Single sperm typing demonstrates that reduced recombination is associated with the production of aneuploid 24,XY human sperm

To account for the increased proportion of paternal nondisjunction in 47,XX Y males as compared to other trisomies, it has been suggested that the XY b ivalent, with its reduced region of homology, is particularly susceptible t o nondisjunction. Molecular studies of liveborn Klinefelter syndrome (47,XX Y) individuals have reported an association between the absence of recombin ation in the pseudoautosomal region and nondisjunction of the XY bivalent. In this study we examined single sperm from a normal 46,XY male to determin e if there is any alteration in the recombination frequency of aneuploid di somic 24,XY sperm compared to unisomic sperm (23,X or Y). Two DNA markers S TS/STS pseudogene and DXYS15 were typed in sperm from a heterozygous man to determine if recombination had occurred in the pseudoautosomal region. Ind ividual unisomic sperm (23,X or Y) were isolated using a FACStar(Plus) flow cytometer into PCR tubes. To identify disomic 24,XY sperm, 3-colour FISH a nalysis was performed with probes for chromosomes X,Y and 1. The 24,XY cell s were identified using fluorescence microscopy, each disomic sperm was scr aped off the slide using a glass needle attached to a micromanipulator and then put into a PCR tube. Heminested PCR analysis of the two markers was pe rformed to determine the frequency of recombination. A total of 329 unisomi c sperm and 150 disomic sperm have been typed. The frequency of recombinati on between the two DNA markers was 38.3% for the unisomic sperm, similar to frequencies previously reported. The 24,XY disomic sperm had an estimated recombination frequency of 25.3%, however, a highly significant decrease co mpared to the unisomic 23,X or 23,Y sperm (chi (2) = 10.7, P = 0.001). This direct analysis of human sperm indicates that lack of recombination in the pseudoautosomal region is a significant cause of XY nondisjunction and thu s Klinefelter syndrome. (C) 2001 Wiley-Liss, Inc.