Molecular Vision 2005; 11:97-110 <http://www.molvis.org/molvis/v11/a11/> Received 15 October 2004 | Accepted 26 January 2005 | Published 2 February 2005

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Genomic structure and organization of the high grade Myopia-2 locus (MYP2) critical region: mutation screening of 9 positional candidate genes

Genaro S. Scavello, Jr.,^1,2 Prasuna C. Paluru,^1,2 Jie Zhou,^1,2 Peter S. White,^3-4 Eric F. Rappaport,² Terri L. Young^1,2
 
 

Divisions of ¹Ophthalmology, ²Genetics, and ³Oncology, The Children's Hospital of Philadelphia, Philadelphia, PA; ⁴Department of Pediatrics, University of Pennsylvania, Philadelphia, PA

Correspondence to: Terri L. Young, MD, Division of Ophthalmology, Children's Hospital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, PA, 19104; Phone: (215) 590-9950; FAX: (215) 590-3850; email: youngt@email.chop.edu

Abstract

Purpose: Myopia is a common complex eye disorder, with implications for blindness due to increased risk of retinal detachment, chorioretinal degeneration, premature cataracts, and glaucoma. A genomic interval of 2.2 centiMorgans (cM) was defined on chromosome band 18p11.31 using 7 families diagnosed with autosomal dominant high myopia and was designated the MYP2 locus. To characterize this region, we analyzed 9 known candidate genes localized to within the 2.2 cM interval by direct sequencing.

Methods: Using public databases, a physical map of the MYP2 interval was compiled. Gene expression studies in ocular tissues using complementary DNA library screens, microarray experiments, reverse transcription techniques, and expression data identified in external databases aided in prioritizing gene selection for screening. Coding regions, intron-exon boundaries and untranslated exons of all known genes [Clusterin-like 1 (CLUL1), elastin microfibril interfacer 2 (EMILIN2), lipin 2 (LPIN2), myomesin 1 (MYOM1), myosin regulatory light chain 3 (MRCL3), myosin regulatory light chain 2 (MRLC2), transforming growth β-induced factor (TGIFβ), large Drosophila homolog associated protein 1 (DLGAP1), and zinc finger protein 161 homolog (ZFP161)] were sequenced using genomic DNA samples from 9 affected and 6 unaffected MYP2 pedigree members, and from 5 external controls (4 unaffected and 1 affected). Gene sequence changes were compared to known variants from public single nucleotide polymorphism (SNP) databases.

Results: In total, 103 polymorphisms were found by direct sequencing; 10 were missense, 14 were silent, 26 were not translated, 49 were intronic, 1 insertion, and 3 were homozygous deletions. Twenty-seven polymorphisms were novel. Novel SNPs were submitted to the public database; observed frequencies were submitted for known SNPs. No sequence alterations segregated with the disease phenotype.

Conclusions: Mutation analysis of 9 encoded positional candidate genes on MYP2 loci did not identify sequence alterations associated with the disease phenotype. Further studies of MYP2 candidate genes, including analysis of putative genes predicted in silico, are underway.

Introduction

Myopia, or nearsightedness, occurs when the focused image falls anterior to the retinal photoreceptor layer of the eye. Myopia is the most common human eye disease affecting 25% of the United States adult population [1-5]. Severe cases (high myopia, defined as exhibiting a spherical refractive error greater than -5.00 D) is a significantly lifestyle debilitating disease and may lead to blinding disorders such as premature cataracts, glaucoma, retinal detachment, and macular degeneration [6-11]. Myopia can occur as an isolated finding, or as a component of specific genetic syndromes [1].

High myopia is a major cause of legal blindness in many developed countries [6-16]. It affects 27% to 33% of all myopic eyes, corresponding to a prevalence of 1.7% to 2% in the general population of the United States [1,2,6]. High myopia is especially common in Asia [12,13,15,17]. In other countries, pathologic myopia has a prevalence rate of 2.3-9.1% [12,13,15,17]. The development of methods to prevent the onset or limit the progression of myopia would be of considerable importance [18]. There is substantive evidence that genetic factors play a significant role in the development of nonsyndromic high myopia. Currently, at least 5 loci for high grade myopia have been determined and mapped to distinct chromosomal regions [19-24].

The genes that contribute to complex or multifactorial disease are notoriously difficult to identify, as they typically exert small effects on disease risks [25]. The magnitude of their effects is likely to be modified by additional genes and environmental factors. We recognize the problem of genetic heterogeneity, phenocopy, and shared environmental factors. Multiplex families with uni-linear transmission of the affected phenotype provide the most unambiguous information for detecting linkage, especially for complex traits [26]. Selectively ascertaining such pedigrees with high disease severity and early age of onset biases towards a strong genetic etiology, minimizing environmental influences to the disease trait. This has been our ascertainment strategy for our mapping studies.

Our laboratory identified the MYP2 locus using 7 families with nonsyndromic, autosomal dominant, high myopia with a refractive error of 6.00 D (spherical equivalent) or greater. We previously demonstrated significant linkage to chromosome 18p11.31 with a maximum cumulative LOD score of 9.59 at θ=0.0 [21]. The 7.6 cM recombinant interval was defined proximally by marker D18S1138 and distally by marker D18S59, which include 9 known genes that map to the MYP2 critical region (Figure 1). The genetic boundaries of the MYP2 region are currently defined by linkage analysis of these 7 existing MYP2 pedigrees. In a subsequent effort to narrow to contract the MYP2 interval, Transmission Disequilibrium Test (TDT) statistics were obtained with the Statistical Analysis for Genetic Epidemiology-TDT (SAGE-TDTEX) and GENEHUNTER-TDT (GH2-TDT) programs, available from the Spielman lab [27-29]. TDT analyses focused on 11 markers on chromosome 18p used for fine mapping in the original study [30]. Markers D18S52 and D18S1138 showed the strongest statistical association, suggesting a refined 2.2 cM interval between D18S52 and D18S481. This locus has since been independently confirmed in additional cohorts by two other research groups [31,32]. These mapping studies support directing further gene identification efforts to the centromeric region of the initial 7.6 cM recombinant interval.

We hypothesize that the identification of myopia disease genes such as the MYP2 gene will not only provide insight into the molecular basis of this significant eye disease, but may identify pathways that are involved in eye growth and development. In addition, this information may implicate other genes as possible myopia disease gene candidates. This effort may lead to effective therapies for severe forms of this potentially blinding eye disease.

Methods

Patients

Probands and affected representatives of the seven MYP2 families were studied (Table 1). Each of the affected individuals had high myopia with a refractive error greater than 6.00 D (spherical equivalent) with elongated axial lengths. Clinical details regarding the complete pedigrees have been described previously [21]. Controls were obtained from family marry-ins, non-myopic family members, and unrelated subjects.

Total genomic DNA was extracted from 10-15 ml of venous blood from each participant after informed consent. DNA was purified from lymphocyte pellets according to standard procedures using the PUREGENE^TM kit (Gentra Systems, Minneapolis, MN). The study protocol was approved by the Children's Hospital of Philadelphia Institutional Review Board on Human Subjects Research and adhered to the tenets of the Declaration of Helsinki.

BLAST searches

The genomic structures of candidate genes (Figure 1 and Figure 2) were elucidated by use of the National Center for Biotechnology Information (NCBI) blastn algorithm [33,34]. Known cDNA, EST, and mRNA sequences were queried against the human genomic, HTGS (high throughput genomic sequence) and GSS (genome survey sequences) databases provided by NCBI. Alignments were scored for quality and length; spliced ESTs were given a higher score. Potential splice variants of all RefSeq genes were studied.

DNA amplification and mutation screening

A total of 229 primer pairs were designed to amplify the 105 identified exons, including 50-200 bp beyond each intron-exon boundary (Table 2). For each amplimer, the polymerase chain reaction (PCR) was performed on 150 ng genomic DNA using standard methods (protocols available upon request). Amplified products were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Amplicons were purified using QIAquick purification columns (Qiagen, Valencia, CA) and sequenced using BigDye^TM Terminator version 3.1 on an Applied Biosystems (ABI3730, ABI377, ABI3700, or ABI3100 Genetic Analyzer; Applied Biosystems, Foster City, CA) sequencer, or with a DYEnamic^TM ET dye terminator kit on a MegaBACE 1000 system (Amersham Biosciences, Piscataway, NJ).

Sequences were trimmed for quality and aligned using Sequencher^TM (Gene Codes, Ann Arbor, MI) or Gap4 (Staden, Cambridge, England). Normal and affected individual DNA sequences were aligned to the known reference genomic sequence (NT_010859), available via the UCSC genome browser and compared for sequence variation.

Reverse transcription-polymerase chain reaction

Pooled total RNAs from retina, cornea, optic nerve, and sclera were extracted from four human donor eyes (Pennsylvania Lions Eye Bank, Philadelphia, PA) using TRIZOL (Invitrogen Corporation, Carlsbad, CA). The eyes were treated by submersion in RNALater solution (Ambion Inc., Austin, TX) within 2-12 h post mortem. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed on the RNA with random hexamers using standard methods to synthesize cDNA with SuperScript II (Invitrogen Corporation, Carlsbad, CA). Total RNA (1 μg) from sclera, optic nerve, retina, cornea, and commercially prepared poly-A RNA (Clontech Inc., Palo Alto, CA; and Ambion Inc., Austin, TX) from various human organs were used as templates for a 20 μl first-strand cDNA synthesis reaction. Gene specific PCR was performed using Platinum Taq polymerase with recommended conditions, using 2 μl of each cDNA sample and 50 pmoles of primer in a final reaction volume of 50 μl. The PCR cycling conditions included an initial denaturation for 120 s at 95 °C, followed by 34 cycles of denaturation for 15 s at 95 °C, annealing for 30 s at 54 °C, extension for 45 s at 68 °C, and a final extension for 4 min at 68 °C. The sense and antisense primers spanned at least 2 exons to distinguish from genomic DNA amplification. The RT-PCR products, along with the amplicon products of the housekeeping gene β-actin, were visualized on 2% agarose gels (Figure 3) after electrophoresis and staining with ethidium bromide.

Results

Transcript identification

We have bias towards analyzing genes with an extracellular matrix associated function, or structural genes such as EMILIN2 and MYOM1. These genes are important for constituent organization and maintenance of connective tissue function and were given priority. The MYP2 gene may also be expressed in the retina and influence scleral growth [35]. This retinal hypothesis emanates mainly from animal studies of experimental myopia. The induction of myopia in juvenile animals by deprivation of form vision demonstrates a visual feedback mechanism in eye growth control. Experimental work indicates that this neural control mechanism is at least partly localized to the retina itself, but how retinal signals directly control the growth of the outer coats of the eye is presently unknown. Transcription factors and regulatory genes expressed in retina such as CLUL1, TGIF, MRLC2, MRCL3, ZFP161, and DLGAP1 may play a role in regulating eye growth.

BLAST searches of ESTs, mRNAs, and cDNA from the 18p11.31 critical region were queried against the human genomic, HTGS, and GSS databases. Search results provided evidence for 109 spliced exons. The genomic structures reported herein are confirmed by data presented in the NCBI MapViewer (build 34) of the reference human genome sequence as outlined in Figure 2. Gene size varied from 2 exons (ZFP161) to 36 exons (MYOM1) and spanned up to 385 kbs of genomic DNA (DLGAP1).

Transcript analysis

There are 9 known and 6 hypothetical genes that are considered candidates based on mapped position within the MYP2 interval. All sequences within this region have been labeled as "finished", and there are no known gaps within the interval. The 9 known genes (Figure 1) that map to the MYP2 critical region include clusterin-like 1 (CLUL1), elastin microfibril interfacer 2 (EMILIN2), lipin 2 (LPIN2), myomesin 1 (MYOM1), myosin regulatory light chain 3 (MRCL3), myosin regulatory light chain 2 (MRLC2), transforming growth β-induced factor (TGIFβ), large Drosophila homolog associated protein 1 (DLGAP1), and zinc finger protein 161 homolog (ZFP161).

The telomeric CLUL1 and centromeric ZFP161 define the genetic boundaries of the larger mapped interval. They were included because of known expression in eye tissues [36-41], and because of their proximity to polymorphic markers with significant LOD scores [21]. CLUL1 is a 9 exon, 1877 bp cone photoreceptor specific gene that is similar in structure to the human clusterin (CLU) gene. CLUL1 expression in canine retinal studies increases significantly at 34 days, which coincides with photoreceptor differentiation. This suggests that clusterin-like 1 may direct differentiation in retinal tissues [42]. It has since been implicated as a candidate gene for bipolar disorder type 1 [43]. Mutation screening of CLUL1 observed no polymorphisms in our families.

EMILIN2 encodes for an elastic fiber interacting protein that confers elasticity to the extracellular matrix [44]. It spans 68 kb and 9 exons encoding a 4009 bp transcript. EMILIN2 has a unique multimodular organization and differs from other elastin associated proteins because it includes a C1q-like globular domain at the C terminus, a short collagen-like region, a long segment of about 650 residues with a high potential for forming coiled-coil α-helices, and a cysteine rich domain at the N-terminus. EMILIN-2 is deposited extracellularly as a fine network; it is broadly expressed in connective tissues, has cell adhesion promoting functions, and is particularly abundant in blood vessels, skin, heart, lung, kidney, and cornea [45,46]. The expression profile, pro-adhesive functions, and the domain characteristics suggest that EMILIN2 likely plays a fundamental role in the process of elastogenesis in association with other extracellular matrix constituents [44]. This may be an important association in scleral wall elasticity seen in high myopia with elongated axial lengths. Mutation screening of EMILIN2 resulted in 8 polymorphisms, 4 silent, 1 missense, and 3 were in the untransilated region (UTR). None of these polymorphisms segregated with the affection status.

LPIN2 is located closest in proximity to the microsatellite marker D18S481 that showed the highest LOD score of 9.59 in linkage analysis [21]. It comprises 20 exons spanning 95 kb region. LPIN2 belongs to a family of nuclear proteins. Three closely related members of the Lipin family, Lipin-1, Lipin-2, and Lipin-3 have been identified in both mouse and human. Lipin 1 (LPIN1) was originally characterized as a candidate gene for mouse lipodystrophy and played an important role in lipid metabolism [47]. Our lab has performed a detailed study on this gene and the data has been submitted for publication [48]. Mutation screening of human LPIN1 in lipodystrophy patients revealed 5 polymorphisms, although none co-segregated with the disease [49]. LPIN2 was identified based on similarity in sequences to LPIN1. There is no systemic characterization of this gene. Mutation screening of LPIN2 resulted in 11 polymorphisms, 2 silent, 1 homozygous deletion, 3 Intronic, and 5 were in the UTR. Eight polymorphisms were novel and none of these segregated with the affection status.

MYOM1 (also known as skelemin) is a 36 exon gene that spans 128 kbps. The protein is a structural constituent of the cytoskeleton thought to integrate the thin and thick filaments while conferring elasticity to the M-band of the sarcomere in striated muscle [50-52]. MYOM1 is a member of the immunoglobin superfamily [53], and binds extracellular matrix proteins [54]. MYOM1 may also play an important role in the assembly and stabilization of myofibrils [55]. Mutation screening of MYOM1 resulted in 39 polymorphisms, 5 silent, 4 missense, 29 Intronic, and 1 in the UTR. Eight polymorphisms were novel and none of these segregated with the affection status.

MRCL3 and MRLC2 are myosin regulatory subunits which share nearly 100% identity at the protein level and greater than 94% identity at the nucleotide level. Each gene is encoded by four exons. It is unknown whether these genes arose from a duplication event or if each plays a specific role in myosin regulation. Both genes have similar calcium binding domains to Recoverin, a retinal specific gene that participates in the recovery phase of visual excitation and in adaptation to light [56]. Diphosphorylation of the myosin regulatory light chain subunit is thought to play a role in regulation of filament assembly and reorganization of muscle cells [57]. Mutation screening of MRLC2 resulted in 13 polymorphisms, 8 Intronic, 3 in the UTR, 1 deletion, and 1 insertion in the 5' UTR. MRCL3 had 4 polymorphisms, 3 intronic, and 1 was in the UTR. None of these polymorphisms segregated with the affection status.

TGIF is a DNA binding homeo-domain protein that belongs to the three amino acid loop extension homeobox family [58,59]. It is a transcription repressor with multiple actions, including a role in retinoid-responsive transcription [60]. TGIF mutations are associated with holoprosencephaly, a congenital craniofacial and brain anomaly disorder [61-64]. TGIF contains 10 exons spanning 46 kb, and has 8 transcript variants encoding four proteins of 402 residues (variant 1), 287 residues (variant 2), 273 residues (variants 3 and 4), and 253 residues (variants 5-8). Mutation screening of TGIF resulted in 21 polymorphisms, 4 missense, 6 Intronic, 9 in the UTR, and 2 homozygous deletions on exon-6 resulting in early truncation. Ten polymorphisms were novel and none of these segregated with the affection status.

DLGAP1 (DISCS large associated protein 1; also known as DAP1 or GKAP) is a member of the PSD95 domain containing family of molecules that are collectively known as "chapsyns" for their function as channel associated proteins. Chapsyns are generally known to have one to three conserved domains: a binding domain found in the amino (NH₂) or the carboxyl (COOH) regions, a sulfhydryl (SH₃) group, and a guanylate kinase domain in the carboxyl region [65]. Mutation screening of DLGAP1 resulted in 3 polymorphisms, 2 silent, and 1 missense. One polymorphisms was novel and none of these segregated with the affection status.

ZFP161 encodes a mature 2896 bp mRNA spanning 2 exons within 4.7 kb of genomic DNA. Zinc finger proteins are structural motifs that usually confer sequence specific DNA binding capability. Nearly all are transcriptional activators involved in tissue differentiation, proto-oncogenic activity, and general gene regulation [66,67]. ZFP161 is a novel transcriptional activator of the dopamine transporter. This retinal neurotransmitter has been implicated in animal models of experimental myopia [35]. Mutation screening of ZFP161 resulted in 5 polymorphisms, 1 silent, and 4 in the UTR. None of these polymorphisms segregated with the affection status.

RT-PCR results confirmed expression of all genes tested in all ocular tissue types (Figure 3). Potential splice variants of CLUL1 were observed in sclera, optic nerve, and retinal cDNA. PCR amplification with the 229 primer pairs produced the expected amplicon products and sequences. In total, 103 polymorphisms were identified by direct sequencing (Table 3). Of these 10 polymorphisms were missense, 14 were silent, 26 were not translated, 49 were intronic, 1 insertion, and 3 were homozygous deletions. Twenty-seven polymorphisms were novel. Observed frequencies for previously described SNPs and novel SNPs were submitted to the dbSNP database. No identified sequence alterations were associated with the disease phenotype.

Discussion

We sequenced the full coding regions of nine positional candidate genes in our patient samples of individuals from pedigrees with MYP2 associated high myopia. No DNA sequence variants were noted that implicated any of the surveyed genes as the causative. We were especially interested in the TGIF candidate gene because of its published association with MYP2 by SNP association studies in a Hong Kong Chinese cohort [68]. TGIF exon 10 (exon 3 in the initial build of this gene) did not show the same number of polymorphic variants in our cohort as in the previous study, as we observed 8 variants rather than the 25 in that report [69]. This may be due to the ethnic differences in our two sample sets, although family-1 of the MYP2 pedigrees studied here was of Chinese descent. All other families were of Northern European origin. The TGIF gene was not fully screened based on the methods described in the Hong Kong cohort publication, and therefore the SNP association observed in the previous report is most likely due to a nearby gene or regulatory element. Given that TGIF mutations are associated with holoprosencephaly, there is less likelihood that it is involved with simplex myopia [64].

Nonsyndromic high myopia is a common complex disorder that is likely to result from alterations of multiple genetic factors. We interpret our negative findings with caution; it is possible that there is MYP2 gene association but with a variant located distally in a promoter region, within an intron, or within an isoform that was not determined.

Information derived from this effort will be useful for submissions to the ever growing SNP database, and to other researchers also exploring candidate genes in this region. Other researchers screening for myopia candidate genes in this interval may wish to avoid repeat screening of those genes that have been excluded. The molecular study of any of these genes requires PCR primers that have been optimized for the gene exonic and intronic area of interest.

Despite the plethora of experimental myopia studies in animal models that demonstrate biochemical factor changes in various eye tissues, and limited human studies utilizing pharmacologic agents to thwart axial elongation, we have little knowledge of the basic physiology that drives myopic development. Identifying the implicated genes for myopia susceptibility will provide a fundamental molecular understanding of how myopia occurs, possibly leading to directed physiologic (e.g., pharmacologic or gene therapy) interventions.

Mutation analysis of 9 encoded positional candidate genes shown to be expressed in ocular tissues for MYP2 autosomal dominant high myopia did not identify sequence alterations associated with the disease phenotype. In silico prediction and analysis of novel genes, and further studies of MYP2 candidate genes are currently underway in our laboratory to determine the causative gene(s) for this potentially blinding disorder.

Acknowledgements

We are grateful to the families for their participation in this research endeavor. The authors gratefully acknowledge the support of Dr. Richard S. Spielman for critical review of the project and manuscript. The authors acknowledge Grant/Financial Support from the Mabel E. Leslie Research Funds, Research to Prevent Blindness, Inc. Physician-Scientist Award, NIH grants NEI-EY00376 and NEI-2PEY01583-26 (TLY).

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