Novel homozygous loss-of-function mutations in RP1 and RP1L1 genes in retinitis pigmentosa patients

ABSTRACT

Background: Retinitis pigmentosa (RP) is a heterogeneous group of ocular dystrophy. It is challenging to identify the underlying genetic defect in individuals with RP due to huge genetic heterogeneity. This study was designed to delineate the genetic defect(s) underlying RP in extended Saudi families and to describe the possible disease mechanism.

Materials and Methods: Fundus photography and a high definition optical coherence tomography (HD-OCT) were performed in order to detect the earlier stages of macular degeneration. Genomic DNA was extracted followed by genome-wide SNP genotyping and whole exome sequencing (WES). Exome data was filtered to identify the genetic variant(s) of interest.

Results: Clinical examination showed that affected individuals manifest key features of RP. The fundus exam shows pale optic disc and bone spicules at the periphery. OCT shows macular degeneration as early as at the age of 4 years. Whole genome scan by SNPs identified multiple homozygous regions. WES identified a 10 bps novel insertion mutation (c.3544_3545insAGAAAAGCTG; p.Ala1182fs) in the RP1 gene in both affected individuals of family A. Affected individual from family B showed a large insertion of 48 nucleotides in the coding part of the RP1L1 gene (c.3955_3956insGGACTAAAGTAATAGAAGGGCTGCAAGAAGAGAGGGTGCAGTTAGAGG; p.Ala1319fs). Sanger sequencing validates the autosomal recessive inheritance of the mutations.

Conclusion: The results strongly suggest that the insertion mutations in the RP1 and RP1L1 genes are responsible for the retinal phenotype in affected individuals from two families. Heterozygous individuals are asymptomatic carriers. We propose that the protective allele in other homozygous regions in heterozygous carriers contribute to the phenotypic variability in asymptomatic individuals.     

Age-dependent prevalence of mutations at the GLC1A locus in primary open-angle glaucoma

PURPOSE: To screen a population with primary open-angle glaucoma for mutations in the gene that encodes the trabecular meshwork inducible glucocorticoid response protein (TIGR), also known as myocilin (MYOC). METHODS: Ophthalmologic information was collected for study subjects with primary open-angle glaucoma and their relatives. Mutation screening of 74 primary open-angle glaucoma probands was conducted by sequencing TIGR/MYOC coding sequence and splice sites. RESULTS: In 23 families we detected 13 nonsynonymous sequence changes, nine of which appear to be mutations likely to cause or contribute to primary open-angle glaucoma. Two mutations, Arg272Gly and Ile499Ser, and one nonsynonymous sequence variant, Asn57Asp, are novel. We found mutations in nine of 25 juvenile glaucoma probands (36%) and two of 49 adult-onset glaucoma probands (4%). Age classification of families rather than individual probands revealed mutations in three of nine families with strictly juvenile primary open-angle glaucoma (33%), and no mutations in 39 families with strictly adult-onset primary open-angle glaucoma (0%). In families with mixed-onset primary open-angle glaucoma containing both juvenile primary open-angle glaucoma and adult-onset primary open-angle glaucoma cases, we found mutations in eight of 26 families (31%). CONCLUSIONS: Our data suggest that Gly252Arg, Arg272Gly, Glu323Lys, Gln368STOP, Pro370Leu, Thr377Met, Val426Phe, Ile477Asn, and Ile499Ser are likely to play roles that cause or contribute to the etiology of autosomal dominant primary open-angle glaucoma. Our finding of more TIGR/MYOC mutations in families with mixed-onset primary open-angle glaucoma than in the families with strictly adult-onset primary open-angle glaucoma implies that the presence of relatives with juvenile primary open-angle glaucoma in a family could be used as a basis for identifying a subset of the population with adult-onset primary open-angle glaucoma with higher prevalence of TIGR/MYOC mutations. To address this issue, and to refine estimations of mutation prevalence in these age-defined subpopulations, prospective study of a larger population ascertained entirely through adult-onset primary open-angle glaucoma probands will be needed.     

Dominant cystoid macular dystrophy associated with mutations in the RP1L1 gene

Aims: To evaluate the effects of intravitreal Conbercept (IVC) as adjunctive treatments before PRP to decrease hyperreflective dots (HRDs) in Chinese PDR patients. Methods: Fifty-nine enrolled patients were categorized into 2 groups: single dose IVC (0.5 mg/0.05 mL) 1wk before PRP (Plus group) or PRP only (PRP group). Six months later, we measured the best corrected visual acuity (BCVA), central macula thickness (CMT) by OCT and counted the number of HRDs in different retina layers. Results: The average CMT significantly decreased in Plus group but increased in PRP group. The average BCVA in the Plus group was also significantly better than that in the PRP group. Total HRDs decreased in the Plus group but increased in PRP group significantly. IVC pre-treatment has beneficial effects on reducing HRDs forming in the inner retina layer while the PRP alone increased the HRDs in the outer retina layer. Conclusion: IVC is a promising adjunctive treatment to PRP in the treatment of PDR. Single dose IVC one week before PRP was suggested to improve retina BRB, decrease lipid exudate and inhibit HRDs development in PDR.     

Disease expression of RP1 mutations causing autosomal dominant retinitis pigmentosa

PURPOSE: To determine the disease expression in heterozygotes for mutations in the RP1 gene, a newly identified cause of autosomal dominant retinitis pigmentosa (adRP). METHODS: Screening strategies were used to detect disease-causing mutations in the RP1 gene, and detailed studies of phenotype were performed in a subset of the detected RP1 heterozygotes using electroretinography (ERG), psychophysics, and optical coherence tomography (OCT). RESULTS: Seventeen adRP families had heterozygous RP1 changes. Thirteen families had the Arg677ter mutation, whereas four others had one of the following: Pro658 (1-bp del), Ser747 (1-bp del), Leu762-763 (5-bp del), and Tyr1053 (1-bp del). In Arg677ter RP1 heterozygotes, there was regional retinal variation in disease, with the far peripheral inferonasal retina being most vulnerable; central and superior temporal retinal regions were better preserved. The earliest manifestation of disease was rod dysfunction, detectable as reduced rod ERG photoresponse maximum amplitude, even in heterozygotes with otherwise normal clinical, functional, and OCT cross-sectional retinal imaging results. At disease stages when cone abnormalities were present, there was greater rod than cone dysfunction. Patients with the RP1 frameshift mutations showed similarities in phenotype to those with the Arg677ter mutation. CONCLUSIONS: Earliest disease expression of RP1 gene mutations causing adRP involves primarily rod photoreceptors, and there is a gradient of vulnerability of retinopathy with more pronounced effects in the inferonasal peripheral retina. At other disease stages, cone function is also affected, and severe retina-wide degeneration can occur. The nonpenetrance or minimal disease expression in some Arg677ter mutation-positive heterozygotes suggests important roles for modifier genes or environmental factors in RP1-related disease.     

Clinical features and mutations in patients with dominant retinitis pigmentosa-1 (RP1)

PURPOSE: To survey patients with dominant retinitis pigmentosa (RP) for mutations in the RP1 gene to determine the spectrum of dominant mutations in this gene, to estimate the proportion of dominant RP caused by this gene, and to determine whether the clinical features of patients with RP1 mutations differ from features of those with rhodopsin mutations. METHODS: A set of 241 patients who did not have mutations in the rhodopsin gene (based on previous work) formed the basis for the study. Of these patients, 117 had also been previously evaluated and were found not to carry mutations in the RDS gene. The single-strand conformation polymorphism (SSCP) method was used to search for sequence variants, which were then directly sequenced. The relatives of selected patients were recruited for segregation analyses. Clinical evaluations of patients included a measurement of Snellen visual acuity, final dark adaptation thresholds, visual fields, and ERGs. Clinical data were compared with those obtained earlier from a study of 128 patients with dominant rhodopsin mutations. RESULTS: Of the 241 patients, all were screened for the most common RP1 mutation (Arg677Ter), and 10 patients were found to have this mutation. In addition, an evaluation of a subset of 189 patients in whom the entire coding sequence was evaluated revealed the following mutations: Gln679Ter (1 case), Gly723Ter (2 cases), Glu729(1-bp del) (1 case), Leu762(5-bp del) (2 cases), and Asn763(4-bp del) (1 case). All of these mutations cosegregated with RP in the families of the index patients. Nine missense mutations that were each found in six or fewer patients were encountered. The segregation of eight of these was evaluated in the respective patients' families, and only one segregated with dominant RP. This cosegregating missense change was in cis with the nonsense mutation Gln679Ter. Although patients with RP1 mutations had, on average, slightly better visual acuity than patients with rhodopsin mutations, there was no statistically significant difference in final dark-adaptation thresholds, visual field diameters, or cone electroretinogram (ERG) amplitudes. Comparably aged patients with RP1 mutations had visual function that varied by approximately two orders of magnitude, based on visual fields and ERG amplitudes. CONCLUSIONS: Dominant RP1 alleles typically have premature nonsense codons occurring in the last exon of the gene and would be expected to encode mutant proteins that are only approximately one third the size of the wild-type protein, suggesting that a dominant negative effect rather than haploinsufficiency is the mechanism leading to RP caused by RP1 mutations. On average, patients with RP1 mutations have slightly better visual acuity than patients with dominant rhodopsin mutations; otherwise, they have similarly severe disease. The wide range in severity among patients with RP1 mutations indicates that other genetic or environmental factors modulate the effect of the primary mutation.     

Molecular analysis of the CYP1B1 gene: identification of novel truncating mutations in patients with primary congenital glaucoma

BACKGROUND: Mutations and polymorphisms have been identified in the CYP1B1 gene; while mutations that affect the conserved core structures of cytochrome P4501B1 result in primary congenital glaucoma (PCG), mutations in other regions hold the potential to define differences in estrogen metabolism. In the present study, we analyzed the CYP1B1 gene in Mexican patients with PCG and described four novel mutations. MATERIALS AND METHODS: The sample included 12 nonrelated cases with PCG. Analysis of coding regions of the CYP1B1 gene was performed through PCR and DNA sequencing analysis from genomic DNA. RESULTS AND DISCUSSION: Molecular analysis of the CYP1B1 gene showed the following molecular defects: (1) a novel single-base pair deletion within codon 370 (1454delC) that produces a substitution of leucine instead of proline and a premature stop codon 57 amino acids after the last original amino acid; this family also harbored a novel polymorphic variant of the cytochrome P4501B1 with six single-nucleotide polymorphisms (142C-->G; 355G-->T; 729G-->C; 4326C-->G; 4360C-->G and 4379C-->T); (2) a novel single-base pair deletion within codon 277 (1176delT) that results in a premature stop codon; (3) a novel single-base pair deletion within codon 179 (880delG) that produces a substitution of arginine instead of alanine and a premature stop codon 17 amino acids downstream from the last original amino acid, and (4) a duplication (or insertion) of ten base pairs within codon 404 (1556dupATGCCACCAC) that results in a premature stop codon 26 amino acids after the last original amino acid. We also observed in 2 nonrelated patients a deletion of 13 bp (1410_1422delGAGTGCAGGCAGA) previously reported for other populations. CONCLUSION: We reported four novel mutations and a novel polymorphic variant in the CYP1B1 gene in PCG in the Mexican population; it has important implications in diagnosis and genetic counseling.     

视网膜色素变性遗传致病基因peripherin/RDS的突变筛选

目的 了解中国视网膜色素变性患者（RP）中peripherin/RDS基因的突变谱及突变率。方法 应用聚合酶链-异源双链-单链构象多态性（PCR-SSCP）及DNA序列分析技术对收集的15个常染色体显性遗传视网膜色谱变性家系和55例散发视网膜色素变性患者peripherin/RDS基因的第一，第二外显子进行检测。结果 15个家系及55例散发患者未检测到peripherin/RDS基因突变。结论 本研究所检测的视网膜色素变性患者与RDS基因无关，显示视网膜色素变性的遗传异质性。     

Autosomal recessive retinitis pigmentosa is associated with mutations in RP1 in three consanguineous Pakistani families

PURPOSE: To localize and identify the gene and mutations causing autosomal recessive retinitis pigmentosa in three consanguineous Pakistani families. METHODS: Blood samples were collected and DNA was extracted. A genome-wide scan was performed by using 382 polymorphic microsatellite markers on genomic DNA from affected and unaffected family members, and lod scores were calculated. RESULTS: A genome-wide scan of 25 families gave an hlod = 4.53 with D8S260. Retinitis pigmentosa in all three families mapped to a 14.21-cM (21.19-Mb) region on chromosome 8 at q11, flanked by D8S532 and D8S260. This region harbors RP1, which is known to cause autosomal dominant retinitis pigmentosa. Sequencing of the coding exons of RP1 showed mutations in all three families: two single-base deletions, c.4703delA and c.5400delA, resulting in a frame shift, and a 4-bp insertion, c.1606insTGAA, all causing premature termination of the protein. All affected individuals in these families are homozygous for the mutations. Parents and siblings heterozygous for the mutant allele did not show any signs or symptoms of RP. CONCLUSIONS: These results provide strong evidence that mutations in RP1 can result in recessive as well as dominant retinitis pigmentosa. The findings suggest that truncation of RP1 before the BIF motif or within the terminal portion results in a simple loss of RP1 function, producing a recessive inheritance pattern. In contrast, disruption of RP1 within or immediately after the BIF domain may result in a protein with a deleterious effect and hence a dominant inheritance pattern.     

Identification and subcellular localization of the RP1 protein in human and mouse photoreceptors.

PURPOSE: Mutations in the RP1 gene account for 6% to 10% of autosomal dominant retinitis pigmentosa (adRP). Previous studies have shown that the RP1 gene is expressed specifically in photoreceptor cells. So far, little is known about the RP1 protein or how mutations in RP1 lead to photoreceptor cell death. The goal of this study was to identify the RP1 protein and investigate its location in photoreceptor cells. METHODS: A combination of RT-PCR and rapid amplification of cDNA ends (RACE) was used to isolate the full-length mouse Rp1 cDNA. Antibodies against different regions of the predicted mouse Rp1 protein were generated. Western blot analyses were used to identify the RP1/Rp1 proteins. The subcellular location of RP1 in human and mouse retinas was determined by immunostaining retinal sections. RESULTS: The full-length mouse Rp1 cDNA is 6944 bp, encoding a predicted protein of 2095 amino acids. Rp1 was found to be a soluble protein of approximately 240 kDa, consistent with predictions based on the cDNA sequence. Immunofluorescence analyses revealed that both the human RP1 and mouse Rp1 proteins are specifically localized in the connecting cilia of rod and cone photoreceptors. CONCLUSIONS: The presence of RP1/Rp1 in connecting cilia suggests that it may participate in transport of proteins between the inner and outer segments of photoreceptors or in maintenance of cilial structure. This study forms the basis for further investigation of the function of RP1 in retina and the mechanism by which mutations in RP1 lead to photoreceptor cell death.     

Novel and de-novo truncating PAX6 mutations and ocular phenotypes in Thai aniridia patients

PURPOSE: To describe the ophthalmic findings and mutation analyses of the PAX6 gene in Thai aniridia patients. METHODS: Ten patients from six unrelated families underwent a comprehensive ophthalmic examination. Mutations in the PAX6 gene were screened by single-strand conformational polymorphism (SSCP) and direct DNA sequencing of the SSCP variants. RESULTS: Seven patients developed cataracts and six developed glaucoma. Mutation analysis demonstrated four different truncating mutations, two of which were de novo. These included one novel insertion/deletion mutation (c.474del12insGA in exon 5) and three nonsense mutations. R203X and R240X are common recurrent mutations, while Q277X in exon 10 is novel. All mutations resulted in loss of function of the PAX6 protein. CONCLUSION: Our data confirm inter- and intrafamilial variable phenotypic manifestations of which the underlying mechanisms may be haploinsufficiency or dominant-negative mutation.     

Putative digenic inheritance of heterozygous RP1L1 and C2orf71 null mutations in syndromic retinal dystrophy

Background: Retinitis pigmentosa (RP) is the most common cause of inherited retinal degeneration and can occur in non-syndromic and syndromic forms. Syndromic RP is accompanied by other symptoms such as intellectual disability, hearing loss, or congenital abnormalities. Both forms are known to exhibit complex genetic interactions that can modulate the penetrance and expressivity of the phenotype.

Materials and methods: In an individual with atypical RP, hearing loss, ataxia and cerebellar atrophy, whole exome sequencing was performed. The candidate pathogenic variants were tested by developing an in vivo zebrafish model and assaying for retinal and cerebellar integrity.

Results: Exome sequencing revealed a complex heterozygous protein-truncating mutation in RP1L1, p.[(Lys111Glnfs*27; Gln2373*)], and a heterozygous nonsense mutation in C2orf71, p.(Ser512*). Mutations in both genes have previously been implicated in autosomal recessive non-syndromic RP, raising the possibility of a digenic model in this family. Functional testing in a zebrafish model for two key phenotypes of the affected person showed that the combinatorial suppression of rp1l1 and c2orf71l induced discrete pathology in terms of reduction of eye size with concomitant loss of rhodopsin in the photoreceptors, and disorganization of the cerebellum.

Conclusions: We propose that the combination of heterozygous loss-of-function mutations in these genes drives syndromic retinal dystrophy, likely through the genetic interaction of at least two loci. Haploinsufficiency at each of these loci is insufficient to induce overt pathology.     

Rapid detection of M1S1 mutations by the protein truncation test

PURPOSE: To determine a method of rapid detection of M1S1 gene mutations in patients with gelatinous droplike corneal dystrophy. METHODS: Forty-one patients from 35 families with gelatinous drop-like corneal dystrophy were studied. The entire coding region of the M1S1 gene was screened using the protein truncation test (PYT), with a polymerase chain reaction fragment amplified from genomic DNA serving as a template of in vitro translation. RESULTS: Homozygous or compound heterozygous mutations were detected in all patients by a single reaction of the PTT. This result matched those obtained using the polymerase chain reaction-restriction fragment length polymorphism and direct sequence analyses. The Q118X mutation was present in 63 of the 70 alleles, accounting for 90% of the disease-associated chromosomes in Japanese patients. CONCLUSIONS: The PTT is useful for detecting mutations in the M1S1 gene. This technique showed that the Q118X mutation is a founder mutation in Japanese patients with gelatinous droplike corneal dystrophy, and it reflects the linkage disequilibrium reported previously.     

Retinal pathology of canine X-linked progressive retinal atrophy, the locus homologue of RP3

PURPOSE: To describe the course of photoreceptor disease in canine X-linked retinal degeneration. METHODS: Retinas from 55 dogs (44 males, 8 carrier females, 3 homozygous females) were obtained by enucleation under general anesthesia. After fixation and dehydration, tissues were embedded in epoxy resin, sectioned at 1 microm for light microscopy and stained with azure II/methylene blue and a paraphenylenediamine counterstain. For electron microscopy, regions identified by light microscopy were selected and cut at 60 nm. Sections were stained with uranyl acetate-lead citrate. Electroretinography from an additional group of normal males, affected males, and carrier females was performed and the rod and cone responses evaluated. RESULTS: The earliest lesion detectable by electron microscopy was vesiculation of rod discs, followed by disruption of outer segments and death of rods. Loss of cones and progressive atrophy of inner retinal layers followed. Lesions were most severe in the peripheral retina and advanced toward the optic disc with disease progression. Significant variation in disease severity was present in males despite the presence of the same disease allele in all affected dogs. Carrier females displayed generalized reduction in photoreceptor density as well as multifocal areas of complete rod loss. The electroretinogram (ERG) findings were compatible with the histopathologic abnormalities. Homozygous females had lesions similar to those seen in affected males. CONCLUSIONS: X-linked retinal degeneration is characterized by initial degeneration of rod photoreceptors, followed by loss of cones and progressive atrophy of the inner retina. Carrier females display a phenotype consistent with random X-chromosome inactivation. Variation in genetic background may alter expression of the disease allele in affected animals, thus accounting for variation in phenotypic expression of the disease.     

A new locus for autosomal recessive RP (RP29) mapping to chromosome 4q32-q34 in a Pakistani family

PURPOSE: To map the disease locus in a six-generation, consanguineous Pakistani family with autosomal recessive retinitis pigmentosa (arRP). All affected individuals had pigmentary retinopathy associated with symptoms of night blindness and the loss of peripheral visual fields by the age of 20 years, loss of central vision between the ages of 25 and 30 years, and complete blindness between the ages of 40 and 50 years. METHODS: Genomic DNA from family members was typed for alleles at known polymorphic genetic markers using polymerase chain reaction. Alleles were assigned to individuals, which allowed calculation of LOD scores using the programs Cyrillic (http://www.cyrillicsoftware.com) and MLINK (Cherwell Scientific Publishing LTD:, Oxford, UK). The genes for membrane glycoprotein (M6a) and chloride channel 3 (CLCN3) were analyzed by direct sequencing for mutations. RESULTS: A new locus for arRP (RP29) has been mapped to chromosome 4q32-q34. A maximum two-point LOD score of 3.76 was obtained for the marker D4S415, with no recombination. Two recombination events in the pedigree positioned this locus to a region flanked by markers D4S621 and D4S2417. A putative region of homozygosity by descent was observed between the loci D4S3035 and D4S2417, giving a probable disease interval of 4.6 cM. Mutation screening of two candidate genes, M6a and CLCN3, revealed no disease-associated mutations. CONCLUSIONS: The results suggest that the arRP phenotype maps to a new locus and is due to a mutated gene within the 4q32-q34 chromosomal region.     

Remodeling of the human retina in choroideremia: rab escort protein 1 (REP-1) mutations

PURPOSE: To characterize in detail the disease expression in choroideremia (CHM), a blinding X-linked disease of the retina caused by loss-of-function mutations in Rab Escort Protein 1 (REP-1). CHM is readily diagnosed in the clinic and by molecular testing but has lacked an animal model to test hypotheses and therapeutics. The recent report of a mouse model for CHM prompts the need for reassessment of the human disease in anticipation of treatment initiatives. METHODS: CHM hemizygotes with REP-1 mutations, spanning an age range of 7 decades, were studied with in vivo microscopy by optical coherence tomography. RESULTS: The disease expression was complex. Earliest stages involved a thickening of the retina that was otherwise normally laminated. Loss of photoreceptors, either independent or associated with retinal pigment epithelium (RPE) depigmentation, was followed by disorganization and further thickening of the retina with interlaminar bridges. The dysmorphic retina then slowly thinned over decades. Laminopathy occurred first in more peripheral rod-rich regions and later in the cone-rich fovea. CONCLUSIONS: The CHM disease sequence involves detectable retinal thickening, which may be due to Müller cell activation and hypertrophy from photoreceptor stress. Photoreceptor degeneration, RPE depigmentation, and retinal remodeling follow. The results represent in vivo evidence in humans for retinal remodeling and provide a marker for the earliest stage of this response to genetic retinal disease. For CHM and other candidate human retinopathies considered for therapy, there is now a framework for making informed decisions about timing, retinal location, and potential value of treatment.     

Molecular genetic analysis of two functional candidate genes in the autosomal recessive retinitis pigmentosa, RP25, locus

PURPOSE: To identify the disease gene in five Spanish families with autosomal recessive retinitis pigmentosa (arRP) linked to the RP25 locus. Two candidate genes, EEF1A1 and IMPG1, were selected from the region between D6S280 and D6S1644 markers where the families are linked. The genes were selected as good candidates on the basis of their function, tissue expression pattern, and/or genetic data. METHODS: A molecular genetic study was performed on DNA extracted from one parent and one affected member of each studied family. The coding exons, splice sites, and the 5' UTR of the genes were amplified by polymerase chain reaction (PCR). For mutation detection, direct sequence analysis was performed using the ABI 3100 automated sequencer. Segregation of an IMPG1 single nucleotide polymorphism (SNP) in all the families studied was analyzed by restriction enzyme digest of the amplified gene fragments. RESULTS: In total, 15 SNPs were identified of which 7 were novel. Of the identified SNPs, one was insertion, two were deletions, five were intronic, six were missense, and one was located in the 5' UTR. These changes, however, were also identified in unaffected members of the families and/or 50 control Caucasians. The examined known IMPG1 SNP was not segregating with the disease phenotype but was correlating with the genetic data in all families studied. CONCLUSIONS: Our results indicate that neither EEF1A1 nor IMPG1 could be responsible for RP25 in the studied families due to absence of any pathogenic variants. However, it is important to notice that the methodology used in this study cannot detect larger deletions that lie outside the screened regions or primer site mutations that exist in the heterozygous state. A role of both genes in other inherited forms of RP and/or retinal degenerations needs to be elucidated.     

Delineation of the plasma membrane targeting domain of the X-linked retinitis pigmentosa protein RP2

PURPOSE: The X-linked retinitis pigmentosa protein RP2 is predominantly targeted to the plasma membrane. This study delineates the exact amino acid sequence requirements for targeting of RP2 through dual N-terminal acyl modification. METHODS: Inhibition of acyl modification with a palmitate analogue was used to confirm the mechanism of intracellular targeting. Mutagenesis of the first 15 residues in a synthetic RP2-green fluorescent protein (GFP) chimera was used to probe the precise requirements for plasma membrane targeting in Chinese hamster ovary (CHO) cells by confocal microscopy and subcellular fractionation. RESULTS: The N-terminal Met-Gly-Cys-X-Phe-Ser-Lys motif of human RP2 is necessary and sufficient for the protein's plasma membrane localization. This motif includes the accepted consensus sequence for N-myristoyl transferase (NMT) and a site for attachment of a palmitoyl moiety. An interesting feature of the motif is an essential phenylalanine at position 5. This is the first report of the requirement of a specific residue at position 5 within the N-terminal acyl modification motif for normal intracellular targeting. Arginine at position 8 is not essential for plasma membrane localization of the protein, but it improves targeting. The motif is highly conserved and is found in all vertebrate orthologues of human RP2, except mouse. In mouse, however, the Ser6Thr change is concordant with the accepted NMT consensus sequence. CONCLUSIONS: Conserved residues mediate the intracellular targeting of RP2, further highlighting the potential significance of the protein's plasma membrane localization. The delineation of this motif identifies residues in which mutations disrupt the dual acylation of RP2 and almost certainly result in disease.