The Effect of Four Pathogenic Mutations in the CHM Gene on Rab Escort Protein-1 Structure

Abstract

Purpose: : Choroideremia (CHM) is a chorioretinal degeneration with an X-linked pattern of inheritance. The CHM gene encodes Rab escort protein-1 (Rep-1), which is involved in trafficking of Rab proteins in the cell. To date, the majority of reported mutations in the CHM gene cause a complete loss of Rep-1 and its function. Here we report 3 mutations in which Rep-1 is absent and a novel missense mutation in which Rep-1 is present.

Methods: : Clinical data were obtained from 4 male patients with CHM (aged 44-76). Mononuclear cells were isolated from peripheral blood of patients and age-matched controls. Skin biopsies from patients and healthy controls were used to establish primary dermal fibroblast (FB) cell lines. Genomic DNA was isolated from the CHM patients and 89 normal human males. PCR-amplification of the exons of the CHM gene was performed with sequencing of the PCR products. A TaqMan MGB genotyping custom assay was developed for the missense mutation.

Results: : Four pathogenic mutations: a novel missense mutation, L550P; a truncation c.1542T>A, STOP; and two deletions (c.525_526delAG, c.1646delC) in the CHM gene were found. To analyze the effect of the mutations, the 3D structure of human REP-1 and the proteins associated with REP-1 function were modeled using sequence homology to rat proteins. In silico analysis of the missense mutation L550P suggests that P550 destabilizes the β-structural elements, and the Rep-1 tertiary structure. Truncation and deletion mutants are associated with a partial or total loss of the REP-1 essential activity and protein-protein interactions as predicted by the analysis of the structure and stability of these protein products. The presumptive loss of protein from three CHM patients was confirmed by the Western blot analysis of protein from mononuclear cells and fibroblasts. A small amount of protein was seen in fibroblasts from the patient with a missense mutation.

Keywords: retinal degenerations: hereditary • genetics • protein structure/function