Isolation and characterization of restriction fragment length polymorphisms for the human X chromosome and gentic mapping of Alport Syndrome

The genetic mapping of many X-linked disease genes has been hindered by the uninformative nature of the majority of the X chromosome DNA probes currently available. In an attempt to identify X chromosome-specific highly polymorphic markers, the X chromosome DOE library LAOXNL01 was screened with three oligonucleotides representative of minisatellite consensus sequences. The Ch35 phage library had been amplified on a recA- host and was considered likely to have maintained an excellent representation of genomic sequences because of the absence of recA and the inhibition of the RecBC nuclease by the Ch35 gam+ gene. A total of 170 clones was isolated by hybridization with oligonucleotide sequences and tested for polymorphism on five random female DNAs with six enzymes. Among the 54 clones demonstrating a polymorphic pattern, 48 different loci were represented. Eleven of these (20%) were determined not to be of X origin. The polymorphisms of the remaining 37 clones were characterized and reported as new restriction fragment length polymorphisms for the X chromosome. A pattern of similar length variation with multiple enzymes, indicative of a variable number tandem repeat (VNTR) polymorphism, was demonstrated in 6/11 (55%) of the non-X clones. However, only 4/37 (11%) of the X clones clearly had VNTR characteristics, suggesting a decreased amount of VNTR polymorphism on the X chromosome. The 37 polymorphic X markers were physically localized by hybridization to a set of rodent-human somatic cell hybrid DNAs representing nine different X breakpoints. An informative subset of 114 individuals from a very large (n > 900) Utah kindred demonstrating X-linked Alport Syndrome was screened with markers from the Xq21.2 - q22 region. Two markers, QST-7 and KZO-33, have demonstrated linkage to the Alport Syndrome gene with a LOD score of 10.0 (theta = 0.07) and 3.5 (theta = 0.05) respectively. Preliminary CEPH family data have been collected and analyzed for contribution to the genetic map of the Alport gene region.