A study of CAG repeat instability of HTT gene following spermatogenesis, by single sperm analysis

Abstract

Microsatellites are among of the most variable DNA sequences. Different factors such as slipped-strand mispairing and incomplete performance of FEN1 enzyme are involved in their instability. All of these mechanisms are also stable during gametogenesis. Numerous mitosis divisions that spermatogonia undertook to evolve to primary spermatocyte as well as the high rate of these divisions give conditions in which the repeats are changed. During meiosis another factor, recombination, and after meiosis, chromatin remodeling in Spermatids also intensifies the microsatellite instability.We considered the normal CAG repeats (14–32 repeats in this study) in HTT gene as a candidate of trinucleotide repeat to investigate whether these repeats change during spermatogenesis. So, we can explain how repeats get to the threshold of being intermediate alleles and eventually disease-causing length. We examined 269 (93 + 81 + 95) single sperm cells from 3 healthy men using nested-PCR followed by polyacrylamide gel electrophoresis and compared the CAG repeat sizes of peripheral blood leukocyte DNA with those in single sperm cells of the same individual.Using single sperm analysis as a rapid method, we showed that the instability of normal CAG repeat tracts during spermatogenesis is higher than those reported in some earlier studies. The variance of CAG repeat changes between three individuals was significant (F-test, P < 0.0001). Since in the present study no changes were found in single sperm cells of the homozygous individual, further investigations are need to study normal repeat instability during spermatogenesis in homozygous individuals. Moreover larger sample size for single sperm cells can also help to get more comprehensive results.