Preliminary Study On Palindromic Sequence-mediated De Novo Chromosomal Mutations In Human Sperms

IntroductionThe recent rapid drop of sperm quality in past decades has attracted much attention worldwide, these become not only one of the reason causing male infertility , but also can cause the raise of birth defect rate. Though the assisted reproduction technologies can help those infertile couples to attain pregnancy, but there is evidence that the intracytoplasmic sperm injection can bring genetic defect to the offspring, thus affect the quality of population. Therefore, study the related genetic factor of spermatogenesis, helping to direct the pre - implantation genetic diagnosis, sound to be more and more important.In the past, the evaluation of sperm quality is dependent on the result (mo-tility, morphology) which is defected under the microscope. In recent years, genetic research on oligo - and azoospermia has attained substantial progress. However, little has been known about the mechanisms of de novo genetic mutations in sperms that may predispose to various birth defects in offspring, t (11;22 )( q23;q11) is the most common human non - Robertsonian translocation. In 2001, Kurahashi and Emanuel discovered unexpectedly high rate of de novo t (11;22) translocations in sperm from normal males, and the translocations are mediated by a symmetrical palindrome from both two chromosome. The t (11;22 ) were occasionally found in carriers" sperm, but somatic DNA from these individuals do not yield PCR junction fragments indicating that this translocation o-riginates during meiosis. In 2002, along with the completion of the whole Y chromosome sequence, researcher also discovered that the breakpoint of Y chromosome AZF microdeletion lie in with a palindrome. Several other translocationsby the similar mechanism, including t ( 1;22 ) ( p21. 2;qll. 21) , t (4;22) (q35.1,qll.2), t(17;22)(qll;qll) and t(X;22) (q27;qll. 21) have also discovered recently. These suggest that palindrome have a wide existence in the genome and can cause genomic instability. As individuals with more mutations may have higher risk to produce offspring affected with dysmorphology, birth defects and mental disabilities. This has urged us to develop a PCR - based system for quantifying aforementioned de novo translocations among sperm samples from genetic counseling. To further discover the relationship between sperm quality and these de novo mutation, we use nested - PCR detecting t (11;22 ) in 60 sperm DNA samples from normal and oligospermic individuals, and found 49 sample have these mutation. The frequency seems to be not associated with age and volume, but density and motility instead. In 4 individuals, we also detected other4 translocations, which included t(l;22), t(4;22), t(17;22) and t(X;22) with various mutation rates. The frequency was not associated with sequence homology. Quantification of relevant mutations within the sperms may provide an important clue for the evaluation of sperm quality.Materials and methodMaterials1. Sperm sample of normal and oligospermia man2. Reagents for sperm DNA abstract3. Reagents for PCR Method1. Sperm sample collection and DNA extraction and quantification2. Nested PCR to detect t(ll;22) rearrangement fragment and analysis3. Multi -Nested PCR to study dynamics of chromosome translocationResultsNested - PCR was applied for detection of t (11;22 ) translocations among 60 sperm DNA samples from 32 normal individual and 28 oligospermic patients,among which 49 were found to have these mutation. The frequency seemed to be not associated with age and volume, but with density and motility instead. In 4 individuals, 3 other chromosome translocations t(1;22) , t(17;22) and t(X;22) were also detected, while t(4;22) , t(5;22) , t(9;22) or t(15;22) was not found. On bioinformatics analysis with BLASTN, the varied mutation rates did not correlate with sequence homology.Conclusion1. t( 11;22) junction fragment is common in sperm sample2. The frequence of t( 11;22) is associated with sperm density and motility , but not associated with age and volume.3. The frequence of palindrome mediated chromosome translocation is not associated with sequence homology.