Mapping of microsatellite markers in the Alagille region and screening of microdeletions by genotyping 23 patients.

Alagille syndrome (AGS) has been assigned to 20p11.23-20p12.2 according to minimum overlap between deletions observed on the chromosome 20 short arm of 9 patients. We report here the localisation of 5 microsatellite markers (D20S41, D20S48, D20S50, D20S56, and D20S58) within the deletion of one AGS patient. This study allows an estimation of the genetic extent of this deletion as being between 30 and 36 cM, and demonstrates its paternal origin. The search for submicroscopic deletions in 23 AGS patients, by typing these 5 markers, failed to reveal allelic loss. However, these results lead to the proposition that the AGS locus lies in one of the seven intervals defined by the six microsatellite markers in the region flanked by D20S5 and D20S18.

Deleted chromosome 20 from a patient with Alagille syndrome isolated in a cell hybrid through leucine transport selection: study of three candidate genes.

Alagille syndrome (AGS) is a well-defined genetic entity assigned to the short arm of Chromosome (Chr) 20 by a series of observations of AGS patients associated with microdeletions in this region. By fusing lymphoblastoid cells of an AGS patient that exhibited a microdeletion in the short arm of Chr 20 encompassing bands p11.23 to p12.3 with rodent thermosensitive mutant cells (CHOtsH1-1) deficient in-leucyl-tRNA synthetase, we isolated a somatic cell hybrid segregating the deleted human Chr 20. This hybrid clone, designated NR2, was characterized by several methods, including PCR, with eight pairs of oligonucleotides mapped to Chr 20: D20S5, D20S41, D20S42, D20S56, D20S57, D20S58, adenosine deaminase (ADA), and Prion protein (PRIP); Restriction Fragment Length Polymorphism (RFLP) analyses with four genomic anonymous probes (D20S5, cD3H12, D20S17, D20S18); and fluorescent in situ hybridization (FISH) with total human DNA and D20Z1, a sequence specific to the human Chr 20 centromere, as probes. The NR2 hybrid allowed us to exclude three candidate genes for AGS: hepatic nuclear factor 3 beta (HNF3 beta), paired box 1 (PAX1), and cystatin C (CST3) as shown by their localization outside of the deletion. The NR2 hybrid is a powerful tool for the mapping of new probes of this region, as well as for obtaining new informative probes specific for the deletion by subtractive cloning of the region. Such markers will be useful for linkage analysis and screening of cDNA libraries.