\set{final} \def\Author{Aldave} \def\author{aldave} \def\vol{11} \def\year{2005} \def\anum{84} \def\pages{713-716} \def\txt_title{Analysis of fifteen positional candidate genes for Schnyder crystalline corneal dystrophy} \def\txt_authors{Anthony J. Aldave, Sylvia A. Rayner, Alexandre H. Principe, John A. Affeldt, Douglas Katsev, Vivek S. Yellore} \def\rcvd{11 March 2005} \def\accept{2 September 2005} \def\publ{2 September 2005} \def\pdfsize{} \def\PMID{} \include{mvstyle.hsm} \| External links \def\ncbi{http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?taxid=9606&chr=1} \def\primer3{http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi} \| Internal defs \article{ \title{Analysis of fifteen positional candidate genes for Schnyder crystalline corneal dystrophy} \authors{\mailto{aldave@jsei.ucla.edu}{Anthony J. Aldave},\sup{1} Sylvia A. Rayner,\sup{1} Alexandre H. Principe,\sup{1} John A. Affeldt,\sup{2} Douglas Katsev,\sup{3} Vivek S. Yellore\sup{1}} \institutions{\sup{1}Cornea Service, Jules Stein Eye Institute, University of California, Los Angeles, CA; \sup{2}Doheny Eye Institute/LA County-USC Medical Center Department of Ophthalmology, Los Angeles, CA; \sup{3}Santa Barbara Medical Foundation, Santa Barbara, CA} \correspondence{Anthony J. Aldave, MD, Assistant Professor, The Jules Stein Eye Institute, 100 Stein Plaza, Los Angeles, CA, 90095; Phone: (310) 206-7202; FAX: (310) 794-7906; email: aldave@jsei.ucla.edu} \abstract \abs_purpose{To identify the genetic basis of Schnyder crystalline corneal dystrophy (SCCD) through screening of positional candidate genes in affected patients.} \abs_methods{Mutation screening of fifteen genes (\i{CORT}, \i{CLSTN1}, \i{CTNNBIP1}, \i{DFFA}, \i{ENO1}, \i{GPR157}, \i{H6PD}, \i{KIF1B}, \i{LOC440559}, \i{LZIC}, \i{MGC4399}, \i{PEX14}, \i{PGD}, \i{PIK3CD}, and \i{SSB1}) that lie within the candidate gene region for SCCD was performed in members of two families affected with SCCD.} \abs_results{No presumed disease-causing mutations were identified in affected patients. Seventeen previously described single nucleotide polymorphisms (SNPs) were identified in eight of the candidate genes. Novel SNPs were identified in both affected and unaffected individuals in \i{GPR157} (c.795C\gt T [Arg218Leu]; c.811C\gt T [Ala223Val]), \i{MGC4399} (c.1024G\gt C [Leu277Leu]), and \i{H6PD} (c.754A\gt C [Asp151Ala]).} \abs_conclusions{No pathogenic mutations were identified in fifteen positional candidate genes in two families with SCCD. As the candidate gene region in each SCCD family previously examined with haplotype analysis has been mapped to the same chromosomal region, the absence of pathogenic mutations in these positional candidates in the families we examined reduces the number of remaining positional candidate genes by half, and the number of remaining candidate genes with a known gene function by two-thirds. We anticipate that screening of the remaining positional candidate genes will lead to the identification of the genetic basis of SCCD.} \introduction \p{Schnyder crystalline corneal dystrophy (SCCD; OMIM \omim{121800}) is associated with corneal stromal cholesterol deposition and elevated serum cholesterol levels in a minority of affected patients [1]. The mechanism of corneal cholesterol deposition is thought to be secondary to a metabolic defect of the corneal keratocytes [1,2], as less than half of affected patients demonstrate elevated serum cholesterol levels, despite evidence of abnormal cholesterol metabolism in their skin fibroblasts [2]. Recently, fine mapping of the SCCD locus in 13 families has narrowed the candidate region to a 2.32 Mbp interval between the D1S1160 and D1S1635 markers [3-5]. Thirty-one genes have been identified between markers \hot{\ncbi}{D1S1160 and D1S1635}; twenty-one are known genes, with the remainder being putative genes. We sought to identify the genetic basis of SCCD through screening of fifteen of these genes in affected and unaffected members of two families with SCCD.} \methods \p{The researchers followed the tenets of the Declaration of Helsinki in the treatment of the subjects reported herein. Study approval was obtained from the institutional review board at The University of California, Los Angeles, CA (UCLA M-IRB number 94-07-243-21).} \subsection{Patient identification} \p{Members of two families diagnosed with SCCD, one of Irish descent (Family 1) and the other of Egyptian origin (Family 2), were referred to one of us (AJA) for evaluation (\figref{1}). The diagnosis was based on the presence of characteristic clinical features, including subepithelial crystalline deposits, central discoid or annular corneal stromal opacification, and associated arcus lipoides in the corneal periphery (\figref{2}).} \subsection{DNA collection and PCR amplification} \p{Informed consent was obtained from each subject after an explanation of the nature and possible consequences of study participation. Genomic DNA was isolated from peripheral blood leukocytes of affected and unaffected members of two families with SCCD. DNA previously collected from greater than 100 unrelated, unaffected individuals without evidence of Schnyder crystalline corneal dystrophy served as control samples. The coding regions of cortistatin (\i{CORT}), calsyntenin 1 (\i{CLSTN1}), catenin, beta-interacting protein 1 (\i{CTNNBIP1}), DNA fragmentation factor, 45 kDa, alpha polypeptide (\i{DFFA}), enolase 1 (\i{ENO1}), G protein-coupled receptor 157 (\i{GPR157}), hexose-6-phosphate dehydrogenase (\i{H6PD}), kinesin family member 1B (\i{KIF1B}), predicted protein LOC440559 (\i{LOC440559}), leucine zipper and CTNNBIP1 domain containing (\i{LZIC}), mitochondrial carrier protein (\i{MGC4399}), peroxisomal biogenesis factor 14 (\i{PEX14}), phosphogluconate dehydrogenase (\i{PGD}), phosphoinositide-3-kinase, catalytic, delta polypeptide (\i{PIK3CD}), and SPRY domain-containing SOCS box protein (\i{SSB1}) were amplified by the polymerase chain reaction (PCR) with custom primers designed using \hot{\primer3}{Primer 3} (sequences available upon request). Each reaction was carried out in a 25 \mu l mixture containing 12.5 \mu l of FailSafe PCR 2X PreMix "D" (100 mM Tris-HCl pH 8.3, 100 mM KCl, 400 \mu M of each dNTP, and proprietary concentrations of MgCl\sub{2} and FailSafe PCR Enhancer; Epicentre, Madison, WI), 0.12 \mu M of each primer, 1 unit of AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA), and approximately 60 ng of genomic DNA. Thermal cycling was performed in an iCycler Thermal Cycler (Bio-Rad, Hercules, CA).} \subsection{DNA sequencing} \p{Purification of the PCR products was achieved by incubating 15-30 ng of DNA with 5 units of Exonuclease I and 0.5 units of Shrimp Alkaline Phosphatase (USB Corp., Cleveland, OH) for 15 min at 37 \deg C. After nuclease inactivation by incubating at 80 \deg C for 15 min, sequencing reactions were performed by the addition of 2 \mu l BigDye Terminator Mix version 3.1 (Applied Biosystems), 2 \mu l of SeqSaver (Sigma-Aldrich, St. Louis, MO) and 0.2 \mu l of primer (10 pM/\mu l). Samples were denatured at 96 \deg C for 2 min, then cycled 25 times at 96 \deg C for 10 s, 50 \deg C for 5 s, and 60 \deg C for 4 min. Unincorporated nucleotides were removed using the CleanSeq reagent and a SPRI plate (Agencourt Bioscience Corporation, Beverly, MA) following the manufacturer's instructions. The PCR products were then analyzed on an ABI-3100 Genetic Analyzer (Applied Biosystems) after resuspension in 0.1 mM EDTA. The nucleotide sequences, read manually and with Mutation Surveyor version 2.2 (Softgenetics, State College, PA), were compared with the published cDNA sequence for each gene; \i{CORT} (\genbankdna{NM_198544}), \i{CLSTN1} (\genbankdna{NM_014944}), \i{CTNNBIP1} (\genbankdna{NM_020248}), \i{DFFA} (\genbankdna{NM_004401}), \i{ENO1} (\genbankdna{NM_001428}), \i{GPR157} (\genbankdna{NM_024980}), \i{H6PD} (\genbankdna{NM_004285}), \i{KIF1B} (\genbankdna{NM_015074}), \i{LOC440559} (\genbankdna{XM_498733}), \i{LZIC} (\genbankdna{NM_032368}), \i{MGC4399} (\genbankdna{NM_032315}), \i{PEX14} (\genbankdna{NM_004565}), \i{PGD} (\genbankdna{NM_002631}), \i{PIK3CD} (\genbankdna{NM_005026}), and \i{SSB1} (\genbankdna{NM_025106}).} \results \p{No presumed pathogenic coding region mutations were identified in affected members in the two families. Seventeen previously described single nucleotide polymorphisms, four associated with missense amino acid substitutions and thirteen resulting in synonymous substitutions, were identified in eight of the candidate genes in these affected patients (\tabref{1}). Two of the four identified missense substitutions (Tyr1087Cys in \i{KIF1B} and Asp246Asn in \i{PGD}) were identified in the proband of Family 2 (\figref{1}{B}, II-1), but not in the proband's affected daughter (\figref{1}{B}, III-1). The other two missense substitutions (Arg453Gln and Pro554Leu) were identified in \i{H6PD} in members of Family 1 (\figref{1}{A}). The Arg453Gln mutation was also identified in an unaffected control individual, and while the Pro554Leu mutation was identified in the proband's affected mother (\figref{1}{A}, II-3), it was not identified in the proband (\figref{1}{A}, III-3).} \p{Four novel SNPs were identified. In \i{GPR157}, a c.795C\gt T (Arg218Leu) substitution was identified in each of the three affected members of Family 2 (\figref{1}{B}) and a c.811C\gt T substitution (Ala223Val) was identified in the affected daughter of the proband of Family 2 (\figref{1}{B}, III-1), but not in the proband (\figref{1}{B}, II-1) or the proband's affected sister (\figref{1}{B}, II-4). The Arg218Leu substitution was identified in 19/102 control chromosomes, and the Ala223Val substitution was identified in 9/102 control chromosomes. A c.1024G\gt C (Leu277Leu) substitution in \i{MGC4399} was identified in the three affected members and one unaffected member (III-2) of Family 2 (\figref{1}{B}). Additionally, a c.754A\gt C (Asp151Ala) substitution was identified in \i{H6PD} in the proband of Family 1 (\figref{1}{A}, III-3), but not in the proband's affected mother (\figref{1}{A}, II-3). This missense substitution was identified in 17/208 control chromosomes.} \discussion \p{Efforts to identify the genetic basis of SCCD began ten years ago when Shearman et al. [4] performed genome-wide linkage analysis in two large Scandinavian families, localizing the SCCD locus to a 16 cM region between the markers D1S2633 and D1S228 on the short arm of chromosome 1, region 34.1-36. Recently, Theendakara et al. [3] have further refined the SCCD candidate gene region, through an analysis of shared haplotype and recombination events in members of these previously reported and 11 additional families, to a 2.32 Mbp interval, reducing the number of identified positional candidate genes to 31. Linkage analysis (using the ten microsatellite markers that Theendakara et al. [3] used for genotyping in all 13 families) performed in the families that we report was not conclusive (data not shown), leaving us to assume that the disease locus in the families that we report is the same as in all previously reported kindreds. However, as there is no evidence for locus heterogeneity in SCCD, the absence of pathogenic mutations in the fifteen candidate genes that we screened strongly suggests that these genes are not involved in the development of SCCD in the families we report, or in other affected families. Although we performed direct sequencing of the coding region in each of the candidate genes, we are not able to definitively exclude the presence of mutations in the promoter region or the 5' or 3' untranslated regions of these genes. However, the absence of disease-causing coding region mutations in any of the positional candidate genes provides strong evidence that other genetic factors are involved in the development of SCCD, and thus we are in the process of screening the remaining positional candidate genes in these families and another recently identified family.} \acknowledgements \p{Support provided by The Emily Plumb Estate and Trust (AJA).} \references \p{1. Bron AJ, Williams HP, Carruthers ME. Hereditary crystalline stromal dystrophy of Schnyder. I. Clinical features of a family with hyperlipoproteinaemia. Br J Ophthalmol 1972; 56:383-99. \pubmed{4537849}} \p{2. Battisti C, Dotti MT, Malandrini A, Pezzella F, Bardelli AM, Federico A. Schnyder corneal crystalline dystrophy: description of a new family with evidence of abnormal lipid storage in skin fibroblasts. Am J Med Genet 1998; 75:35-9. \pubmed{9450854}} \p{3. Theendakara V, Tromp G, Kuivaniemi H, White PS, Panchal S, Cox J, Winters RS, Riebeling P, Tost F, Hoeltzenbein M, Tervo TM, Henn W, Denniger E, Krause M, Koksal M, Kargi S, Ugurbas SH, Latvala T, Shearman AM, Weiss JS. Fine mapping of the Schnyder's crystalline corneal dystrophy locus. Hum Genet 2004; 114:594-600. \pubmed{15034782}} \p{4. Shearman AM, Hudson TJ, Andresen JM, Wu X, Sohn RL, Haluska F, Housman DE, Weiss JS. The gene for schnyder's crystalline corneal dystrophy maps to human chromosome 1p34.1-p36. Hum Mol Genet 1996; 5:1667-72. \pubmed{8894705}} \p{5. Riebeling P, Polz S, Tost F, Weiss JS, Kuivaniemi H, Hoeltzenbein M. [Schnyder's crystalline corneal dystrophy. Further narrowing of the linkage interval at chromosome 1p34.1-p36?]. Ophthalmologe 2003; 100:979-83. \pubmed{14669035}} \endreferences } \beginfigures \figfile{1}{ \figtitle{1}{Pedigrees of two families with Schnyder crystalline corneal dystrophy} \p{Pedigrees of Schnyder crystalline corneal dystrophy Family 1 (\panel{A}) and Family 2 (\panel{B}) showing multiple affected members in consecutive generations. The black symbols represent individuals who were examined and found to be affected, gray symbols represent individuals considered affected based on history, and unfilled symbols represent unaffected individuals based on examination or history. The proband is identified with an arrowhead; family members who were examined and in whom DNA analysis was performed are identified with an asterisk. Individuals with an unknown affected status are identified with question marks.} \ctr{\gifimage{1}{550}{683}{18}} } \figfile{2}{ \figtitle{2}{Slit lamp examination of affected from Family 1} \p{\panel{A}: The 38-year-old proband demonstrating peripheral arcus and annular central corneal opacification with focal, superficial crystalline deposition. \panel{B}: Peripheral arcus and round central corneal opacification with crescentic crystalline deposition remaining following laser phototherapeutic keratectomy in the proband's 56-year-old mother. \panel{C}: Irregular, subepithelial, linear and polymorphic opacifications in the proband's 11-year-old son.} \ctr{\jpgimage{2}{500}{1149}{72}} } \begintables \tabfile{1}{ \tabtitle{1}{Candidate gene sequence variants in SCCD patients} \p{The c.754A\gt C change was identified in 17/104 control individuals (17/208 chromosomes) and the c.1024G\gt C change was identified in an unaffected member of Family 2.} \box{\pre{ Nucleotide Amino acid refSNP ID Gene change change (dbSNP build 123) ------- ---------- ---------- ----------------- CORT c.803C\gt T Ala129Ala rs628462 DFFA c.239T\gt G Leu61Leu rs1057017 GPR157 c.795C\gt T Arg218Leu None c.811C\gt T Ala223Val None H6PD c.754A\gt C Asp151Ala None c.938G\gt A Ala212Ala rs7524046 c.1043T\gt C Ala247Ala rs11121350 c.1660G\gt A Arg453Gln rs6688832 c.1963C\gt T Pro554Leu rs17368528 c.2321T\gt C Tyr673Tyr rs9434742 c.2420A\gt G Ser706Ser rs9434743 KIF1B c.1412G\gt A Thr409Thr rs17034660 c.3445A\gt G Tyr1087Cys rs2297881 c.4346A\gt G Pro1387Pro rs12125492 MGC4399 c.1024G\gt C Leu277Leu None PEX14 c.161T\gt C Phe52Phe rs12375 c.1037G\gt T Gly344Gly rs11539794 PGD c.822C\gt T Asp244Asp rs2229687 c.826G\gt A Asp246Asn rs2229688 PIK3CD c.3003T\gt C Tyr936Tyr rs11121484 SSBI c.686C\gt T Leu132Leu rs3795309 }} }