Abstract
purpose. High myopia is a common complex-trait eye disorder, with implications for blindness due to increased risk of retinal detachment, macular degeneration, premature cataracts, and glaucoma. Mapping studies have identified at least four loci for nonsyndromic autosomal dominant high myopia at 18p11.31, 12q22-q23, 17q21-q23, and 7q36. The smallest haplotyped interval for these loci is that of the MYP2 locus on 18p11.31. Recently, the transforming growth β-induced factor (TGIF) gene was reported to be a candidate gene for MYP2-associated high myopia in single-nucleotide polymorphism studies. The purpose of this study was to determine whether DNA sequence variants in the human TGIF gene are causally related to MYP2-associated high myopia.
methods. The protein coding regions and intron-exon boundaries of the human TGIF gene were sequenced using genomic DNA samples from MYP2 individuals (affected, unaffected) and external control subjects. The TGIF model used was the April 20, 2003, human genome National Center for Biotechnology Information (NCBI) build 33, which has 10 exons and encodes eight transcript variants. Polymorphic sequence changes were compared to those in the previous report. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to validate TGIF gene expression in ocular tissues.
results. A total of 21 polymorphisms of TGIF were found by direct sequencing: 3 were missense, 2 were silent, 10 were not translated, 4 were intronic, and 2 were homozygous deletions. The 3 missense allelic variants were localized to exon 10 at positions 236C→T(Pro→Leu), 244C→T(Pro→Ser), and 245C→T(Pro→Leu). Silent mutations were observed in exon 10 at positions 177A→G, 333C→T. Ten polymorphisms were novel. No sequence alterations were exclusively associated with the affected disease phenotype. RT-PCR results confirmed expression of TGIF in RNA samples derived from human sclera, cornea, optic nerve, and retina.
conclusions. TGIF is a known candidate gene for MYP2-associated high myopia, based on its mapped location within the MYP2 interval. Mutation analysis of the encoded TGIF gene for MYP2 autosomal dominant high myopia did not identify sequence alterations associated with the disease phenotype. Further studies of MYP2 candidate genes are needed to determine the gene that causes of this potentially blinding disorder.
Myopia affects approximately 25% of the adult population of the United States
1 2 3 4 5 and is a significant public health problem, especially in Asian populations such as Chinese or Indians, as it is associated with increased risk for visual loss.
1 6 7 8 9 10 Myopic chorioretinal degeneration due to high myopia is the fourth most frequent cause of blindness, leading to registration for visual services and disability and accounting for 8.8% of all causes.
11 It has been estimated that 5.6% of blindness among school children in the United States is attributable to high myopia.
11 Substantial resources are required for optical correction of myopia with spectacles, contact lenses, and, more recently, surgical procedures such as photorefractive keratectomy. The market for optical aids in the United States was estimated to exceed $8 billion in annual sales in 1990; most dollars were spent for the correction of myopia.
11 The development of methods for preventing the onset or limiting the progression of myopia would be of considerable importance.
Our laboratory identified the
MYP2 locus in seven families with nonsyndromic autosomal dominant (AD) high myopia of −6.00 D or greater. We demonstrated significant linkage to the short arm of chromosome 18, region 11.31, with a maximum cumulative LOD score of 9.59 at θ = 0.0.
12 The 7.6 cM recombinant interval was defined distally by marker
D18S59 and proximally by marker
D18S1138, with recombinants in pedigrees 1, 4, and 5
(Table 1) . The genetic boundaries of the
MYP2 region are currently defined by linkage analysis of these seven existing MY
P2 pedigrees, which represent the group of
MYP2-affected families we have screened for mutations at the
MYP2 locus.
In an effort to contract the
MYP2 interval, transmission disequilibrium test (TDT) statistics
13 were obtained with the Statistical Analysis for Genetic Epidemiology Transmission Disequilibrium Test (SAGE-TDTEX)
14 and Genehunter-TDT (GH2-TDT)
15 programs. Both programs examine each allele separately to look for increased frequency of disequilibrium or nonrecombination events on disease-bearing chromosomes over normal chromosomes, using a standard one-sided test (Fisher exact test). The SAGE program also calculates a summary χ
2 for each marker, as it examines the degree of linkage disequilibrium at the marker. TDT analysis was focused on eleven 18p markers used for fine mapping in the original study.
16 The significance values determined by both programs are listed in
Table 1 for each marker locus in marker order for the 18p11.31 region. Markers
D18S52 and
D18S1138 show the strongest statistical association with the disease phenotype. These data suggest that the
MYP2 gene is likely within a 2.2-cM interval between
D18S52 and
D18S481.
Critically important are the recent independent confirmations of the
MYP2 locus with an Italian patient population with AD high myopia by Heath et al.
17 and six families of Hong Kong Chinese descent by Lam et al.
18 The mapping studies of both laboratories support directing further gene identification efforts to the centromeric region of the initial 7.6-cM recombinant interval. These results, combined with our studies, provide a basis for focused positional candidate gene analysis at the
MYP2 locus, as the interval of interest has likely contracted significantly from the initial 7.6 cM.
We constructed a physical bacterial artificial chromosome (BAC) contig map across the
MYP2 critical region, shown in
Figure 1 , by taking advantage of the multiple databases available in conjunction with the Human Genome Project (HGP). Integration was obtained by mapping markers of different types (monomorphic, polymorphic, genes and expressed sequence tags [ESTs]) from different sources (e.g., NCBI, Genethon, Whitehead Institute, UCSC- “Golden Path”, Celera; see listing at end of article). The core region extends from marker
D18S481 to
D18S52. It ranges in depth from 1 to 9 BACs, with an average depth of −4 BACs and requires 19 overlapping BACs, averaging 150 to 200 kb, to span the
MYP2 region. The
MYP2 critical region on the short arm of chromosome 18 is now fully sequenced and is a 1.2-Mb region on contig NT_010859.13. There are six known and nine hypothetical genes that map within the
MYP2 interval. All the sequences in this region are now labeled “finished” sequences.
One gene that maps within the 18p11.3 interval is the transforming growth β-induced factor (
TGIF) gene.
TGIF is a DNA-binding homeo-domain protein that belongs to the TALE homeobox family.
19 20 It is a transcription repressor with multiple actions, including a role in retinoid-responsive transcription.
21 TGIF mutations are associated with holoprosencephaly, a congenital craniofacial and brain anomaly disorder.
22 23 24 25
The direct analysis of sequence within a critical region can be the most accurate, precise, and efficient approach to disease gene identification. This is particularly true for instances where the “perfect” candidate gene (based on function or expression) does not exist within a defined critical region. It is also true for a disorder such as myopia, in which the temporal and spatial expression of the disease gene is not known, and could be restricted to early development and to any eye component. All genes that map within the MYP2 critical region are candidate disease genes based on position. TGIF for example, therefore, is a candidate gene for the MYP2-associated high myopia based on map position alone. Sequence variants must be uncovered only in affected subjects compared to unaffected subjects for a fully penetrant dominant disorder such as MYP2-linked high myopia.
A recent report by Lam et al. describes a TGIF sequence variation study of the 3-exon transcript variant 4 using conformation specific gel-electrophoresis.
26 They found 25 single-nucleotide polymorphism (SNPs) on exon 3 (exon 10 in our study). Six SNPs showed significant high myopia association with univariate analysis, and one showed significance with multivariate analysis.
We sought to determine whether the TGIF gene is causally related to MYP2-associated high myopia by direct DNA sequencing, using DNA samples from the original MYP2 pedigrees. One consideration is that the TGIF genetic structure studied by Lam et al.
26 had 3 exons—the current sequence build is a 10-exon gene structure. Exons 1, 2, and 3 are now exons 5, 9, and 10, respectively, according to the reference sequence build 33 (http://www.ncbi.nlm.nih.gov/genome/guide/human/HsStats.html) of
TGIF, which corresponds to transcript variant 4.
Nonsyndromic high myopia is a common, complex disorder that is likely to result from alterations of multiple genetic factors. Indeed, several loci have been mapped for the disorder.
We sequenced the full
TGIF gene in our patient samples of individuals from pedigrees with
MYP2-associated high myopia. No DNA sequence variants were noted that implicated
TGIF as the causative gene.
TGIF exon 10 (exon 3 in the initial build of this gene) did not show the same level of polymorphic variants in our cohort, as we observed 8 variants rather than the 25 reported by Lam et al.
26 This may be due to the ethnic differences in our two sample sets, although family 1 of the
MYP2 pedigrees studied was of Chinese descent. All other families were of Northern European descent.
In conclusion, TGIF is a known candidate gene for MYP2-associated high myopia based on its mapped location within the MYP2 interval. Mutation analysis of the encoded TGIF gene for MYP2 AD high myopia did not identify sequence alterations associated with the disease phenotype. Further studies of MYP2 candidate genes are needed to determine the molecular genetic factors that cause this potentially blinding disorder.
Electronic databases (listed below) were used for developing a physical map of the
MYP2 critical region. The Genethon, Whitehead, and NCBI websites were queried for microsatellite marker data. The NCBI, UCSC, and Celera websites were used to align BACs and close gap regions. The hypothetical genes were determined by the GENSCAN and OTTO websites. All listed links are free to the public except for OTTO and Celera.
-
Celera: http://cds.celera.com/cds
-
Genethon: http://www.genethon.fr/php/index_us.php
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GENSCAN: http://genes.mit.edu/GENSCAN.htm
-
MapViewer: http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi
-
NCBI: http://www.ncbi.nlm.nih.gov/
-
OTTO: http://cds.celera.com/biolib/info
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Public SNP repository: http://www.ncbi.nlm.nih.gov/SNP
-
UCSC “Golden Path”: http://genome.ucsc.edu/
-
Whitehead Institute: http://www-genome.wi.mit.edu/
Supported by National Eye Institute Grant EY00376-03, Research to Prevent Blindness, Mabel E. Leslie Research Endowment Funds, and the Lions Eye Bank of Delaware Valley.
Submitted for publication August 25, 2003; revised January 7, 2004; accepted February 10, 2004.
Disclosure:
G.S. Scavello, None;
P.C. Paluru, None;
W.R. Ganter, None;
T.L. Young, None
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: Terri L. Young, Division of Ophthalmology, 1st Floor, Wood Building, Children’s Hospital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, PA 19104;
[email protected].
Table 1. MYP2 Locus Marker Recombinants and TDT Allelic Association Analysis
Table 1. MYP2 Locus Marker Recombinants and TDT Allelic Association Analysis
Marker Distance (cM) | Pedigree Marker (Telomere) | 1 | 4 | 5 | SAGE-TDTEX P | GH2-TDT P |
| D18S1140 | + | − | − | 0.036 | 0.083 |
0.1< | | | | | | |
| D18S59 | + | − | − | 0.013 | 0.317 |
1.5< | | | | | | |
| D18S476 | − | − | − | 0.007 | 0.045 |
0.1< | | | | | | |
| D18S1146 | − | − | − | 0.227 | 0.083 |
4.5< | | | | | | |
| D18S481 | − | − | − | 0.001 | 0.108 |
1.4< | | | | | | |
| D18S63 | − | − | − | 0.062 | 0.034 |
0.1< | | | | | | |
| D18S1138 | − | − | + | 3.9 × 104 | 0.011 |
0.7< | | | | | | |
| D18S52 | − | − | + | 1.79 × 106 | 0.007 |
9.4< | | | | | | |
| D18S62 | − | + | + | 0.141 | 0.479 |
18.6< | | | | | | |
| D18S1150 | + | + | + | 0.018 | 0.096 |
4.1< | | | | | | |
| D18S1116 | + | + | + | 0.214 | 0.683 |
Table 2. Subject DNA Samples Used in the Study
Table 2. Subject DNA Samples Used in the Study
Original MYP2 Pedigree-Individual | Refractive Error | |
| OD | OS |
1-15 | −10.50 + 2.50 × 82 | −11.50 + 2.75 × 97 |
1-16 | −11.00 + 0.75 × 60 | −12.25 + 1.00 × 70 |
1-19 | −6.00 + 1.50 × 50 | −6.50 + 0.75 × 80 |
1-21 | Plano | Plano |
1-23 | Plano | Plano |
2-6 | −16.00 sph | −16.00 sph |
2-10 | Plano | Plano |
3-6 | −19.25 + 2.25 × 135 | −21.00 + 3.00 × 60 |
4-7 | −6.25 sph | −6.00 sph |
4-8 | Plano | Plano |
5-6 | −10.00 + 1.50 × 46 | −8.25 + 1.75 × 105 |
5-9 | +0.25 + 0.50 × 170 | +0.25 + 0.25 × 170 |
6-4 | −7.25 + 1.25 × 180 | −7.25 + 1.25 × 20 |
6-6 | Plano | Plano |
7-5 | −10.75 + 2.00 × 10 | −10.50 + 2.50 × 70 |
External control-1 (C1) | Plano | Plano |
External control-2 (C2) | Plano | Plano |
External control-3 (C3) | Plano | Plano |
External control-4 (C4) | Plano | Plano |
External Myopic control-5 (C5) | −9.00 sph | −9.00 sph |
Table 3. TGIF Gene Primers Designed for Mutation Screening
Table 3. TGIF Gene Primers Designed for Mutation Screening
Exon | Size (bp) | Primer Sequence 5′–3′ | Primer |
1 | 754 | CAGGAAACACAGACCAGCTTATCTT | TGIF1F |
| | AATGCAGTCATGCCACTCGATATA | TGIF1R |
2 | 249 | CCCAAATTGTCTATCGGTGA | TGIF2F |
| | ATGACTAGGTTCAAGCCAATG | TGIF2R |
3 | 449 | TTTTGAGGCTGTTCCCTTTGTTACG | TGIF3F |
| | TGGACTTGGCTAATGACTGCGATT | TGIF3R |
4 | 632 | GAGGCGGCTGTCTCGTGCGGCTAGA | TGIF4F |
| | GATCCCAGGCGCCCGCTCCTT | TGIF4R |
5 | 662 | CCGCCCCCGAGGGACGAGT | TGIF5F |
| | AGCCGCTGCCGTTTCAGACGC | TGIF5R |
6 | 1061 | GTCCCCCGAGTGTTCCGCTGT | TGIF6F |
| | CCCACCCCAGACGACTCGC | TGIF6R |
7 | 711 | CCTCTGAGAAGGTGGGATTCACG | TGIF7F |
| | CCTTTGAGCTGGGAGCAATGTCTCT | TGIF7R |
| 796 | GAATTCCTGCTTTGGGATTGC | TGIF7AF |
| | CACAAGCTTCTTTCACGCTATCCAC | TGIF7AR |
8 | 329 | AGAACGTGCAGGAACAAGGTCG | TGIF8F |
| | CACTGCAGTAAAACCACGTTTGCTG | TGIF8R |
9 | 361 | CTCGCTCTCAGTTGTTGG | TGIF9F |
| | TCACGCTCTCTTTCTTTA | TGIF9R |
| 562 | TCTTCGGGATTGGCTGTATGA | TGIF9AF |
| | TCCCCATTTTAACCTATCACCTACT | TGIF9AR |
10 | 427 | TCAATTAGGTACCCCATAGAACAT | TGIF10F |
| | TCCCACACCGACCGACTG | TGIF10R |
| 385 | TCTGCCATACCACTGTGACTG | TGIF10AF |
| | AATATATAAAATAATGCAATTCATCTCTTG | TGIF10AR |
| 284 | GGGAATGAGAAGATGCTAT | TGIF10BF |
| | GGTGAAGGCAAGAGATGAAT | TGIF10BR |
Table 4. Observed Sequence Polymorphisms in the TGIF Gene
Table 4. Observed Sequence Polymorphisms in the TGIF Gene
NT_010859.13 Position | Wild-Type Nucleotide | Base Pair Change Observed | SNP rs | Original MYP2 Family-Individual | Exon Position | AA Change |
3402167 | C | C→T, T→T | Novel | 1-23, -15, -16, and -19, 2-10, 5-6, C2 and 3 | bp96 of exon 1 | 5′ UTR |
3402386 | C | C→T | rs8092903 | C2 | Intron 1 | |
3402523 | C | C→T, T→T | Novel | 5-6, 7-5, C2 | Intron 1 | |
3408219 | T | C→T | Novel | 7-5 | Intron 2 | |
3437725 | C | C→T | 238137 | 1-16, 1-21, C2 | bp118 of exon 3 | 5′ UTR |
3437871 | T | C→T | rs151472 | 1-15, 1-19, 4-7, 5-6, 7-5, 1-21, 1-23, 2-10, C2 | Intron 3 | |
3440455 | C | C→A | rs238132 | 4-8, 6-4, C2, -3, and -4 | bp284 of exon 5 | 5′ UTR |
3441762 | C | C→T, T→T | rs238533 | 4-7, 6-4, 4-8, C4 | bp172 of exon 6 | 5′ UTR |
3441896 | G | G→A, A→A | Novel | 4-7, 6-4, 4-8 | bp306 of exon 6 | 5′ UTR |
3442978 | C | C→T, T→T | 2238536 | 4-7, 6-4, 4-8 | bp374 of exon 7 | UTR |
3442216 | C | C→C Deletion | Novel | C1 | bp626 of exon 6 | frameshift |
3442223 | T | T→T Deletion | Novel | 3-6, C1 | bp633 of exon 6 | frameshift |
3443789 | T | G→T | Novel | 1-16 and 21, 2-6 | bp18 of exon 8 | 5′ UTR |
3447539 | A | G→A, G→G | rs2229337 | 4-7, 6-4, C2, -4, and C5 | bp177 of exon 10 | NONE |
3447598 | C | C→T | Novel | 6-6, C1 | bp236 of exon 10 | PRO→LEU |
3447606 | C | C→T | rs4468717 | 3-6 | bp244 of exon 10 | PRO→SER |
3447607 | C | C→T, T→T | rs2229333 | 4-7, 6-4, 4-8 | bp245 of exon 10 | PRO→LEU |
3447695 | C | C→T | rs2229335 | C1 and -4 | bp333 of exon 10 | NONE |
3447776 | T | T→G | rs2229336 | C1 and -4 | bp414 of exon 10 | 3′ UTR |
3448161 | G | G→A | Novel | 5-9 | bp799 of exon 10 | 3′ UTR |
3448260 | G | G→A | Novel | 4-7, 5-6 and -9, 6-4, 7-5, C2 | bp898 of exon 10 | 3′ UTR |
The authors thank the families for their participation.
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