Mutations in SCO2 Are Associated with Autosomal-Dominant High-Grade Myopia
2013; Elsevier BV; Volume: 92; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2013.04.005
ISSN1537-6605
AutoresKhanh-Nhat Tran-Viet, Caldwell Powell, Veluchamy A. Barathi, Thomas Klemm, Sebastian Maurer‐Stroh, Vachiranee Limviphuvadh, Vincent Soler, Candice Ho, Tammy L. Yanovitch, Georg Schneider, Yi‐Ju Li, Erica B. Nading, Ravikanth Metlapally, Seang‐Mei Saw, Liang Kee Goh, Steve Rozen, Terri L. Young,
Tópico(s)Retinal Diseases and Treatments
ResumoMyopia, or near-sightedness, is an ocular refractive error of unfocused image quality in front of the retinal plane. Individuals with high-grade myopia (dioptric power greater than −6.00) are predisposed to ocular morbidities such as glaucoma, retinal detachment, and myopic maculopathy. Nonsyndromic, high-grade myopia is highly heritable, and to date multiple gene loci have been reported. We performed exome sequencing in 4 individuals from an 11-member family of European descent from the United States. Affected individuals had a mean dioptric spherical equivalent of −22.00 sphere. A premature stop codon mutation c.157C>T (p.Gln53*) cosegregating with disease was discovered within SCO2 that maps to chromosome 22q13.33. Subsequent analyses identified three additional mutations in three highly myopic unrelated individuals (c.341G>A, c.418G>A, and c.776C>T). To determine differential gene expression in a developmental mouse model, we induced myopia by applying a −15.00D lens over one eye. Messenger RNA levels of SCO2 were significantly downregulated in myopic mouse retinae. Immunohistochemistry in mouse eyes confirmed SCO2 protein localization in retina, retinal pigment epithelium, and sclera. SCO2 encodes for a copper homeostasis protein influential in mitochondrial cytochrome c oxidase activity. Copper deficiencies have been linked with photoreceptor loss and myopia with increased scleral wall elasticity. Retinal thinning has been reported with an SC02 variant. Human mutation identification with support from an induced myopic animal provides biological insights of myopic development. Myopia, or near-sightedness, is an ocular refractive error of unfocused image quality in front of the retinal plane. Individuals with high-grade myopia (dioptric power greater than −6.00) are predisposed to ocular morbidities such as glaucoma, retinal detachment, and myopic maculopathy. Nonsyndromic, high-grade myopia is highly heritable, and to date multiple gene loci have been reported. We performed exome sequencing in 4 individuals from an 11-member family of European descent from the United States. Affected individuals had a mean dioptric spherical equivalent of −22.00 sphere. A premature stop codon mutation c.157C>T (p.Gln53*) cosegregating with disease was discovered within SCO2 that maps to chromosome 22q13.33. Subsequent analyses identified three additional mutations in three highly myopic unrelated individuals (c.341G>A, c.418G>A, and c.776C>T). To determine differential gene expression in a developmental mouse model, we induced myopia by applying a −15.00D lens over one eye. Messenger RNA levels of SCO2 were significantly downregulated in myopic mouse retinae. Immunohistochemistry in mouse eyes confirmed SCO2 protein localization in retina, retinal pigment epithelium, and sclera. SCO2 encodes for a copper homeostasis protein influential in mitochondrial cytochrome c oxidase activity. Copper deficiencies have been linked with photoreceptor loss and myopia with increased scleral wall elasticity. Retinal thinning has been reported with an SC02 variant. Human mutation identification with support from an induced myopic animal provides biological insights of myopic development. Myopia is a common ocular disorder primarily resulting from globe axial elongation.1Curtin B.J. The Myopias: Basic Science and Clinical Management. Harper & Row, Philadelphia1985Google Scholar, 2Curtin B.J. Karlin D.B. Axial length measurements and fundus changes of the myopic eye.Am. J. Ophthalmol. 1971; 71: 42-53Abstract Full Text PDF PubMed Scopus (295) Google Scholar Its extreme form, high-grade myopia (refractive error greater than −6.00 diopters [D]) (MIM 160700, MIM 613969, MIM 60995, MIM 608367, MIM 614167, MIM 603221, MIM 608474, MIM 612554, and MIM 609994), is highly heritable and associated with ocular morbidities such as retinal detachment, maculopathy, cataracts, and glaucoma.3Saw S.M. Gazzard G. Shih-Yen E.C. Chua W.H. Myopia and associated pathological complications.Ophthalmic Physiol. Opt. 2005; 25: 381-391Crossref PubMed Scopus (708) Google Scholar Myopia prevalence rates vary worldwide. The highest prevalence rates are those in Asian countries, particularly in urban settings. Over 80% of school children in Taiwan develop myopia by adulthood, and similar rates are seen in children aged between 13 and 15 years in Hong Kong.4Lin L.L. Shih Y.F. Hsiao C.K. Chen C.J. Lee L.A. Hung P.T. 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Cai L. et al.Exome sequencing identifies ZNF644 mutations in high myopia.PLoS Genet. 2011; 7: e1002084Crossref PubMed Scopus (152) Google Scholar In 2011, Shi et al. identified mutations in zinc finger protein 644 isoform 1 (ZNF644) in a Chinese family with autosomal-dominant high-grade myopia by using exome sequencing, which was replicated in four cases in our European descent cohort.16Shi Y. Li Y. Zhang D. Zhang H. Li Y. Lu F. Liu X. He F. Gong B. Cai L. et al.Exome sequencing identifies ZNF644 mutations in high myopia.PLoS Genet. 2011; 7: e1002084Crossref PubMed Scopus (152) Google Scholar, 17Tran-Viet K.N. St Germain E. Soler V. Powell C. Lim S.H. Klemm T. Saw S.M. Young T.L. Study of a US cohort supports the role of ZNF644 and high-grade myopia susceptibility.Mol. Vis. 2012; 18: 937-944PubMed Google Scholar Herein, we describe the identification of pathogenic mutations in the SCO2 cytochrome c oxidase (COX) assembly protein (SCO2) on chromosome 22q13.33 (NM_001169111.1). Gene expression studies in an experimentally induced myopic mouse model suggest that SCO2 may play a role in myopic development. A large three-generation index family (11 members) of European descent with nine affected individuals with high-grade myopia (average spherical refractive error of −22.00D) participated in the study (Figure 1). Informed consent was obtained from all participants, with approval by the Institutional Review Board according to the principles of the Declaration of Helsinki. DNA was extracted from blood and/or saliva from all participating family members. The affected phenotype was determined as those with high myopia (refractive error greater than −6.00D) with no systemic abnormalities. To identify the genetic etiology of disease in our family, we employed exome sequencing. We selected four individuals (III:1, III:2, IV:1, IV:2) for sequencing and analysis (Figure 1). An additional 60 ethnically matched exomes and 1,172 ethnically matched controls (500 samples ascertained internally, and 672 samples purchased commercially; The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Canada) were available for subsequent validation studies to analyze allelic frequencies for candidate variants. Exome sequencing was performed by Beijing Genomics Institute (BGI), and data analyses were conducted internally. Seven micrograms (μg) of DNA were submitted with independent sample processing by using the Agilent SureSelect XT Human All Exon 38 Mb kit (Agilent, Santa Clara, CA). High throughput sequencing was performed with 91 base pair (bp) paired-end runs on a HiSeq2000 (Illumina, San Diego, CA). Read alignment was conducted by using Burrows-Wheeler Aligner (v.0.5.6) and potential duplicate reads were removed with Picard v.1.40.18Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26642) Google Scholar Filtering and detection of reads were conducted by using SAMtools (v.0.1.7).19Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (31547) Google Scholar Single nucleotide variants and microindels were annotated by using the Genome Analyzer Toolkit (GATK- v1.4). We generated an average of 37.8 gigabases (Gb) of sequence and coverage of 34× for each individual. An average of 95.7% of targeted bases was covered in the four subject samples, and 87.5% of the target had at least 5× coverage (see Supplement 1 available online). Variants in dbSNP132 with a minor allele frequency greater than 3% and those present in more than 1% of public exomes (NHLBI and 1000 Genomes) were excluded. In addition, heterozygous variants present in at least one affected individual were kept in the finalized list. In conjunction with filtering, Integrated Genome Viewer (IGV) visualization software was utilized to confirm corresponding reads and read depth to verify false positives or negatives. We identified 49 variants shared among the exome-sequenced affected members only. To minimize false positives due to batch effects, we verified all 49 variants as unique in silico relative to 60 exomes previously sequenced by our group. We performed Sanger sequencing of all 49 variants and demonstrated 100% validation (49/49) in the four index DNA samples. All microindels were eliminated based on our filtering criteria. To confirm the segregation of variants with the disease phenotype, we used Sanger sequencing for the remaining 7 family member DNA samples against all 49 variants. A rare nonsense mutation of c.157C>T (rs74315510) within exon 2 of SCO2 cytochrome c oxidase assembly protein (SCO2, NM_001169111.1) segregated with high-grade myopia in the pedigree. The SCO2 mutation converts the amino acid glutamine to a premature stop codon on base 53 (p.Gln53*). The minor allele was not present in 1,000 control samples for the SCO2 variant. The MERLIN program using a dominant parametric model was employed to estimate the linkage of this variant.20Abecasis G.R. Cherny S.S. Cookson W.O. Cardon L.R. Merlin—rapid analysis of dense genetic maps using sparse gene flow trees.Nat. Genet. 2002; 30: 97-101Crossref PubMed Scopus (2780) Google Scholar A two-point LOD score of 1.49 for c.157C>T was calculated for the family.20Abecasis G.R. Cherny S.S. Cookson W.O. Cardon L.R. Merlin—rapid analysis of dense genetic maps using sparse gene flow trees.Nat. Genet. 2002; 30: 97-101Crossref PubMed Scopus (2780) Google Scholar PCR sequencing of the SCO2 coding exon 2 was conducted in an additional 140 high-grade myopia cases. The average spherical equivalent refraction of the cases was −11.00D (OD) and −10.50D (OS), respectively. We identified two additional rare variants (rs74315511 and rs8139305) and one variant in three unrelated cases that were heterozygous (Table 1). Rs74315511 (c.418G>A) is a missense mutation predicted to cause a p.Glu140Lys substitution. Rs8139305 (c.776C>T) missense mutation causes a p.Ala259Val substitution. The missense mutation (c.341G>A) causes a substitution of p.Arg114His. These variants were not seen in the same 1,000 control DNA samples. ANNOVAR was used to assess functional annotation and at least one in silico software predicted the mutations to be deleterious or damaging (Table 1).21Wang K. Li M. Hakonarson H. 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Methods. 2010; 7: 575-576Crossref PubMed Scopus (2123) Google Scholar By using the Fisher exact test, the likelihood for identifying the 4 functional variants in 141 individuals with high-grade myopia relative to 1,000 nonmyopic controls was significant (p = 0.000248).Table 1Summary of Identified SCO2 MutationsFamily/Case NameEthnicityRefractive Error (Diopters)Nucleotide ChangeAmino Acid ChangeChromsome 22 Location (bp)bGRCh37.p5.rs NumberLJB_PhyloPLJB_GERP++AVSIFTLJB_SIFTLJB_PolyPhen2LJB_MutationTasterODOSCHP-6aRefraction of CHP-6 depicts the average refractive error among the affected individuals.European descent−22.00−22.00c.157C>Tp.Gln53*50962684rs743155100.9983.130.710.9110.6501CHP-150Middle Eastern−15.25−12.00c.341.G>Ap.Arg114His50962500NA0.9984.440.030.860.9950.930CHP-162European descent−14.25−15.00c.418G>Ap.Glu140Lys50962423rs743155110.9703.060110.908CHP-84African-American−11.00−10.25c.776C>Tp.Ala259Val50962065rs81393050.8700.2790.080.90.0780.329Annotations were derived with ANNOVAR. Evolutionary site conservation scores for PhyloP and GERP++ were calculated as described in dbNSFP and bolded when above the threshold for being classified as “conserved.” Predicted mutation effect scores are shown for SIFT, PolyPhen2, and MutationTaster. Scores above the threshold for predictions as “deleterious” are bolded if close to the threshold. OD, right eye; OS, left eye.a Refraction of CHP-6 depicts the average refractive error among the affected individuals.b GRCh37.p5. Open table in a new tab Annotations were derived with ANNOVAR. Evolutionary site conservation scores for PhyloP and GERP++ were calculated as described in dbNSFP and bolded when above the threshold for being classified as “conserved.” Predicted mutation effect scores are shown for SIFT, PolyPhen2, and MutationTaster. Scores above the threshold for predictions as “deleterious” are bolded if close to the threshold. OD, right eye; OS, left eye. SCO2 consists of two exons of which only the second exon is protein coding. Given the mutation location, c.157C>T truncates the protein before the catalytic domain, rendering it nonfunctional. The protein changes (p.Arg114His, p.Glu140Lys, and p.Ala259Val) by using BLAST are all located within the functional catalytic domain and are predicted to affect the protein structure. The p.Glu140Lys amino acid substitution results in removal of a salt bridge between Glu140 and Lys143 and changes the electrostatic potential of the copper binding site, which can moderately to strongly affect SCO2 function (Supplements 2 and 3). Moreover, p.Arg114His and p.Ala259Val are predicted to destabilize the structure based on FoldX, with mild-to-moderate influence on SCO2 function (Supplement 4).28Van Durme J. Delgado J. Stricher F. Serrano L. Schymkowitz J. Rousseau F. 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Refractive error genetics has proven to be complex, as demonstrated by mapping and large association studies. Although SCO2 did not colocalize in any reported myopia loci, our findings provide evidence that SCO2 may play an important role in eye growth and development, particularly in those who become highly myopic. A myopic phenotype should not be overlooked in studies involving a heterogeneous group of rare disorders involving SCO2. This research was funded by the National Institutes of Health Grant R01 EY014685, the Lew Wasserman Award from Research to Prevent Blindness (Chicago, IL), and a Duke-National University of Singapore core grant to T.L.Y. V.S. was supported by the Toulouse Hospital Young Researcher Fellowship, the Fondation pour la Recherche Médicale, and Fondation de France. The animal model experiments were supported by the National Medical Research Council of Singapore (NMRC/IRG/1117/2008 and NMRC/CG/T1/2010) to V.A.B. The sponsors or funding organizations had no role in the design or conduct of this research. The authors also thank Felicia Hawthorne, Xiaoyan Luo, and Kwan Jia Lin for their helpful insights regarding gene expression and immunohistochemical experiments. Download .pdf (1.01 MB) Help with pdf files Document S1. Supplements 1–7 The URLs for data presented herein are as follows:1000 Genomes, http://browser.1000genomes.orgAnnovar, http://www.openbioinformatics.org/annovar/BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgiBroad Institute Integrated Genomics Viewer, http://www.broadinstitute.org/igv/NHLBI Exome Sequencing Project (ESP) Exome Variant Server, http://evs.gs.washington.edu/EVS/Online Mendelian Inheritance in Man (OMIM), http://www.omim.org/Picard, http://picard.sourceforge.net/PolyPhen, www.genetics.bwh.harvard.edu/pph2/SIFT, http://sift.jcvi.org/
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