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 >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.
Zhang
Yun
Y
Department of Experimental Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA. yzhang@ciit.org
Monckton
Darren G
DG
Siciliano
Michael J
MJ
Connor
Thomas H
TH
Meistrich
Marvin L
ML
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000633:toxicity*; D015139:Blotting, Southern; D003520:Cyclophosphamide / Q000633:toxicity*; D004252:DNA Mutational Analysis; D017931:DNA Primers; D006801:Humans; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation / Q000187:drug effects* / Q000528:radiation effects*; D009223:Myotonic Dystrophy / Q000235:genetics; D065636:Myotonin-Protein Kinase; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000235:genetics; D013094:Spermatozoa / Q000187:drug effects* / Q000528:radiation effects*; D019680:Trinucleotide Repeat Expansion / Q000235:genetics*; D014916:Whole-Body Irradiation
D018906:Antineoplastic Agents, Alkylating; C585517:DMPK protein, human; C585516:DMPK protein, mouse; D017931:DNA Primers; D011506:Proteins; D003520:Cyclophosphamide; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases