Retinitis pigmentosa: mutation analysis of RHO, PRPF31, RP1, and IMPDH1 genes in patients from India

Abstract

Purpose: To screen for possible disease-causing mutations in rhodopsin (RHO), pre-mRNA processing factor 31 (PRPF31), retinitis pigmentosa 1 (RP1), and inosine monophosphate dehydrogenase 1 (IMPDH1) genes in Indian patients with isolated and autosomal dominant forms of retinitis pigmentosa (adRP). Information on such data is not available in India and hence this study was undertaken.

Methods: Blood samples were obtained from 48 isolated and 53 adRP patients, who were recruited for the study. Each patient underwent a detailed clinical examination. Genomic DNA was extracted from the blood samples and screened for mutations in four genes using an ABI3100 Avant genetic analyzer. Reverse transcriptase polymerase chain reaction was performed to amplify the mutated (IVS6+1G/A) mRNA of PRPF31 in a two-generation adRP family.

Results: Of the 101 probands analyzed, three harbored possible disease-causing mutations. Pathogenic changes were observed in RHO and PRPF31. A RHO mutation, p.Gly106Arg, was found in an isolated RP patient with sectoral RP. Two novel, heterozygous mutations were identified in PRPF31: p.Lys120GlufsX122 in an isolated RP patient and a splice site mutation, IVS6+1G/A in an adRP patient. However, no disease-causing changes were observed in RP1 and IMPDH1.

Conclusions: We screened RHO, PRPF31, RP1, and IMPDH1 and identified causative mutations in 4% of isolated and 2% of adRP patients from India. To the best of our knowledge, this is the first report to identify frequencies of mutations in isolated and adRP patients in India.

Figure 1

Clinical and molecular features of the proband (L2:50) with the p.Gly106Arg mutation. A: Pedigree showing the isolated form of the disease. B: Genomic DNA sequences (reverse) of a part of the RHO gene of L2:50 displaying the p.Gly106Arg mutation (top) and of a normal subject (bottom). The rectangular box shows the position of a heterozygous change at nucleotide 316 (c.316G/A, but the sequence shows the reverse sequence boxed as Y). C: Visual field test reveals a sectoral form of RP. D: Fundus (right eye) photograph showing mild retinitis pigmentosa changes.

Figure 2

Electropherograms displaying a novel PRPF31 mutation, p.Lys120GlufsX122. Genomic DNA sequences (reverse primer) of a part of PRPF31 from a patient with the p.Lys120GlufsX122 mutation (top) and from a normal subject (bottom). The rectangular box shows the position of a heterozygous deletion of two nucleotides at codon 120 (c. c.358_359 del AA, but the sequence shows the reverse sequence boxed as TT).

Figure 3

Pedigree of family A harboring a IVS6+1G/A mutation in PRPF31. The change in the genomic DNA sequence was observed in two affected patients and an asymptomatic individual. Normal individuals are shown as clear circles (females) or squares (males), and affected individuals are shown as solid symbols. The clear square with the symbol “?” indicates an asymptomatic carrier.

Figure 4

Electropherograms displaying intron 6 retention. A comparison of a portion of PRPF31 cDNA sequences between the unaffected (top: I-2) and the affected (bottom: II- 1) revealed intron 6 retention in all affected and asymptomatic individuals of family A. The green hatched box represents exonic region, and the red rectangular box indicates the start position of intron 6 retention (non-hatched) in the proband.

Figure 5

Fundus images of I-1, I-2, II-1, and II-2 of family A. The fundus pictures from unaffected members I-2 and II-2 were normal. However, typical features of retinitis pigmentosa could be well appreciated in affected members I-1 and II-1.The other details of the members of family A are as follows; Individual I-1 was a 24-year-old female with a history of night blindness since 8 years. Her visual acuity was counting fingers at 4 meters, and was not improving with glasses (NIG). Fundus examination revealed arteriolar attenuation, bony spicules with degenerative macular changes, and disc pallor. The electroretinogram (ERG) was nonrecordable, fields were grossly defective. Individual, I-2 was a 32-year-old male with a vision of 6/6 and normal fundus. Individual II-1 (proband) was a 5-year-old female with a complaint of night blindness since 6 months. Fundus revealed attenuated vessels, normal disc, dull foveal reflex, and altered retinal sheen. The ERG was nonrecordable in both the eyes. Individuals II-2 was a 3.5-year-old male with normal vision and fundus. Individual II-3 is a 2-year-old female with normal fundus. The ERG and visual field test could not be done due to pediatric age.

Figure 6

Alignment of protein sequences of the PRPF31 protein The wild-type sequence and two identified novel mutants, IVS6+1G/A and p.Lys120GlufsX122, are shown. Both the mutations resulted in a premature truncation of the protein.

Figure 7

Schematic representation of PRPF31 and its protein structure. Illustrated are positions of reported mutations (including large deletions) in human PRPF31 and domain features of predicted PRPF31 protein. Two novel PRPF31 mutations reported in this study are depicted in bold. Distance between exons and domains are not drawn to scale.