DNA from 203 adRP, 55 arRP, 7 isolated RP, 17 MD, 20 isolated LCA, and 4 multiplex LCA patients was tested for mutations in the 14 coding exons and flanking intron-exon junctions of IMPDH1. No probable disease-causing mutations were identified in any of the arRP, isolated RP, or MD patients tested.
As expected, several IMPDH1 variants were identified in the adRP samples, including the previously described Asp226Asn mutation that was found in the UTAD030, UTAD177, and UTAD557 families ( and ).2,13
Analysis of DNA from nine additional members of the UTAD557 family demonstrated that the mutation segregated with disease. Samples were not available from additional members of the UTAD030 and UTAD177 families.
IMPDH1 Variants Seen in Patients with 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 (more ...)
Five novel, potentially disease-causing IMPDH1 variants were also identified in the adRP cohort (). One of these mutations, Val268Ile, was reported previously in our original report characterizing mutations in IMPDH1 as the cause of RP10. We report it again here for accurate frequency calculations but did not include it in other analyses.2,8
A Thr116Met mutation was identified in the UTAD083 proband and in her affected son. This mutation alters a residue in the first CBS domain of the flanking region. A His372Pro was identified in the UTAD026 proband. This mutation alters a highly conserved residue in the core region of the protein (). Unfortunately, no additional family members were available for testing from the UTAD026 family.
A Gly324Asp variant was identified in the adRP proband from the UTAD067 and UTAD904 families. This variant alters a residue adjacent to the active site of the enzyme (). DNAs from three additional UTAD904 family members were tested and found to also contain the Gly324Asp mutation. One of these family members was diagnosed with RP and another with fundus flavimaculatus. The third family member was reported to be unaffected, although definitive clinical testing, such as fundus examination and electroretinogram (ERG), had never been done. No additional UTAD067 family members were available at this time.
Two novel heterozygous IMPDH1 variants were identified in the isolated LCA patients (). An Arg105Trp was found in UTAD463 and an Asn198Lys was found in UTAD391 (). No mutations in AIPL1, CRB1, CRX, GUCY2D, LRAT, MERTK, or RPE65 were identified in either of these patients when tested by Asper Ophthalmics (Tartu, Estonia) with their LCA microarray chip. Additional family members were not available from UTAD463. DNA was obtained from the unaffected sister and parents of the UTAD391 proband. None of these individuals had the Asn198Lys change, indicating that Asn198Lys is a new mutation or the result of germ-line mosaicism. Parentage of the affected child was confirmed by SNP and STR markers (data not shown). PCR and subcloning was used to determine which of the informative SNP rs2288550 genotypes carried the mutant allele. This analysis suggests that the mutation occurred on the allele inherited from the patient’s mother ().
A His296Arg change was found in one of the arRP probands. This variant was heterozygous in this patient, and no other protein-altering variants could be identified. Subsequent testing in our diagnostic laboratory identified this variant in an adRP patient who had a rhodopsin mutation, further substantiating reports that this variant is nonpathogenic.15
DNA samples from 116 unrelated individuals from the Centre d’Etude du Polymorphisme Humain were sequenced to check for the presence of the novel IMPDH1 variants and to determine the background variation found in IMPDH1. None of the variants described above were found in these samples. Only one rare amino-acid substitution, Ala285Thr, was found in this population, further demonstrating the high conservation of IMPDH1 () and increasing the likelihood that most of the observed amino acid changes in retinal degeneration patients are pathogenic.
IMPDH1 Variants Seen in CEPH Controls
Wild-type and mutant IMPDH1 were expressed in E. coli
strain H712 (which lacks endogenous IMPDH) and purified to >95% homogeneity. The enzyme activity of each IMPDH1 protein was determined by monitoring NADH production. Each mutant IMPDH1 protein tested showed a specific activity similar to wild type (). This is consistent with reports of other IMPDH1 mutations associated with retinal degeneration.7,8
Enzymatic Activity and Nucleic Acid Binding of Novel IMPDH1 Variants Identified in This Study
Nucleic Acid Binding
Previous studies have demonstrated that IMPDH1 binds singlestranded nucleic acids with nanomolar affinity.9
Several adRP-associated IMPDH1 mutations have been shown to alter this property, thereby suggesting a common functional abnormality that can aid in classifying variants as pathogenic or benign.8
Each novel IMPDH1 mutant identified in our cohort of patients was tested to determine single-stranded nucleic acid binding affinity and specificity.
Wild-type IMPDH1 binds random pools of ssDNA oligonucleotides with a Kd = 6 nM ( and ; IMPDH concentrations refer to tetramers). The Arg105Trp, Thr116Met, and Asn198Lys mutations show 8-fold, 17-fold, and 12-fold reductions in binding affinity, respectively. The His372Pro mutant also shows a decrease in binding affinity, although only 2-fold, much less than the other mutants. The Gly324Asp did not affect the affinity of IMPDH1 for ssDNA.
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.
In our current studies, wild-type IMPDH1 binds approximately 7% of the random pool of nucleic acids, which is consistent with previous findings.8
Just as with other reported adRP mutants, most of the IMPDH1 mutants tested in this study affect ssDNA binding specificity.9
This is true for the Arg105Trp, Thr116Met, and Asn198Lys mutants, which bind 2.9-, 2.3-, and 3.6- more of the random oligonucleotide pool, respectively. The binding specificity decreased most with the His372Pro mutant that bound 4-fold more of the random pool than wild-type IMPDH1. As with binding affinity, the Gly324Asp mutant was indistinguishable from wild-type and did not show loss of binding specificity.
Determining pathogenicity of novel protein variants is a frequent problem due to small family size or a lack of DNA samples from additional family members. This study used a combination of segregation analysis, absence in unaffected controls, cross-species comparisons, and alteration of the nucleic acid binding property to assess pathogenicity of IMPDH1 variants. Grading of a variant as benign, probably pathogenic, or pathogenic was based on the number of criteria that each mutant fulfilled and is summarized in .
Assessing Pathogenicity of IMPDH1 Variants
There is little doubt as to the pathogenicity of the Asp226Asn mutation. This mutation has been identified in many families, including two of the three very large families originally used to identify and refine the RP10 locus (Wada Y, IOVS
2005;45:ARVO E-Abstract 2456).2,15
Furthermore, this mutation has never been seen in unaffected controls, is 100% conserved in other IMPDH proteins, and alters the ability of the protein to bind single-stranded nucleic acids.2,8
Only one additional family member was available to test segregation of the Thr116Met mutation, and no additional family members were available from the His372Pro family. As with previously reported IMPDH1 mutations, neither of these mutants affected IMPDH1 enzyme activity, but they do decrease the affinity and the specificity of single-stranded nucleic acid binding. This, in conjunction with the high sequence conservation of each residue () and the lack of these variants in unaffected controls (), leads us to conclude that the Thr116Met and His372Pro mutations are likely to be pathogenic, as is the previously described and tested Val268Ile.2,8
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 (more ...)
The glycine at residue 324 is also 100% conserved in other IMPDH proteins, but segregation analysis of the Gly324Asp mutation shows a probably unaffected individual with the variant, which raises doubts as to pathogenicity. Functional analysis of the Gly324Asp mutation shows no effect on enzyme activity or nucleic acid binding, suggesting that this is a rare, benign variant and not a likely cause of retinal degeneration.
Pedigree and functional analysis demonstrates that the Asn198Lys mutation in the CBS domain is pathogenic and the result of a new mutational event. This residue is conserved in the mammalian IMPDH type I and type II proteins and in Drosophila and C. elegans IMPDH (). Like Asn198Lys, the Arg105Trp mutation is located at the junction of the CBS subdomain and alters the nucleic acid binding properties of IMPDH1. This residue is found in a large number of IMPDH proteins but is not as conserved as the Asn198 residue. This leads us to conclude that the Arg105Trp mutation is likely to be pathogenic. Furthermore, the isolated nature of the UTAD463 patient suggests the possibility that this could be the result of a new mutation.
Phenotypic Description of Patients with adRP
The proband from the UTAD030 family was seen in the clinic at age 32 years, after an initial diagnosis of RP. At that time, she had been an insulin-dependent diabetic for 4 years, had 20/20 corrected vision OU, and complained of night blindness. Biomicroscopy was normal, except for mild posterior subcapsular and paracentral cortical haze. The anterior vitreous showed syneresis with +1 pigmented cells. Fundus examination demonstrated granular pigmentary changes, pigment clumping, and vascular attenuation. The macula was intact in each eye. Goldmann visual fields showed concentric constriction to ~20° in all meridians (). One year later, her visual fields had constricted to 15° and were essentially the same 2 years later.
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 (more ...)
The proband from the UTAD177 family was initially diagnosed with RP at 10 years of age. When examined at 41 years of age, he had diffuse RPE atrophy, bone spicules at the equator and central RPE hypertrophy in the macula. ERGs were nonrecordable, and visual fields were 3° OD and 2° OS with the IV-4e isopters.
Clinical details are unavailable for most of the UTAD557 family. Affected members of this family were diagnosed with RP between 9 and 54 years of age. One affected male who was diagnosed at age 14 years was blind by age 39 years; while another was diagnosed at age 23 years and still had significant vision at age 65 years, although optic disc pallor, vascular attenuation and RPE abnormalities were present on examination.
The proband from UTAD083 began experiencing night blindness in her early 40’s and was first seen in the clinic at age 50 years. At this time, her visual acuity was 20/20 OU. Goldman visual fields were severely and symmetrically contracted OU with the I-4-e isopter subtending 8° and her IV-4-e isopter approximately 20° (). Fifteen years later the visual acuity was 20/40 OU. The visual fields were decreased to 2° with the I-4-e isopter, and 12° with the IV-4-e isopter.
The proband began experiencing night blindness around age 42 years. When examined at age 67 years, she had severe loss of vision, with the visual acuity of OD 20/80 and OS hand motions at 3 feet. Retinal examination showed RPE dropout like choroidal sclerosis. Optic disc pallor and bull’s-eye maculopathy were present (). Geographic atrophy was seen in the fluorescein angiography.
Phenotypic Description of Patients with LCA
The affected child in the UTAD463 family was first seen at 8 months of age when he was diagnosed with LCA and developmental delay with severe hypotonia. He had roving nystagmus with no fixation to light. Macular reflex was present in both eyes with the retina showing diffuse RPE mottling. No pigmentary deposits were present.
The affected female in the UTAD391 family was seen after referral at age 33 months. The parents had noted the child could not see things in her peripheral vision, and she could not find her food in dimly lighted conditions. Refractive error was OD +3.50+1.50 × 85, and OS +3.50+1.50 × 95. By Allen cards, her vision was 20/40 ().