Molecular Vision 2006; 12:1283-1291 <>
Received 10 March 2006 | Accepted 28 August 2006 | Published 26 October 2006

Mutations in the gene encoding the α-subunit of rod phosphodiesterase in consanguineous Pakistani families

S. Amer Riazuddin,1,2 Fareeha Zulfiqar,2 Qingjiong Zhang,1 Wenliang Yao,1 Shouling Li,1 Xiaodong Jiao,1 Amber Shahzadi,2 Muhammad Amer,2 Muhammad Iqbal,2 Tayyab Husnain,2 Paul A. Sieving,1 Sheikh Riazuddin,2 J. Fielding Hejtmancik1
(The first three and last two authors contributed equally to this publication).

1Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, MD; 2Center of Excellence in Molecular Biology, University of the Punjab, Lahore Pakistan

Correspondence to: J. Fielding Hejtmancik, M.D., PhD, OGVFB/NEI/NIH, Building 10, Room 10B10, 10 Center Drive MSC 1860, Bethesda, MD, 20892-1860; Phone: 301-496-8300; FAX: 301-435-1598; email:


Purpose: To localize and identify the gene and mutations causing autosomal recessive retinitis pigmentosa (RP) in consanguineous Pakistani families.

Methods: Families were ascertained and patients underwent complete ophthalmological examinations. Blood samples were collected and DNA was extracted. A genome-wide scan was performed 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 50 families gave a lod score of 7.4172 with D5S2015 using HOMOG1. RP in all 4 linked families mapped to a 13.85 cM (14.87 Mb) region on chromosome 5q31-33 flanked by D5S2090 and D5S422. This region harbors the PDE6A gene, which is known to cause autosomal recessive RP. Sequencing of PDE6A showed a homozygous single base pair change; c.889C->T, single base pair insertion; c.2218-2219insT, and single base pair substitution in the splice acceptor site; IVS10-2A->G in each of three families. In the fourth family linked to this region, no disease-causing mutation was identified in the PDE6A gene.

Conclusions: These results provide strong evidence that mutations in PDE6A result in recessive RP in three consanguineous Pakistani families. Although a fourth family was linked to markers in the 5q31-33 interval, no mutation was identified in PDE6A.


Retinis pigmentosa (RP) is the most common inherited retinal dystrophy, affecting approximately 1 in 5000 individuals worldwide [1,2]. RP primarily affects the rod photoreceptors, whereas the function of the cone receptors is compromised as the disease progresses [3]. Ocular findings comprise atrophic changes of the photoreceptors and retinal pigment epithelium (RPE) followed by appearance of melanin-containing structures in the retinal vascular layer. Typical fundus appearance includes attenuated arterioles, bone-spicule pigmentation, and waxy pallor of the optic disc. Affected individuals often have severely abnormal or nondetectable rod responses in the electroretinograms (ERG) recordings even in the early stage of the disease [3].

RP may be inherited as an autosomal recessive, autosomal dominant, or as an X-linked recessive trait. To date, 39 loci have been implicated in nonsyndromic RP, of which 30 genes are known [4]. These include genes encoding components of the phototransduction cascade, proteins involved in retinoid metabolism, cell-cell interaction proteins, photoreceptor structural proteins, transcription factors, intracellular transport proteins and splicing factors [4]. Autosomal dominant RP (adRP) represents 15-20% of all cases; autosomal recessive RP (arRP) comprises 20-25% of cases (syndromic and nonsyndromic); X-linked recessive RP makes up 10-15%, and the remaining 40-55% of cases, in which family history is absent, are called simplex RP (SRP), but many of these may represent autosomal recessive RP [5-8].

cGMP phosphodiesterase in the retinal rod cells is a key phototransduction enzyme. It is a heterotetrameric protein consisting of an α, β, and two γ subunits. As RP is primarily a disease of rods, the components of the phototransduction cascade are a primesite suspect as for the defect in RP. Huang et al. identified mutations (one homozygous and one compound heterozygous) in the cGMP phosphodiesterase (NM_000440, PDE6A, MIM 180071) gene in 2 autosomal recessive pedigrees while screening 340 unrelated patients with RP [9].

Here we report four consanguineous Pakistani families with multiple individuals affected by arRP. Clinical findings in these families are typical of early onset RP. Linkage analysis showed linkage to chromosome 5q31-33, a region including PDE6A. Sequencing of PDE6A showed a homozygous single base pair change (c.889C->T), single base pair insertion (c.2218-2219insT), and a single base pair substitution in the splice acceptor site (IVS10-2A->G) in each of three families. In the fourth family, in which the RP was linked to this region, we did not identify any disease-causing mutation in the PDE6A gene.


Patient ascertainment

We recruited 50 consanguineous Pakistani families with nonsyndromic RP to participate in a collaborative study between the Center of Excellence in Molecular Biology, Lahore, Pakistan and the National Eye Institute, Bethesda, Maryland. Our plan was to identify new disease loci causing inherited vision diseases. Institutional review board (IRB) approval was obtained for this study from the National Eye Institute, Bethesda, MD, USA and the Center of Excellence in Molecular Biology, Lahore, Pakistan. The participating subjects gave informed written consent, consistent with the tenets of the Declaration of Helsinki. The families described in this study are from the Punjab province of Pakistan. A detailed medical history was obtained by interviewing family members. Fundus photographs of affected individuals showed changes typical of RP, including waxy pale optic discs, attenuation of retinal arteries, and bone-spicule pigment deposits in the mid periphery of the retina. Affected individuals had typical RP changes on ERG including loss of both the rod and cone responses. Blood samples were collected from affected and unaffected family members. DNA was extracted by following a nonorganic method described by Grimberg et al. [10].

Genotyping and linkage analysis

A genome-wide scan was performed with 382 highly polymorphic fluorescent markers from the ABI PRISM Linkage Mapping Set MD-10 (PE Applied Biosystems, Foster City, CA) having an average spacing of 10 cM. Multiplex polymerase chain reactions (PCR) were carried out by following guidelines given in the reference [11]. PCR products from each DNA sample were pooled and mixed with a loading cocktail containing HD-400 size standards (PE Applied Biosystems) and loading dye. The resulting PCR products were separated on a 5% Long Ranger denaturing urea-polyacrylamide gel in an ABI 377 DNA sequencer and analyzed by using GENESCAN 3.1 and GENOTYPER 2.1 software packages(PE Applied Biosystems).

Two point linkage analyses were performed using the FASTLINK version of MLINK from the LINKAGE Program Package [12,13]. Maximum lod scores were calculated using ILINK. Autosomal recessive RP was analyzed as a fully penetrant trait with an affected allele frequency of 0.001. The marker order and distances between the markers were obtained from the Genethon database and the National Center for Biotechnology Information (NCBI) chromosome 5 sequence maps. For the initial genome scan equal, allele frequencies were assumed, while for fine mapping, allele frequencies were estimated from 96 unrelated and unaffected individuals from the Punjab province of Pakistan. Admixture analysis was carried out using the HOMOG1 program [14] comparing linkage to D5S2015 at θ's of 0.001, 0.01 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, and 0.4 with absence of linkage.

Mutation screening

Individual exons of PDE6A were amplified by PCR using primer pairs as shown in Table 1. Amplifications were carried out as described [15]. The PCR products were analyzed on 2% agarose gel and were purified by vacuum filtration manifold plate (Millipore, Billerica, MA). The PCR primers for each exon were used for bidirectional sequencing using BigDye Terminator Ready reaction mix according to the manufacturer's instructions (Applied Biosystems). Sequencing products were resuspended in 10 ml of formamide (Applied Biosystems) and denatured at 95 °C for 5 min. Sequencing was performed on an ABI PRISM 3100 Automated sequencer (Applied Biosystems). Sequencing results were assembled and analyzed using the Seqman program of DNASTAR Software (DNASTAR Inc, Madison, WI).


All affected individuals examined in all three families fit the diagnostic criteria of RP. Fundus photographs of affected individuals showed typical changes of RP including a waxy pale optic disc, attenuation of retinal arteries, and bone-spicule pigment deposits in the mid periphery of the retina as shown in Figure 1A-C. None of the unaffected individuals in either of four families complained of night blindness. Affected individuals had typical RP changes on ERG including loss of both the rod and cone responses (Figure 2), while parents showed no changes consistent with RP. As these families reside in remote parts of Punjab province of Pakistan, the clinical records pertaining to the disease onset were not available. However, the clinical records of the affected individuals available to us did suggest there was no intrafamilial variability.

A genome-wide scan was performed using a set of 382 polymorphic markers spanning the human genome at approximately 10 cM intervals (ABI Linkage Mapping Set MD10, Version 2.5). During the genome-wide scan, cosegregation of RP with alleles of a microsatellite marker, D5S410, was noted in families 61019, 61021, 61074, and 61081 and marker, D5S436 in family 61074 and 61081. In the genome-wide scan significantly positive lod scores were obtained with D5S410, showing lod scores of 3.96, 2.54, 1.19, and 3.68 at θ=0 with families 61019, 61021, 61074 and 61081, respectively and lod scores of 3.39 and 3.50 at θ=0 with D5S436, an adjacent marker with families 61074 and 61081, respectively.

For fine mapping additional markers, D5S2090, D5S812, D5S2015, D5S2013 and D5S1469 from the Genethon database were analyzed as shown in Table 2 (panels A-D) and Figure 3A-D. Two point linkage analyses gave further evidence for linkage to markers on chromosome 5q31-33 with maximum lod scores of 3.96 with D5S2013, and D5S1469 at θ=0 and 3.75 with D5S2090 at θ=0 for family 61019; 3.21 with D5S2015 at θ=0 and 3.01 with D5S1469 at θ=0 for family 61021; 3.39 with D5S1469 at θ=0 and 3.23 with markers D5S812 and D5S2015 at θ=0 for family 61074 and 4.0 with D5S2015 at θ=0 and 4.18 with markers D5S2013 and D5S1469 at θ=0 for family 61081.

When linkage results from the entire data set were subjected to admixture analysis, heterogeneity was suggested with a=0.05 and a maximum ln (likelihood) for linkage with heterogeneity of 17.0596, corresponding to an hlod=7.4172. These values give a likelihood ratio R=37,750:1 favoring linkage, and a c2=34.119, corresponding to a p>0.0001 if an asymptotic c2 distribution is assumed. The conditional probabilities of linkage of families 61019, 61021, 61074, 61081 are 0.9922, 0.9944, 0.9947, and 0.9991, respectively, while conditional probabilities of linkage for the remaining families are >0.005. Taken together, the four linked families localize the arRP gene to a 13.85 cM (14.87 Mb) region on chromosome 5q31-33 flanked by D5S2090 and D5S422.

Visual inspection of the haplotypes in this region supported the linkage analysis (Figure 3), localizing the disease to a region of chromosome 5q31-33 flanked by D5S2090 and D5S410. Recombination events at D5S436 in affected individual 9 of family 61019 and at D5S2090 in affected individual 9 of family 61021 identify marker D5S2090 as the proximal flanking marker.

Similarly, recombination events at D5S422 in affected individual 13 of family 61074, and at D5S400 in affected individual 15 of family 61021, and affected individuals 11 and 13 of family 61074 identified D5S422 as the distal flanking marker. Lack of homozygosity at D5S422 and D5S400 in affected individual 9 of family 61019, individuals 9, 12, and 15 of family 61021 and affected individuals 7, 14, and 17 of family 61081 also suggested that the disease-causing gene lay proximal to marker D5S422. Alleles for D5S812, D5S2013, D5S2015, D5S1469, and D5S410 were homozygous for all affected individuals in families 61019, 61021, 61074, and 61081.

The linked region on chromosome 5q31-33 harbored PDE6A (NM_000440), which has been described as a cause of arRP. The PDE6A gene contains 22 exons and encodes an 860 amino acid protein (NP_000431). Sequencing of PDE6A showed changes in three of the four families studied (Figure 4). The affected individuals of family 61019 showed a single base change in exon 4: c.889C>T, resulting in a premature termination (p.R256X). The affected individuals of family 61021 showed a single base pair insertion in exon 17; c.2218-2219insT, leading to a frame shift and premature termination of the protein; p.Y700LfsX714. In family 61074, the PDE6A gene showed a single base pair substitution in conserved splice acceptor site in exon 11; IVS10-2A>G. In addition, sequencing of PDE6A in family members disclosed nine new polymorphisms (Table 3). Sequencing of the 22 coding exons and intron-exon junctions of PDE6A in affected members of family 61081 did not show a disease-causing mutation. In addition, sequencing of 462 bp upstream of the coding sequences of the gene did not disclose any sequence variations segregating with the disease phenotype in the family.


Here we report characterization of four consanguineous Pakistani families with multiple family members affected with autosomal recessive RP, the localization of the arRP gene in these families to a 13.85 cM (14.87 Mb) region on chromosome 5q31-33 flanked by D5S2090 and D5S422, and association of RP with mutations in the PDE6A gene in three of the families. These mutations include a single base pair change (c.889C>T), a single base pair insertion (c2218-2219insT), and in the third family a single base substitution in the splice acceptor site (IVS10-2A>G). Two of the three point mutations (c.889C>T and c2218-2219insT) are predicted to results in premature terminations p.R256X and p.Y700LfsX714, respectively, whereas the third mutation (IVS10-2A>G), would result in skipping of exon 11 during mRNA processing.

The PDE6A gene consists of 22 exons spanning approximately 45-50 kb on chromosome 5q31-33 and encoding for a protein of 860 amino acids. Nonsense mutations in mammalian genes generally lead to unstable mRNAs that are degraded by nonsense mediated decay, unless the mutation is present in the last exon [16]. Hence, two of three mutations reported here (p.R256X and p.Y700LfsX714) are expected to produce unstable transcripts that might be degraded by nonsense mediated decay. Conversely, the mRNA carrying the splice acceptor site mutation (IVS10-2A>G) is expected to be stable and processed by the cellular splicing machinery. The processed mRNA transcript is expected to skip exon 11. This should not shift the reading frame of the protein, but is predicted to result in an internal deletion of 22 amino acids of the PDE6A protein (p.K470_L491del). As the individuals heterozygous for acceptor site mutation are unaffected, it seems unlikely that the mutant protein interferes with the proper functioning of the wild-type protein.

As described, family 61081 showeed significant linkage independently to chromosome 5q31-34, but no sequence variations segregating with the disease phenotype in the family were detected in the 22 coding exons of PDE6A or in the 462 bases directly upstream of the gene. We cannot rule out the possibility that there might be a sequence change internal to one of the introns affecting splicing. Family 61081 is linked to the region with a proximal marker D5S2115 and distal marker D5S422, a linked region of 25.55 cM (27.34). This region does not contain any previously reported RP-causing genes except PDE6A. Further efforts are being made to identify the mutation as well as the gene causing RP in family 61081. Identification of additional genes involved in causing RP will greatly enhance our understanding of the retinal biology at a molecular level and the physiology of retinal photoreceptors.


We acknowledge the family members who donated samples to make this work possible. The authors are thankful to Amir Niazi (optometrist, Layton Rahmatullah Benevolent Trust Hospital, Lahore) for his expert help in the identification of families. We thank the Ministry of Science and Technology, Islamabad Pakistan and National Institutes for Health, Bethesda forr funding.

This study was supported, in part by Higher Education Commission, Ministry of Science and Technology Islamabad, Pakistan and by funds from the intramural program of National Eye Institute, National Institutes of Health, Bethesda, MD.


1. Bunker CH, Berson EL, Bromley WC, Hayes RP, Roderick TH. Prevalence of retinitis pigmentosa in Maine. Am J Ophthalmol 1984; 97:357-65.

2. Bundey S, Crews SJ. A study of retinitis pigmentosa in the city of Birmingham. J Med Genet 1986; 23:188.

3. Bird AC. Retinal photoreceptor dystrophies LI. Edward Jackson Memorial Lecture. Am J Ophthalmol 1995; 119:543-62.

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

5. Boughman JA, Conneally PM, Nance WE. Population genetic studies of retinitis pigmentosa. Am J Hum Genet 1980; 32:223-35.

6. Boughman JA, Caldwell RJ. Genetic and clinical characterization of a survey population with retinitis pigmentosa. Prog Clin Biol Res 1982; 82:147-66.

7. Jay M. Figures and fantasies: the frequencies of the different genetic forms of retinitis pigmentosa. Birth Defects Orig Artic Ser 1982; 18:167-73.

8. Inglehearn CF. Molecular genetics of human retinal dystrophies. Eye 1998; 12:571-9.

9. Huang SH, Pittler SJ, Huang X, Oliveira L, Berson EL, Dryja TP. Autosomal recessive retinitis pigmentosa caused by mutations in the alpha subunit of rod cGMP phosphodiesterase. Nat Genet 1995; 11:468-71.

10. Grimberg J, Nawoschik S, Belluscio L, McKee R, Turck A, Eisenberg A. A simple and efficient non-organic procedure for the isolation of genomic DNA from blood. Nucleic Acids Res 1989; 17:8390.

11. Riazuddin SA, Yasmeen A, Zhang Q, Yao W, Sabar MF, Ahmed Z, Riazuddin S, Hejtmancik JF. A new locus for autosomal recessive nuclear cataract mapped to chromosome 19q13 in a Pakistani family. Invest Ophthalmol Vis Sci 2005; 46:623-6.

12. Schaffer AA, Gupta SK, Shriram K, Cottingham RW Jr. Avoiding recomputation in linkage analysis. Hum Hered 1994; 44:225-37.

13. Lathrop GM, Lalouel JM. Easy calculations of lod scores and genetic risks on small computers. Am J Hum Genet 1984; 36:460-5.

14. Ott J. Linkage analysis and family classification under heterogeneity. Ann Hum Genet 1983; 47:311-20.

15. Riazuddin SA, Yasmeen A, Yao W, Sergeev YV, Zhang Q, Zulfiqar F, Riaz A, Riazuddin S, Hejtmancik JF. Mutations in betaB3-crystallin associated with autosomal recessive cataract in two Pakistani families. Invest Ophthalmol Vis Sci 2005; 46:2100-6.

16. Hentze MW, Kulozik AE. A perfect message: RNA surveillance and nonsense-mediated decay. Cell 1999; 96:307-10.

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