A YEAST ARTIFICIAL CHROMOSOME (YAC) CONTIG ENCOMPASSING THE CRITICAL REGION OF THE X-LINKED LYMPHOPROLIFERATIVE DISEASE (XLP) LOCUS

X-linked lymphoproliferative disease (XLP) is characterized by a marke d vulnerability to Epstein-Barr virus (EBV) infection. Infection of XL P patients with EBV invariably results in fatal mononucleosis, agammag lobulinemia, or malignant lymphoma. Initially the XLP gene was assigne d to a 10-cM region in Xq25 between DXS42 and DXS37. Subsequently, an interstitial, cytogenetically visible deletion in Xq25 was identified in one XLP family, 43. In this study we estimated the deletion in XLP patient 43-004 by dual-laser flow karyotyping to involve 2% of the X c hromosome, or approximately 3 Mb of DNA sequence. From a human chromos ome Xq25-specific yeast artificial chromosome (YAC) sublibrary, five Y ACs containing DNA sequences deleted in patient 43-004 have been isola ted. Sequence-tagged sites (STSs) fi om these YACs have been used to i dentify interstitial deletions in unrelated XLP patients. Three more f amilies with interstitial deletions were found. Two of the patients (6 3-003 and 73-032) carried an interstitial deletion of 3.0 Rib overlapp ing the 43-004 deletion. In one XLP patient (30-011) who exhibited the characteristic postinfectious mononucleosis phenotype of XLP with hyp ogammaglobulinemia and malignant lymphoma, a deletion of approximately 250 kb was detected overlapping the deletion detected in patients 43- 004, 63-003, and 73-032. A YAC contig of 2.2 Mb spanning the XLP criti cal region, whose orientation on chromosome X was determined by double color fluorescence in situ hybridization and which consists of 15 ove rlapping YAC clones, has been constructed. A detailed restriction enzy me map of the region has been constructed. YAC insert sizes were deter mined by counter-clamped homogenous electric held gel electrophoresis. Chimerism of YACs was determined by FISH and restriction mapping. On the basis of lambda subclones, YAC end-derived plasmids, and STSs with an average spacing of 100 kb, a long-range physical map was construct ed using 5 rare-cutter restriction enzymes. The STSs and lambda subclo nes were used in Southern hybridization and PCR analyses. The work pre sented here substantially refines the critical region for XLP. The YAC contig with the overlapping interstitial deletions constitutes the ba sis for the construction of a transcriptional map of the critical regi on and facilitates the identification of the XLP gene. (C) 1997 Academ ic Press