OPTOMETRY INVITED REVIEW. A review of current approaches to identifying human genes involved in myopia

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1 C L I I C A L A D E X P E R I M E T A L OPTOMETRY IVITED REVIEW A review of current pproches to identifying humn genes involved in myopi Clin Exp Optom 2008; 91: 1: 4 22 Wing Chun Tng Sc(Hons) Murice KH Yp PhD MCOptom FAAO She Ping Yip MPhil PhD FIMS School of Optometry nd the Deprtment of Helth Technology nd Informtics, The Hong Kong Polytechnic University, Hong Kong SAR, Chin E-mil: she.ping.yip@polyu.edu.hk Submitted: 13 Mrch 2007 Revised: 9 My 2007 Accepted for publiction: 17 My 2007 DOI: /j x The prevlence of myopi is high in mny prts of the world, prticulrly mong the Orientls such s Chinese nd Jpnese. Like other complex diseses such s dibetes nd hypertension, myopi is likely to be cused by both genetic nd environmentl fctors, nd possibly their interctions. Owing to multiple genes with smll effects, genetic heterogeneity nd phenotypic complexity, the study of the genetics of myopi poses complex chllenge. This pper reviews the current pproches to the genetic nlysis of complex diseses nd how these cn be pplied to the identifiction of genes tht predispose humns to myopi. These pproches include prmetric linkge nlysis, non-prmetric linkge nlysis like llele-shring methods nd genetic ssocition studies. sic concepts, dvntges nd disdvntges of these pproches re discussed nd explined using exmples from the literture on myopi. Microstellites nd single nucleotide polymorphisms re common genetic mrkers in the humn genome nd re indispensble tools for gene mpping. High throughput genotyping of millions of such mrkers hs become fesible nd efficient with recent technologicl dvnces. In turn, this mkes the identifiction of myopi susceptibility genes relity. Key words: genetics, linkge, linkge disequilibrium, myopi, polymorphism Myopi is the most common eye disorder in the world. The prevlence of myopi is estimted to be 17, 26 nd 27 per cent in Austrli, the United Sttes nd Western Europe, respectively. 1 7 It is especilly high (71 to 96 per cent) in Asin regions nd countries including Hong Kong, Tiwn nd Singpore Severely myopic eyes re prone to degenertive chnges, such s retinl degenertion nd glucom, which cn led to visul impirment. In the long term, such problems will impose hevy burden on the helth cre system nd the economy of the society concerned. The development of myopi is usully due to the excessive growth of the eyebll, s evident from mny biometric studies on the eyes of humns nd other nimls. 12 The etiology or mechnism underlying this bnorml oculr growth is still uncler, while the debte bout the role of nture nd nurture in myopic development continues. 13,14 From simplistic viewpoint of genetics, myopi cn pper s prt of rre disese syndrome, s rre monogenic trit (or disese), or more commonly, s complex trit (or disese) (Figure 1). Myopi, usully high myopi, is sometimes presented s one of the fetures in wide vriety of rre heritble disese syndromes. 15 Mny of these uncommon syndromes re single-gene disorders, such s Stickler syndrome nd Mrfn syndrome, nd some re chromosoml in nture such s Down syndrome. There re lso rre cses of myopi, typiclly high myopi, tht show simple Mendelin inheritnce ptterns (dominnt, recessive or sex-linked). 16 y definition, such cses probbly represent the monogenic form of myopi, which is cused by muttions in single gene. It is very likely tht there is hndful of such myopi genes nd muttions in ech of these genes re expected to cuse directly the monogenic form of myopi. 4

2 Methods for mpping genes Prmetric linkge nlysis on-prmetric linkge nlysis nd Assocition study Gene or locus Mrfn syndrome (15q) Stickler syndrome (6p & 12q) ornholm eye disese (MYP1, Xq28) MYP2 (18p) MYP3 (12q) MYP4 (7q) MYP5 (17q) MYP6 (22q) Locus 1 Locus 2 Locus 3 Locus 4 Figure 1. Myopi cn pper s prt of syndrome, in rre monogenic form or more commonly s complex trit/disese. The boxes represent syndromes (with myopi s one of the presenting fetures) or myopi (monogenic or complex forms). The size of the boxes serves s very crude indiction of the frequency of the syndromes or different forms of myopi. The reltive thickness of the rrows serves to roughly indicte the reltive contribution of genetic nd environmentl fctors to the genesis of different forms of myopi. The influence of environmentl fctors on the monogenic form is probbly miniml, s indicted by the question mrk. The complex form of myopi is produced by the ction of mny genes (locus 1, locus 2 et ceter) nd mny environmentl fctors. Also indicted on the left re the pproprite methods for mpping genes involved in monogenic nd complex forms of myopi. ote tht the first myopi locus MYP1 ws ssigned to the clinicl syndrome ornholm eye disese mnifested s mblyopi, myopi nd deuternopi. MYP1, MYP2 et ceter re stndrd symbols of myopi genes pproved by the HUGO Gene omenclture Committee. The pproximte chromosoml loctions (15q, 18p et ceter) re indicted within brckets fter the nmes of the syndromes or myopi genes. On the other hnd, there is evidence tht both heredity nd environment ply role in the development of common myopi (Figure 1). Common myopi is like mny other common diseses tht re complex or multifctoril in nture Myopi? Environmentl fctors Environmentl fctors erwork Form deprivtion Monogenic Complex (Multifctoril) nd result from the effects of both genetic nd environmentl fctors Environmentl fctors such s excessive ner work nd form-deprivtion my interct with genetic fctors to produce bnorml eye growth. y definition, complex disese/ trit is cused by multiple genes, multiple environmentl fctors nd possibly genegene nd gene-environment interctions. Therefore, complex disese/trit tends to run in fmilies but does not show typicl Mendelin inheritnce pttern. A single susceptibility gene is neither necessry nor sufficient to cuse complex disese/trit nd genetic effects involve probbilistic predisposition rther thn predetermined progrmming. Complex diseses re exemplified by dibetes, hypertension nd coronry hert disese, while common exmples of complex trits include height, body weight nd blood cholesterol concentrtion. As n nlogy with myopi, non-insulindependent (type 2) dibetes shows the chrcteristics of complex disese. It lso hs rre monogenic form showing typicl Mendelin inheritnce, which is cused by muttions in one of severl different genes. Twin studies hve presented strong evidence for genetic inheritnce in the control of eye size nd refrctive errors. Monozygotic twins hve much higher concordnce of myopi nd its relted oculr components, such s xil length, nterior chmber depth nd cornel curvture, thn dizygotic twins Fmily studies indicte tht children re more likely to develop myopi nd hve longer nterior nd vitreous chmbers even before becoming myopic, if their prents re myopic. 18,27,28 In generl, estimtes of heritbility indicte tht xil length, nterior chmber depth nd cornel curvture re predominntly geneticlly determined This suggests role for genetic influences in eye shpe nd development of myopi. Most myopi is due to excessive eye growth nd individuls with n pprently inherited form of the condition exhibit excessive elongtion of the eye. 26,34 Thus, xil length nd refrctive error re similr to height, which is continuous nd complex trit nd is determined by both genetic fctors nd environmentl fctors (like diet nd exercises). Insted of single gene, multiple genes exhibit effects on the vritions in both eyebll size nd height. 5

3 Heterogeneity nd complexity in phenotypic expression mke the study of complex diseses much more chllenging. 35 Heterogeneity refers to the sitution in which disese is contributed by more thn one gene or different genetic vritions in the sme gene or different genes. The expression of phenotype cn be vrible in tht the sme genetic vrition cn pprently result in different degrees of severity for disese. In ddition, some individuls with prticulr genetic vrition my not express the phenotype, while others without the genetic vrition my somehow express the phenotype becuse of some other cuses. Despite these complexities, mny reserchers mke serious ttempts t mpping the genes of myopi in humns by different pproches of genetic nlysis. Gene mpping refers to the process of locting or identifying gene or group of genes within our genome for subsequent studies. As the study of complex diseses identifies significnt contribution of heredity to their development, it is likely tht more genes will be found to influence susceptibility to complex diseses rther thn to cuse disese directly. Genetic susceptibility refers to the bility of genetic fctors to ffect the risk of contrcting disese. Exposure to pproprite environmentl risk fctors my trigger the expression of the genetic effects on complex disese, lter its mnifesttion or excerbte its severity. Identifiction of susceptibility genes of myopi will shed light on the pthophysiologicl mechnism underlying their genesis. Undoubtedly such informtion is importnt for the design of preventive nd therpeutic mesures in the future. One cn imgine tht, in the not too distnt future, it my be possible to use genetic tests to identify geneticlly susceptible individuls t very young ge even before myopi develops. Such individuls my be dvised to modify their lifestyle (such s reding hbits) or treted to dely the onset of myopi, slow down the progression or even prevent it from developing. Susceptible individuls with different bckgrounds of genetic predisposition my develop myopi on exposure to different environmentl triggers nd need different kinds of dvice nd different tretment modlities. One tretment my be effective for those of one prticulr genetic bckground but not those of nother. This form of long-wited personlised medicine cn be relised only with the elucidtion of the moleculr pthwys involved in the trigger nd development of myopi. In turn, this relies on the identifiction of myopi genes nd the subsequent study of their functions. The purpose of this rticle is to elucidte the pproches of genetic epidemiology to identifying the genes involved in myopi. This review is intended to help optometrists in the understnding nd interprettion of reports on genetics of myopi nd other complex eye diseses. efore going through these pproches, different types of genetic mrkers tht re useful tools in disese gene mpping re first introduced. Such informtion is essentil for understnding the bsic concepts of genetic nlysis. GEETIC MARKERS Deoxyribonucleic cid (DA) contins the genetic instructions for the biologicl processes of cellulr form of life nd is responsible for the genetic propgtion of inherited trits. In humns, these trits rnge from eye colour to disese susceptibility. Muttion is heritble ltertion of this genetic mteril. It cn result from errors tht occur during the repliction of DA before cell divides nd tht escpe the repir mechnisms of the cell. It cn lso be induced by substnces known s mutgens nd cn be trnsmitted to subsequent genertions. Muttion is the ultimte source of genetic vritions mong individuls of the sme species nd mong difference species. If vrition in DA sequence occurs by chnce nd reches frequency of t lest one per cent in popultion, it is clssiclly clled polymorphism. ot ll polymorphisms hve phenotypic effects or direct effects on the genesis of disese. Some my hve no effect t ll nd some my just ply role in whether person hs high or low risk of prticulr disese. The term polymorphism is neutrl term nd cn simply men sequence vrition. y contrst, prticulrly in clinicl setting, muttion is used typiclly in more restricted nd negtive sense, implying disese-cusing genetic vritions. A locus is specific physicl loction in chromosome of the genome. A gene is stretch of DA sequence tht instructs cell to produce prticulr product, usully protein. Alleles re lterntive forms (two or more forms) of ny defined gene or DA sequence t given locus, nd usully differ from one nother by one or few bses in their DA sequences. In humns, ll cells except sex cells (sperm cells nd eggs) re diploid nd hence they ech contin two sets of chromosomes, one from ech prent. The terms homozygous nd heterozygous re used to describe the presence of the sme llele nd different lleles, respectively, t locus for given pir of homologous chromosomes. Genotype is the specific genetic mke-up of n individul, in the form of DA. Together with the environmentl fctors tht influence the individul, it codes for the phenotype (observble trit) of tht individul. A genetic mrker is n inherited genetic trit with different possible forms tht re redily recognisble nd help reserchers tell prt different individuls in n experiment. Any DA sequences existing s two or more distinguishble lterntive forms (lleles) cn be used s genetic mrkers. If disese is inherited in simple pttern, the inheritnce of disese lleles or genetic vrints cn be followed in pedigrees. Known polymorphisms re useful genetic mrkers for genetic studies or gene mpping. Rre muttions usully contribute to rre Mendelin genetic diseses. In recent yers, mny reserchers believe tht common genetic vritions contribute to the genetic risk of developing complex diseses. 36,37 Mny studies relte common genetic vritions to the clinicl phenotypes of complex diseses, drug responses nd environmentl fctors. There is no doubt tht the development of high-resolution mp of genetic vritions is very importnt prt of the Humn Genome Project (HGP), in which the DA sequences nd the genes of the 6

4 entire humn genome re determined. The complete sequence is now known nd this provides severl million polymorphisms spreding cross the genome for genetic studies Two types of genetic mrkers re used commonly in gene mpping of complex diseses: microstellites nd single nucleotide polymorphisms (SPs). They re bundntly present in the humn genome. The single nucleotide polymorphism dtbse (dbsp) t the tionl Centre for iotechnology Informtion (CI) provides detiled informtion of these genetic vritions in humns nd mny other different orgnisms. The informtion includes the surrounding sequence context of the polymorphism, the llele frequency of the polymorphism nd the experimentl methods, protocols nd conditions used to ssy the vrition. Microstellites Microstellites re lso clled short tndem repets or simple sequence length polymorphisms nd re rrys of short repet sequences (usully di-, tri- nd tetr-nucleotide repets) tht disply length vritions with different lleles crrying different numbers of repet units (Figure 2A). They re multi-llelic, usully between five nd 10 or more lleles t ech locus. For exmple, there my be five lleles t certin locus of GT dinucleotide tndem repet polymorphism nd ech llele hs GT repet for 11, 12, 13, 14 or 15 times, giving length differences in the multiples of two bses (or nucleotides). Such DA length differences cn be detected by utomtic DA sequencers vi cpillry gel electrophoresis, which seprtes microstellite-contining frgments tht re first mplified by obel prize-winning technique clled polymerse chin rection. Genetic mps, which re composed of severl hundred evenly spced microstellites with known positions, llow loctions of unknown genes to be determined in reltion to those of microstellites. Such informtion is esily ccessed in CI dtbses. Microstellites re usully pplied to linkge nlysis, which cn mp gene of interest to lrge region of chromosoml segment (A) Microstellite (GT repets)..tac(gtgtgtgtgtgtgtgtgtgtgt)acc....tac(gtgtgtgtgtgtgtgtgtgtgtgt)acc....tac(gtgtgtgtgtgtgtgtgtgtgtgtgt)acc....tac(gtgtgtgtgtgtgtgtgtgtgtgtgtgt)acc....tac(gtgtgtgtgtgtgtgtgtgtgtgtgtgt )ACC.. () Single nucleotide polymorphism (SP) GCCTCCGTCAGTGCTGCCT GCCTCCATCAGTGCTGCCT Figure 2. Exmples of microstellite nd single nucleotide polymorphism. A: The top pnel shows multi-llelic microstellite with ech llele crrying t lest 11 GT dinucleotide repets (plced within brckets for the ske of clrity). : The bottom pnel shows di-llelic single nucleotide polymorphism with G llele nd n A llele (underlined). Short stretches of DA sequences flnking these genetic mrkers re lso shown. extending few thousnd kilobses (kb). Mny loci for high nd low myopi were successfully mpped by linkge nlysis using microstellites (Tble 1) Single nucleotide polymorphisms (SPs) A SP (pronounced s snip) is DA sequence vrition due to the chnge in single bse (Figure 2). About 99.9 per cent of the DA sequences in the genome of ny two unrelted individuls re identicl. Of the 0.1 per cent difference, SPs ccount for over 80 per cent of the sequence vritions in the humn genome. Although they re distributed unevenly in the genome, on verge they cn be found t intervls of 500 to 1,000 bsepirs. 39 The simple bi-llelic nture of SPs llows gret potentil for utomted genotyping t different levels of throughput Indeed, the vilbility of mny different SP genotyping methods (detils not mentioned here) fcilittes nd ccelertes the gene mpping process of complex eye diseses. Vritions occurring in the coding sequences (exons) of gene re termed coding SPs. DA sequence vritions tht re not expressed t the protein level re clled non-coding SPs. on-coding SPs cn be found in introns (non-coding sequences between exons of gene), the promoter region, 5 untrnslted region (5 UTR), 3 untrnslted regions (3 UTR) of gene nd the lrge mount of DA sequences between ny two genes. Some coding SPs my hve functionl consequences nd hence phenotypic effects, if they chnge the mino cid sequence of the protein encoded by the gene nd they re clled non-synonymous. For exmple, if codon sequence GGC is chnged to GAC, the encoded mino cid will be chnged from glycine to sprtic cid nd this is non-synonymous chnge. Some SPs in exons do not chnge encoded mino cid. For exmple, if codon GGC is chnged to GGA, the sme mino cid (glycine) is encoded nd this is clled synonymous chnge. In ddition, not ll of the functionl chnges lie within exons. 7

5 Myopi Authors (yers) Inheritnce/ Loction Ethnicity of subjects Types of fmilies Linkge locus QTL nlysis (PL or PL) Affected sttus Mx LOD MYP1 Schwrtz et l (1990) 41 XR Xq28 Dnish Lrge pedigrees PL 4.80 MYP2 Young et l (1998) 42 AD 18p11.31 Americn nd Chinese Moderte to lrge PL D SE 9.59 multigenertionl fmilies MYP2 Lm et l (2002) 43 AD 18p11.31 Hong Kong Chinese Moderte pedigrees PL D 2.10 MYP3 Young et l (1998) 44 AD 12q21-23 Germn/Itlin A lrge pedigree PL D SE 3.85 MYP3 Frbrother et l (2004) 45 AD 12q21-23 UK popultion ucler fmilies PL, PL D in the lest negtive meridin of both eyes MYP4 iglin et l (2002) 46 AD 7q36 French nd Algerin Lrge to moderte pedigrees PL, PL D both eyes 2.81 MYP5 Pluru et l (2003) 47 AD 17q21-22 English/Cndin A lrge pedigree PL D SE 3.17 MYP6 Stmbolin et l (2004) 48 AD 22q12 Americn fmilies of Ashkenzi Jewish descent Lrge pedigrees PL, PL D in ech meridin for both eyes MYP6 Stmbolin et l (2006) 49 AD 22q12 Additionl Jewish descent Pedigrees PL, PL D in ech meridin for both eyes MYP6 Klein et l (2007) 50 QTL 22q12 Americns of orthern Europen nd/or Germn ncestry Sib-pirs PL Men D SE; rnge: to D MYP7 Hmmond et l (2004) 51 QTL 11p13 UK popultion Dizygotic twin pirs PL Men SE < 0 D 6.10 MYP8 Hmmond et l (2004) 51 QTL 3q26 UK popultion Dizygotic twin pirs PL Men SE < 0 D 3.70 MYP9 Hmmond et l (2004) 51 QTL 4q12 UK popultion Dizygotic twin pirs PL Men SE < 0 D 3.30 MYP10 Hmmond et l (2004) 51 QTL 8p23 UK popultion Dizygotic twin pirs PL Men SE < 0 D 4.10 MYP10 Stmbolin et l (2005) 52 AD 8p23 Old Order Amish Fmilies with ffected sibs PL, PL D in ech meridin for both eyes MYP11 Zhng et l (2005) 53 AD 4q22-27 Hn Chinese in smll villge of centrl Chin P vlue = A lrge pedigree PL Rnge: to D 3.11 MYP12 Pluru et l (2005) 54 AD 2q37.1 US fmily of northern Europe A lrge pedigree PL D SE; rnge: to D MYP13 Zhng et l (2006) 55 XR Xq23-25 Chinese A lrge pedigree PL D SE; rnge: to D MYP13 Zhng et l (2007) 56 XR Xq Chinese A lrge pedigree PL D SE; rnge: to D MYP14 Wojciechowski et l (2006) 57 QTL 1q36 Ashkenzi Jewish Moderte to lrge multigenertionl fmilies MYP15 Klein et l (2007) 50 QTL 1q41 Americns of orthern Europen nd/or Germn ncestry MYP16 Klein et l (2007) 50 QTL 7p21 Americns of orthern Europen nd/or Germn ncestry MYP17 llsmy et l (2007) 58 AD 10q21.2 Hutterite popultion from South Dkot PL Men D SE; D in ech meridin for both eyes Sib-pirs PL Men D SE; rnge: to D Sib-pirs PL Men D SE; rnge: to D A lrge pedigree PL Men D; rnge to D P vlue = P vlue = Myopi loci re shown in stndrd gene symbols pproved by Humn Gene omenclture Committee but MYP15, MYP16 nd MYP17 re newly identified nd the gene symbols re still tenttive Inheritnce is indicted s X-linked recessive (XR), utosoml dominnt (AD). QTL represents quntittive trit locus. PL represents prmetric linkge nlyses wheres PL represents non-prmetric linkge nlyses, usully ffected sibpir nlysis SE represents sphericl equivlent in dioptres (D) Mx. LOD stnds for mximum logrithm of the odds score. Only P vlues re given for MPY6, MYP15 nd MYP16 in Klein s study. Tble 1. Myopi loci mpped by linkge nlysis 8

6 on-coding SPs my hve functionl consequences, if they ffect the regultion of gene trnscription, mra splicing or mra stbility. Promoters nd regultory elements ffect gene trnscription nd re usully locted t 5 UTR nd the upstrem region of gene. Some regultory elements re found t 3 UTR or in introns where they my be close to the splice sites. 62 Therefore, efforts to identify functionl SPs should include the proximl nd distl regultory sequences in the 5 nd the 3 ends of the gene. SPs re present in helthy norml individuls, who cn be recruited esily for ny study. SP screening within cndidte genes (smll-scle) or throughout the whole genome (lrge-scle) cn be conducted before the collection of ptient smples is completed. Thus, SPs hve become the most populr genetic tools for gene mpping of complex diseses. The CI mintins the dbsp dtbse s the mjor public repository for SP informtion, lthough mny other SP dtbses (with or without chrges) re lso vilble in both public nd privte sectors. More thn five million SPs with minor llele frequency greter thn 10 per cent re expected to exist in the humn genome. 63 In fct, bout four million SPs cross the humn genome in four different ethnic popultions (igerin, Americn, Jpnese nd Chinese) hve now been genotyped in the Interntionl Hplotype Mp (HpMp) Project (see below). SP llele frequencies nd their linkge disequilibrium (LD; see below) ptterns cn be esily ccessed in the HpMp dtbse. Comprison of microstellites nd SPs Microstellites re more informtive when compred with SP mrkers. It is becuse, on verge, mny microstellites re heterozygous in bout 70 per cent of humn smples tested. 64 They re often locted between genes rther thn within genes. Clssiclly, genetic studies of linkge nlysis use microstellites to locte disese gene to corse chromosoml region. SPs within nd flnking genes in the cndidte region cn then be used s genetic mrkers for further studies. Although single SP mrker is less informtive thn microstellite mrker, nlysis of multiple SPs, nd hence hplotypes, enriches the informtion content nd improves the efficiency for gene mpping. Hplotype is the combintion of lleles of two or more loci closely linked on the sme chromosome. SPs re lso more stble, especilly for those tht re not involved in mino cid chnge becuse they re less likely to be subjected to nturl selection nd thus, do not chnge much from genertion to genertion. 65 This mkes them esier to follow in popultion studies. PHEOTYPE DEFIITIO A phenotype refers to specific feture of n individul tht cn be observed, ctegorised or mesured by some mens. From the perspective of gene mpping, phenotypes cn be clssified into two brod types: qulittive (or discontinuous) nd quntittive (or continuous) trits. A qulittive trit is one with two or more distinct ctegories nd ech ctegory cn be esily seprted from ll other ctegories (for exmple, the AO blood group of n individul). In most medicl conditions, disesed trits re recognised by their stte of being significnt devitions from the norm. Signs nd symptoms indicte whether disese stte is present or bsent. On the other hnd, quntittive trit exhibits wide rnge of possible ctegories tht show continuous spectrum of vrition (for exmple, body weight nd blood pressure). It does not follow typicl Mendelin inheritnce. Very likely, it is controlled by mny genes with smll effects tht re influenced by the environment nd their interction with other loci. Myopi is refrctive error tht is mesured in continuous scle nd in the unit of dioptre (D). The refrctive error of n eye is the result of the totl refrctive power of oculr components, which include xil length, nterior chmber depth, cornel power nd lens thickness, which re lso continuous trits. Trits with well-defined Mendelin ptterns of phenotypic expression re optiml for linkge nlysis (Figure 3). Therefore, continuous trits re usully converted into discontinuous or dichotomous trits by defining threshold for the disese of interest for genetic studies. Different definitions of myopi hve been dopted in different clinicl nd genetic studies nd this leds to difficulties in compring the results mong studies. The most common definition of myopi is refrctive error with equivlent sphericl power less thn D. In most studies of linkge nlysis, low myopi is defined s refrctive error between D nd D, wheres high myopi refers to refrctive error equl to or over D. A few genetic studies used more minus refrctive power s the cutoff threshold for high myopi to increse the sttisticl power to detect modest nd smll gene effects. 41,66,67 Most myopic eyes hve longer xil length. 26,34,67 While heredity hs strong impct on xil length, the role of heredity on nterior chmber depth nd cornel power is still controversil 26,29 33,70 but profiles of these oculr components vry mong myopes. Strict inclusion criteri nd creful phenotyping of myopic subjects re importnt for the success of genetic studies of myopi. Axil length cn be tken into ccount in myopic subject recruitment to reduce the complexity of the genetic fctors potentilly involved. As lrge mounts of stigmtism will hve n effect on the finl equivlent sphere, myopic subjects with high stigmtism (-2.00 D or worse is suggested but there is no consensus) 68,69 should be excluded. This is becuse the genetic nd environmentl influences of stigmtism my be different from those of myopi. To illustrte this potentil clssifiction error, consider n eye with D of sphericl error nd D of stigmtism. The finl equivlent sphere will be D, qulifying it to be clssified s n eye with high myopi. APPROACHES TO FIDIG GEES IVOLVED I MYOPIA Although there re two mjor clssicl pproches to finding genes involved in humn trits nd diseses, one of these pproches known s genetic mpping is 9

7 Gene effect size more pproprite for identifying genes for myopi. This genetics-bsed pproch relies only on the behviour of genes during trnsmission from genertion to genertion in fmily. It cn be pplied even though the underlying moleculr mechnism of the disese of interest myopi is not known. In fct, the only requirement for pplying this pproch is tht the disese or trit cn be clerly defined. Indeed, myopi cn be defined objectively nd unmbiguously by the mount of refrctive error. With genetic mpping, there re two strtegies: linkge nlysis nd ssocition study (Figure 3). For ech strtegy, there re different methods tht del with different scenrios nd issues. Linkge nlysis cn be prmetric or non-prmetric. Linkge nlysis cses Frequency in popultion controls Assocition study Figure 3. Gene mpping strtegies for diseses different in popultion frequency nd in gene effect size. Genes involved in rre Mendelin diseses show lrge effects nd re best mpped by prmetric linkge nlysis of lrge fmilies. Genes involved in common complex diseses exhibit smll effects nd re best identified by genetic ssocition studies like cse-controlled studies nd fmily-bsed ssocition studies. on-prmetric linkge nlysis (not shown) flls between these two pproches. Prmetric linkge nlysis requires the ssumption of correct genetic model, while non-prmetric linkge nlysis does not ssume ny genetic model. On the other hnd, ssocition studies cn be popultion-bsed or fmily-bsed. Popultion-bsed ssocition studies work on unrelted individuls with or without the disese of interest, while fmily-bsed ssocition studies work on ffected children from smll nucler fmilies. LIKAGE AALYSIS Prmetric linkge nlysis If disese gene nd genetic mrker locus re locted on different chromosomes, the lleles of the disese gene re trnsmitted to the next genertion independently of the lleles of the mrker locus. In other words, these two sets of lleles re free to form new combintions during trnsmission to the next genertion sitution s dictted by Mendel s second lw (the principle of independent ssortment). If disese gene nd genetic mrker locus re locted fr prt on the sme chromosome, the chnce of exchnging genetic mteril nd hence the lleles (tht is, recombintion) between these two loci is very likely to occur s result of crossing-over between pir of homologous chromosomes during meiosis. In other words, the trnsmission of the lleles of these two loci still obeys Mendel s second lw. Thus, the trnsmission of lleles t ech locus from genertion to genertion is rndom nd independent of the other locus under these two conditions. These loci re sid to be unlinked. In this cse, the rtios of lleles of the two loci observed in ech genertion will be close to those predicted by the Mendelin principle of independent ssortment nd the rte of recombintion is 50 per cent. If the mrker locus is close to the disese gene on the sme chromosome, they re more likely to segregte together (without crossing-over) rther thn independently during meiosis. Linkge between these two loci occurs nd the recombintion rte is less thn 50 per cent. 71 In other words, the recombintion rte becomes smller when two loci re much closer to ech other. y tking dvntge of these properties of meiosis, mesurement of genetic linkge cn tke plce in fmily studies for disese gene mpping. Linkge nlysis is method for determining if there is significnt evidence for co-segregtion of lleles t mrker locus nd lleles t hypotheticl disese locus. It involves explining the inheritnce ptterns of genotypes observed in pedigree ffected by disese. This nlysis is useful only when the disese model is correctly specified nd usully is pplied to simple Mendelin diseses or trits, which show strong effects of the muttions involved. Thus, this method is clled model-bsed 10

8 (or prmetric) linkge nlysis. If disese is cused by dominnt muttion (M) t gene G on chromosome, ll ffected members in pedigree should inherit this chromosoml region round the mutnt llele (Figure 4). Assume tht two genetic mrkers re close to ech other (linked) on the sme chromosome. When the disese muttion is locted between these two linked genetic mrkers, ffected members in the pedigree re expected to inherit two prticulr lleles of these two mrkers (A nd b) together with the mutnt llele (M) of the disese gene from n ffected prent. The known position of the linked mrkers indictes the pproximte chromosoml loction of the disese-cusing gene. The linkge of mrkers to disese muttion is usully quntified by the logrithm of the odds (LOD) score, which provides sttisticl evlution of the cosegregtion of the mrker nd the muttion. Under specific mode of inheritnce (for exmple, utosoml dominnt), the likelihood of getting specific mrker lleles nd disese (tht is, the disese muttion) in lrge fmily under two hypotheses re compred. The hypothesis tht the mrker is linked to the disese locus t prticulr recombintion rte (θ) is compred to the null hypothesis tht there is no linkge between the mrker nd the disese locus (tht is, θ = 0.5). Therefore, likelihood rtio cn be clculted s the rtio of the likelihood of the dt, if the loci re linked t prticulr θ to the likelihood of the dt, if the loci re unlinked (θ = 0.5). The log 10 of this rtio is the LOD score. The LOD score serves to indicte whether there is linkge t prticulr vlue of θ between the mrker nd the disese locus. A LOD score of 3 (n odds rtio of 1000 : 1) is trditionlly greed s evidence of linkge. 35 The LOD score pproch hs the dvntge tht linkge studies cn be compred nd nlysed together. It is becuse LOD scores cn be simply dded together (tht is, odds rtios multiplied together) when the linkge for different fmilies with the sme genetic mrkers is investigted. For exmple, if the LOD scores of fmilies A A M* b A M* b Unffected femle Unffected mle Figure 4. Linkge nd recombintion. Allele M is the disese llele cused by muttion in the gene G, wheres llele is the norml llele t the sme locus. ote tht llele M is mrked by n sterisk for esy recognition. The ffected fther (blck squre in the first genertion) crries the disese llele M on chromosome together with mrker lleles A nd b. Two ffected offspring (shown on the left in the second genertion) lso inherit the lleles A nd b from the fther becuse these two lleles re linked to the disese-cusing llele M on the sme chromosome. The youngest ffected dughter (shown on the right) inherits the mrker llele (insted of llele b) from the fther s result of recombintion between the disese locus (M/) nd the mrker locus (/b) during meiosis. The disese locus is linked to these two mrker loci nd is closer to mrker locus A/ thn to mrker locus /b. A M* b A M* Affected femle Affected mle nd re 1.5 nd 2.5, respectively, t θ = 0.01, neither shows convincing evidence of linkge on its own. If two LOD scores re dded together, the dt become significnt with LOD score of 4.0 but the sme dignostic criteri nd genetic model must be used for both fmilies. Moreover, lrge pedigree with severl genertions is more informtive thn severl smll fmilies ech with fewer genertions nd hence more powerful to detect linkge. To increse the power of detecting linkge, severl mrker loci cn be simultneously tested nd nlysed for linkge with the disese: multipoint linkge nlysis. The combintion of lleles of these genetic mrkers tht inherit together in fmily with the disese is clled the linked hplotype. For exmple, the disese muttion (llele M) nd the mrkers (lleles A nd b) re inherited together on the sme chromosome, s hplotype unit (A-M-b) in two ffected offspring (Figure 4). The recombinnt hplotype A-M- is trnsmitted to one ffected offspring (Figure 4). In pst decdes, mny eye disese or eye-relted loci were successfully mpped 11

9 Prmetric linkge nlysis on-prmetric linkge nlysis Fmily smples Lrge pedigrees with mny disesed subjects At lest two ffected members for ech fmily est when prents vilble Mode of inheritnce Assumption of mode of inheritnce Model-free (AD, AR nd X-linked) Sttisticl power High for Mendelin diseses Low for complex diseses Moderte for Mendelin diseses nd lrge genetic effects of complex diseses Advntges Disdvntges Highest power for Mendelin diseses More efficient using genome scn pproch eed to scertin pedigrees tht re difficult to find Specific genetic model required Limited by heterogeneity if present AD stnds for utosoml dominnt nd AR utosoml recessive Model-independent High power to detect moderte nd lrge genetic effects of complex diseses Genome scn possible Problems of genotyping errors, non-pternity nd smple mix-up detectble only when prents nd/or siblings vilble ot sensitive to detect smll genetic effects Tble 2. Comprison between prmetric nd non-prmetric linkge nlyses by the study of lrge pedigrees with linkge nlysis (Figure 3). These eye diseses were usully monogenic, polygenic or syndromic (syndromes ssocited with oculr mnifesttions), such s Stickler syndrome 72 nd Mrfn syndrome. 73 They follow typicl modes of inheritnce: utosoml dominnt (AD), utosoml recessive or X-linked. On the other hnd, gene mpping for complex eye disorders including myopi is still in its infncy. Linkge nlysis is usully used s the first step in the study of complex eye diseses, s there is very little knowledge of the disese mechnism. For lrge-scle studies, whole genome scn is crried out with severl hundred (400 to 800) microstellites spced evenly throughout the genome. This mens systemtic serch for genetic effects t different loctions long ll humn chromosomes but it is very expensive nd time-consuming. Tble 1 summrises the myopi loci mpped to dte. Updted informtion cn be ccessed esily t Online Mendelin Inheritnce in Mn (OMIM). The first myopi locus MYP1 ws mpped to Xq28 in 1990 nd ws responsible for X-linked high myopi (pthologicl myopi) ssocited with mblyopi nd deuternopi. 41 More loci for high myopi were found subsequently, using linkge nlysis with the ssumption of AD models in most cses. These non-syndromic high myopi loci were locted t chromosome 18p11, 42,43 12q21-23, 44,45 7q36, 46 17q21-22, 47 4q22-27, 53 2q37, 54 Xq ,56 nd 10q The symbols p nd q represent the short nd the long rms of chromosome, respectively, nd the numbers before nd fter p or q indicte the chromosome number nd the sub-region in the corresponding rm respectively. The loci for low myopi were mpped t chromosome 22q12 nd 1q36 by Stmbolin s group nd Wojciechowski s group, respectively. 48,49,57 Different myopi loci mpped for different ethnic groups support the presence of genetic heterogeneity. Complex eye disorders clerly do not follow Mendelin inheritnce ptterns. Stndrd linkge nlysis hs limited power in detecting such smll genetic effects in these disorders. If wrong model is specified, it is likely to miss the true linkge. Moreover, it is usully very difficult to recruit lrge pedigrees. Mpping genes for complex eye disorders ws menble to non-prmetric linkge nlysis nd ssocition study. oth prmetric nd non-prmetric nlyses were dopted for the sme set of fmily smples in few studies on myopi to increse the chnces of identifying the myopi genes. 45,46,48,49,52 on-prmetric linkge nlysis: llele-shring methods The llele-shring method, lso known s ffected pedigree member method, is n lterntive to the prmetric linkge nlysis for identifying disese locus nd hs some dvntges over clssicl linkge nlysis (Tble 2). First, no ssumption bout the mode of inheritnce is needed. Second, smll nucler fmilies insted of lrge pedigrees re used nd re esier to recruit. Third, the frction of geneticlly heterogeneous phenotype tht is determined by given locus cn be estimted. This method is more robust thn prmetric linkge nlysis but less powerful thn the linkge nlysis with correctly specified mode of inheritnce. The lleleshring method is still very useful when the disese models re uncler, especilly for complex diseses. The llele-shring method ims t determining if ffected reltives in pedigree inherit specific copies of chromosoml region more often thn expected by chnce of rndom segreg- 12

10 A M* b tion (Figure 5). 35 In other words, it studies how often prticulr llele t mrker locus is shred identicl-by-descent (ID) within the pedigree. ID mens the inheritnce of the sme llele from common ncestor. If there is disese-cusing muttion in specific chromosoml region in high proportion of fmilies, two ffected individuls from the sme fmily will shre n llele of the mrker locus more often thn expected by chnce (50 per cent for sibling pirs). Affected sibpir (ASP) nlysis is the simplest type of llele-shring method nd is commonly used for gene mpping of complex eye diseses or trits. ASP nlysis ignores the ffection sttus of the sibs prents nd requires ffected sibling pirs. Thus, this pproch is very useful if prentl genotype informtion is missing. In ASP nlysis, the men proportion of mrker lleles shred by two ffected siblings in nucler fmily is compred with 50 per cent shred proportion expected by rndom Mendelin segregtion. Under rndom segregtion, the expected distribution of shring of 0, 1 or 2 lleles ID is 25, 50 nd 25 per cent, respectively, between the sibling pirs of nucler fmily. If enough sibling pirs from mny nucler fmilies re compred, excess shring of lleles ID is mesured by simple chi-squre (χ 2 ) test. Significnt χ 2 indictes lrge devition from the expected distribution nd implies the linkge between the disese nd the mrker. 35 ASP nlysis mps genes to corse chromosoml region nd is not very powerful in detecting smll genetic effects of complex diseses. Further investigtion of positive loci should be followed by the lterntive genetic pproch: ssocition studies. As huge number of genetic mrkers nd dvnced genotyping technologies is now vilble, llele shring methods combined with the whole-genome scn pproch hve been used frequently to identify corse chromosoml regions of mny multifctoril eye diseses. Hmmond nd collegues 51 mpped new myopi loci to chromosomes 11p13, 3q26, 4q12 nd 8p23 by using ASP with twin pirs (Tble 1). The locus on chromosome 8p23 ws confirmed lter in the Old Order Amish by Stmbolin s group using the ASP method. 52 Recently, using sibpir nlysis, Klein nd ssocites 50 lso identified novel regions of suggestive linkge to oculr refrction on 1q41 nd 7q21 (Tble 1). The sme group lso confirmed the MYP6 locus t 22q12 previously mpped by Stmbolin s group owdys, mny softwre pckges for ASP nlysis re vilble, such s MAP- MAKER/SIS within GEEHUTER. Mny on-line resources provide the softwre for genetic linkge nlysis, such s the Lbortory of Sttisticl Genetics t Rockefeller University. ASSOCIATIO STUDY A M* b Assocition studies represent n lterntive pproch to identifying susceptibility genes for complex diseses. It is more powerful thn linkge nlysis in detecting genes with smll effects in complex A M* b Figure 5. Allele shring method. The disese locus hs disese llele M nd norml llele. ote tht llele M is mrked by n sterisk for esy recognition. The disese locus is flnked by two linked mrker loci crrying lleles A/ nd /b. The muttion M nd lso the hplotype A-M-b trnsmits to the three ffected offspring from the ffected fther (the ncestor), nd thus re shred identicl-by-descent (ID) between the ffected offspring. Similrly, the norml nd hence the hplotype -- re shred ID between the two unffected offspring. ote tht only one of the two hplotypes is shown for ech offspring. The menings of the symbols re s shown in Figure 4. A M* b diseses (Figure 3). 36 This method is bsed on mesuring linkge disequilibrium (LD) or llelic ssocition between mrker nd custive genetic vrition. When the mrker nd the custive genetic vrition for disese re physiclly close together on the sme chromosome with tight linkge, they re likely to be inherited together s prticulr combintion of lleles from genertion to genertion in popultion. This sitution is known s LD nd the set of ssocited SP lleles in region of the sme chromosome is clled hplotype. Allelic ssocition refers to significntly incresed or decresed frequency of mrker llele in disese nd represents devitions from the rndom occurrence of the lleles with respect to disese phenotype. Allelic ssocition cn be due to LD mintined by tight linkge. The HpMp Project of the humn genome provides tremendous mount of informtion on the ptterns of vrition in the genome of four different 13

11 ethnic popultions. 74 It includes the chromosoml regions with sets of strongly ssocited SPs, the hplotypes in those regions nd the SPs (clled tg SPs) tht re representtive of those hplotypes. Thus, one of the pplictions of the HpMp dtbse is to provide tg SPs tht ct s representtives for mny other surrounding sequence vritions. The reserchers cn genotype much smller number of tg SPs to determine the collection of hplotypes present in ech subject. This cn help in the quest of complex disese genes by reducing the number of SPs to be tested in ssocition studies. Popultion-bsed ssocition study Popultion-bsed ssocition study (or cse-controlled study) is the most widely pplied strtegy of ssocition studies for complex diseses/trits. It compres prticulr genetic mrker or set of mrkers between group of ffected or disesed individuls (cses) nd group of unffected or norml individuls (controls) within popultion. oth cse nd control groups should be unrelted to ech other (tht is, without ny blood reltion with ech other). A greter percentge of mrker llele (or genotype) in ffected individuls is considered s evidence of ssocition between the disese phenotype nd the mrker llele (or genotype). The chi-squre (χ 2 ) test is used for mesuring the sttisticl significnce of such differences between the llele (or genotype) frequency distributions in cses nd controls. In cse-controlled ssocition studies, unrelted individul smples rther thn fmily smples re required. Subject recruitment for this method is esier thn for linkge nlysis or fmily-bsed ssocition studies (see next section). Csecontrolled ssocition study becomes better pproch for gene mpping, if the eye diseses re lte-onset or prents of the cses re not vilble for study, such s ge-relted mculr degenertion nd dult-onset glucom. A few myopi susceptibility genes, including trnsforming growth fctor β-induced fctor (TGIF), 75 trnsforming growth fctor bet 1 (TGF1) 76 nd lumicn (LUM) 77 hve been identified by this pproch in Chinese popultions. Positive ssocition cn rise in three situtions. 35 First, it is due to the direct effect of genetic vrint on the risk of suffering from n eye disorder. In this cse, the sme positive ssocition is expected in ll popultions tested. Second, positive ssocition cn occur if the mrker SP being tested is in LD with the custive genetic vrint (tht is, certin llele of the mrker SP tends to occur on the sme chromosome tht crries the custive genetic vrint). Different lleles of the mrker my be ssocited with the disese in different popultions becuse the pttern of LD mong different SPs cn vry ccording to the history of the popultions under study. The third scenrio is worrying becuse positive ssocition cn be flsely obtined if the popultion under study is not homogeneous. In other words, this flse positive result is n rtifct of popultion dmixture, which refers to popultion with multiple subgroups tht hve different llele frequencies. This cn be due to either recent dmixture of different popultions or to inpproprite mtching of ptients nd controls. 35 For illustrtion, consider popultion tht is mix of two subgroups. Assume tht subgroup 2 hs higher prevlence of disese under study, higher llele frequency of SP thn subgroup 1 nd subjects with the disese (tht is, cses) re preferentilly recruited from subgroup 2 in n unknowingly bised mnner. This will give higher llele frequency of the SP in the cses thn in the controls nd, in turn, produce spurious ssocition between the SP nd the disese. Even if no differences in disese prevlence between different subgroups re present, spurious ssocition my still rise if the probbility of cses or controls being selected into the study is not independent of the originl popultion subgroups (selection bis). The extent to which popultion dmixture or strtifiction cretes problems for ssocition studies hs been extensively discussed Tht mny genetic ssocition studies cnnot be replicted my be due to this problem. Therefore, crefully mtched cses nd controls to void popultion strtifiction re essentil for high-qulity ssocition study. Fmily-bsed ssocition study As discussed bove, filure to mtch cses nd controls in popultion-bsed ssocition studies cn led to spurious ssocition due to popultion strtifiction. This problem cn be overcome if the fmilybsed ssocition study pproch is dopted. Insted of unrelted individuls, nucler fmilies hve to be used nd ech fmily consists of ffected offspring nd their prents. These fmily-bsed studies re bsed on the ide tht non-trnsmitted lleles from the prents of the ffected offspring ct s internl controls, nd trnsmitted lleles re the cses (Figure 6). Therefore, prents cn provide both the cses nd the controls, nd the issue of ethnic mismtches cn be voided. The price to py for this dvntge is tht the mount of genotyping work is incresed by 50 per cent when compred with csecontrolled ssocition studies. Recent reserch lso suggested tht involvement of unffected siblings would increse the power of studies of mpping loci for quntittive trits The trnsmission disequilibrium test (TDT) is the most widely used version of fmily-bsed ssocition tests. In essence, it tests for distortion (or disequilibrium) in the trnsmission of lleles from heterozygous prent to n ffected child by compring the frequency of the llele trnsmitted to the ffected child to the frequency of the llele not trnsmitted. 85 As n illustrtion, consider four heterozygous (A) prents (Figure 6). If there is no ssocition between the mrker nd the disese, lleles A nd re likely to be trnsmitted eqully to the ffected children. If llele A increses the risk of contrcting the disese, it will be preferentilly trnsmitted (three times out of four) to the ffected children. In TDT nlysis, Mcemr χ 2 test is used to test the null hypothesis tht ech mrker llele is trnsmitted from heterozygous prents to n ffected child with probbility of 50 per cent expected by rndom Mendelin seg- 14

12 A A A A AA Figure 6. Trnsmission disequilibrium test (TDT). Four trio fmilies re shown, ech with n ffected child. Ech fmily hs heterozygous prent with genotype A, who cn trnsmit either llele A or to the ffected child. In three fmilies out of four, llele A is trnsmitted to ffected children from their heterozygous prents nd llele cn ct s n internl control. Apprently, llele A is trnsmitted to ffected children more frequently thn expected. In one fmily (second from the right), llele is trnsmitted to the ffected child from the heterozygous prent nd llele A cn ct s n internl control. Homozygous prents (AA or ) re not informtive. For TDT study, heterozygous prents re needed nd the frequencies of trnsmitted nd non-trnsmitted lleles to ffected offspring re compred. The Mcemr chi-squre test is used to test the sttisticl significnce of the observed difference. If there re m ffected children receiving llele A from their heterozygous prents nd n ffected children receiving llele from their heterozygous prents, then the Mcemr chi-squre sttistic is simply (m - n) 2 /(m + n) with one degree of freedom. regtion. The TDT cn lso be extended to fmilies with more thn one ffected child (myopic offspring) becuse, under the null hypothesis of no linkge/ssocition, every child in fmily hs n independent 50 per cent probbility of inheriting ech of the two lleles from heterozygous prent. 86 Recently, the TDT pproch hs been used successfully to identify two myopi susceptibility genes in Chinese popultions: the heptocyte growth fctor (HGF) nd the myocilin (MYOC) genes. 67,87 The TDT is less useful when the disese is due to the contribution of rre lleles. It is becuse of the smll proportion of heterozygotes crrying the rre lleles within the popultion. Another disdvntge is tht it is much more difficult to recruit fmilies thn unrelted individuls. The comprison of cse-controlled ssocition tests nd fmily-bsed ssocition tests is summrised in Tble 3. SCALE OF GEE MAPPIG EFFORTS o mtter which of the bove-mentioned genetic pproches is used, the scle of A A A gene mpping efforts cn vry tremendously. It cn rnge from systemticlly investigting lrge number of genetic mrkers throughout the humn genome (genome-wide scn) to strtegiclly testing only few mrkers within smll cndidte region (cndidte-gene pproch). Obviously, the time, cost nd lbour involved vry ccordingly. Genome-wide pproches One of the common pproches to mpping genes implicted in complex diseses is genetic linkge nlysis using whole genome scn. 35 It entils systemtic serch in lrge fmilies for genetic effects t different loctions long ll humn chromosomes even though the mechnisms underlying disese re not known. Although the time nd cost of this pproch re relly tremendous, this pproch is useful for mpping gene of interest to lrge region of chromosoml segment extending bout few thousnd kb in length. Genome-wide linkge scns usully employ severl hundred microstellite mrkers (in the rnge of 400 to 800) spced t regulr intervls in the humn genome. Mny myopi loci were identified using this pproch in the recent decde (Tble 1). 42,44,46 58 As there is n explosion of high-throughput nd dvnced SP genotyping technologies, 61,88 90 efficient genome-wide linkge scns cn now be chieved esily with 5,000 to 10,000 SPs within reltively short period. High-throughput genotyping pltforms hve lso recently mde genome-wide ssocition studies relity We need to do ssocition tests on ll lleles for n extremely lrge number of mrkers (in the rnge of 0.1 to 1.0 million), n enterprise resulting in over one million dt points for just single individul. Correction for mny different sttisticl comprisons is needed. It cn be imgined tht n stronomicl number of smples is required to detect ssocitions tht re sttisticlly significnt even for moderte or smll gene effects. Cndidte-gene pproches According to the estimtion of the Humn Genome Project, there re bout 30,000 genes in the humn genome. It is very chllenging, perhps impossible for smllscle reserch studies, to test ll the genes with ssocition methods. Therefore, cndidte-gene pproch insted of genome-wide pproch cn be used for identifying susceptibility genes of complex eye diseses. This is lso widely pplied to other complex diseses such s dibetes nd hypertension. This mens tht some cndidte genes re first selected nd DA sequence vritions (s genetic mrkers) within nd flnking the cndidte genes re then chrcterised for subsequent use in ssocition studies. Cndidte genes re usully selected on the bsis of their biology nd function. 95 For studies of myopi, the genes re expected to be expressed in the eye. We cn lso choose some cndidte genes tht re expressed in the specific oculr tissue relted to the process of myopic development. Exmples include genes encoding the components of sclerl tissues or enzymes relted to sclerl thinning or remodelling in myopic eyes; 96,97 genes underlying genetic oculr or systemic diseses usully with high myopi s one of 15