Spectrum and frequency of mutations in IMPDH1 associated with autosomal dominant retinitis pigmentosa and leber congenital amaurosis

Abstract

Purpose: The purpose of this study was to determine the frequency and spectrum of inosine monophosphate dehydrogenase type I (IMPDH1) mutations associated with autosomal dominant retinitis pigmentosa (RP), to determine whether mutations in IMPDH1 cause other forms of inherited retinal degeneration, and to analyze IMPDH1 mutations for alterations in enzyme activity and nucleic acid binding.

Methods: The coding sequence and flanking intron/exon junctions of IMPDH1 were analyzed in 203 patients with autosomal dominant RP (adRP), 55 patients with autosomal recessive RP (arRP), 7 patients with isolated RP, 17 patients with macular degeneration (MD), and 24 patients with Leber congenital amaurosis (LCA). DNA samples were tested for mutations by sequencing only or by a combination of single-stranded conformational analysis and by sequencing. Production of fluorescent reduced nicotinamide adenine dinucleotide (NADH) was used to measure enzymatic activity of mutant IMPDH1 proteins. The affinity and the specificity of mutant IMPDH1 proteins for single-stranded nucleic acids were determined by filter-binding assays.

Results: Five different IMPDH1 variants, Thr116Met, Asp226Asn, Val268Ile, Gly324Asp, and His 372Pro, were identified in eight autosomal dominant RP families. Two additional IMPDH1 variants, Arg105Trp and Asn198Lys, were found in two patients with isolated LCA. None of the novel IMPDH1 mutants identified in this study altered the enzymatic activity of the corresponding proteins. In contrast, the affinity and/or the specificity of single-stranded nucleic acid binding were altered for each IMPDH1 mutant except the Gly324Asp variant.

Conclusions: Mutations in IMPDH1 account for approximately 2% of families with adRP, and de novo IMPDH1 mutations are also rare causes of isolated LCA. This analysis of the novel IMPDH1 mutants substantiates previous reports that IMPDH1 mutations do not alter enzyme activity and demonstrates that these mutants alter the recently identified single-stranded nucleic acid binding property of IMPDH. Studies are needed to further characterize the functional significance of IMPDH1 nucleic acid binding and its potential relationship to retinal degeneration.

Figure 1

IMPDH1 variants found in retinal degeneration cohort. (A) Pedigrees of families with IMPDH1 variants. Closed symbols represent affected individuals, open symbols represent unaffected individuals, arrows indicate proband. Genotypes for each tested family member are listed below: +, wild-type allele; -, mutant allele. (B) Localization of variants in the human IMPDH1 monomer crystal structure. The catalytic domain is in gray and the subdomain is in blue (some of which is disordered). Variants found in adRP patients are in yellow, and LCA-associated variants are in purple. (C) UTAD391 pedigree with SNP and STR genotypes used to determine parental origin of allele with de novo Asn198Lys mutation. Based on rs2288550, the mutant allele (shaded in gray) must have come from the patient’s mother.

Figure 2

Nucleic acid binding affinity of IMPDH1 variants in a filter binding assay. Each graph is representative of three experiments. IMPDH1 concentration is shown as tetramers.

Figure 3

Alignment of IMPDH proteins from multiple species. Proteins were aligned by using ClustalW and were formatted with ESPrpt 2.0. Sequence numbering is based on the human IMPDH1 sequence. Completely conserved residues are shown as white letters on black background; highly conserved residues are boxed. CBS domains are shown with gray bars, and IMPDH1 variants identified in this study are shown with arrows.

Figure 4

Selected fundus photographs from patients with IMPDH1 mutations. (A) Asp226Asn; 32-year-old female with moderately advanced paucipigmentary RP. Each eye manifested vitreous syneresis, cells and condensations, vascular attenuation, and pigment epithelial atrophy concentrically from the periphery. The macula in each eye was spared, consistent with residual, formal visual fields of approximately 20° in all meridians. Left photograph shows optic disc pallor and spared macula; right eccentric view shows vascular attenuation, RPE thinning, and pigment clumping. (B) His372Pro; 67-year-old female with advanced RP. The disease is symmetric between eyes. The left photograph shows the left macular with severe loss of the macula, scleorotic-appearing loss of the adjacent retina, vessel attenuation, and pallor of the optic nerve-head. Right photograph shows typical pigmentary retinal degeneration in the equatorial retina. (C) Thr116Met; 54-year-old female demonstrating a relatively normal posterior pole with mild choroidal sclerotic changes (left photo) while the equatorial regions has heavy pigmentary bone spicule-like deposits (right photo). (D) Asn198Lys; photographs, right eye (on left) and left eye (on right) of 33-month-old female with severe visual loss in periphery and 20/40 vision OU. There is a generalized depigmentation of the fundus, vascular attenuation, pallor of the optic nerve, and a diffuse hypopigmented ring around the discs.