Mutations in RLBP1 associated with fundus albipunctatus in consanguineous Pakistani families

doi:10.1136/bjo.2010.189076

Br J Ophthalmol. Author manuscript; available in PMC 2012 September 27.

Published in final edited form as:

Br J Ophthalmol. 2011 July; 95(7): 1019–1024.

Published online 2011 March 28. doi: 10.1136/bjo.2010.189076

PMCID: PMC3459316

NIHMSID: NIHMS407687

Mutations in RLBP1 associated with fundus albipunctatus in consanguineous Pakistani families

Shagufta Naz,¹ Shahbaz Ali,¹ S Amer Riazuddin,^1,² Tahir Farooq,³ Nadeem H Butt,⁴ Ahmad U Zafar,¹ Shaheen N Khan,¹ Tayyab Husnain,¹ Ian M MacDonald,^5,⁶ Paul A Sieving,⁶ J Fielding Hejtmancik,⁶ and Sheikh Riazuddin^1,⁴

¹National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan

²The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

³Layton Rahmatulla Benevolent Trust Hospital, Lahore, Pakistan

⁴Allama Iqbal Medical College, University of Health Sciences, Lahore, Pakistan

⁵Department of Ophthalmology, The University of Alberta, Edmonton, Alberta, Canada

⁶Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA

SN, SA, JFH and SR contributed equally to this study.

Correspondence to Sheikh Riazuddin, National Centre of Excellence in Molecular Biology, 87 West Canal Bank Road, Lahore 53700, Pakistan; Email: riaz/at/aimrc.org

The publisher's final edited version of this article is available at Br J Ophthalmol

Abstract

Objective

To identify disease-causing mutations in two consanguineous Pakistani families with fundus albipunctatus.

Methods

Affected individuals in both families underwent a thorough clinical examination including funduscopy and electroretinography. Blood samples were collected from all participating members and genomic DNA was extracted. Exclusion analysis was completed with microsatellite short tandem repeat markers that span all reported loci for fundus albipunctatus. Two-point logarithm of odds (LOD) scores were calculated, and coding exons and exon–intron boundaries of RLBP1 were sequenced bi-directionally.

Results

The ophthalmic examination of affected patients in both families was consistent with fundus albipunctatus. The alleles of markers on chromosome 15q flanking RLBP1 segregated with the disease phenotype in both families and linkage was further confirmed by two-point LOD scores. Bi-directional sequencing of RLBP1 identified a nonsense mutation (R156X) and a missense mutation (G116R) that segregated with the disease phenotype in their respective families.

Conclusions

These results strongly suggest that mutations in RLBP1 are responsible for fundus albipunctatus in the affected individuals of these consanguineous Pakistani families.

INTRODUCTION

Fundus albipunctatus (FA) is a rare form of apparently stationary night blindness that is also characterised by the presence of symmetrical round white dots in the fundus with a greater concentration in the midperiphery but with normal retinal arterioles, disc and visual fields.¹ In FA patients, electroretinogram (ERG) recordings yield poor responses after retinal bleaching and profoundly delayed dark adaptation improves upon prolonged exposure to darkness.¹ A clinically distinct disorder, retinitis punctata albescens (RPA) is characterised by nyctalopia, reduced visual acuity, multiple round white deposits in the retina, progressive attenuation of retinal arterioles, and non-detectable or severely reduced ERG recordings.²

RPA and FA are genetically heterogeneous disorders: mutations in RLBP1 and occasionally in RHO, RDS and RDH5 have been causally associated with RPA, whereas mutations in RLBP1 and RDH5 have been identified with patients diagnosed with FA.^3–10 Previously, Katsanis and colleagues reported a missense mutation in RLBP1 (R150Q) that segregated with the disease phenotype in a Saudi family with both RPA and FA.¹⁰

RLBP1 encodes a 317 amino acid cytosolic cellular retinaldehyde binding protein (CRALBP) that has been reported in the developing ciliary body and iris.^11,12 CRALBP has been localised in adult species to the retinal pigment epithelium, Müller cells of neural retina and ocular ciliary epithelium.^13–15 It can select 11-cis-retinaldehyde from a mixture of retinoids and protect it from photoisomerisation, suggesting a possible role in the generation of 11-cis-retinoids in the visual cycle.¹⁶

Here we report on two consanguineous families from the Punjab province of Pakistan that included multiple affected individuals diagnosed with FA. Exclusion analyses with closely spaced short tandem repeat (STR) markers localised the critical interval to a region on chromosome 15q that harbours RLBP1. Sequencing identified novel mutations that segregated with the disease phenotype in their respective families and were not present in ethnically matched controls.

METHODS

Clinical assessment

In total, 125 consanguineous Pakistani families with inherited retinal dystrophies were recruited to participate in a collaborative study between the National Centre of Excellence in Molecular Biology (NCEMB), Lahore, Pakistan, and the National Eye Institute, Bethesda, Maryland, USA. A detailed medical history was obtained by interviewing family members. Fundus photographs were obtained at the Layton Rahmatulla Benevolent Trust Hospital, Lahore. ERG measurements were recorded with equipment manufactured by LKC (Gaithersburg, Maryland, USA). Rod responses were determined through an incident flash attenuated by −25 dB and the rod–cone response was measured at 0 dB. Isolated cone responses were recorded at 0 dB with a 30 Hz flicker over a background illumination of 17–34 cd/m². Blood samples were collected from all participating members and genomic DNA was extracted by the non-organic method described previously.^17,18

Genotype analysis

Exclusion analyses were performed with highly polymorphic fluorescent STR markers spanning all reported loci for FA. A multiplex PCR was completed in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Foster city, CA, USA) as described previously.^19,20 PCR products from each DNA sample were pooled and mixed with a loading cocktail containing HD-400 size standards (Applied Biosystems). The resulting PCR products were separated in an ABI 3100 DNA Analyzer (Applied Biosystems) and genotypes were assigned with GeneMapper software (Applied Biosystems).

Linkage analysis

Two-point linkage analyses were performed using the FASTLINK version of MLINK from the LINKAGE Program Package (provided in the public domain by the Human Genome Mapping Project Resources Centre, Cambridge, UK).^21,22 Maximum two-point LOD scores were calculated using ILINK. Autosomal recessive FA was analysed 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 Marshfield database (http://research.marshfieldclinic.org/genetics/) and the National Center for Biotechnology Information chromosome 15 sequence maps.

Mutation screening

Primer pairs for individual exons were designed using the Primer3 program (http://primer3.sourceforge.net/). The sequences and annealing temperatures are available upon request. Amplifications were performed in 25 μl reactions as described previously.^19,20 PCR products were separated on a 2% agarose gel and purified by ethanol precipitation. The PCR primers for each exon were used for bidirectional sequencing using BigDye Terminator Ready reaction mix (Applied Biosystems), according to the manufacturer's instructions. Sequencing products were resuspended in 10 μl of formamide (Applied Biosystems) and denatured at 95°C for 5 min. The sequencing products were separated in an ABI PRISM 3100 DNA Analyser (Applied Biosystems). The results were assembled with ABI PRISM sequencing analysis software version 3.7 and analysed with SeqScape software (Applied Biosystems).

RESULTS

Family PKRP064 recruited from the Punjab province of Pakistan included six affected individuals in three consanguineous loops (figure 1). This family resides in remote villages of the Punjab province of Pakistan, and so the exact age of disease onset is not known. However, the medical records available to us along with communications with the family elders were suggestive of an early, most probably a congenital onset. The fundus photographs of affected individuals illustrate multiple round white deposits in the mid and peripheral regions of the retina (figure 2). The scotopic ERG responses were non-detectable, whereas the photopic response was severely reduced in affected individuals (figure 3). Taken together, the ophthalmological examination in PKRP064 identified typical features of FA with an early onset form of night blindness (table 1).

Figure 1

Pedigree drawing of family PKRP064 with haplotypes of six adjacent 15q markers and segregation of c.466C/T variation with the disease phenotype. Squares are males, circles are females; filled symbols are affected individuals; a double line between individuals (more ...)

Figure 2

Fundus photographs of individuals of family PKRP064. (A) Individual 11: OD and OS; (B) unaffected individual 14: OD and OS. The fundus photographs show multiple round white deposits with relative absence in the central macula. OD, oculus dexter (right (more ...)

Figure 3

Electroretinographic (ECG) responses of family PKRP064. Individual 11: (A) OD combined rod and cone response, and cone flicker response; (B) OS combined rod and cone response, and cone flicker response. Unaffected individual 14: (C) OD combined rod and (more ...)

Table 1

Clinical characteristics of affected individuals of family PKRP064

Genomic DNA of affected individuals was investigated with closely spaced STR markers that span all reported loci associated with FA (data not shown). Two-point linkage analysis with alleles of chromosome 15q STR markers was suggestive of linkage with significant LOD scores. Maximum two-point LOD scores of 4.77 and 3.19 were obtained with markers D15S1045, and D15S202, at θ=0, respectively (table 2).

Table 2

Two point limit of detection (LOD) scores of chromosome 15q markers of families PKRP064 and PKRP107

Haplotype analysis supported linkage to the chromosome 15q. There are recombination events in individuals 12 and 22 at D15S152 and D15S979, respectively, which defines the proximal boundary (figure 1). The distal boundary is defined by recombination in individual 19 at marker D15S207 (figure 1). In addition, lack of homozygosity at D15S116 in individual 22 suggests that the pathogenic mutation resides proximal to marker D15S116 in a 2.24 cM (1.18 Mb) region flanked by D15S979 proximally and D15S116 distally.

The critical interval harbours RLBP1, a gene previously associated with RPA and FA, and sequencing of the coding exons of RLPB1 identified a C to T transition c.466C→T (figure 4A), which leads to premature termination of the protein R156X. This variation segregated with the disease phenotype in family PKRP064: all affected individuals were homozygous for the mutation, whereas unaffected individuals were either heterozygous carriers or were homozygous for the wild type allele (figure 1). The mutation was not present in 192 ethnically matched control chromosomes.

Figure 4

Forward and reverse sequence chromatograms of family PKRP064. (A) Individual 15 harbouring the wild type allele; (B) individual 14 heterozygous and (C) affected individual 12 homozygous for C to T transition c.466 C→T, resulting in a p.R156X. (more ...)

Subsequently, we identified a second family with typical clinical characteristics of FA (figure 5). Exclusion analyses localised the disease phenotype to chromosome 15q with suggestive LOD scores. Two-point scores of 3.04, 3.09, and 3.01 at θ=0 were obtained with markers D15S152, D15S979 and D15S1045, respectively (table 2). Bi-directional sequencing identified a missense mutation c.346G/C, resulting in glycine to arginine substitution: p.G116R (figure 5). The mutation segregated with the disease phenotype in the family (figure 6) and was not present in 192 ethnically matched control chromosomes. The mutation segregated with the disease phenotype in the family and was not present in 192 ethnically matched control chromosomes. We investigated the evolutionary conservation of glycine 116 in other RLBP1 orthologues using the UCSC genome browser (http://genome.ucsc.edu/cgi-bin/ hgGateway) and as shown in figure 6, the amino acid glycine at position 116 is conserved in other RLBP1 orthologues.

Figure 5

Sequence chromatograms of family PKRP107 and alignment of G116 in RLBP1 orthologues. (A) Individual 15 harbouring the wild type allele; (B) individual 7 heterozygous and (C) affected individual 8 homozygous for G to C transition c.346G→C, resulting (more ...)

Figure 6

Pedigree drawing of family PKRP107 with haplotypes of six adjacent 15q markers and segregation of c.346G→C variation with the disease phenotype. Symbols are as described in figure 1. The alleles forming the risk haplotype are shaded black, and (more ...)

DISCUSSION

Here, we report pathogenic mutations in RLBP1 associated with FA in two consanguineous Pakistani families. Exclusion analysis localised the critical interval to chromosome 15q harbouring RLBP1 and subsequent sequencing identified causal mutations in RLBP1. These mutations segregated with the disease phenotype in their respective families and were not present in ethnically matched controls. These results strongly suggest that pathogenic mutations in RLBP1 are responsible for FA in both Pakistani families. Identification of novel pathogenic mutations reaffirms the diverse allelic heterogeneity of RLBP1.

The p.R156X mutation is expected to produce an unstable transcript that will be degraded by nonsense-mediated decay.⁹ If somehow the mutant mRNA escapes nonsense mediated decay, the protein thus produced will lack the 161 amino acids of the C-terminal domain. On the other hand, the evolutionary conservation of amino acid glycine 116 in other RLBP1 orthologues suggests that p.G116R will have a deleterious effect on RLBP1 structure; however, the molecular mechanism of causality still remains unknown. Additional functional analysis will help us better understand how these mutations affect the ability of RLBP1 protein and lead to the disease phenotype.

Acknowledgements

The authors are grateful to all family members for their participation in this study.

Funding This work was supported in part by Higher Education Commission, Islamabad, Pakistan and Ministry of Science and Technology, Islamabad, Pakistan.

Footnotes

Competing interests None to declare.

Patient consent Obtained.

Ethics approval This study was conducted with the approval of the Institutional Review Board of National Centre of Excellence in Molecular Biology and the National Eye Institute.

Provenance and peer review Not commissioned; externally peer reviewed.

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