Molecular Vision 2004; 10:884-889 <>
Received 15 June 2004 | Accepted 11 November 2004 | Published 17 November 2004

Autosomal recessive retinitis pigmentosa in a Pakistani family mapped to CNGA1 with identification of a novel mutation

Qingjiong Zhang,1 Fareeha Zulfiqar,2 S. Amer Riazuddin,1,2 Xueshan Xiao,1 Zahoor Ahmad,2 Sheikh Riazuddin,2 J. Fielding Hejtmancik1
(The first two authors contributed equally to this publication)

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

Correspondence to: J. Fielding Hejtmancik, MD, PhD, OGCSB/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 map the locus for autosomal recessive retinitis pigmentosa in a large Pakistani family and to determine the causative mutation.

Methods: A large family with multiple individuals affected by autosomal recessive retinitis pigmentosa was ascertained in the Punjab province of Pakistan as part of an ongoing project between the CEMB, Lahore, Pakistan and the NEI to identify genetic causes of eye diseases. After initial analysis of previously identified autosomal recessive retinitis pigmentosa loci, a genome wide scan was performed using microsatellite markers at about 10 cM intervals. Two point lod scores were calculated and haplotypes were analyzed in order to define disease locus. Bidirectional dideoxynucleotide sequencing was used to screen for mutations in candidate genes.

Results: In the genome wide scan, autosomal recessive retinitis pigmentosa in this Pakistani family showed linkage to an 11.7 cM region of chromosome 4p12 between D4S405 and D4S1592 with a maximum lod score of 2.90 with D4S405 at θ=0.01 Sequence analysis of CNGA1 identified a 2 bp deletion in exon 8: c.626_627delTA resulting in a frameshift, p.Ser209fsX26 in the translated protein. This mutation results in deletion of the COOH terminal 482 of 690 total amino acids in CNGA1 and their replacement by 25 novel amino acids before a premature termination. The mutation is found in a homozygous state in all 7 affected individuals and was heterozygous in all 15 unaffected family members examined. The mutant allele of CNGA1 itself shows linkage to the disease with maximum lod score of 4.43 at θ=0.

Conclusions: The autosomal recessive retinitis pigmentosa in this family is caused by a mutation in CNGA1 gene. To our knowledge, this is the first report in which both linkage analysis and identification of a mutation support CNGA1 as a cause for autosomal recessive retinitis pigmentosa.


Retinitis pigmentosa (RP; OMIM 268000), the most common hereditary retinal disease causing blindness worldwide, refers to a heterogeneous group of progressive retinal degenerations. Clinically, RP is usually characterized by night blindness, progressive constriction of the visual fields, pigment deposit on mid-peripheral retina, and gradually reduced visual acuity resulting from degeneration of rods. RP in different families can be transmitted as an autosomal dominant, autosomal recessive, X-linked recessive, mitochondrial or digenic trait. A large number of loci or genes responsible for RP have been reported (RetNet).

Twenty-one loci have been reported to cause autosomal recessive retinitis pigmentosa (arRP), which is the most frequent form [1-22]. Of these, causative mutations have been identified in genes at 17 loci, including ABCA4 [2], CERKL [3], CNGA1 [4], CNGB1 [5], CRB1 [6], LRAT [7], MERTK [8], NR2E3 [9], PDE6A [10], PDE6B [11], RGR [12], RHO [13], RLBP1 [14], RPE65 [15], SAG [16], TULP1 [17], and USH2A [18]. Mutations in each individual gene identified so far are responsible for arRP in only a small fraction of families, between 2 and 5%. Overall, causative mutations have been identified in a small fraction of arRP families, possibly as low as 15% [3].

To date, six mutations in CNGA1 have been identified through candidate gene screening of arRP patients, but arRP has not been mapped to this locus by linkage studies [4,23]. Mutations in CNGA1 are of particular interest in studying molecular pathogenesis as at least in one case the mutant protein still preserves its functional property [4]. In vitro studies suggest that abnormal targeting of the mutant protein rather than loss of function might be the molecular mechanism of pathogenesis [24,25]. This pathogenic mechanism is usually seen in dominant rather than recessive diseases, and it would be interesting to know why abnormal targeting of this mutant protein does not cause an abnormal phenotype in carrier members in families with CNGA1 mutations. Conversely, there might be other mechanisms contributing to the disease process.

Accumulating more information about CNGA1 mutations and their relation to arRP may provide useful clues in understanding the mechanism of arRP resulting from CNGA1. In addition, mapping arRP to CNGA1 provides additional assurance that the mutations seen in this gene are causative. In this study we described a large Pakistani family with multiple members affected by arRP in which a genome wide scan mapped the disease gene to 4p12 and consequent mutational screening identified a novel frameshift variation in the CNGA1 gene.


Patient samples, pedigrees, and diagnostic criteria

This family (61039) from Punjab province of Pakistan with arRP was one of 25 ascertained as part of a collaborative project between the CEMB, Lahore, Pakistan and the NEI to identify genetic causes of eye diseases. It includes five consanguineous marriages with 11 affected individuals across four generations (Figure 1). Seven affected and 15 unaffected individuals from the family participated in this study (individuals 30, 39, and 40 were not involved in the genome wide scan but did participate in fine mapping studies). Diagnosis of RP was based on night blindness in early childhood, progressive loss of peripheral visual fields, and decreased visual acuity with age, as well as on typical signs observed under funduscopic examination, including waxy-pale optic discs, attenuation of retinal arteries, and bone spicule pigment deposits in the mid-peripheral retina. Informed consent was obtained from all participating individuals consistent with the tenets of Declaration of Helsinki. This project was approved by the IRBs of the National Eye Institute (Bethesda, MD) and the Centre of Excellence in Molecular Biology (Lahore, Pakistan).

Genotyping and linkage analysis

Genomic DNA was prepared from white blood cells as previously described [26]. After initial analysis of arRP candidate loci, a full genome wide scan was carried out using panels 1 to 27 of the ABI (Foster City, CA) PRISM linkage Mapping Set Version 2, which includes 382 markers spaced at intervals averaging about 10 cM. PCR was conducted at 94 °C for 8 min, followed by 10 cycles of amplification at 94 °C 15 s, 55 °C 15 s, and 72 °C 30 s; then 20 cycles at 89 °C 15 s, 55 °C 15 s, 72 °C 30 s; finally at 72 °C for 10 min. After mixing with GENESCANTM 400HD ROXTMROXTM standard (ABI) and deionized formamide, PCR products were denatured at 95 °C for 5 min and then immediately placed on ice for 5 min. The amplicons were separated on Long Ranger sequencing gels (Cambrex Bio Science; Rockland, ME) on an ABI 377 DNA sequencer. Genotyping data were collected by using GeneScan Analysis 3.0 and analyzed by Genotyper 2.5 of the software package from ABI. Two point linkage analysis was performed using the MLINK program of the FASTLINK implementation of the LINKAGE program package [27,28]. RP in the family was analyzed as autosomal recessive trait with full penetrance and with a disease gene allele frequency of 0.0001. For fine mapping, the markers in candidate locus were arranged according to National Center for Biotechnology Information (NCBI) website. All linkage analyses of family 61039 were carried out with 4 loops broken at individuals 9, 16, 34, and 44. Haplotypes were generated using the Cyrillic 2.1 program and confirmed by inspection. Marker allele frequencies were arbitrarily set as equal for the genome wide scan and fine mapping.

DNA sequencing

Ten pairs of primers (Table 1) were used to amplify the 8 coding exons (exon 2 to exon 9) and the adjacent intronic sequence of the CNGA1 gene (NCBI human genome build 34.3, NT_006238.10 for genomic DNA, NM_000087.1 for cDNA). The amplicons were sequenced with ABI BigDye Terminator cycle sequencing kit version 3.1, according to the manufacturer's instructions and electrophoresed on an ABI 3100 Genetic Analyzer. Sequencing results were analyzed using the SeqManII program of the Lasergene package (DNASTAR Inc., Madison, Wisconsin).

Restriction endonuclease analysis

Exon 8 of the CNGA1 gene, which contains the identified mutation, c.626_627delTA, was amplified by using the same pair of PCR primers used for sequencing. PCR products (409 bp for the wild type and 407 bp for the mutant, 20 μl) were digested with 3 units of HpyCH4III at 37 °C for 4 h and then separated by 1.8% agarose gel electrophoresis and visualized with ethidium bromide staining.


Clinical examination

All affected individuals examined in the family (Figure 1) fit the diagnostic criteria. Figure 2 shows the typical fundus changes for affected members in this family.

Linkage analysis

An initial scan of arRP candidate loci performed without samples number 30, 39, and 40, failed to establish linkage to any known RP loci except for a suggestive lod score of 2.66 at θ=0 for D4S405, near the CNGA1 gene. Additional markers in the CNGA1 region of chromosome 4 were either uninformative or yielded suggestive but indeterminate lod scores. A genome wide scan of chromosome 1 through 22, give lod scores above 1.5 only for markers D4S405 and D4S1592. This region was excluded as a candidate locus for arRP in an additional 24 Pakistani families by linkage analysis.

Fine mapping suggests the disease gene lies between D4S2950 and D4S428 on chromosome 4p12 (Table 2), even though the lod scores for microsatellite markers are somewhat lower than expected for a family of this size (Table 2). When the CNGA1 sequence change itself was used as a marker, a lod score of 4.43 was obtained at θ=0, close to the maximum expected from this family.

Haplotype analysis supports the fine mapping results, showing cosegregation of alleles at markers between D2S405 and D2S428 with the disease (Figure 1). Obligate recombinants are seen distally at D4S428 in unaffected individual 12 and at D4S1592 in unaffected individual 39. The presence of the same homozygous allele for D4S428 in unaffected individual 12 as in affected sibling 11 gives a lod score of -∞ for this marker at θ=0. Lack of homozygosity is seen at D4S1592 in affected individual 11. Lack of homozygosity at D4S405 in individuals 30, 36, and 41 sets the proximal boundary. Lack of homozygosity is also seen at D4S2950 in individuals number 18, 19, 34, and 41. While these are not obligate recombinants, one would expect homozygosity near the disease locus in this highly consanguineous family.

Mutation detection

CNGA1 lies in the linked region and is an obvious candidate gene. Sequencing the 8 coding exons of CNGA1 in this family identified a c.626_627delTA mutation at codon 209 in exon 8 (Figure 3), which results in p.Ser209fsX26, a frameshift producing 25 novel downstream amino acids followed by a premature stop at codon 234. This deletion also creates a new HpyCH4III recognition site.

The mutation was homozygous in all 7 affected individuals and heterozygous in all 15 unaffected individuals studied in the family. Restriction endonuclease HpyCH4III digestion of the CNGA1 exon 8 PCR products from the family further supports the sequencing results (Figure 3). The mutation cosegregates with the disease in the family, and it is not detected in 178 chromosomes from unrelated controls from Pakistan. When used as a marker in linkage analysis, the mutation itself yields a lod score of 4.43 at θ=0 in this family (Figure 1 and Table 2).


Here we described a large Pakistani family with multiple members affected by arRP associated with a novel frameshift variation in the CNGA1 gene. A full genome wide scan showing linkage only to this region, characteristics of the mutant protein, and absence of this mutation from the general Pakistani population all suggest that this mutation is the cause of RP in this family. To our knowledge, this is the first arRP family assigned to the CNGA1 locus by a linkage study. The relatively low lod score generated from microsatellite markers around CNGA1 results from the limited number of chromosomes transmitted in this family (Figure 1), where only 3 major chromosome haplotypes are present in 22 individuals across three generations. This limited the information provided by many matings.

The c.626_627delTA or p.Ser209fsX26 variation in CNGA1 identified in the Pakistan family results in deletion of 5 out of 6 transmembrane helices, deletion of the cyclic nucleotide binding domain, and deletion of the phosphorylation site involved in modulation [29,30]. This mutant protein would be unable to form heterotetramers with CNGB1, which form the functional units of CNG channels. Thus, the mutant protein functionally would be expected to be a functionally null mutation, explaining the recessive nature of RP in this family [24,25,31-33].

Mutations in the CNGA1 were detected in 4 out of 173 families [4], 1 out of 46 families [23], and 1 out of 25 families screened in this study, totally accounting for 2.46% of families with arRP. Of the 7 mutations detected, such as p.Arg28X, p.Glu70X, p.Lys139X, p.Ser209fsX26, p.Asp654fsX2, a deletion encompassing most of the transcriptional unit, and p.Ser316Phe, 6 mutations resulted in absence of part or all of the encoded protein. The protein with the p.Asp654fsX2 mutation, when expressed in vitro, was found to be retained inside the cell, but maintained normal channel activity [4,24,25] and this was suggested to be the molecular mechanism causing retinal degeneration in an isolated patient with arRP [4,24]. Unfortunately, there are no experimental data describing whether heterotetramers formed between mutant and normal proteins are functional in heterozygous individuals.

In summary, arRP in a Pakistan family was mapped to a region on chromosome 4p12 containing the CNGA1 gene and is associated with a c.626_627delTA mutation resulting in a p.Ser209fsX26 frameshift in the CNGA1 protein. To our knowledge, while a number of mutations have been identified by mutation screening in RP patients, this is the first instance in which the mutation is supported by linkage data. Understanding characteristics of CNGA1 mutations and their relation to arRP not only will facilitate genetic diagnosis of this disease, but also can provide clues into the molecular mechanism causing disease and function of the protein itself. Accumulating more information about CNGA1 mutations and their relation to arRP may provide useful clues in understanding the mechanism of arRP resulting from CNGA1.


We are grateful to the families for their participation in this study. We sincerely thank the staff of Lyton Rehmatullah Benevolent Trust (LRBT) Hospital for the identification of the families, their cooperation and expert clinical evaluation of affected individual and for the cooperation and help. We also thank Ms. Afshan Yasmeen, Ms. Shagufta Begum, and Mr. Farooq Sabar for their technical help. A part of the study carried out in Pakistan was supported by Higher Education Commission, Islamabad, Pakistan and Ministry of Science and Technology, Islamabad, Pakistan. Dr. Qingjiong Zhang gratefully acknowledges support from the National 863 Plan of China (Z19-01-04-02).


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