HUMAN MUTATION Mutation in Brief #208 (1998) Online

MUTATION IN BRIEF

Severe Autosomal Dominant Retinitis Pigmentosa

Caused by a Novel Rhodopsin Mutation (Ter349Glu)
David A. R. Bessant1,2, Shagufta Khaliq3, Abdul Hameed3, Khalid Anwar3, Annette M. Payne1, S.
Qasim Mehdi3, and Shomi S. Bhattacharya1*
Department of Molecular Genetics, Institute of Ophthalmology1, and Moorfields Eye Hospital2, London, UK.;
and Dr. A.3 Q. Khan Research Laboratories, Biomedical and Genetic Engineering Division, Islamabad,
Pakistan.

*Correspondence to Prof. S.S. Bhattacharya, Department of Molecular Genetics, Institute of Ophthalmology,

London, EC1V 9EL. Tel. +44 (0)171 608 6806. Fax +44 (0)171 608 6863. E-mail: sbhatach@hgmp.mrc.ac.uk
Contract grant sponsor: Wellcome Trust ; Contract grant number: 049571/Z/96/Z.
Contract grant sponsor: Medical Research Council of the U.K.; Contract grant number: G9301094.

Communicated by R.G.H. Cotton

ABSTRACT : Mutations in the rhodopsin gene are reported to be responsible for

approximately 25% of all cases of autosomal dominant Retinitis pigmentosa (adRP).
Affected individuals from a large family with an unusually severe form of adRP were
screened for mutations in the rhodopsin gene. Direct sequencing of exon 5 revealed a TAA
to GAA transversion at nucleotide 5276 / codon 349, which was confirmed by Dde1
restriction digest analysis. This change would replace the normal termination codon with a
glutamic acid residue (Ter-349-Glu, or X349E). The next predicted termination codon
(TAA) lies 153bp downstream at nucleotides 5429 to 5431. Termination of transcription at
this point would add an additional 51 amino-acid residues to the carboxy terminus of the
rhodopsin molecule. This mutation is unique in producing a mutant rhodopsin in which all
of the normal 348 amino-acid residues remain intact. It produces one of the most severe
adRP phenotypes ever observed in a family with a rhodopsin mutation. In view of this the
Ter-349-Glu mutation is worthy of further investigation to determine how the presence of
this particular mutant opsin leads to rod photoreceptor degeneration. © 1998 Wiley-Liss, Inc.

KEY WORDS: rhodopsin; retinitis pigmentosa, autosomal dominant; retinal dystrophies; candidate genes.

INTRODUCTION
Retinitis pigmentosa (RP; MIM# 268000) is the term applied to a clinically and genetically heterogenous
group of retinal degenerations, which primarily affect the rod photoreceptors, and have an overall prevalence of
about 1:4000 (Bundey and Crews, 1984). RP is characterized by progressive loss of vision, initially manifesting
as night blindness and reduction in the peripheral visual field, and later involving loss of central vision (Bird,
1995). It may be inherited as an autosomal dominant, autosomal recessive, digenic, or X-linked trait. Autosomal
dominant RP (adRP) accounts for around 22% of all cases of RP (Jay, 1982). There are currently nine mapped

Received 1 June 1998; accepted 27 August 1998.

© 1998 WILEY-LISS, INC.

2 Bessant et al.

loci for adRP (Retnet: http://www.sph.uth.tmc.edu/RetNet/).

Rhodopsin (RHO; MIM# 180380) is the photopigment molecule of the rod photoreceptors. Mutations in the
rhodopsin gene are reported to be responsible for approximately 25% of all cases of adRP in the U.K., Europe,
and the U.S.A. (Inglehearn et al., 1992; Bunge et al., 1993). Over 90 pathogenic mutations have been identified
(for review see Gal et al. 1997). The great majority have been reported in patients with adRP, but two mutations
(when present in the homozygous state) have been observed to cause autosomal recessive RP, and two
heterozygous missense mutations are described as responsible for Congenital stationary night blindness (Gal et
al., 1997).
Of the many mutations reported in rhodopsin around 90% are single base substitutions (Gal et al., 1997).
Three mutations have been described which alter the reading frame in exon 5 of rhodopsin (Horn et al., 1992; Al-
Maghtheh et al., 1994). Two of these result in translation continuing for a variable distance beyond the normal
stop signal.
A large adRP family which is of particular interest because of the unusual severity of the RP phenotype was
screened for mutations in the rhodopsin gene.

PATIENTS AND METHODS

A three generation family with eight living members affected by severe adRP was ascertained in Pakistan
(Figure 1A). All affected subjects had affected parents indicating an autosomal dominant mode of inheritance.
Informed consent was obtained by local clinicians.

A FIGURE 1 : A. Partial pedigree of dominant

retinitis pigmentosa Family 16RP. B. Restriction
digest analysis using the enzyme Dde1 performed
upon affected subject II:4 and her six children. The
B presence of an undigested band, indicating
heterozygosity of the substituted allele, was
observed all affected subjects (II:4, III.5, III:6 and
III:10). This contrasts with the complete digestion
observed in the unaffected siblings.

Oligonucleotide primers to the five exons of the rhodopsin gene (Genbank: U49742) were designed from the
published genomic sequence (Nathans and Hogness, 1984). Polymerase chain reaction PCR was performed using
standard conditions and an annealing temperature of 58°C. The resulting product was subjected to heteroduplex
analysis (Keen et al., 1991).
Once detected sequence variants were directly sequenced using an automated fluorescent sequencer (ABI
Biosystems model 373). PCR products were purified and then reamplified using the FS kit (Perkin Elmer).
Variants were sequenced in both the forward and reverse directions to confirm the location of sequence changes.
Restriction digests were performed directly on 20µl of exon 5 PCR product. Dde1 digests were incubated
overnight at 37°C to ensure complete digestion. The resultant bands were photographed after running on a 2%
agarose gel and staining with ethidium bromide.

RESULTS
A heteroduplex band was observed following PCR amplification of exon 5 of the rhodopsin gene. Direct
sequencing of this PCR product revealed a TAA to GAA transversion at nucleotide 5276 / codon 349. This
substitution would replace the normal termination codon with a glutamic acid residue. Since this nucleotide
change abolishes a recognition site for Dde1 restriction enzyme, Dde1 restriction analysis of the exon 5 product
 Severe Retinitis Pigmentosa caused by Rhodopsin Mutation Ter349Glu 3

was used to confirm the T to G sequence change in all affected members of this family and its absence in their
unaffected siblings (Figure1B).
Examination of the genomic sequence of rhodopsin reveals that the next predicted termination codon
(TAA) lies 153bp downstream at nucleotides 5429 to 5431. Termination of transcription at this point would add
an additional 51 amino-acid residues to the carboxy terminus of the rhodopsin molecule.

Clinical findings
A summary of the clinical findings in the adRP family 16RP (Ter-349-Glu) is presented in Table 1. The
first symptom in affected individuals was the development of night blindness (nyctalopia) in early childhood.
Deterioration of visual acuity was observed in even the youngest subject, a girl aged 6 years. Patients over the age
of 18 years all had severely reduced central vision (though this may in part be due to the presence of cataract and
macular oedema). Fundoscopy revealed pigment migration into the retina, optic nerve head pallor, and the
attenuation of retinal vessels as early as 16 years of age. These clinical signs indicate extensive photoreceptor cell
death and are not usually seen at such an early age in adRP.

TABLE 1 : Clinical features of affected members of adRP family 16RP (CF = counting fingers).

Patien Age Visual Acuity Intra- Optic Attenuation Cataract Macular

t No. (yrs) retinal disc of vessels oedema
Right / Left pigment pallor

II:4 40 3/60 2/60 + + + + +

III:1 16 6/18 6/12 + + +

III:4 6 6/9 6/18

III:5 20 CF CF + + + + +

III:6 19 CF CF + + + + +

III:10 9 6/18 6/18 + +

The early onset of central visual loss makes this one of the most severe adRP phenotypes ever reported in a
family with a rhodopsin mutation. It is more severe than the phenotype described in patients with the codon 296
rhodopsin mutation, which abolishes the retinal binding site (Owens et al., 1994), or the Cys187Tyr mutation
which disrupts the disulphide bond (Richards et al., 1995). Loss of visual acuity in childhood was reported in a
family with an Arg135Trp mutation, but the children in that family had acuities no worse than 6/12 (Pannarale et
al., 1996).

DISCUSSION
The Ter-349-Glu mutation segregating with disease in this family abolishes the normal rhodopsin
termination codon and replaces it with a glutamic acid residue. This would generate a mutant rhodopsin with all
4 Bessant et al.

of the normal 348 amino-acid residues intact, but with an additional 51 amino-acid residues added to the carboxy
terminus of the molecule (Figure 2).

FIGURE 2. A two-dimensional
= hydrophilic 300 schematic representation of the
carboxy terminus of the rhodopsin
molecule illustrating the additional 51
amino-acids. These are predicted to
= hydrophobic form a segment of β-pleated sheet, a
short α-helix, and then two domains,
the first of which is hydrophilic and
350 the second strongly hydrophobic.

COOH

Wild-type rhodopsin inserts into the photoreceptor outer segment disc membrane with its carboxy-terminus
located within the cytoplasm. Whilst post-translational processing of the mutant rhodopsin would not be expected
to be adversely affected by the additional residues, it is difficult to predict what effect they may have on the
tertiary structure of the molecule. It is possible that this mutant rhodopsin may become incorporated into the
photoreceptor outer segment disc membrane and bind retinal to form a light-absorbing chromophore, in which
case the additional C-terminal hydrophobic domain would probably become embedded in the disc membrane.
Alternatively, the additional polar amino-acids may prevent the normal folding of the molecule and prevent its
export from the endoplasmic reticulum.
The severity of the RP phenotype in the family with the Ter-349-Glu mutation indicates that this mutation
has a more detrimental effect on rod photoreceptor function and longevity than most other rhodopsin mutations.
Marked deterioration of central vision in childhood is very unusual in adRP. Since rhodopsin is not expressed in
cone photoreceptors, this finding implies that the adverse effect on cone function in the central retina (macular
region) is secondary to the primary degenerative process taking place in the rod photoreceptors.
The Ter-349-Glu mutation is a particularly fascinating one and it is worthy of further investigation to
determine how the presence of this particular mutant opsin leads to rod and cone photoreceptor degeneration.

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 Severe Retinitis Pigmentosa caused by Rhodopsin Mutation Ter349Glu 5

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6 Bessant et al.