Cytogenetic and molecular characterization of a human chromosomal region which renders in vitro phenotypic correction of xeroderma pigmentosum complementation group D

Abstract

Xeroderma pigmentosum (XP) has been a focus of extensive investigation since its clinical description in 1874 and the subsequent demonstration of a DNA repair defect in 1968. However, identification of the mutant genes responsible for XP has been hindered by numerous experimental limitations and extensive genetic heterogeneity. The obvious in vitro phenotypes of UV sensitivity and defective DNA repair observed in XP fibroblasts can be complemented following transfer of chromosomes from normal cells. This strategy offers an excellent approach for gene mapping and cloning of the defective genes. Recently, a rearranged single human chromosome (Tneo) has been isolated which restores both UV light resistance and DNA repair activity in XP complementation group D (XP-D) cells. The cytogenetic and molecular characterization of Tneo was the focus of this dissertation. The results provided preliminary mapping of the XP-D locus and serve as a critical first step in the isolation of the gene(s) defective in XP-D cells. Classical cytogenetic analysis revealed that Tneo is a monocentric chromosome of undefined human origin. "Reverse in situ hybridization" by Alu-PCR indicated that this chromosome involves a complex rearrangement consisting of chromosome segments from 16q, 17p and 19. "Painting" with chromosome-specific DNA libraries was used to define the orientation of the chromosomal segments present in Tneo. Southern blot hybridizations using chromosome-specific probes further delineated the specific breakpoints involved in the Tneo rearrangement, as consistent with a der(16)(19pter{dollar}\to{dollar}19p13.2::17p?{dollar}\to{dollar}17p?::19p13.1{dollar}\to{dollar}19p13.1::16cen{dollar}\to{dollar}16q23q24?:: 19q13.2{dollar}\to{dollar}19q13.3::17p?{dollar}\to{dollar}17p?). Deletions of the complementing chromosome were generated and revealed that the restoration of UV resistance in XP-D cells resides in chromosomal material in the distal q-arm of Tneo. Molecular characterization demonstrated that the XP-D complementing region involves DNA derived from 19q13.2{dollar}\to{dollar}19q13.3 and 17p. Transfer of a normal human chromosome 17 into XP-D cells failed to correct the defective phenotypes, implying that the XP-D gene lies within or near 19q13.2{dollar}\to{dollar}19q13. It is of particular interest that this region includes a previously described human DNA repair gene cluster. The experiments described have generated materials which should prove valuable for the molecular cloning of the XP-D gene(s). Additionally, the data illustrate the power of combined cytogenetic and molecular mapping techniques for use in the isolation of genes defective in a variety of human genetic disorders which exhibit in vitro phenotypes.