Molecular Vision 2006; 12:177-183 <>
Received 14 July 2005 | Accepted 7 March 2006 | Published 15 March 2006

Novel variants in the hotspot region of RP1 in South African patients with retinitis pigmentosa

Lisa Roberts, Lecia Bartmann, Rajkumar Ramesar, Jacquie Greenberg

Division of Human Genetics, Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa

Correspondence to: Associate Professor Jacquie Greenberg, UCT/MRC Human Genetics Unit, Division of Human Genetics, Level 3, Wernher and Beit North, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Observatory, 7925, South Africa; Phone: 27 21 406-6299; FAX: 27 21 406-6826; email:


Purpose: Mutations in the hotspot of RP1 are reportedly responsible for 4-7% of autosomal dominant retinitis pigmentosa (ADRP) in the United States, Canada, and Europe. South Africa (SA) has unique subpopulations and a comparatively low observed frequency of rhodopsin mutations, which lead to this investigation of the contribution of RP1 mutations to the ADRP disease burden in SA.

Methods: Fifty-seven affected, unrelated South African individuals with ADRP were selected for mutation screening of the RP1 hotspot, using denaturing high performance liquid chromatography (HPLC). Variants were identified by direct sequencing, after which cosegregation analysis and population frequency studies were performed using restriction fragment length polymorphism analysis, nondenaturing HPLC, or denaturing HPLC.

Results: Three mutations were identified, including two novel sequence variations and the common Arg677X mutation. A wide spectrum of disease severity was observed in the families with these RP1 gene mutations. Two nondisease-associated polymorphisms were also detected, with the frequency of one of these variants being significantly low in Black African individuals.

Conclusions: Mutations were only found in Caucasian families with origins in the British Isles. The observed RP1 mutation frequency of 5.3% in SA ADRP patients is comparable to the frequency reported in other populations.


Defects of the eye account for approximately one-third of all human inherited diseases [1]. One in 3,500 people live with retinitis pigmentosa (RP), the most common inherited retinal dystrophy [1], characterized by nightblindness and progressive constriction of the visual fields. Total blindness occurs in approximately 30% of cases.

Approximately 15% of RP is inherited in an autosomal dominant manner [2]. The major genes contributing to autosomal dominant retinitis pigmentosa (ADRP) described to date are Rhodopsin (RHO), RDS, and RP1. In the United States, United Kingdom, and Europe, mutations in RHO account for 20-25% of all ADRP cases [3]. RDS mutations were identified in 8% of ADRP cases in the United States [4]. Mutations in a hotspot region of RP1 are reported to be responsible for 4-7% of ADRP in the United States, Canada, and Europe [4,5].

In 1977, RP1 on chromosome 1 was the first locus reported to be associated with RP [6]. This finding was later retracted, and the locus reassigned to chromosome 8 [7]. Twenty years later, the gene "Oxygen-regulated protein 1" (ORP-1 or RP1) was identified, and mutations therein were found to correlate with the ADRP phenotype [8,9]. The RP1 protein is expressed in the photoreceptor connecting cilia, and appears to be required for the correct orientation and stacking of discs during photoreceptor outer segment morphogenesis [10]. In addition, it is reported to be modulated by retinal oxygen levels [8].

Although the RP1 gene has four exons (GenBank accession number NM_006269), reports have indicated that pathogenic mutations cluster in a small region of exon 4 [10,11]. These mutations include deletions, insertions, and substitutions and cause a wide spectrum of disease severity [12]. The Arg677X mutation in RP1 accounts for 2% of ADRP, making it the third major contributor to this disease after the two RHO mutations, Pro23His and Pro347Leu [8,13]. South Africa (SA) has subpopulations including Black (people of indigenous Black African origin); mixed ancestry (representing an admixture of San, Khoi-Khoi, West African, Madagascan, Javanese, and Western European populations), and Caucasian (in SA, immigrants mainly of Western European origin, including Dutch, French, German, and British). This provides a novel gene pool with which one can study inherited disorders [14]. Given these population demographics, detection of mutations previously identified in Europe and the United Kingdom is expected in some of the subpopulations. The surprisingly low frequency of the two common RHO mutations in the SA population with ADRP [15], despite a strong immigrant history, lead to this investigation of the contribution of RP1 mutations to the ADRP disease burden in SA.



Patients with inherited retinal degenerative disorders and their families are referred to the Division of Human Genetics at the University of Cape Town from throughout South Africa by ophthalmologists, health care professionals, nurses from the Department of Health, and the lay organization, Retina South Africa (from whom most of the referrals are received). Affected individuals and their families are recruited into a research program investigating the genetics of retinal degenerative disorders in this country, in an attempt to define the genetic profile of all subjects who suffer from inherited forms of retinal degeneration. Informed consent is obtained using the tenets of the most recent Declaration of Helsinki (2000). Fifty-seven affected, unrelated individuals from known families with ADRP were selected for mutation screening of the RP1 hotspot.

The ethnic breakdown of these families was as follows: thirty-six Caucasian, fifteen Black, three mixed ancestry, two Indian, and one of unknown origin. A general population control cohort of 103 individuals was selected for population frequency studies of novel RP1 polymorphisms. These individuals were not assessed specifically for the presence of disease, and were classified ethnically as belonging to Caucasian or Black population groups based on the same system used to classify the sample cohort.

Peripheral blood samples were obtained and genomic DNA was isolated from white blood cells using standard methods.

Polymerase chain reaction (PCR) amplification

The RP1 region of interest was amplified using three overlapping sets of primers [5]. All polymerase chain reaction (PCR) amplification was undertaken using the following conditions, unless otherwise stated in Table 1: 0.2 mM dNTPs, 1X InvitrogenTM Buffer (InvitrogenTM Life Technologies, Paisley, UK), 1.5 mM magnesium chloride, 1 U InvitrogenTM Taq polymerase, 200 ng genomic DNA. Final reaction volumes were made up to 50 μl with distilled water, and thermal cycling conditions were as follows: 95 °C for 5 min, followed by 30 or 35 cycles of 94 °C for 5 min, 50 °C for 30 s, 72 °C for 40 s, and a final single incubation at 72 °C for 7 min.

An aliquot of 5 μl of PCR product was subjected to agarose gel electrophoresis on 2% agarose gel to confirm successful amplification, prior to denaturing high performance liquid chromatography (dHPLC).

denaturing High Pressure Liquid Chromatography

Mutation screening was performed according to the manufacturer's instructions, using the WAVE® Nucleic Acid Fragment Analysis System (Transgenomic Inc., Omaha, NE). An aliquot of 10-20 μl of each PCR product was heat-denatured at 95 °C for 5 min, and heteroduplex formation was promoted by allowing the tube temperature to reach room temperature over 45 min. PCR products were then separated into hetero- and homoduplexes on the WAVE®, using an acetonitrile gradient. In the partially denaturing mode, in which heteroduplexes emerge prior to homoduplexes, separation chemistry is based on sequence, size of the fragment, and analysis temperature. Gradient parameters, temperatures, and flow rates were developed empirically based on the size and melting profile of the fragments (Table 2).

Nondenaturing conditions on the WAVE® (in which the acetonitrile gradient is determined by the number of base pairs of the fragment) allowed size-based separation of PCR products.

When numerous samples appeared to have the same heteroduplex profiles, these samples were tested using a second dHPLC analysis to confirm whether they were due to the same sequence variation. This was done by mixing equal volumes of the PCR product being queried with that of a sample of a known DNA sequence variant. This mixture was subjected to heteroduplex formation and dHPLC analysis with the appropriate gradient condition. Lack of additional peaks indicated that sequences of the PCR product being queried and the known sample were identical. This highly effective secondary dHPLC was found to be cost efficient when compared to direct sequencing of multiple samples with the same variant. It is a convenient and rapid way of testing for common sequence variants that do not alter restriction enzyme sites.

Direct sequencing

DNA samples that exhibited variant elution profiles on dHPLC were characterized by direct sequencing on an ABI PrismTM 3100 Automated Sequencer (Applied Biosystems, Foster City, CA) using the above described standard procedures and the same primers.

Restriction fragment length polymorphism (RFLP) analysis

Where possible, sequence variations were confirmed and tested for cosegregation with disease by restriction fragment length polymorphism (RFLP) analysis. Restriction enzyme digests were performed for approximately 15 h, in a final volume of 20-25 μl. Five units of the appropriate enzyme were used to digest 10 μl of PCR product. Incubation temperatures of 65 °C, 37 °C, and 50 °C were used for TaqI, Hsp92II, and AcsI enzymes, respectively.

Proof of disease association

Disease association of novel variants was judged by the following criteria: (1) whether the protein would be significantly altered and thus likely to modify the phenotype; (2) whether there was co-segregation of the variant and the phenotype; and (3) whether the variant occurred in less than one percent of the population tested [16].


Of the 57 affected, unrelated individuals screened for variants in the hotspot of RP1, three were determined to carry disease associated mutations (5.26%) and two of these mutations are novel. These results and the frequencies of variant sequences are summarized in Table 3.


RP1 mutations are reported to cause 4-7% of ADRP in the United States, Canada, and Europe [4,5] and thus could be considered one of the major identifiable causes of this form of retinal degeneration. There are no reports of the mutation spectrum nor disease burden of the RP1 gene in Africa. This led to the investigation of the role of RP1 in this cohort.

Several factors resulted in mutation screening being taken as a direct approach, rather than linkage analysis. These factors include: first, the lack of comprehensive family information, and second, the fact that all disease associated mutations reported to date, have been found in a hotspot region encompassing 553 codons of exon 4 of RP1 [10].

The hotspot region of RP1 was investigated in 57 SA individuals with pedigrees exhibiting an autosomal dominant mode of inheritance of retinitis pigmentosa. Variations were detected by dHPLC, and these were confirmed by sequencing or restriction enzyme analysis. Further investigation revealed that three of the changes contributed toward disease, while two of them were nondisease associated. The findings indicate that RP1 mutations are responsible for approximately 5.26% of ADRP cases in the SA population screened to date. It is interesting to note that all three families identified with RP1 mutations have origins in either the United Kingdom or Ireland, and two of these families carry novel mutations.

Disease associated variants

The Arg677X mutation has previously been reported as the most common RP1 disease associated variant [8,13]. Arg677X was detected in a Caucasian family of British origin (Figure 1). The presence of the mutation was confirmed in two affected individuals by RFLP analysis using TaqI as the mutation (c.2029C>T) destroys a cutting site of this enzyme.

Ser911X was detected in a Caucasian family of British origin (Figure 2). This novel mutation is predicted to truncate the protein by 1,246 amino acids, and was not detected in 102 chromosomes investigated in the Caucasian controls or 104 chromosomes investigated in the Black indigenous controls. Ser911X (c.2732 C>A) creates a cutting site for AcsI, and its presence was thus confirmed by a restriction enzyme digest. The novel Ser911X mutation co-segregates with the disease in the family: It was detected in two affected individuals (IV:4 and III:2 of Figure 2, aged 43 and 69, respectively), and two at-risk individuals (IV:7 and IV:5, aged 34 and 40, respectively), who showed no signs of the disorder at the time of recruitment.

A novel 10 bp deletion (c.2590-2599delATAACTTTAA) was detected in a Caucasian family originating from Dublin (Figure 3). The presence of the mutation was confirmed by using nondenaturing, size-based separation on the WAVE. The mutation is predicted to cause a frameshift at codon 864, truncating the protein by 1,280 amino acids. Due to these great effects on RP1, it was not necessary to perform population frequency analysis. The mutation cosegregates with the disease in this family: it is present in four affected members in four successive generations of the family; these are individuals III:4 (deceased), IV:2 (age 71), VI (age 51), and VI:4 (age 26). This mutation was also detected in one at-risk individual VI:2 (age 28) clinically unaffected at the time of recruitment.

Clinical categorization of families with RP1 mutations is confounded by the variability of disease severity in the RP1 form of ADRP, ranging from nyctalopia at age 11 to asymptomatic carriers at age 66, and variability within families has been reported [11,12]. This phenomenon of variable expressivity was also evident within the three families identified in this study, as described in Table 4. It should be noted that we often only have access to individuals' clinical test results via their ophthalmologists and only when they have had a routine check-up. We do not ask asymptomatic individuals to have any additional evaluations done unless they request molecular testing as part of our genetic service and are then informed about their mutation-carrier status, accompanied by appropriate genetic counseling. The policies and guidelines used for the genetic testing of retinal degenerative disorders are available on the Services page of our division's website, under the link "Genetic testing protocols and policy guidelines," Genetics.

This wide spectrum of severity of disease in these families will be an important issue for genetic counseling when providing diagnostic advice to the individuals concerned.

Polymorphic variants

Two nondisease-associated polymorphisms were detected in the cohort screened, and these were further investigated to determine the frequencies in the general population. Over 100 chromosomes were screened for these two polymorphisms in each ethnic group (Caucasian and Black) of unaffected, unrelated individuals.

A heterozygous polymorphism, Thr752Met was detected in 4 of the 57 ADRP individuals screened. Two subjects were Caucasian, one was Black, and one was of mixed ancestry (4/114 chromosomes=3.51%). The presence of this polymorphism was confirmed by performing an Hsp92II restriction enzyme digest, as the mutation (c.2255C>T) creates a cutting site for this enzyme. This sequence variation was found to be nonpathogenic as it did not cosegregate with the disease. The variant was detected in 1 of 102 chromosomes investigated in the Caucasian controls (0.98%) and 3 of 104 chromosomes investigated in the Black controls (2.83%).

Arg872His was previously reported as a nonpathogenic variant occurring in 25% of the population [9]. This variant (c.2615 G>A) was investigated in several cohorts, as described in Table 3, and was found to occur in 20/114 chromosomes (17.54%) of the total ADRP cohort; 19.44% of the Caucasian individuals with ADRP, and 23.53% of the Caucasian control chromosomes. These frequencies correlate with those reported previously; however, this variant was only detected in 11.5% of Black control chromosomes. There is thus a significant (p=0.037) association between ethnicity and the prevalence of this polymorphism. The difference in gene variant frequencies between these South African (SA) ethnic groups has been noted previously [17] and should be investigated further.

In conclusion, this is the first report describing the role of the RP1 gene in SA patients with ADRP. We have found that the rapid, large-scale screening of the hotspot region of RP1 using the WAVE is a viable initial approach in the routine screening of ADRP families for defects in candidate genes, as this technique is reported to have a specificity and sensitivity greater than 96% [18]. The results of this study could also indicate merit in screening sporadic individuals for the hotspot of RP1 as suggested previously [13]. The observed RP1 mutation frequency of 5.3% in SA ADRP patients is comparable to the 4-7% frequency reported elsewhere [4,5], however, mutations were only found in Caucasian families with origins in the British Isles. The frequency did not compensate for, nor did it explain, the reported low frequency of RHO mutations in SA ADRP families.


We are grateful to Annapurna Hazra for her assistance with the statistical calculations. This research was supported by grants from the University of Cape Town, Retina South Africa, the South African Medical Research Council, and Technology and Human Resources for Industry Programme (THRIP, South Africa). We are indebted to all RD family members in SA for their participation in this research program.


1. Hims MM, Diager SP, Inglehearn CF. Retinitis pigmentosa: genes, proteins and prospects. Dev Ophthalmol 2003; 37:109-25.

2. Phelan JK, Bok D. A brief review of retinitis pigmentosa and the identified retinitis pigmentosa genes. Mol Vis 2000; 6:116-24 <>.

3. van Soest S, Westerveld A, de Jong PT, Bleeker-Wagemakers EM, Bergen AA. Retinitis pigmentosa: defined from a molecular point of view. Surv Ophthalmol 1999 Jan-Feb; 43:321-34.

4. Sohocki MM, Daiger SP, Bowne SJ, Rodriquez JA, Northrup H, Heckenlively JR, Birch DG, Mintz-Hittner H, Ruiz RS, Lewis RA, Saperstein DA, Sullivan LS. Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies. Hum Mutat 2001; 17:42-51.

5. Bowne SJ, Daiger SP, Hims MM, Sohocki MM, Malone KA, McKie AB, Heckenlively JR, Birch DG, Inglehearn CF, Bhattacharya SS, Bird A, Sullivan LS. Mutations in the RP1 gene causing autosomal dominant retinitis pigmentosa. Hum Mol Genet 1999; 8:2121-8.

6. Spence MA, Sparkes RS, Heckenlively JR, Pearlman JT, Zedalis D, Sparkes M, Crist M, Tideman S. Probable genetic linkage between autosomal dominant retinitis pigmentosa (RP) and amylase (AMY2): evidence of an RP locus on chromosome 1. Am J Hum Genet 1977; 29:397-404. Erratum in: Am J Hum Genet 1977; 29:592.

7. Blanton SH, Heckenlively JR, Cottingham AW, Friedman J, Sadler LA, Wagner M, Friedman LH, Daiger SP. Linkage mapping of autosomal dominant retinitis pigmentosa (RP1) to the pericentric region of human chromosome 8. Genomics 1991; 11:857-69.

8. Pierce EA, Quinn T, Meehan T, McGee TL, Berson EL, Dryja TP. Mutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pigmentosa. Nat Genet 1999; 22:248-54.

9. Sullivan LS, Heckenlively JR, Bowne SJ, Zuo J, Hide WA, Gal A, Denton M, Inglehearn CF, Blanton SH, Daiger SP. Mutations in a novel retina-specific gene cause autosomal dominant retinitis pigmentosa. Nat Genet 1999; 22:255-9.

10. Liu Q, Lyubarsky A, Skalet JH, Pugh EN Jr, Pierce EA. RP1 is required for the correct stacking of outer segment discs. Invest Ophthalmol Vis Sci 2003; 44:4171-83.

11. Jacobson SG, Cideciyan AV, Iannaccone A, Weleber RG, Fishman GA, Maguire AM, Affatigato LM, Bennett J, Pierce EA, Danciger M, Farber DB, Stone EM. Disease expression of RP1 mutations causing autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci 2000; 41:1898-908.

12. Berson EL, Grimsby JL, Adams SM, McGee TL, Sweklo E, Pierce EA, Sandberg MA, Dryja TP. Clinical features and mutations in patients with dominant retinitis pigmentosa-1 (RP1). Invest Ophthalmol Vis Sci 2001; 42:2217-24.

13. Schwartz SB, Aleman TS, Cideciyan AV, Swaroop A, Jacobson SG, Stone EM. De novo mutation in the RP1 gene (Arg677ter) associated with retinitis pigmentosa. Invest Ophthalmol Vis Sci 2003; 44:3593-7.

14. Ramesar R, Roberts L, Rebello G, Goliath R, Vorster A, September A, Ehrenreich L, Gama D, Greenberg J. Retinal degenerative disorders in Southern Africa: a molecular genetic approach. In: LaVail MM, Hollyfield JG, Anderson RE, editors. Retinal degenerations: mechanisms and experimental therapy. Proceedings of the 10th International Symposium on Retinal Degenerations; 2002 Sep 30-Oct 5; Burgenstock, Switzerland. New York: Kluwer Academic/Plenum Publishers; 2003. p.35-40.

15. Roberts L, Ramesar R, Greenberg J. Low frequency of rhodopsin mutations in South African patients with autosomal dominant retinitis pigmentosa. Clin Genet 2000; 58:77-8.

16. Cotton RG, Scriver CR. Proof of "disease causing" mutation. Hum Mutat 1998; 12:1-3.

17. Greenberg J, Roberts L, Ramesar R. Unusual frequencies of Rhodopsin mutations and polymorphisms in Southern African patients with retinitis pigmentosa. In: Anderson RE, LaVail MM, Hollyfield JG, editors. New insights into retinal degenerative diseases. Proceedings of the 9th International Symposium on Retinal Degeneration; 2000 Oct 9-14; Durango, CO. New York: Kluwer Academic/Plenum Publishers; 2001. p.329-33.

18. Xiao W, Oefner PJ. Denaturing high-performance liquid chromatography: A review. Hum Mutat 2001; 17:439-74.

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