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High Myopia Caused by a Mutation in LEPREL1, Encoding Prolyl 3-Hydroxylase 2

2011; Elsevier BV; Volume: 89; Issue: 3 Linguagem: Inglês

10.1016/j.ajhg.2011.08.003

1537-6605

Shikma Mordechai, Libe Gradstein, Annika Pasanen, Rivka Ofir, Khalil El Amour, Jaime Levy, Nadav Belfair, Tova Lifshitz, S. Weitz Joshua, Ginat Narkis, Khalil Elbedour, Johanna Myllyharju, Ohad S. Birk,

Connective tissue disorders research

Autosomal-recessive high-grade axial myopia was diagnosed in Bedouin Israeli consanguineous kindred. Some affected individuals also had variable expressivity of early-onset cataracts, peripheral vitreo-retinal degeneration, and secondary sight loss due to severe retinal detachments. Through genome-wide linkage analysis, the disease-associated gene was mapped to ∼1.7 Mb on chromosome 3q28 (the maximum LOD score was 11.5 at θ = 0 for marker D3S1314). Sequencing of the entire coding regions and intron-exon boundaries of the six genes within the defined locus identified a single mutation (c.1523G>T) in exon 10 of LEPREL1, encoding prolyl 3-hydroxylase 2 (P3H2), a 2-oxoglutarate-dependent dioxygenase that hydroxylates collagens. The mutation affects a glycine that is conserved within P3H isozymes. Analysis of wild-type and p.Gly508Val (c.1523G>T) mutant recombinant P3H2 polypeptides expressed in insect cells showed that the mutation led to complete inactivation of P3H2. Autosomal-recessive high-grade axial myopia was diagnosed in Bedouin Israeli consanguineous kindred. Some affected individuals also had variable expressivity of early-onset cataracts, peripheral vitreo-retinal degeneration, and secondary sight loss due to severe retinal detachments. Through genome-wide linkage analysis, the disease-associated gene was mapped to ∼1.7 Mb on chromosome 3q28 (the maximum LOD score was 11.5 at θ = 0 for marker D3S1314). Sequencing of the entire coding regions and intron-exon boundaries of the six genes within the defined locus identified a single mutation (c.1523G>T) in exon 10 of LEPREL1, encoding prolyl 3-hydroxylase 2 (P3H2), a 2-oxoglutarate-dependent dioxygenase that hydroxylates collagens. The mutation affects a glycine that is conserved within P3H isozymes. Analysis of wild-type and p.Gly508Val (c.1523G>T) mutant recombinant P3H2 polypeptides expressed in insect cells showed that the mutation led to complete inactivation of P3H2. Myopia is the most common human eye disorder and constitutes a significant public health concern.1Hornbeak D.M. Young T.L. Myopia genetics: A review of current research and emerging trends.Curr. Opin. Ophthalmol. 2009; 20: 356-362Crossref PubMed Scopus (103) Google Scholar Specifically, high myopia is a leading cause of visual impairment and blindness worldwide because of its associated ocular comorbidities of retinal detachment, macular degeneration, premature cataract, and glaucoma.1Hornbeak D.M. Young T.L. Myopia genetics: A review of current research and emerging trends.Curr. Opin. Ophthalmol. 2009; 20: 356-362Crossref PubMed Scopus (103) Google Scholar, 2Tano Y. Pathologic myopia: Where are we now?.Am. J. Ophthalmol. 2002; 134: 645-660Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Childhood myopia is primarily due to growth in axial length of the eyeball; most cases are either congenital (usually high myopia) or juvenile (onset between 7 and 16 years of age). In general, the earlier the onset of myopia occurs, the greater the degree of progression.1Hornbeak D.M. Young T.L. Myopia genetics: A review of current research and emerging trends.Curr. Opin. Ophthalmol. 2009; 20: 356-362Crossref PubMed Scopus (103) Google Scholar, 2Tano Y. Pathologic myopia: Where are we now?.Am. J. Ophthalmol. 2002; 134: 645-660Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Twin studies and heritability measures suggest a strong genetic component in myopia.3Morgan I.G. The biological basis of myopic refractive error.Clin. Exp. Optom. 2003; 86: 276-288Crossref PubMed Scopus (100) Google Scholar, 4Feldkämper M. Schaeffel F. Interactions of genes and environment in myopia.Dev. Ophthalmol. 2003; 37: 34-49Crossref PubMed Google Scholar Myopia is mostly regarded as a multifactorial polygenic disorder.1Hornbeak D.M. Young T.L. Myopia genetics: A review of current research and emerging trends.Curr. Opin. Ophthalmol. 2009; 20: 356-362Crossref PubMed Scopus (103) Google Scholar Genome-wide association studies of the common presumably polygenic multifactorial myopia in large cohorts have recently identified association with genomic loci at chromosomes 13q12,5Shi Y. Qu J. Zhang D. Zhao P. Zhang Q. Tam P.O.S. Sun L. Zuo X. Zhou X. Xiao X. et al.Genetic variants at 13q12.12 are associated with high myopia in the Han Chinese population.Am. J. Hum. Genet. 2011; 88: 805-813Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar 15q14,6Solouki A.M. Verhoeven V.J. van Duijn C.M. Verkerk A.J. Ikram M.K. Hysi P.G. Despriet D.D. van Koolwijk L.M. Ho L. Ramdas W.D. et al.A genome-wide association study identifies a susceptibility locus for refractive errors and myopia at 15q14.Nat. Genet. 2010; 42: 897-901Crossref PubMed Scopus (180) Google Scholar and 15q25,7Hysi P.G. Young T.L. Mackey D.A. Andrew T. Fernández-Medarde A. Solouki A.M. Hewitt A.W. Macgregor S. Vingerling J.R. Li Y.J. et al.A genome-wide association study for myopia and refractive error identifies a susceptibility locus at 15q25.Nat. Genet. 2010; 42: 902-905Crossref PubMed Scopus (178) Google Scholar as well as with genetic variants in CTNND2 (MIM 604275) at 5p15.8Li Y.J. Goh L. Khor C.C. Fan Q. Yu M. Han S. Sim X. Ong R.T. Wong T.Y. Vithana E.N. et al.Genome-wide association studies reveal genetic variants in CTNND2 for high myopia in Singapore Chinese.Ophthalmology. 2011; 118: 368-375Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar There are more than 150 defined monogenic syndromes in which familial high myopia is one of the features.1Hornbeak D.M. Young T.L. Myopia genetics: A review of current research and emerging trends.Curr. Opin. Ophthalmol. 2009; 20: 356-362Crossref PubMed Scopus (103) Google Scholar, 3Morgan I.G. The biological basis of myopic refractive error.Clin. Exp. Optom. 2003; 86: 276-288Crossref PubMed Scopus (100) Google Scholar These syndromes are associated with a range of other phenotypic changes, and most are associated with severe congenital myopia.3Morgan I.G. The biological basis of myopic refractive error.Clin. Exp. Optom. 2003; 86: 276-288Crossref PubMed Scopus (100) Google Scholar Some syndromes affect processes, such as retinal processing, that are likely to be important in the regulation of eye size or affect the properties of connective-tissue macromolecules such as collagen.3Morgan I.G. The biological basis of myopic refractive error.Clin. Exp. Optom. 2003; 86: 276-288Crossref PubMed Scopus (100) Google Scholar Pedigrees of apparently monogenic nonsyndromic hereditary myopia were previously described and were mostly found to be autosomal dominant; rarely, they were found to be X linked or recessive.1Hornbeak D.M. Young T.L. Myopia genetics: A review of current research and emerging trends.Curr. Opin. Ophthalmol. 2009; 20: 356-362Crossref PubMed Scopus (103) Google Scholar, 9Yang Z. Xiao X. Li S. Zhang Q. Clinical and linkage study on a consanguineous Chinese family with autosomal recessive high myopia.Mol. Vis. 2009; 15: 312-318PubMed Google Scholar At least 17 susceptibility loci have been described for nonsyndromic monogenic myopia.1Hornbeak D.M. Young T.L. Myopia genetics: A review of current research and emerging trends.Curr. Opin. Ophthalmol. 2009; 20: 356-362Crossref PubMed Scopus (103) Google Scholar, 9Yang Z. Xiao X. Li S. Zhang Q. Clinical and linkage study on a consanguineous Chinese family with autosomal recessive high myopia.Mol. Vis. 2009; 15: 312-318PubMed Google Scholar However, no clear causative gene mutations have been identified so far in these loci. Large consanguineous Israeli Bedouin kindred presented with an apparently autosomal-recessive phenotype of nonsyndromic severe myopia with variable expressivity of cataract and vitreo-retinal degeneration (Figure 1). Thirteen affected and 32 unaffected individuals (M1–M45, Figure 1) of the kindred underwent complete ophthalmic examination, including slit-lamp biomicroscopy, indirect ophthalmoscopy, measurement of visual acuity and refractive errors, and examination of eye movements and ocular alignment. The study was approved by the Soroka Medical Center institutional review board and the Israeli Helsinki Committee for genetic studies, and informed consent was obtained from all individuals studied. The affected individuals (seven males and six females, ages 7 to 65 years) were examined by a clinical geneticist and had no apparent dysmorphology. They all appeared normal at birth and presented with poor sight in childhood. In addition to myopia, many affected individuals developed cataracts and peripheral vitreo-retinal degeneration, and some had subluxated lenses. The onset of myopia usually preceded that of the cataracts and the retinal degeneration. Most affected individuals had high myopia between −5 and −18 diopters (Table 1), but two had mild myopia. Preoperative refractive error was not available in two patients and in one eye of other three patients. Table 1 shows the spherical equivalent of myopia, calculated for each eye as a sphere plus half of the cylinder of the refractive error. Mean refractive error was −11.3 diopters. All patients had axial myopia, as evidenced by an increased axial length of the eye (the length ranged between 25.1 and 30.5 mm; Table 1). Eleven patients developed cataracts that were significant enough to warrant surgery in one or both eyes, usually in the first or second decade of life. In three patients, subluxated lenses were detected and were associated with cataracts in two patients and lens coloboma in one of them. In an additional five patients, lens instability due to weak or partially missing lens zonules was found during cataract surgery. This caused surgical complications, including rupture of the lens capsule and decentration of the inserted intraocular lens, which required reoperation. In four patients, lens instability made it impossible to insert the intraocular lens at all, which left them aphakic. Peripheral vitreo-retinal degenerative changes were found in nine patients (Table 1), four of whom developed retinal tears (mostly dialyses) causing retinal detachment in one or both eyes. In three patients (23% of affected individuals), in spite of multiple surgical procedures, intractable retinal detachments led to blindness in one eye.Table 1Clinical Data of Affected IndividualsNumberSexAge (y)Myopia (diopters)aSpherical equivalent of myopia is shown, calculated for each eye as a sphere plus half of the cylinder of the refractive error.Axial length (mm)CataractCataract surgery age (y)Vitreo-Retinal ChangesCommentsODOSODOSODOSODOSM14M178.005.0027.726. 3yesyes913OU vitreous opacities OD retinal dialysis and detachment OS retinal tearsOU rupture of lens capsule in cataract surgery, aphakia OD glaucoma, and blindnessM15F231.00NA25.125.5yesyes2320OD pigmented retinal scarM35M1314.5014.5028.928.8yesyes98OD vitreous opacities, OS lattice degenerationOU zonulolysis and phacodonesis seen in cataract surgery, aphakiaM11M128.0011.00NANAnononononoOU subluxated lensesM12F1711.5013.50NANAnoyesno17noM17M281.25NANANAyesyesno13OD retinal tears and detachment OS retinal dialysis and total detachmentOS blindM18F25NANANANAyesyes2015OU fine vitreous opacitiesOU rupture of lens capsule in cataract surgery, OS subluxated IOLM29F712.0010.50NANAnononononoM2F65NANANANAyesyes4146OD lattice degeneration, OS retinal dialysis & detachmentOD aphakia OS subluxated IOLM25F24NA11.0026.027.3yesyes7noOD retinal dialysis and detachment, OS vitreous opacitiesOD blind OS subluxated lens, lens colobomaM28M1316.0018.0028.427.6yesyes77noOU subluxated lenses, aphakia, OS rupture of lens capsule in cataract surgeryM36M1615.0017.0030.529.5yesyesno12OU lattice degeneration and vitreous veilsOS reoperation for subluxated IOL due to rupture of lens capsuleM8M2913.0014.00>26.5>26.5yesyes87OU vitreous opacitiesdeafnessAbbreviations are as follows: OD, right eye; OS, left eye; OU, both eyes; F, female; M, male; NA, data not available; and IOL, intraocular lens.a Spherical equivalent of myopia is shown, calculated for each eye as a sphere plus half of the cylinder of the refractive error. Open table in a new tab Abbreviations are as follows: OD, right eye; OS, left eye; OU, both eyes; F, female; M, male; NA, data not available; and IOL, intraocular lens. Assuming a founder effect, we studied 11 affected individuals and seven healthy individuals by performing a genome-wide linkage analysis with 400 fluorescently end-labeled polymorphic markers (ABI PRISM Linkage Mapping Set MD10 [Applied Biosystems]) as previously described.10Khateeb S. Flusser H. Ofir R. Shelef I. Narkis G. Vardi G. Shorer Z. Levy R. Galil A. Elbedour K. Birk O.S. PLA2G6 mutation underlies infantile neuroaxonal dystrophy.Am. J. Hum. Genet. 2006; 79: 942-948Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar PCR products were separated by electrophoresis on an ABI PRISM 377 DNA Sequencer (Applied Biosystems) and analyzed with Gene-Scan software (detailed results in Document S1). Fine-mapping was carried out using polymorphic markers as previously described.10Khateeb S. Flusser H. Ofir R. Shelef I. Narkis G. Vardi G. Shorer Z. Levy R. Galil A. Elbedour K. Birk O.S. PLA2G6 mutation underlies infantile neuroaxonal dystrophy.Am. J. Hum. Genet. 2006; 79: 942-948Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar Haplotypes were manually constructed and analyzed. Noninformative regions were excluded by the use of additional adjacent microsatellite markers. Analysis of the results with SUPERLINK software11Fishelson M. Geiger D. Optimizing exact genetic linkage computations.J. Comput. Biol. 2004; 11: 263-275Crossref PubMed Scopus (73) Google Scholar yielded two loci with LOD scores above two: a LOD score of 4.1764 at marker D3S3530 and a LOD score of 3.04 at marker D7S484. Fine mapping at the chromosome 7 locus ruled out its association with the disease (Document S2). We performed fine mapping surrounding D3S3530 by using the following additional polymorphic markers to test DNA samples of the 45 available family members (13 affected and 32 unaffected): D3S1580, D3S3549, D3S2398, D3S_A, D3S1294, D3S1314, D3S1288, D3S2747, D3S3043, D3S_B, D3S2455, and D3S1601 (D3S_A and D3S_B are polymorphic markers designed with Tandem Repeats Finder; primer sequences are given in Document S3). As seen in Figure 2, an interval of homozygosity common to all affected individuals in the family was demonstrated between markers D3S3530 and D3S2455. Six affected individuals (M2, M14, M15, M17, M18, and M25) lacked homozygosity at D3S3530, setting the upper limit of the homozygosity interval at D3S3530 (Figure 2). Ten affected individuals (M2, M8, M14, M11, M12, M15, M17, M18, M29, and M25) presented with a recombination event at D3S2455, setting the lower limit of the homozygosity interval at D3S2455. The common haplotype indicated a minimal candidate interval of 1.7 Mbp (∼4 cM) that contained six known or predicted genes. Linkage was demonstrated by two-point analysis with SUPERLINK,11Fishelson M. Geiger D. Optimizing exact genetic linkage computations.J. Comput. Biol. 2004; 11: 263-275Crossref PubMed Scopus (73) Google Scholar which provided a LOD score of 11.5 at θ = 0 for Marker D3S1314 (data available upon request). On the basis of data from the UCSC Genome Browser, only six genes were located within the identified interval. Their coding sequences and exon-intron boundaries were amplified from genomic DNA with PCR primers designed with the Primer3 program; all primer sequences are given in Document S3). PCR products were subject to agarose-gel electrophoresis and gel extraction (QIAGEN), followed by sequencing with either the forward or reverse primer on an ABI PRISM 377 DNA Sequencer (Applied Biosystems). Results were analyzed with ChromasPro software, and the obtained DNA sequences were compared with published sequences (BLAST software). No mutations were found in the coding regions or intron-exon boundaries of five genes within the 3q28 locus. As shown in Figures 3A–3C , sequence analysis of LEPREL1 (RefSeq number NM_018192.3; MIM 610341) revealed that exon 10 contained a c.1523G>T mutation, which led to the replacement of glycine at position 508 by valine (p.Gly508Val; c.1523G>T) in the encoded protein, LEPREL1 (Leprecan-Like 1, termed also P3H2). It should be noted that LEPREL1 was suggested by our S2G software12Gefen A. Cohen R. Birk O.S. Syndrome to gene (S2G): In-silico identification of candidate genes for human diseases.Hum. Mutat. 2010; 31: 229-236Crossref PubMed Scopus (20) Google Scholar to be the top candidate gene of the six genes within the locus. To test for the mutation in a larger cohort, we amplified genomic DNA from 45 individuals of the pedigree and from 200 unrelated controls by using the following PCR primers: 5′-TGGAATAAAATGGGGATGGT-3′ and 5′-TCTGCCATTTACAGAAGCCTTA-3′. We analyzed PCR products by denaturing high-performance liquid chromatography (DHPLC, WAVE Nucleic Acid Fragment Analysis System, Transgenomics). Analysis of all 45 DNA samples of the kindred was compatible with association of the mutation with the disease phenotype, implying full penetrance with variable expressivity. The mutation was not found in any of 400 chromosomes from ethnically matched controls tested by DHPLC. It should be noted that the glycine at position 508 of LEPREL1 is conserved throughout evolution (Figure 3D). As seen in Figure 3E, the p.Gly508Val mutation is predicted to alter the three-dimensional structure of the prolyl-hydroxylase encoded by LEPREL1 (NNpredict).13Kneller D.G. Cohen F.E. Langridge R. Improvements in protein secondary structure prediction by an enhanced neural network.J. Mol. Biol. 1990; 214: 171-182Crossref PubMed Scopus (647) Google Scholar To test for functional consequences of the LEPREL1 mutation, we analyzed wild-type and p.Gly508Val mutant recombinant human P3H2 polypeptides expressed in insect cells. The P3H2 p.Gly508Val mutation was introduced into the pVL1392-P3H2 plasmid14Tiainen P. Pasanen A. Sormunen R. Myllyharju J. Characterization of recombinant human prolyl 3-hydroxylase isoenzyme 2, an enzyme modifying the basement membrane collagen IV.J. Biol. Chem. 2008; 283: 19432-19439Crossref PubMed Scopus (62) Google Scholar with the QuickChangeXL site-directed mutagenesis kit (Stratagene). The recombinant vector was cotransfected into Spodoptera frugiperda (Sf9) insect cells with modified Autographa californica nuclear polyhedrosis virus DNA (BaculoGold, PharMingen) by calcium phosphate transfection, and the recombinant viruses were amplified.15Crossen R. Gruenwald S. Baculovirus Expression Vector System. PharMingen, San Diego, CA1998Google Scholar Monolayer cultures of Sf9 cells in TNM-FH medium (Sigma) supplemented with 10% fetal bovine serum (HyClone) were infected with the recombinant viruses coding for wild-type or mutant P3H2 at a multiplicity of 5 and a cell density of 5 × 106 per 100 mm plate. The cells were harvested 72 hr after infection, washed with a solution of 0.15 M NaCl and 0.02 M phosphate (pH 7.4; PBS), and homogenized in a solution of 0.1 M NaCl, 0.1 M glycine, 2 mM CaCl2, 10 μM dithiothreitol, 0.1% Triton X-100, 0.01 M Tris (pH 7.8), and the complete EDTA-free protease inhibitor cocktail (Roche Diagnostics), and centrifuged at 13,000 x g for 20 min. The remaining pellets were solubilized in 1% SDS, and aliquots of both fractions were analyzed by 15% SDS-PAGE under reducing conditions followed by Coomassie Blue staining or ECL-immunoblotting (Amersham Biosciences) with a polyclonal antibody against P3H214Tiainen P. Pasanen A. Sormunen R. Myllyharju J. Characterization of recombinant human prolyl 3-hydroxylase isoenzyme 2, an enzyme modifying the basement membrane collagen IV.J. Biol. Chem. 2008; 283: 19432-19439Crossref PubMed Scopus (62) Google Scholar at a 1:500 dilution. Equal amounts of soluble wild-type and mutant P3H2 polypeptides were found in the Triton X-100-soluble fractions (not shown) that were assayed for P3H activity by a method based on the hydroxylation-coupled decarboxylation of 2-oxo-[1-14C]glutarate16Kivirikko K.I. Myllylä R. Posttranslational enzymes in the biosynthesis of collagen: Intracellular enzymes.Methods Enzymol. 1982; 82: 245-304Crossref PubMed Scopus (324) Google Scholar with differing amounts of a peptide substrate (Gly-Pro-4Hyp)5, (Innovagen). In additional experiments, the peptide substrate was replaced by the synthetic peptides Ser-Lys-Gly-Glu-Gln-Gly-Phe-Met-Gly-Pro-4Hyp-Gly-Pro-Gln-Gly-Gln-4Hyp-Gly-Leu-4Hyp-Gly or Pro-Thr-Gly-Pro-Arg-Gly-Phe-Pro-Gly-Pro-4Hyp-Gly-Pro-Asp-Gly-Ley-4Hyp-Gly-Ser-Met-Gly, corresponding to two known prolyl 3-hydroxylation sites in the collagen IV α1 chain17Schuppan D. Glanville R.W. Timpl R. Covalent structure of mouse type-IV collagen. Isolation, order and partial amino-acid sequence of cyanogen-bromide and tryptic peptides of pepsin fragment P1 from the alpha 1(IV) chain.Eur. J. Biochem. 1982; 123: 505-512Crossref PubMed Scopus (38) Google Scholar or by Leu-Asn-Gly-Leu-4Hyp-Gly-Pro-Ile-Gly-Pro-4Hyp-Gly-Pro-Arg-Gly-Arg-Thr-Gly-Asp-Ala-Gly, corresponding to the only prolyl 3-hydroxylation site in the collagen I α1 chain at position 986 of the triple helix.18Morello R. Bertin T.K. Chen Y. Hicks J. Tonachini L. Monticone M. Castagnola P. Rauch F. Glorieux F.H. Vranka J. et al.CRTAP is required for prolyl 3- hydroxylation and mutations cause recessive osteogenesis imperfecta.Cell. 2006; 127: 291-304Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar Triton X-100 extracts of insect cells expressing human collagen prolyl 4-hydroxylase isoenzyme II (C-P4H-II)19Annunen P. Helaakoski T. Myllyharju J. Veijola J. Pihlajaniemi T. Kivirikko K.I. Cloning of the human prolyl 4-hydroxylase alpha subunit isoform alpha(II) and characterization of the type II enzyme tetramer. The alpha(I) and alpha(II) subunits do not form a mixed alpha(I)alpha(II)beta2 tetramer.J. Biol. Chem. 1997; 272: 17342-17348Crossref PubMed Scopus (110) Google Scholar were used as negative controls. A large amount of P3H activity was obtained in the P3H2 sample when the synthetic peptide (Gly-Pro-4Hyp)5 was used as the substrate, whereas the activity of the p.Gly508Val mutant P3H2 never exceeded the background values (Table 2). Also, the mutant P3H2 polypeptide did not show any activity when synthetic peptides representing the known prolyl 3-hydroxylation sites in human type I and type IV collagen polypeptides were used as substrates (data not shown).Table 2P3H Activities of Triton X-100-Soluble Fractions of Insect Cells Expressing Wild-Type P3H2, p.Gly508Val Mutant P3H2, or C-P4H-II with the peptide (Pro-4Hyp-Gly)5 as the SubstrateEnzymePeptide Concentration (μM)Activity (dpm/100 μlaThe values are means ± SD from at least three independent experiments.)P3H2504570 ± 3201008170 ± 610P3H2Gly508Val50<100100<100C-P4H-IIbThe Triton X-100-soluble fraction of insect cells expressing C-P4H-II was used as a negative control.100<100100<100a The values are means ± SD from at least three independent experiments.b The Triton X-100-soluble fraction of insect cells expressing C-P4H-II was used as a negative control. Open table in a new tab LEPREL1 is localized to the ER and the Golgi network.20Järnum S. Kjellman C. Darabi A. Nilsson I. Edvardsen K. Aman P. LEPREL1, a novel ER and Golgi resident member of the Leprecan family.Biochem. Biophys. Res. Commun. 2004; 317: 342-351Crossref PubMed Scopus (32) Google Scholar The 708 amino acid protein contains a signal sequence, four tetra-tricopeptide repeats (TPRs), a leucine zipper, a P loop, and a C-terminal region that contains the catalytically critical residues that are required for binding of Fe2+ and 2-oxoglutarate and are shared with the collagen-modifying lysyl hydroxylases and prolyl 4-hydroxylases, as well as other 2-oxoglutarate-dependent dioxygenases and a C-terminal KDEL ER-retention motif.19Annunen P. Helaakoski T. Myllyharju J. Veijola J. Pihlajaniemi T. Kivirikko K.I. Cloning of the human prolyl 4-hydroxylase alpha subunit isoform alpha(II) and characterization of the type II enzyme tetramer. The alpha(I) and alpha(II) subunits do not form a mixed alpha(I)alpha(II)beta2 tetramer.J. Biol. Chem. 1997; 272: 17342-17348Crossref PubMed Scopus (110) Google Scholar, 20Järnum S. Kjellman C. Darabi A. Nilsson I. Edvardsen K. Aman P. LEPREL1, a novel ER and Golgi resident member of the Leprecan family.Biochem. Biophys. Res. Commun. 2004; 317: 342-351Crossref PubMed Scopus (32) Google Scholar The mutation results in a glycine-to-valine substitution at amino acid 508 within the C-terminal region.19Annunen P. Helaakoski T. Myllyharju J. Veijola J. Pihlajaniemi T. Kivirikko K.I. Cloning of the human prolyl 4-hydroxylase alpha subunit isoform alpha(II) and characterization of the type II enzyme tetramer. The alpha(I) and alpha(II) subunits do not form a mixed alpha(I)alpha(II)beta2 tetramer.J. Biol. Chem. 1997; 272: 17342-17348Crossref PubMed Scopus (110) Google Scholar The mutated glycine is a highly conserved residue within the P3H family (Figure 3D), suggesting its importance: the p.Gly508Val variant is predicted (via PolyPhen algorithms) to be probably damaging with a position-specific independent counts (PSIC) score difference of 2.423; moreover, substitution of glycine with valine leads to putative changes in the secondary structure of the protein (Figure 3E).13Kneller D.G. Cohen F.E. Langridge R. Improvements in protein secondary structure prediction by an enhanced neural network.J. Mol. Biol. 1990; 214: 171-182Crossref PubMed Scopus (647) Google Scholar Accordingly, the p.Gly508Val mutation led to complete inactivation of recombinant P3H2 polypeptide expressed in insect cells, both when analyzed with a (Gly-Pro-4Hyp)5 peptide substrate that fulfills the substrate requirements of P3H and when analyzed with synthetic peptides representing known prolyl 3-hydroxylation sites in human type I and type IV collagen polypeptides. The large kindred presented here is distinctive in its recessive heredity of myopia. Interestingly, a study of juvenile cataract with myopia in a Chinese family demonstrated linkage to a locus between D3S3606 and D3S1262, approximately 3 Mbp from the region highlighted in our kindred;21Zhang Q. Guo X. Xiao X. Yi J. Jia X. Hejtmancik J.F. Clinical description and genome wide linkage study of Y-sutural cataract and myopia in a Chinese family.Mol. Vis. 2004; 10: 890-900PubMed Google Scholar however, the genomic loci in the two kindreds do not overlap. In contrast with that of the Bedouin kindred, the phenotype in the Chinese family was dominantly inherited, manifested as cataracts with likely secondary myopia that appeared in only some of the affected individuals, and was found to be associated with a heterozygous mutation in BFSP2 (MIM 603212), a gene known to be mutated in dominant cataracts.21Zhang Q. Guo X. Xiao X. Yi J. Jia X. Hejtmancik J.F. Clinical description and genome wide linkage study of Y-sutural cataract and myopia in a Chinese family.Mol. Vis. 2004; 10: 890-900PubMed Google Scholar The homozygous LEPREL1 mutation identified in affected individuals of the Bedouin kindred was associated with full penetrance and variable expressivity of the disease. The main clinical phenotype was severe myopia. However, cataracts, vitreo-retinal degeneration, and subluxated lenses were also found in some patients. These led to severe ocular morbidity and a high rate of surgical complications, which resulted in blindness in 23% of our patients. We clearly demonstrate that the myopia is due to increased axial eye length, although one cannot rule out the possibility that changes in the lens might also contribute to the myopia phenotype. Although the cataract and vitreo-retinal degeneration might be related to the myopia, it is plausible that the myopia, cataracts, vitreo-retinal degeneration, and lens subluxation are all independent consequences of the LEPREL1 mutation. The association of the LEPREL1 mutation with ocular abnormalities is in line with previous findings: LEPREL1 is found in a wide variety of tissues, including the eye;22Saika S. Kawashima Y. Miyamoto T. Okada Y. Tanaka S.I. Ohmi S. Minamide A. Yamanaka O. Ohnishi Y. Ooshima A. Yamanaka A. Immunolocalization of prolyl 4-hydroxylase subunits, alpha-smooth muscle actin, and extracellular matrix components in human lens capsules with lens implants.Exp. Eye Res. 1998; 66: 283-294Crossref PubMed Scopus (108) Google Scholar specifically, it was demonstrated in the iris, lens, trabecular meshwork, and retinal pericyte cells (NEIBank). Recent protein-localization studies of the P3H isoenzymes in mouse demonstrated that P3H2 appears to be coordinately expressed with and therefore recognizes basement membrane collagens such as type IV collagen.23Vranka J. Stadler H.S. Bächinger H.P. Expression of prolyl 3-hydroxylase genes in embryonic and adult mouse tissues.Cell Struct. Funct. 2009; 34: 97-104Crossref PubMed Scopus (26) Google Scholar Furthermore, it was suggested that other basement-membrane-associated collagens may be targets of this enzyme as well.23Vranka J. Stadler H.S. Bächinger H.P. Expression of prolyl 3-hydroxylase genes in embryonic and adult mouse tissues.Cell Struct. Funct. 2009; 34: 97-104Crossref PubMed Scopus (26) Google Scholar It is noteworthy that collagen type IV is found in various ocular structures, including the lens, iris, retina, retinal pigment epithelium, and trabecular meshwork (NEIBank). Interestingly, P3H2 is expressed in the developing lens capsule of mouse embryos (E11–E15).23Vranka J. Stadler H.S. Bächinger H.P. Expression of prolyl 3-hydroxylase genes in embryonic and adult mouse tissues.Cell Struct. Funct. 2009; 34: 97-104Crossref PubMed Scopus (26) Google Scholar Because the lens capsule is a basement membrane whose main component is type IV collagen, these findings further suggest that defective collagen hydroxylation as a result of P3H2 inactivation might cause abnormality of the lens capsule, possibly leading to the development of cataracts and possibly contributing to the myopia phenotype by affecting distensibility of the lens capsule. An abnormally weak lens capsule can also account for a capsular rupture during cataract surgery; such a rupture occurred in many of our patients. Because type IV collagen has been demonstrated in the ciliary zonules of the eye (suspensory ligament of the lens),24Los L.I. van der Worp R.J. van Luyn M.J. Hooymans J.M. Presence of collagen IV in the ciliary zonules of the human eye: An immunohistochemical study by LM and TEM.J. Histochem. Cytochem. 2004; 52: 789-795Crossref PubMed Scopus (11) Google Scholar its abnormality may cause a defective zonular structure. Weakening of the zonules probably underlies lens instability, as evidenced by spontaneous lens subluxation in some of our patients, as well as pathological zonulolysis during cataract surgery in others. Another basement membrane in the eye is the inner limiting membrane (ILM) of the retina. It has been shown that ILM is important for proper retinal development and in the early regulation of eye size: ILM disruption resulted in marked eye enlargement in chick embryos, implicating that defective ILM might lead to congenital high myopia.25Halfter W. Winzen U. Bishop P.N. Eller A. Regulation of eye size by the retinal basement membrane and vitreous body.Invest. Ophthalmol. Vis. Sci. 2006; 47: 3586-3594Crossref PubMed Scopus (41) Google Scholar Inactivation of P3H2 as a result of the LEPREL1 mutation in our patients might cause a weakened retinal ILM and thus lead to excessive eye growth and development of myopia. Likewise, a defective retinal basement membrane harbors greater risk for retinal tears and dialyses, which can lead to retinal detachments such as those observed in some of our patients. This predisposition, in addition to complicated cataract surgery, might have further aggravated the prognosis of retinal detachments in our patients. It is worth noting that defects in collagen hydroxylation have been previously demonstrated to cause phenotypes similar to those caused by collagen mutations: autosomal-recessive variants of osteogenesis imperfecta, a disease known to be caused by dominant collagen mutations, can be caused by defects in collagen prolyl 3- hydroxylation.18Morello R. Bertin T.K. Chen Y. Hicks J. Tonachini L. Monticone M. Castagnola P. Rauch F. Glorieux F.H. Vranka J. et al.CRTAP is required for prolyl 3- hydroxylation and mutations cause recessive osteogenesis imperfecta.Cell. 2006; 127: 291-304Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 26Cabral W.A. Chang W. Barnes A.M. Weis M. Scott M.A. Leikin S. Makareeva E. Kuznetsova N.V. Rosenbaum K.N. Tifft C.J. et al.Prolyl 3-hydroxylase 1 deficiency causes a recessive metabolic bone disorder resembling lethal/severe osteogenesis imperfecta.Nat. Genet. 2007; 39: 359-365Crossref PubMed Scopus (353) Google Scholar, 27Barnes A.M. Chang W. Morello R. Cabral W.A. Weis M. Eyre D.R. Leikin S. Makareeva E. Kuznetsova N. Uveges T.E. et al.Deficiency of cartilage-associated protein in recessive lethal osteogenesis imperfecta.N. Engl. J. Med. 2006; 355: 2757-2764Crossref PubMed Scopus (264) Google Scholar Development of myopia has been shown to be associated with decreased rates of collagen synthesis in the sclera.28Gentle A. Liu Y. Martin J.E. Conti G.L. McBrien N.A. Collagen gene expression and the altered accumulation of scleral collagen during the development of high myopia.J. Biol. Chem. 2003; 278: 16587-16594Crossref PubMed Scopus (164) Google Scholar Moreover, various clinical syndromes such as Stickler syndrome (MIM 108300), Marshall syndrome (MIM 154780), and Weissenbacher-Zweymuller syndrome (MIM 277610), in which myopia is a prominent feature, are caused by defects in collagens.3Morgan I.G. The biological basis of myopic refractive error.Clin. Exp. Optom. 2003; 86: 276-288Crossref PubMed Scopus (100) Google Scholar, 4Feldkämper M. Schaeffel F. Interactions of genes and environment in myopia.Dev. Ophthalmol. 2003; 37: 34-49Crossref PubMed Google Scholar Thus, whereas mutations in collagen genes cause autosomal-dominant myopia syndromes, defects in collagen hydroxylation might cause autosomal-recessive myopia. It of course yet remains to be tested whether P3H2 modifies the collagens known to act in those processes. Our findings demonstrate a significant role of LEPREL1 in the molecular pathways of ocular growth, as well as in the development of myopia and cataracts. Further studies will need to explore whether LEPREL1 mutations or polymorphisms are associated with ocular abnormalities in a wider population. An improved understanding of the molecular basis of pathological myopia might allow early diagnosis and treatment and thus prevent irreversible visual loss, as developed in our patients. We deeply thank the Morris Kahn Family Foundation for making this study possible. J.M. was supported by the Academy of Finland and the Sigrid Juselius Foundation. Download .pdf (8.19 MB) Help with pdf files Document S1. Results of Genome-Wide Scan Download .pdf (.12 MB) Help with pdf files Document S2. Exclusion of Chromosome 7 Locus Download .pdf (.11 MB) Help with pdf files Document S3. DNA Sequences of Primers Used The accession number and URLs for data presented herein are as follows:BLAST, http://www.ncbi.nlm.nih.gov/blast/GenBank, http://www.ncbi.nlm.nih.gov/Genbank/NEIBank, http://neibank.nei.nih.gov/cgi-bin/runSearch.cgi/Online Mendelian Inheritance in Man (OMIM), http://www.omim.orgPolyPhen, http://genetics.bwh.harvard.edu/pph2/Primer3, http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi/SUPERLINK, http://bioinfo.cs.technion.ac.il/superlink/Tandem Repeats Finder, http://tandem.bu.edu/trf/trf.html/UCSC Genome Browser, http://www.genome.ucsc.edu/ [accession number NM_018192])