(Investigative Ophthalmology and Visual Science. 2001;42:1436-1438.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
A New Locus for Autosomal Recessive RP (RP29) Mapping to Chromosome 4q32-q34 in a Pakistani Family
Abdul Hameed1,2,
Shagufta Khaliq1,2,
Muhammad Ismail1,
Khalid Anwar1,
S. Qasim Mehdi1,
David Bessant3,
Annette M. Payne3 and
Shomi S. Bhattacharya3
1 From the Biomedical and Genetic Engineering Division, Dr. A. Q. Khan Research Laboratories, Islamabad, Pakistan; and the
3 Department of Molecular Genetics, Institute of Ophthalmology, University College London, United Kingdom.
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Abstract
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PURPOSE. To map the disease locus in a six-generation, consanguineous Pakistani
family with autosomal recessive retinitis pigmentosa (arRP). All
affected individuals had pigmentary retinopathy associated with
symptoms of night blindness and the loss of peripheral visual fields by
the age of 20 years, loss of central vision between the ages of 25 and
30 years, and complete blindness between the ages of 40 and 50 years.
METHODS. Genomic DNA from family members was typed for alleles at known
polymorphic genetic markers using polymerase chain reaction. Alleles
were assigned to individuals, which allowed calculation of LOD scores
using the programs Cyrillic (http://www.cyrillicsoftware.com)
and MLINK (Cherwell Scientific Publishing Ltd., Oxford, UK). The genes
for membrane glycoprotein (M6a) and chloride
channel 3 (CLCN3) were analyzed by direct sequencing for
mutations.
RESULTS. A new locus for arRP (RP29) has been mapped to
chromosome 4q32-q34. A maximum two-point LOD score of 3.76 was obtained
for the marker D4S415, with no recombination. Two recombination events
in the pedigree positioned this locus to a region flanked by markers
D4S621 and D4S2417. A putative region of homozygosity by
descent was observed between the loci D4S3035 and D4S2417,
giving a probable disease interval of 4.6 cM. Mutation screening of two
candidate genes, M6a and CLCN3, revealed
no disease-associated mutations.
CONCLUSIONS. The results suggest that the arRP phenotype maps to a new locus and is
due to a mutated gene within the 4q32-q34 chromosomal
region.
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Introduction
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Retinal photoreceptor dystrophies are a clinically and
genetically heterogeneous group of retinal degenerations that together
form the most frequent cause of inherited visual disorders, with an
estimated prevalence of 1 in 4000.1
2
Retinitis pigmentosa
(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.1
Ophthalmoscopic examination typically reveals pigmentary disturbances
of the midperipheral retina. RP may be inherited as an autosomal
recessive, autosomal dominant, digenic, or X-linked trait. Autosomal
recessive RP (arRP) accounts for approximately 20% of all cases of RP,
whereas sporadic RP, which is presumed to be recessive in most cases,
accounts for a further 50%.2
To date, approximately 17 loci for arRP have been
reported.3
In this study we identified a novel locus for
arRP (RP29) on chromosome 4q32-q34.
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Methods
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The study protocol adhered to the tenets of the Declaration of
Helsinki. We studied 16 members of a six-generation,
consanguineous Pakistani family in which RP segregated as an autosomal
recessive trait (Fig. 1)
. This pedigree contained six affected individuals in two branches.
Fundus examination of all affected individuals revealed the typical
clinical features of retinitis pigmentosa, including pigment
deposition, attenuation of blood vessels, and pallid disc. The
peripheral blood vessels were completely obliterated in individuals
V:3, V:6, and VI:7 (Fig. 1)
. The early stage of the disease was
clinically characterized by bone corpuscle–type pigmentation,
deposited mainly in the equatorial region, and macular involvement in
the later stages of the disease. Affected subjects experienced night
blindness from the age of approximately 20 years and deterioration of
visual acuity (central vision) between 25 and 30 years of age. By the
age of 40 to 50 years affected subjects had no perception of light in
either eye. Biomicroscopy showed the presence of cells in vitreous of
individuals V:7 and VI:4. Anterior polar and posterior capsular lens
opacities were also observed in individuals V:3 and V:6 (Fig. 1)
.
Subjects were designated as unaffected if they showed no clinical
evidence of RP by the age of 30 years.
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Figure 1. Pedigree of an arRP family with analysis of genotypic data for
microsatellite markers. Circles: women;
squares: men; filled symbols: affected
individuals; open symbols: unaffected individuals.
Double line connecting the spouses represents
consanguinity.
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DNA Extraction
Peripheral blood samples were collected with informed consent
from the affected and unaffected members of the family (Fig. 1)
. For
comparison, 100 ethnically matched individuals with no personal or
family history of retinopathy were selected to serve as control
subjects. Genomic DNA was extracted from whole blood using an
extraction kit (Nucleon II; Scotlab Bioscience, Strathclyde, Scotland,
UK).
Microsatellite and Linkage Analysis
Genomic DNA from family members was amplified by using primers
for microsatellite markers for the known arRP loci3
and
whole-genome search4
by polymerase chain reaction (PCR).
PCR products were separated by nondenaturing polyacrylamide gel
electrophoresis (Protogel; National Diagnostics, Manville, NJ) and
visualized under UV illumination after staining with ethidium bromide.
Alleles were assigned to individuals, and genotypic data were used to
calculate the LOD scores using the programs Cyrillic and MLINK. Allele
frequencies were calculated from the spouses in this family and a
control ethnically matched population. The phenotype was analyzed as an
autosomal recessive trait, with complete penetrance, and a frequency of
0.0001 for the affected allele.
Mutation Screening
Intronic forward and reverse primers were designed for the exons
of the candidate genes. The PCR reactions (25 or 50 µl) were
performed using DNA samples of unaffected parents for heteroduplex
analysis under standard conditions with Taq DNA polymerase
(Bio-Line; London, UK). Annealing was at the exon-specific temperature.
To identify any heteroduplexes, the amplified exons were analyzed by
electrophoresis using MDE gel (FMC, Rockland,
ME).5
The gels were run at 180 V overnight on a
commercial apparatus (model 600S; Hoefer, San Francisco, CA). Products
of PCR amplification were sequenced using a commercially available kit
(Prism Ready Reaction Sequencing Kit; Perkin Elmer–ABI, Warrington,
UK), and the products were analyzed on an automated sequencer (model
377; Perkin Elmer–ABI). All PCR products were sequenced in the forward
and reverse directions.
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Results
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Linkage analysis was performed on this family as described
earlier. The known arRP loci3
were tested for
linkage to the disease in this family using two microsatellite markers
centered on the critical region of each locus. Linkage to any of the
known arRP loci was not observed.
A genome-wide search was undertaken using 300 polymorphic markers
spanning the entire human genome at approximately 20-cM intervals
(Research Genetics, Huntsville, AL). Significant exclusion was obtained
for all markers except those located on chromosome 4q32-q34.
Haplotypes for these markers are shown in Figure 1
. Two-point
LOD scores between arRP and the markers in this region (D4S1629,
D4S2368, D4S2979, D4S621, D4S2431, D4S3028, D4S3035, D4S3030, D4S415,
and D4S2417) are summarized in Table 1
. The maximum LOD score of 3.76 was obtained for the marker
D4S415. A positive LOD score of 3.48 at 
= 0.00 was also
obtained with D4S3030. Recombination events involving markers D4S621 in
individual V:3 and D4S2417 in individuals V:6 and VI:6 define the
centromeric and telomeric boundaries, respectively, of the
6-cM5
disease locus between these markers (Fig. 1)
.
Because this was a consanguineous family, a region of homozygosity
would be expected to surround the associated gene. All the patients in
both branches of this family were homozygous for alleles of the
microsatellites D4S3030 and D4S415. This most probably indicates an
area of homozygosity by descent, and it is therefore probable that the
disease gene lies in this smaller interval, flanked by the markers
D4S3035 and D4S2417. This interval spans only 4.6 cM (Fig. 1)
. These
results have permitted us to identify a novel locus for arRP
(RP29) at 4q32-q34. The locus does not overlap any
previously identified retinal dystrophy locus; therefore, this
represents the identification of a novel locus for a gene that could be
important for normal retinal functioning. This 18th autosomal recessive
locus illustrates genetic heterogeneity of the arRP phenotype.
The genetic databases6
7
8
were searched to identify the
candidate gene(s) in the region. The genes M6a and
CLCN3, which were physically mapped within the disease
region, were analyzed for mutation by heteroduplex analysis and direct
sequencing. All the exons were amplified using primers designed for the
intronic regions of the genes to permit detection of any splice site
mutations. M6a is a cell surface glycoprotein, mainly
expressed on neurons in the murine central nervous system (CNS), which
plays significant roles in neural cell adhesion and some aspects of
neurite growth.9
10
11
CLCN3 is a member of the
voltage-gated chloride channel family and is expressed primarily in
tissues derived from the neuroectoderm.12
Sequencing of both these genes revealed no disease-associated
mutations. However, a T
G substitution was found in the noncoding
region of exon 5 at nucleotide 1307 of the M6a
gene.10
No disease-associated segregation for this change
was observed.
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Discussion
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Examination of the GeneMap’996
and analysis of the
genomic sequence available from this region did not identify any
candidates in addition to the two we have screened. The genomic
sequencing of the region is unfinished as yet, and, in addition, many
gaps remain that could explain the lack of identifiable candidates.
Further isolation and characterization of novel transcripts from this
region will aid the identification of this disease gene. Further effort
is required to identify the disease-causing gene in this region of
chromosome 4q, which is syntenic with a region of mouse
chromosome 8. There are, however, no reports of a mouse model with
retinal degeneration mapping to this locus.
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Footnotes
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2 AH and SK contributed equally to this work. 
Supported by The Wellcome Trust.
Submitted for publication September 19, 2000; revised December 15, 2000; accepted January 8, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Shagufta Khaliq, Dr. A. Q. Khan Research Laboratories, Biomedical and Genetic Engineering Division, PO Box 2891, 25 Mauve Area, Islamabad, Pakistan. sqmehdi{at}isb.comsats.net.pk
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References
|
|---|
-
Bird, AC (1985) Retinal photoreceptor dystrophies Am J Ophthalmol 119,543-562
-
Jay, M. (1982) Figures and fantasies: the frequencies of the different genetic forms of retinitis pigmentosa Birth Defects Orig Artic Ser 18,167-173
-
RetNet. University of Texas-Houston Health Science Center; available in the public domain at http://www.sph.uth.tmc.edu/Retnet/disease.htm
-
Marshmed. Marshfield Laboratories, Marshfield, WI; available in the public domain at http://www.marshmed.org/genetics
-
Keen, J, Lester, D, Inglehearn, C, Curtis, A, Bhattacharya, SS (1991) Rapid detection of single base mismatches as heteroduplexes on Hydrolink gels Trends Genet 7,5[Medline][Order article via Infotrieve]
-
National Center for Biotechnology Information (NCBI), National Institutes of Health, Bethesda, MD; available in the public domain at http://www.ncbi.nlm.nih.gov
-
Ensembl Genome Server. Sanger Centre, Hinxton Hall, UK; available in the public domain at http://www.ensembl.org
-
The Genome Database. Hospital for Sick Children, Toronto, Ontario, Canada; available in the public domain at http://www.gdb.org
-
Lagenaur, C, Kunemund, V, Fischer, G, Fushiki, S, Schachner, M. (1992) Monoclonal M6 antibody interferes with neurite extension of cultured neurons J Neurobiol 23,71-88[Medline][Order article via Infotrieve]
-
Shimizu, F, Watanabe, TK, Fujiwara, T, Takahashi, E, Nakamura, Y, Maekawa, H. (1996) Isolation and mapping of the human glycoprotein M6 gene (GPM6A) to 4q33–q34 Cytogenet Cell Genet 74,138-139[Medline][Order article via Infotrieve]
-
Olinsky, S, Loop, BT, DeKosky, A, et al (1996) Chromosomal mapping of the human M6 genes Genomics 33,532-536[Medline][Order article via Infotrieve]
-
Taine, L, Coupry, I, Boisseau, P, et al (1998) Refined localisation of the voltage-gated chloride channel, CLCN3, to 4q33 Hum Genet 102,178-181[Medline][Order article via Infotrieve]
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Abstract
Introduction
