Download Genetic and physical mapping of human recoverin: a gene

Genetic and Physical Mapping of Human Recoverin:
A Gene Expressed in Retinal Photoreceptors
Allan F. Wiechmann,*§ Gita Akots,^ James A. Hammarback,* Mark J. Pettenati,X
P. Nagesh Rao,% and Donald W. Bowden\
Purpose. Recoverin is a calcium-binding protein thai may be involved in phototransduciion in
mammalian retinal photoreceptors, and is considered to be a candidate gene for retinitis
pigmeniosa. This study was undertaken to develop the recoverin locus into a polymorphic
marker for future linkage studies on retinitis pigmeniosa families.
Methods. A human genomic cosmid clone was isolated and used to map the recoverin gene to a
human chromosome through hybridization to a panel of somatic hybrid cell line DNAs, and lo
human metaphase chromosomes by fluorescence in situ hybridization. A dinucleotide repeat
polymorphism located within the coding region of the recoverin gene was identified, and used
to genetically map the recoverin gene relative to index markers. In addition, three restriction
fragment length polymorphisms revealed by the cosmic! clone were identified and characterized.
Results. Hybridization to the somatic hybrid cell line DNAs localized the recoverin gene to
chromosome I 7. Recoverin was further localized to .17p.l 2-pl 3 by fluorescence in situ hybridization. The dinucleotide repeat polymorphism and restriction fragment length polymorphisms at the recoverin locus have a cumulative polymorphic information content = 0.71.
Conclusions. These polymorphic markers and additional closely linked markers will be useful
for linkage analysis of families with retinitis pigmeniosa. Invest Ophthalmol Vis Sci.
1994;35:32:>-331.
I n the process of visual phototransduction, absorption of light by rhodopsin initiates an enzymatic cascade that leads to hydrolysis of cyclic guanosine monophosphate (cGMP) and closure of cGMP-gated sodium/calcium channels resulting in photoreceptor
hyperpolarization.1 Recoverin is a calcium-binding
protein that until recently was believed to reverse the
effects of light on cGMP levels by activating guanylate
cyclase at low calcium concentration.2-3 Recent findings, however, have cast, some doubt on the ability of
recoverin to affect guanylate cyclase activity.4-5
From thi; Departments of "Nenrobiology and Anatomy, •[Biochemistry, and
%Pedialrics, and the Molecular ('•enetics I'rogram and §Wake Forest University
Eye Center, Bowman (Way School of Medicine, Wake Forest University, WimtonSalem, North Carolina.
This work was supported try grants from the National Eye Institute (EY08006 and
EY1008I) to A.F.W., and North Carolina Baptist Hospital Developmental
Technology Grant A-007-92 and Brenner Children's Hospital (I'.N.R. ami
M.J.P.).
Submitted for publication: July 6, 1993; accepted August 13, 1993.
Proprietary interest category: N.
Reprint requests: Allan F. Wiechmann, Department of Neurobiology and Anatomy,
Bowman Gray School of Medicine, Medical Ce?iter Boulevard, Wimton-Salem,
NC 27157-1010.
Investigative Ophihahnology & Visual .Science, February 1904, Vol. 'tf>. No.
Copyright © Association for Research in Vision and Ophthalmology
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The complementary DNA (cDNA) encoding human retinal recoverin has been cloned and sequenced.6"8 The human nucleotide sequence is 88%
identical to the bovine sequence,910 and contains a
600 base pair open reading frame encoding 200
amino acids. Recoverin-like immunoreactivity has
been localized to the photoreceptors and a subpopulation of bipolar cells in the human retina.11-8 The human recoverin gene is a 9 to 10 kb single-copy gene
with three exons (which together contain the entire
coding sequence) and two introns, and has been
mapped to human chromosome .17 by hybridization to
a panel of human/rodent hybrid DNAs.6 Northern
blot analysis of human retina, brain, lung, liver, and
skin fibroblasts reveals a transcript only in the retina.6
The most likely candidates for gene mutations that
result in retinal degeneration are those that serve crucial functions within the photoreceptor cell. It is
known that abnormal levels of cyclic nucleotides lead
to photoreceptor cell death.12 Because recoverin was
thought to be involved in regulating cGMP levels in
the photoreceptor, it therefore was a candidate for
325
326
Investigative Ophthalmology & Visual Science, February 1994, Vol. 35, No. 2
involvement in retinitis pigmentosa. Although the precise role of recoverin in the human retina is not well
understood, it remains a potentially important molecule in photoreceptor physiology. To evaluate the possible role of the recoverin molecule in inherited forms
of retinal degenerations, we cloned a genomic cosmid
containing the recoverin gene, which was used to physically localize the recoverin gene. In addition, multiple
recoverin polymorphisms were identified and used to
genetically map the gene.
MATERIALS AND METHODS
DNA Probes
A full-length recoverin cDNA probe isolated from human retina was prepared as described previously.8
Briefly, a set of oligonucleotide primers complementary to the 5' and 3' ends of the human recoverin cDN A
sequence were used with a recoverin bacteriophage
clone as a template for polymerase chain reaction
(PCR) amplification of recoverin cDNA. The probe
was labeled with 35S-dATP (1000 Ci/rnmol; Amersham, Arlington Heights, IL) using a Random Primer
labeling kit (Promega, Madison, WI).
Using the recoverin cDNA as a probe, a genomic
cosmid clone, designated 2A/S.110, was isolated from
a human genomic library prepared from peripheral
blood lymphocytes as described previously.13 The cosmid contained approximately 40 kb of human genomic DNA.
Double-stranded DNA sequencing (USB Sequenase kit; United States Biochemical, Cleveland, OH) was
performed using two different oligonucleotides
(5'[AAGTGCTGGAGATCGTCATG]3' and 5'[TGGAGGGAGGACAGCTGJ3') complementary to different
regions of the recoverin cDNA as primers.
Hybridization With Somatic Cell Hybrid Panel
The recoverin cosmid was radiolabeled with 32P-dCTP
(3000 Ci/mmol; New England Nuclear, Boston, MA)
using Random Primer labeling (Promega), and hybridized to a Southern blot containing a panel of genomic
DNAs from somatic cell hybrid cell lines, NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panel # 1 ,
containing different complements of human chromosomes.
In Situ Hybridization
Fluorescence in situ hybridization of the cosmid to
metaphase chromosomes was performed as described
previously.1415 Briefly, peripheral blood leukocytes
from chromosomally normal donors were cultured
and chromosomes were prepared using colcemid and
ethidium bromide.15 The recoverin cosmid probe was
labeled by nick-translation with biotin-14-dATP
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(Gibco-BRL; Gaithersburg, MD) using the BioNick Labeling System (Gibco-BRL). Fluorescence in situ hybridization was carried out with the recoverin probe
singly or in combination with the chromosome 17 centromere-specific probe D17Z1 (ONCOR; Gaithersburg, MD) using modifications of the manufacturer's
Blockit.
Restriction Fragment Length Polymorphism
Identification
Restriction fragment length polymorphisms (RFLPs)
identified in this study were found by hybridizing
EcoRI fragments derived from the parent cosmid 2A/
SI 10 to Southern blots containing panels of five different human genomic DNAs digested with one of
eight different restriction enzymes (BamHl, Bgl II,
EcoRI, Hinc II, Hind III, Msp I, Pst I, Rsa I, and Taq I).
RFLP loci were evaluated using methods for purification and radiolabeling of probe DNAs, restriction enzyme digests of human DNAs, agarose gel electrophoresis and Southern blot transfer of the human DNAs,
hybridization, and autoradiography as described previously.1617 Polymorphic information content (PIC) values were calculated using the LINKAGE utility program PIC version 1.3,18 using data from the genotyping of Centre d'Etude du Polymorphisme Humain
(CEPH) parents. The procedures followed the tenets
of the Declaration of Helsinki. Informed consent was
obtained from all subjects, and institutional human
experimentation committee approval was granted for
this study.
Identification of Recoverin Dinucleotide
Repeat Polymorphism
A dinucleotide repeat sequence was identified in the 3'
noncoding region of the recoverin cDNA with the interrupted sequence: (CA)7TG(CA)5(CG)4(CA)9, beginning at nucleotide position 666.8 Oligonucleotides
corresponding to nucleotide position 640-657
(5'[TCACATGACACCCGTGAG]3'; primer 1), and
position 839-819 (5'[AGTAGTGGAG GGAATGCTGAA]3'; primer 2) of the human recoverin sequence8
were synthesized for PCR amplification of the dinucleotide repeat sequence. Primer 1 (640-657) was endlabeled with 32P-ATP (4500 Ci/mmol; ICN) and polynucleotide kinase (Promega), and used in the PCR reaction with the other primer, and the genornic DNA of
the CEPH family members. PCR reactions were
carried out in a 15 fi\ volume containing 250 ng of
genomic DNA, 6 ng of primer 1 end-labeled with 32PATP, 30 ng of unlabeled primer 1, 30 ng of primer 2,
and components from a Gene-Amp PCR kit (Perkin
Elmer Cetus, Norwalk, CT). Samples were processed
through 30 temperature cycles consisting of 1 minute
at 95°C, 1 minute at 50°C, and 2 minutes at 72°C, and
327
Mapping of Recoverin Gene
the products were analyzed on standard sequencing
gels (6%) as described by Weber and May.19
clone. Sequencing of the 2A/S110 cosmid revealed a
100% identity with the corresponding regions of the
recoverin cDNA clone over the entire region analyzed
(data not shown). The regions analyzed in this study
were two 200 base pair segments of the coding region
(nucleotides 362 to 571, and 631 to 4308). This confirmed that the 2A/S110 human cosmid contains the
recoverin gene.
Genetic Mapping
Genotypic data for the recoverin dinucleotide repeat
sequence was collected by genotyping the CEPH mapping families. Genotyping was carried out in the standard panel of 40 CEPH families. These data were
combined with the data from the CEPH version 6 genetic database for chromosome 17 for genetic mapping of recoverin. Map construction was carried out as
described elsewhere in detail2015 using the CRI-MAP
linkage analysis program (version 2.4). Recoverin was
mapped relative to the collection of index markers on
chromosome 17 described by O'Connell et al.21 Location of the recoverin polymorphism was calculated using the ALL option of CRI-MAP. Two-point LOD
scores of recoverin to other chromosome 17 markers
were calculated using the TWOPOINT option of CRIMAP. Map distances were calculated using the Kosambi mapping function of this program.
Physical Mapping of Recoverin
Genomic DNAs from the NIGMS panel (#1) of rodent/human somatic cell hybrid cell lines were digested with Pst I restriction enzyme, and a Southern
blot was prepared from the digests. The Southern blot
was hybridized with the radiolabeled recoverin cosmid
2A/S110. As summarized in Table 1, evaluation of the
concordances and discordances map the gene to chromosome 17. The recoverin cosmid probe hybridized
only to the 15 cell line DNAs containing human chromosome 17.
This assignment of the recoverin gene to human
chromosome 17 was confirmed by fluorescence in situ
hybridization (Fig. 1). The recoverin cosmid hybridized to the short arm of chromosome 17 as identified
by the centromere-specific probe Dl 7Z1. Localization
was to band 17pl2-pl3.
RESULTS
Isolation and Characterization of a Recoverin
Cosmid Clone
Identification of Polymorphisms and Genetic
Mapping of Recoverin
DNA fragments from restriction enzyme digestion of
the cosmid 2A/S110 were used to screen for RFLPs
The genomic cosmid clone, designated 2A/S110, was
isolated from a human genomic library with the recoverin cDNA as a probe, and was sequenced to confirm the presence of the recoverin gene in the cosmid
l. Mapping of Human Recoverin in NIGMS Human/Rodent
Somatic Cell Hybrid Mapping Panel
TABLE
Human Chromosome* Hybrid
Line
GM/NA09925
GM/NA09926
GM/NA09927
GM/NA09928
GM/NA09929
GM/NA09930A
GM/NA09931
GM/NA09932
GM/NA09933
GM/NA09934
GM/NA09935A
GM/NA09936
GM/NA09937
GM/NA09938
GM/NA09940
GM/NA10324
GM/NA10567
GM/NA10611
Discordant
hvbrids
Recoverin
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
/
2
3
4
7
5
++-
10
11
12
13
H
15
16
17
18
19 20
21
22
X
+ -
+ + - + +
+ + <
- - - - +
- + +
+ - - + <
- - +
- + +
- < +
_ + _
_ _ _
11 9
8
5
4
+ +
- +
+ + + +
+ +
+ +
+ +
- +
- +
+ +
- _
_ _
5
3
- + - < - - <
+
< + - + + - +
+ < - - <
+ + +
+ - + - + - +
- + - <
+ + - + + - < + + +
< + - + + - <
- - - < - + + +
+ < + + - <
+
+ + - < + - +
+ < - + + - +
+ + _ _ _ _ _ _
_ _ _ _ _ _ _ _
3
2
16
6
8
+ - +
< +
+ - +
- +
- +
+ - +
+ - +
- +
< +
+ - +
< +
+ _ +
_ _ _ _
14
0
+
+
+
+
+
-
+
+
+
< +
+ + +
- + +
+ < +
+ + - - <
+ +
_ _ _ _
_ _ _ _
<
+
6
+
+
+
+ - <
+
<
- - + < +
+
- +
_ _
+
6
+
<
+
+
<
_
_
13 10
* Cell lines with >10% of the cells containing the chromosome are designated +, <10% are designated <, and not containing the chromosome are designated —. Only chromosomes present in >10% of the cells were used to calculated discordances (e.g., Yu et al, 1993).
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Investigative Ophthalmology & Visual Science, February 1994, Vol. 35, No. 2
FIGURE 1. Fluorescence in situ hybridization of the recoverin
cosmid probe to human metaphase chromosomes. The fluorescent tag (small arrows) identifies the recoverin locus (one
copy of the gene) to the short arm of an E Group chromosome (17 or 18). Inset: Hybridization with both the recoverin
cosmid probe (small arrows) and the specific chromosome
marker D17Z1 {large arrows) confirms the assignment of recoverin to chromosome 17.
on panels of genomic DNAs of five unrelated persons
digested with eight different enzymes. As summarized
in Table 2, three different RFLPs were identified and
characterized. PICs were determined from genotyping the RFLPs on CEPH parents.
The recoverin cosmid was also evaluated for the
presence of dinucleotide repeat sequences. A Southern blot prepared from EcoRl restriction enzyme digested cosmid clone was hybridized with the radiolabeled dinucleotide (CA)n. Two EcoKl fragments (4.8
TABLE 2.
and 1.5 kb) from the cosmid hybridized to (CA)n, suggesting that there are at least two dinucleotide repeat
sequences in the cosmid clone. One of these dinucleotide repeat sequences was recognized as being a dinucleotide repeat sequence located in the 3' untranslated
region of the recoverin cDNA. Oligonucleotide
primers derived from sequences flanking the dinucleotide repeat were prepared for PCR amplification of
human genomic DNA. These experiments revealed a
3-allele dinucleotide repeat polymorphism with the
characteristics outlined in Table 2.
The dinucleotide repeat was used to collect genotypic data in the CEPH families. Parents in 26 of 40
CEPH families were at least partially informative and
these families were analyzed with the dinucleotide repeat polymorphism. The recoverin mapping data were
merged with the CEPH Version 6 database for chromosome 17, and both two-point and multipoint linkage analysis were carried out using the CRI-MAP linkage analysis programs. Three markers from 17p, loci
D17S505, D17S510, and D17S66, had maximum twopoint LOD scores greater than 15 with recoverin as
summarized in Table 3. In addition, Table 3 shows the
two-point LOD scores for recoverin linkage to MYH2
(the myosin heavy chain gene) and D17S1.
D17S1 and MYH2 flank the recoverin locus based
on multipoint linkage analysis as summarized in Figure
2. The uniquely placed index markers on chromosome
17 described by O'Connell et al.21 were set as a fixed
order of markers and recoverin was mapped relative to
that fixed order using the ALL option of CRI-MAP.
Recoverin was uniquely placed between the two 17p
arm markers D17S1 and MYH2. This order is favored
with odds of at least 1000:1 over any other placement
Recoverin Polymorphisms
RFLPs
Probe
Enzyme
Number of
Alleles
Allele
Size (kb)
Allele
Frequency
Constant
Fragments (kb)
2A/S110/2
BamHI
*
0.37/0.50
TaqI
%
0.52
0.48
0.73
0.24
0.03
0.39
0.61
72
2A/S110
12.3
5.8
9.5
7.5
6.3
1.6
1.3
2.7, 2.2
0.35/0.41
2A/S110/4
Mspl
2
PIC/
Heterozygosity
1,4
—
0.36/0.48
Dinucleotide Repeat
Number of
Alleles
Allele
Size (bp)
Allele
Frequency
640-657 TCACATGACACCCGTGAG
S
830-819 AGTAGTGGAGGGAATGCTGAA
Cumulative
—
200
208
206
—
0.79
0.18
0.03
—
Oligonucleotides
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PIC/
Heterozygosity
0.30/0.34
0.71/0.73
Mapping of Recoverin Gene
329
TABLE 3.
Two-Point Linkage Analysis With Recoverin Microsatellite
and Polymorphic 17p Markers
LOD Score at 0
Locus
0.001
0. 05
0.01
0.15
0.20
0.25
0.30
0.35
0.40
0.45
t'max
e
D17S505
D17S510
D17S66
D17S1
MYH2
-12.81
-25.40
5.45
9.93
5.95
14 .98
12 .25
15 .47
12 .02
7 .19
17.40
16.14
15.56
11.24
6.81
17.42
16.86
14.67
10.15
6.19
16.35
16.17
13.30
8.90
5.42
14.58
14.61
11.61
7.53
4.55
12.27
12.43
9.65
6.06
3.59
9.52
9.75
7.45
4.51
2.54
6.39
6.66
5.04
2.90
1.47
3.04
3.30
2.48
1.31
0.51
17.56
.16.85
15.71
12.12
8.67
0.12
0.15
0.08
0.03
0.05
and the local odds for the recoverin location are
greater than 105:l for this location.
DISCUSSION
Recoverin is a 23 kD calcium-binding protein expressed in photoreceptoi s and some cone bipolar cells
in the human retina. 8 " It has been reported to stimulate guanylate cyclase in response to the light-generated low calcium concentration in the rod photoreceptor outer segment,211 although new reports suggest
that recoverin may not affect guanylate cyclase activity.45 The cGMP-gated calcium/sodium channels in
the outer segment membrane open in response to the
resulting higher cGMP levels, and the photoreceptor
depolarizes. Recoverin therefore was believed to promote recovei-y of the photoreceptor from light stimulation.
The cDNA encoding human retinal recoverin has
been cloned and sequenced.6"8 It contains a 78 base
pair base pair 5' untranslated region, a 600 base pairopen reading frame encoding 200 amino acids, and a
415 base pair 3' untranslated region.8 The nucleoiide
sequence of the coding region of human recoverin
shows 88% identity to the bovine910 and mouse sequences.22 Murakami and coworkers6 reported that
the human recoverin gene is a 9 to 10 kb single-copy
gene with three exons and two introns, and have
mapped the gene to human chromosome 17 using a
panel of human/rodent hybrid DNAs.
Using a human recoverin cDNA probe, we have
isolated a genomic clone for the recoverin gene. The
cosmid clone has been used as a probe to physically
map recoverin to the short arm of chromosome 1 7,
and to search for polymorphic loci in or near the recoverin gene. Hybridization of the radiolabeled recoverin cosmid with a Southern blot of a panel of genomic DNAs from somatic cell hybrid cell lines indicates
that the recoverin gene is located on human chromosome 17, consistent with the results of iMurakami and
coworkers.6 Fluorescence in situ hybridization localized the recoverin cosmid to the short arm of chromo-
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some 17, with sublocalization of the recoverin gene to
17pl2-pl3 on human metaphase chromosomes.
The recoverin genomic clone contained a polymorphic dinucleotide repeat with a PIC = 0.30. This
PIC value correlates well with the values reported by
Weber,23 where it was found that the informativeness
of (CA)n polymorphisms generally increases with an
increasing number of repeats, and decreases as the
number of interruptions of the repeat sequence increases. The (CA)n repeat sequence identified in this
13
D17S34
D17S450
D17S28
4.8
4.3
7.4
3.4
12
11.2
11.1
11.1
E
RECOVERIN
YH2
D17S124
—
7.3
18.0
11.2
12
3.1
1.7
5.3
2.0
D17S331
D17S82
NF1
0.6
3.4
10.0
D17S135
D17S83
8.3
FIGURE 2. Mapping of recoverin relative to index markers on
chromosome I 7 by multipoint analysis. The figure shows the
genetic map of index markers from O'Connell el alai present in the CEPH version 6 database. These are related to the
cytogenelic map of the short arm and proximal long arm of
chromosome 17. Through multipoint linkage analysis, recoverin maps between D17S1 and MYH2, 2 cM from the
MYH2 locus. Genetic distances between markers are shown
to the right expressed in cM as calculated using the FIXED
option of CRI-MAP.
330
Investigative Ophthalmology 8c Visual Science, February 1994, Vol. 35, No. 2
study is located in the 3' untranslated region of the
recoverin cDNA, and was used to genetically map the
recoverin locus between D17S1 and MYH2. D17S1
and MYH2 have both been assigned to 17pl3, 24 " 27
suggesting that recoverin also lies in this region. Another (CA)n repeat was determined to be located
within the cosmid clone, and may be useful for future
linkage analysis.
In addition to the dinucleotide repeat polymorphisms, three RFLPs revealed by the cosmid clone
were identified, with PICs in the 0.3 to 0.4 range. Together with the (CA)n repeat we have characterized,
these markers have a cumulative PIC = 0.71. These
polymorphic markers associated with the recoverin
gene will be useful in studies investigating the possible
relationship of the recoverin gene to retinitis pigmentosa. Retinitis pigmentosa is a group of inherited retinal degenerations characterized by progressive loss of
peripheral vision and night blindness as a result of rod
photoreceptor cell death. 28 Recent advances in identification of genes involved in retinitis pigmentosa have
suggested that mutations in retina-specific genes that
have crucial functions in photoreceptor physiology
might result in photoreceptor degeneration. 29 Because of its potential role in photoreceptor physiology,
recoverin has been considered to be a candidate gene
for retinitis pigmentosa. With multiple polymorphicmarkers at the recoverin locus, a large proportion of
families will be informative in linkage analyses with
recoverin. These markers, in conjunction with other
closely linked polymorphisms such as at DJ7S506
should allow rapid evaluation of recovering relationship to retinitis pigmentosa.
Key Words
recoverin, retina, genetic mapping, retinitis pigmentosa
Acknowledgments
The authors thank Dr. Cynthia Rothschild for helpful advice, and Felicia Lamm and Rosa Hayworth for technical
assistance.
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