Mutations in the CMV UL97 gene associated with ganciclovir resistant retinitis

Purpose: To analyze vitreal isolates from patients with CMV retinitis in order to identify mutations in the CMV UL97 gene associated with clinical resistant disease. Methods: Vitreal isolates from patents with CMV retinitis, clinically resistant to ganciclovir, were obtained in the course of otherwise scheduled ocular surgery. CMV strain AD169 and vitreous from patients with responsive CMV retinitis served as controls. A 715 bp fragment of the UL97 gene, spanning the entire portion of the UL97 gene in which drug resistant mutations have been found (codons 420-640), was PCR amplified from clinical vitreal isolates, cloned and sequenced as double stranded DNA. Results: Mutations at codons 460 or 594 of the CMV UL97 gene were found in 5/6 of the vitreous samples from patients with ganciclovir resistant CMV retinitis. These mutations cause predicted amino acid changes in the UL97 gene product and have been shown by others to confer ganciclovir resistance. Further analysis revealed that each of these same 5 isolates had one or more additional mutations capable of disrupting the predicted amino acid sequence of the wild type UL97 gene product. Our analysis included two isolates, taken seven months apart, from the same eye. Two point mutations that give rise to predicted amino acid changes in the U_L97 gene product and three polymorphisms that do not give rise to amino acid changes were noted in both of these isolates. Three additional point mutations were found only in the second of these two isolates. Conclusions: These data provide direct evidence that clinically resistant CMV retinitis may be due to the emergence of resistant viral strains with mutations in the UL97 gene.