct single genome-equivalent PCR of the sperm DNA of an individual male appears to predict the distribution of mutant allele sizes that might be inherited by its offspring. In further contrast to a recent report, the sex of the offspring had no detectable effect on the direction of the mutational length change