Catarata Congenita Hereditaria

Embed Size (px)

Citation preview

  • 8/3/2019 Catarata Congenita Hereditaria

    1/13

    Comprehensive Mutational Screening in a Cohort ofDanish Families with Hereditary Congenital Cataract

    Lars Hansen,1,2 Annemette Mikkelsen,2 Peter Nurnberg,3,4 Gudrun Nurnberg,3

    Iram Anjum,1,2 Hans Eiberg,2 and Thomas Rosenberg5

    PURPOSE. Identification of the causal mutations in 28 unrelated

    families and individuals with hereditary congenital cataractidentified from a national Danish register of hereditary eyediseases. Seven families have been published previously, andthe data of the remaining 21 families are presented togetherwith an overview of the results in all families.

    METHODS. A combined screening approach of linkage analysisand sequencing of 17 cataract genes were applied to mutationanalyses of total 28 families.

    RESULTS. The study revealed a disease locus in seven of eightfamilies that were amenable to linkage analysis. All loci repre-sented known genes, and subsequent sequencing identifiedthe mutations. Mutations were found in eight genes, amongthem crystallins (36%), connexins (22%), and the transcriptionfactors HSF4 and MAF (15%). One family carried a complex

    CRYBB2 allele of three DNA variants, and a gene conversion isthe most likely mutational event causing this variant. Tenfamilies had microcornea cataract, and a mutation was identi-fied in eight of those. Most families displayed mixed pheno-types with nuclear, lamellar, and polar opacities and no appar-ent genotypephenotype correlation emerged.

    CONCLUSIONS. In total, 28 families were analyzed, and mutations were identified in 20 (71%) of them. Despite considerablelocus heterogeneity, a high mutation identification rate wasachieved by sequencing a limited number of major cataractgenes. Provided these results are representative of WesternEuropean populations, the applied sequencing strategy seemsto be suitable for the exploration of the large group of isolatedcataracts with unknown etiology. ( Invest Ophthalmol Vis Sci.

    2009;50:32913303) DOI:10.1167/iovs.08-3149

    Congenital cataract (CC) is among the most common devel-

    opmental anomalies of the eye. It occurs as an isolatedtrait, in association with other ocular dysmorphology as well assystemic malformations. The etiology of isolated CC is un-known in approximately 50% of cases, and approximately 30%is monogenetic, with autosomal dominant transmission as themost common mode of inheritance. The knowledge of thegenetic background has increased considerably during the pastdecennia, mainly based on linkage strategies in large families(for review, see Refs. 1 and 2 and references therein). Exten-sive locus heterogeneity has been documented, and more than40 cataract-associated loci are known, of which 25 representidentified genes, and the number of mutations exceeds morethan 100.1,2 Mutations causing developmental cataracts mainlyinvolve proteins with structural and chaperone functions, in-

    cluding

    -,

    -, and

    -crystallins. Another group includes thelens-specific transmembrane gap junction protein genes GJA3and GJA8, and the membrane protein genes MIP and LIM2. Athird group of genes represents the lens-associated transcrip-tion factors HSF4, PITX3, MAF, PAX6, and FOXE3. Mutationsin HSF4 have mainly been associated with nonsyndromic cat-aract, whereas MAFmutations often involve the microcorneacataract phenotype. Structural proteins as the lens-specificbeaded filament protein genes BFSP1 and BFSP2 represent anadditional group of proteins that may have mutations leading tocataract formation. For most of these genes, cataract is the onlydisease phenotype observed.1,2

    Dominantly inherited mutations are mainly missense muta-tions that lead to amino acid substitutions. Only a few exam-ples of nonsense mutations or frame shift mutations have been

    described1,2

    (see dbCCM, http://www.wjc.ku.dk/ccmd1.html/Congenital Cataract Mutation Database, provided in the publicdomain by the Panum Institute, University of Copenhagen,Copenhagen, Denmark). With a few exceptions, such as thehyperferritinemia-cataract syndrome,3,4 no consistent geno-typephenotype relations have become evident to facilitate theidentification of the involved gene.

    We initiated an investigation of hereditary isolated CC andCC with microcornea in the Danish population, to trace thegenetic background in selected families.57

    M ATERIAL AND METHODS

    Patients

    Families and patients were recruited from The National Danish Regis-

    ter of Hereditary Eye Diseases at the former National Eye Clinic for the

    Visually Impaired, now The Kennedy Center (www.kennedy.dk/). Of

    97 families with congenital or infantile cataract with or without mi-

    crocornea we contacted members of 11 families suited for linkage

    analyses. A sufficient number of participants from eight families at-

    tended the study. DNA from an additional 20 unrelated individuals

    belonging to smaller families collected during clinical examinations

    was retrieved from the DNA bank of the Eye Clinic. Except microcor-

    nea cataract, families with syndromic cataract including congenital

    cataract and mental retardation and cataract in aniridia syndrome were

    From 1The Wilhelm Johannsen Centre for Functional GenomeResearch, Institute of Cellular and Molecular Medicine, and the 2Insti-tute of Cellular and Molecular Medicine, Section IV, Panum Institute,University of Copenhagen, Copenhagen, Denmark; the 3Cologne Cen-ter for Genomics (CCG) and Institute for Genetics, and the 4CologneExcellence Cluster on Cellular Stress Responses in Aging-AssociatedDiseases (CECAD), University of Cologne, Cologne, Germany; and the5Gordon Norrie Centre for Genetic Eye Diseases, Kennedy Center,Hellerup, Denmark.

    Supported by grants from The Danish Association of the Blind andThe Danish Eye Health Society. RC-Link is supported by The DanishMedical Research Council. The Wilhelm Johannsen Centre for Func-tional Genomics and the Genome Group/RC-LINK hosted the project;

    the Danish National Research Foundation funds the Wilhelm Jo-hannsen Centre for Functional Genomics.

    Submitted for publication November 12, 2008; revised January 1,2009; accepted April 16, 2009.

    Disclosure: L. Hansen, None; A. Mikkelsen, None; P. Nurn-berg, None; G. Nurnberg, None; I. Anjum, None; H. Eiberg, None; T. Rosenberg, None

    The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be marked advertise-ment in accordance with 18 U.S.C. 1734 solely to indicate this fact.

    Corresponding author: Lars Hansen, Section IV, ICMM, PanumInstitute, University of Copenhagen, Blegdamsvej 3b, DK-2200 Copen-hagen N, Denmark; [email protected].

    Investigative Ophthalmology & Visual Science, July 2009, Vol. 50, No. 7Copyright Association for Research in Vision and Ophthalmology 3291

  • 8/3/2019 Catarata Congenita Hereditaria

    2/13

    excluded. The results of mutational analysis of patients with aniridia

    syndrome have been reported earlier.8 A single two-generation family

    with possible X-linked transmission and clinical signs of Nance-Horan

    syndrome, and two families with probable autosomal recessive inher-

    itance did not participate in the present project. Genomic DNA was

    extracted from whole blood by standard procedures. Mutation controls

    in the background population were performed with DNA from unre-

    lated normal individuals retrieved from the Copenhagen Family Bank.9

    The study adhered to the tenets of the Declaration of Helsinki and was

    approved by the Copenhagen Scientific Ethics Committee. After being

    informed, all subjects gave written consent to participate in the study.

    Linkage Analysis

    Four families were analyzed using one or several locus-specific STS

    markers close (1 cM) to known cataract disease genes. Haplotypes

    were drawn for each locus (Cyrillic ver. 2.1.3; Cherwell Scientific,

    Oxford, UK) and checked by visual examination. Initial locus screening

    was performed with markers for 12 different known cataract loci

    (Table 1A). Additional STS markers were included for fine mapping of

    candidate loci (Table 2) and two-point LOD scores for initial exclusion

    were calculated with the program LIPED.10 The STS marker analyses

    were performed using P-33 radioactive labeled oligonucleotide primers

    followed by PCR. Endlabeling of oligonucleotides was performed ac-

    cording to a standard protocol with T4-DNA-polynucletide kinase (Fer-

    mentas, Vilnius, Lithuania) and -33P-ATP (Hartmann Analytic, Braun-

    schweig, Germany) and the labeled oligonucleotides were used for

    PCR without further purification. Taq-DNA polymerase was purchased

    from New England Bio Laboratories (Ipswich, MA), and PCR primers

    were purchased from TAG Copenhagen A/S (Copenhagen, Denmark).

    A complete genomic scan (data not shown) was performed with a 10K

    SNP array analysis in two families (CC00116 and CC00162) in which

    the STS marker analyses failed to show linkage either due to incom-

    plete penetrance (CC00162) or inconclusive results (CC00116).

    Direct Genomic DNA Sequencing

    Direct DNA sequencing of amplified PCR products of all exons, exon

    intron borders, and parts of the 5 and 3 UTR was performed in 17

    known cataract disease genes (Table 1B; BigDye version 1.1 sequenc-

    ing technology and 3130xlsequencing apparatus; Applied Biosystems,

    Inc. [ABI], Foster City, CA). PCR and sequencing primers were de-signed by Primer 3 and purchased from TAG Copenhagen A/S (Copen-

    hagen, Denmark). (PCR primer sequences are found in Supplementary

    Table S1, online at http://www.iovs.org/cgi/content/full/50/7/3291/

    DC1.) The sequence data for the coding regions were aligned to

    GenBank reference sequences, and genomic intron sequences were

    aligned to human reference assembly hg17 (NCBI Build 35 from UCSC,

    http://genome.ucsc.edu/ provided in the public domain by UCSC Ge-

    nome Bioinformatics, University of California at Santa Cruz, Santa Cruz,

    CA).11 Taq DNA polymerases were purchased from three sources

    (New England Biolabs; HotStartTaq DNA Polymerase from Qiagen,

    Hilden, Germany; and Platinum TaqDNA polymerase from Invitrogen,

    Carlsbad, CA). PCR and sequencing were performed according to

    standard protocols and analyzed (Chromas software; Technelysium

    Pty. Ltd., Tewantin, Australia).

    TABLE 1. Cataract Disease Loci and Genes

    A. Disease Loci

    Chromosomal Band STS Marker Locus

    1pter-p36.13 D1S243 CCV, Volkmann cataract1q21.1 D1S2612 GJA82q33.3 D2S2208 CRYGA, CRYGB, CRYGC, CRYGD

    3q22.1 D3S1290 BFSP210q24.32 D10S1697 PITX311q23.1 D11S4192 CRYAB12q13.3 D12S1691 MIP13q12.11 D13S175 GJA316q22.1 D16S3086 HSF417q11.2 D17S841 CRYBA121q22.3 D21S1890 CRYAA22q11.23-12.1 D22S421 CRYBB2, CRYBA4, CRYBB3, CRYBB1

    B. Genes

    Gene Symbol GenBank Ref. Seq. Gene Name

    CRYAA NM_000394.2 Crystallin -ACRYAB NM_001885.1 Crystallin -B

    CRYBB1 NM_001887.3 Crystallin -B1CRYBB2 NM_000496.2 Crystallin -B2CRYBB3 NM_004076.3 Crystallin -B3CRYBA4 NM_001886.1 Crystallin -A4CRYBA1 NM_005208.3 Crystallin -A1CRYGC NM_020989.2 Crystallin -CCRYGD NM_006891.2 Crystallin -DGJA3 NM_021954.3 Gap junction protein, 3GJA8 NM_005267.3 Gap junction protein, 8

    HSF4 NM_001538.2 Heat shock factor 4MIP NM_012064.2 Major intrinsic protein of lens fiberBFSP1 NM_001195.2 Beaded filament structural protein 1, FilensinBFSP2 NM_003571.2 Beaded filament structural protein 2, PhakininMAF NM_005360.3 v-Maf musculoaponeurotic fibrosarcomaPITX3 NM_005029.3 Paired-like homeodomain transcription factor

    3292 Hansen et al. IOVS, July 2009, Vol. 50, No. 7

  • 8/3/2019 Catarata Congenita Hereditaria

    3/13

    Restriction Enzyme Digests

    Identified mutations were analyzed by restriction enzymes digests in

    accordance with the manufacturers protocol (New England Biolabs)

    in a 20-L volume with 2- to 4-L PCR product and 5 to 10 units

    enzyme. The cleaved PCR products were analyzed by 2% agarose or

    20% acrylamide gel electrophoresis with 1 TBE, and the DNA was

    visualized by staining with ethidium bromide.

    Subcloning of PCR Products

    PCR products were subcloned by TA cloning (pCR-XL-TOPOII vector;

    Topo XL PCR cloning procedure; Invitrogen). The cloned PCR frag-

    ments were screened by direct colony PCR in conditions identical with

    those used for the genomic DNA PCR.

    RESULTS

    The present study includes the remaining 21 families from aninvestigation of 28 families of Northern European decent withhereditary congenital cataract. The results from seven of thesefamilies have been presented in previous publications.57 Over-all, likely pathogenic mutations were identified in 20 (71%) ofthe 28 families. The affirmative results implicated a total ofeight genes involving four crystallin genes, CRYAA (six fami-

    lies), CRYBB2 (two families), and CRYBB3 and CRYGD (onefamily each); two connexin genes, GJA3 and GJA8 (threefamilies each); and two transcription factors, HSF4 and MAF(two families each; Table 3). In addition to the identifiedmutations a genome-wide scan of a large family (CC00116)revealed two provisional novel loci with equal LOD scores(Z 2.7, 0). The two regions, 2q32.2-33.3 and 17q11.2-q21.2, are bordered by the SNPs rs952242 and rs1551443 forchromosome 2 and the SNPs rs952581 and rs1846043 forchromosome 17 (data not shown). The remaining seven incon-clusive families underwent sequencing of the 17 examinedcataract genes without identifying any pathogenic mutation.Figures 1 to 5 illuminate the hitherto unpublished results ac-cording to the implicated genes. Seven novel polymorphismswere identified and are presented in Table 4.

    GenotypePhenotype Relations

    Most patients had their cataracts surgically removed, and thephenotypes were therefore retrieved retrospectively from thefiles of various ophthalmology departments. Many of the notes,however, dated 30 to 50 years ago and the available informa-tion was often too insufficient for an adequate classificationresulting in only fragmentary data. Cases demanding early sur-gical intervention were frequently accompanied by nystagmus,which persisted after surgery.

    The study included six families with CRYAA mutations,

    CC00105, CC00124, CC00174, CCMC0101, CCMC0106, andCCMC0106. Among these identical mutations, p.Arg21Trp, were found in three families, CC00105, CC00124, andCCMC0108. Affected members of CC00105 and CC00124 hadanterior polar cataracts and various nuclear and lamellar corti-cal involvements with highly variable impact on visual func-tion. Furthermore, one member of CC00105 had a congenital,unilateral, inferior iris coloboma, and another individual in thesame family had reduced corneal diameters of 9 mm. A thirdmutation carrier had completely clear lenses when examinedat 24 years of age. Four of six individuals in family CC00124 stillhad their lenses left at the ages of 5, 13, 45, and 59the latter with optical iridectomies in both eyes. The cataracts weremainly of the anterior polar type with different involvement of

    nucleus, posterior pole, and cortex in six individuals. (See theDiscussion section for further details.) Information was avail-able for two families with a GJA3 mutation (CC00162) and aGJA8mutation (CC00145). Three members of family CC00162showed a lamellar cataract type with moderate opacity of thefetal nucleus and Y-shaped condensations in the anterior su-ture. In three members of family CC00145 the lens morphol-ogy was described as dense and star-shaped with various loca-tions in the nucleus or the poles. One individual with ananterior polar opacity developed a nuclear opacity during earlyinfancy. Family CC00128 harbored a HSF4 mutation. In thisfamily, four individuals had lamellar cataracts involving faintopacities of the fetal nucleus with condensation along theanterior Y-suture and various, partly progressive opacities in

    TABLE 2. Two-Point LOD Score Calculations

    Mb*

    Z()

    0.00 0.01 0.05 0.10 0.20 0.30 0.40

    CC00105CRYAAD21S1411 43.03 1.73 1.77 1.82 1.76 1.41 0.91 0.36c.61CT 43.46 3.38 3.38 3.29 3.07 2.46 1.69 0.79

    D21S1890 43.67 1.29 1.32 1.37 1.35 1.13 0.76 0.29CC00124CRYAAc.61CT 43.46 3.01 2.96 2.74 2.46 1.85 1.16 0.43D21S1890 43.67 0.36 0.34 0.30 0.26 0.22 0.17 0.09

    CC00128HSF4D16S3086 65.49 2.77 2.70 2.43 2.09 1.44 0.83 0.29c.341TC 65.75 5.61 5.52 5.14 4.66 3.62 2.50 1.28D16S3095 68.50 3.86 3.78 3.43 2.99 2.10 1.25 0.46

    CC00145GJA8D1S442 143.12 2.25 2.21 2.04 1.82 1.39 0.95 0.50c.218CT 144.61 5.34 4.99 4.55 3.58 2.51 1.32 5.42D1S3466 147.00 3.43 3.39 3.22 2.98 2.40 1.72 0.93

    CC00162GJA3c.227G A 2.50 2.51 2.47 2.32 1.88 1.32 0.69

    * Physical distances in mega base pairs according to UCSC Marts 2006, NCBI Build 36.1.

    Penetrance in the calculations for Z() is 0.95. Penetrance in the calculations for Z() is 1.00.

    IOVS, July 2009, Vol. 50, No. 7 Hereditary Congenital Cataract Mutations 3293

  • 8/3/2019 Catarata Congenita Hereditaria

    4/13

    the cortices. One of these patients also developed a denseanterior polar cataract.

    Two families with MAF mutations (CCMC0102 and

    CCMC0113; Table 3) had small corneae. One member of thelatter family was reported to be unaffected but showed up tocarry the mutation. An examination, however, showed micro-corneae with 10-mm horizontal diameters and only scatteredpunctuate stromal opacities, which otherwise would not havebeen noted. His visual acuity was 1.0 on both eyes. Anothermember of the same family showed dense nuclear opacitieswith a clear periphery as documented by a photograph from1967 when the patient was 26 years of age (Fig. 6).

    DISCUSSION

    Apart from mutation screening projects in South Indian fami-lies1719 and Australian families,20 our study is among the first

    to report on the mutation spectrum in a relative large cohort ofpatients with CC. Although there is a sizeable and still expand-ing genetic heterogeneity, we show that a relative high successrate would have been obtained with a sequencing strategyinvolving rather few major genes. Although this is true of thepresent family collection, it is not necessarily true of newunselected families. However, it suggests that these genes maybe particularly useful for screening in samples of similar origin.In family CC00162 the initial linkage analysis failed to point outa locus. A whole-genome screening, however, led to the iden-tification of a known cataract gene, GJA3. The family investi-gations disclosed the presence of two instances of nonpen-etrance (CC00105 and CC00162) that explained the failedlinkage analysis. Without the whole-genome screening, thereduced penetrance in this family supposedly would have

    passed unnoticed. Many of the included families were too smallto decide about the hereditary mode. Except for one family(CC00116) linkage analyses identified only the already known

    loci representing known genes, which implies that the sameresults would have been obtained by the sequencing of a singleaffected individual.

    SNP-array analysis in family CC00116 identified two differ-ent loci representing a 15.6-Mbp region on chromosome 2 anda 9.3-Mbp region on chromosome 17. The results suggest atleast one novel cataract-associated locus. Known cataractgenes are located close to both regions, on chromosome 2 the-crystallin cluster (CRYGA to CRYGD) is found 1.4 Mbp distalto the linkage region and on chromosome 17, the CRYBA1gene is located 2.4 Mbp proximal to the linkage region. Boththese loci are outside the mapped regions and the genes havein addition been sequenced before the genome scan. Whetherone of the two linkage regions harbors a mutation in a long-distance regulatory element or a new cataract gene awaitsdisclosure and is under investigation.

    In total, 20 mutations were identified among the 28 familiesincluded in the study, and it is noteworthy that five of thesemutations were reported earlier. We document that a foundereffect seems very unlikely with one exception (CC00124 andCCMC0108).

    The DNA sequence analyses included 17 of the known atleast 25 cataract-associated genes. The remaining genes wereexcluded due to a weighing of workload against the possiblerelevance for our samples. The newly identified gene EPHA221

    was published after the conclusion of this study, but will beincluded in future analyses.

    In our study, most of the involved genetic variants weremissense mutations; only two nonsense mutations were en-

    TABLE 3. Accumulated Results of the Danish Congenital Cataract Mutation Study

    Family Gene Nucleotide Change* Amino Acid

    Change*Number of Analyzed

    Control Persons References

    CCMC0101 CRYAA c.34CT p.Arg12Cys 170 This study, 6CC00105, CC00124, CCMC0108 CRYAA c.61CT p.Arg21Trp 170 This study, 6CC00174 CRYAA c.155CT p.Arg49Cys None This study, 12CCMC0106 CRYAA c.337G A p.Arg116His 170 This study, 6CC00156 CRYBB2 c.498C A p.Tyr159X None NovelCC00133 CRYBB2 c.[433CT; 440AG;

    449CT]p.[Arg145Trp;

    Gln147Arg;Thr150Met]

    100 Novel (rs2330991,rs2330992,rs4049504)

    CCMC0102 CRYBB3 c.224G A p.Arg75His 238 NovelCCMC0109 CRYGD c.418C A p.Tyr134X None This study, 6CC00103 GJA3 c.32TC p.Leu11Ser 60 This study, 5CC00129 GJA3 c.176CT p.Pro59Leu None This study, 13CC00162 GJA3 c.227G A p.Arg76His None This study, 14CC00145 GJA8 c.218CT p.Ser73Phe 60 NovelCCMC0103 GJA8 c.565CT p.Pro189Leu 170 This study, 6CC00110 GJA8 c.836C A p.Ser259Tyr 170 NovelCC00128 HSF4 c.341TC p.Leu114Pro None This study, 15CC00171 HSF4 c.355CT p.Arg119Cys None This study, 15CCMC0112 MAF c.895C A p.Arg299Ser 52 This study, 7CCMC0113 MAF c.958AG p.Lys320Glu 173 NovelCCMC0107 No mutation

    CCMC0110 No mutation CC00109 No mutation CC00117 No mutation CC00155 No mutation CC00159 No mutation CC00805 No mutation CC00116 Locus chr2q32.233.3 or

    chr17q11.2q21.2 Novel

    * DNA and protein variation nomenclature is according to Human Genome Variation Society recommendations (http://www.hgvs.org/).16

    3294 Hansen et al. IOVS, July 2009, Vol. 50, No. 7

  • 8/3/2019 Catarata Congenita Hereditaria

    5/13

    countered and no insertions or deletions were found, which isin accordance with the finding of other investigators. Themajority of genetic analyses of congenital cataract includeAD-CC families and very few cases of AR-CC forms or sporadiccases are reported. These results infer that mainly missense andnonsense mutations are found for AD-CC, whereas AR-CC alsois due to frameshift mutations.

    The finding of only one mutation in the Danish familiesindirectly supports an assumption of autosomal dominanttransmission. The present paper includes the remaining un-

    published mutational results from 21 families and includes 12mutations in 13 families.

    Mutations in the Lens-Specific Crystallins

    Eight mutations were found in 10 families corresponding to36% of the analyzed families, which is in the same magnitudeas the percentage of crystallin mutations in South India (30%) when corrected for the share of 53% autosomal dominantfamilies.19

    FIGURE 1. Pedigrees and restrictiondigests of three families with CRYAA

    mutations. (A) TheMspI restriction en-zyme digest of exon 1 identified allaffected individuals as carriers of themutation c.61CT in family CC00105.Note that person IV:7a was a healthycarrier. Wild-type allele, 205, 117, and107 bp; mutant allele, 321 bp. (B) Themutation and the sizes of the MspI re-striction enzyme digest in familyCC00124 were identical with the di-gest of family CC00105. (C) The pedi-gree and the AciI restriction enzymedigest of family CC00174 showed thetwo affected individuals to be carriersof the mutation CRYAA c.155CT.

    Wild-type allele: 15, 98, 124, and 191

    bp; mutant allele: 98, 124, and 206 bp(only the 191- and 206-bp fragmentsare shown). Filled symbols: affectedindividuals; open symbols: unaffectedindividuals; circles: females; squares:males; M: 50-bp DNA ladder; (C ) Di-gest of a normal unrelated individual.U: uncut PCR products. (D) The DNAsequence and the translation of thefirst 70 nucleotides of the CRYAA cod-ing region show the SNP rs872331(c.6CT) and the CRYAA mutation(c.61CT) together with the haplo-types of the families CC00105 andCC00124 and family CCMC0108.6 Allthree families carried the CRYAA mu-

    tation c.61C

    T, and the haplotypesexcluded a common founder forCC00105 and CC00124.

    IOVS, July 2009, Vol. 50, No. 7 Hereditary Congenital Cataract Mutations 3295

  • 8/3/2019 Catarata Congenita Hereditaria

    6/13

    3296 Hansen et al. IOVS, July 2009, Vol. 50, No. 7

  • 8/3/2019 Catarata Congenita Hereditaria

    7/13

    FIGURE 3. Sequence analyses of families carrying CRYBB3 mutations. (A) The CRYBB3 mutation c.224GA was found in individual II:1 in familyCCMC0102 and confirmed by a SacII restriction enzyme digest. Only individual II:1 was available for analyses. Wild-type allele (C): 130 and 270bp; mutant allele and uncut (U): 400 bp; M: 100-bp DNA ladder. (B) The -B crystallin proteins share a common secondary and tertiary structureof two crystallin domains, each composed of two Greek key motifs. A Greek key motif is inserted in the top left corner. The mutations found inthe families CC00133 and CC00156 are denoted. The amino acid numbers for intron positions are shown. (C) The protein sequence alignment ofthe third and the fourth Greek key motif for the human -B crystallin-1, -2, and -3 showed conservation or semiconservation for p.Arg145, p.Gln147,p.Thr150, p.Gln155, and p.Tyr159 (positions highlighted in yellow). Protein Ref. Seq.: NP_001878, NP_000487, and NP_004067. (D) The CRYBB3c.224GA position was highly conserved among several mammalian genomes as shown by the DNA sequence alignment (UCSC Human genome

    browser).

    11

    FIGURE 2. Pedigrees and analyses of two families with CRYBB2 mutations. (A) In family CC00156, only individual II:2 was available for analysis.The DNA chromatogram shows the nonsense mutation CRYBB2 c.498CA, which changes the tyrosine codon TAC into TAA. (B) Alignment ofthe DNA sequence for exon 6 for the wild-type CRYBB2 (NM_000496) and the corresponding sequence for individual II:2 and for the homologouspseudogene CRYBB2P1 (BC037884) shows variation for c.475C (green ), for position c.483C (blue ), and position c.489C (yellow ). The mostabundant CRYBB2 mutation c.475CT may be due to a gene conversion, whereas the alignment of positions c.483 and c.489 excludes a geneconversion for the novel mutation c.489C A. Redundant nucleotide M: C and A. (C ) The pedigree and the haplotypes of the two affectedindividuals and the inferred haplotype of one healthy individual of family CC00133. The DNA chromatogram shows the three DNA polymorphismsfor individual I:1. (D) Alignment of the genomic sequences of exon 5 and the border to intron 5 shows wild-type CRYBB2 (top ), the analyzedsequence of individual II:1 (middle), and the CRYBB2P1 pseudogene (bottom) suggesting that the three nonsynonymous changes in the diseaseallele was a result of a gene conversion. The converted region was a minimum of 80 bp long (green ); the maximum length (gray) could not bepredicted due to missing sequence information. Redundant nucleotides Y: C and T; R: A and G; and S: C and G. Exons are shown in capital letters,introns in lower case; the SNP rs57112959 (GA) refers to the wild-type CRYBB2 gene.

    IOVS, July 2009, Vol. 50, No. 7 Hereditary Congenital Cataract Mutations 3297

  • 8/3/2019 Catarata Congenita Hereditaria

    8/13

    3298 Hansen et al. IOVS, July 2009, Vol. 50, No. 7

  • 8/3/2019 Catarata Congenita Hereditaria

    9/13

    Among 32 families with autosomal dominant inheritanceand four families of uncertain inheritance from southeasternAustralia, only two (5%) crystallin mutations were identified.20

    We have no explanation for this difference. However, therewere few tissue samples, and selection bias for larger familiesmay influence the results. It is also noteworthy that the Aus-tralian and the Danish source populations are of similar mag-nitude, while the total number of ascertained families wasmore than twofold in the Danish population.

    The mutations in our cohort were found in four of the ninelens-expressed crystallin genes that we have analyzed (Table3). One previously published mutation6 was found in twofamilies (CRYAA, c.61CT, p.Arg21Trp; Fig. 1) and a differentmutation in the same codon (p.Arg21Leu) has been reportedbefore in association with cataract,22 which suggests the Arg21residue to be of crucial importance for the protein function.Sequencing of exon 1 of CRYAA using the SNP rs872331located 55 nt upstream of the mutant nucleotide c.61T dem-onstrated the haplotype c.[6T;61C] for family CC00105 and thehaplotype c.[6C;61C] for family CC00124 (Fig. 1D).This sug-gests that the mutation arose independently in the two fami-lies. The third family (CCMC0108) with cataract and microcor-nea carried the same mutation and sequence analysis of

    subcloned separated PCR products from one affected individ-ual from CCMC0108 revealed the haplotype c.[6T;61C] identi-cal with family CC00105 (Fig. 1D). Subsequent genealogicalstudies confirmed a common ancestral founder for the twofamilies. According to our knowledge, incomplete penetranceas documented in family CC00105 (Fig. 1A, IV:7) has not beenreported before in families with a CRYAA mutation.

    The novel CRYBB2 mutation p.Tyr159X (Table 3, Figs. 2A,2B, 3B, 3C) presumably terminates the reading frame of exon6 before the authentic stop codon. The mutant mRNA willpresumably avoid the nonsense-mediated RNA decay pathwayand be translated into a truncated protein. Another nonsensemutation (p.Gln155X, Fig. 2B, 3B, 3C) has been reported in fiveunrelated families with dominantly inherited cataract.20,2327

    Two of these mutations25,27 has been shown to be a conse-

    quence of gene conversions between a region of 9 to 104 bpsurrounding the mutation and the homologous region in theCRYBB2P1 pseudogene. The remaining three mutations seemto have occurred by point substitutions.23,24,26 By alignment ofthe wild-type CRYBB2, the corresponding sequence from II:2-CC00156, and the CRYBB2P1 exon 6 sequence (Fig. 2B), it isobvious that the p.Tyr159X mutation found in family CC00156is a point mutation and not a result of gene conversion, whichis further shown by the chromatogram that demonstrates thesequence [CCCCGGCTAC/A], which should have been[CCCC/TGGTAC/A] if both a point mutation and gene conver-sion have taken place. Identification of the two nonsensemutations in the same fourth Greek key motif of CRYBB2suggests a crucial region for cataract-associated mutations, and

    the pathogenic mechanism is presumably the same for the twomutations.

    A complex CRYBB2 allele with three nucleotide changes inexon 5 was detected in both affected individuals of familyCC00133 (Table 3, Fig. 2C). The DNA variations rs2330991,rs2330992, and rs4049504 (dbSNP, http://www.ncbi.nlm.nih-.gov/, provided in the public domain by the National Center forBiotechnology Information [NCBI]National Institute of Health,Bethesda, MD) are affirmed as nonsynonymous polymor-phisms. PCR products from individual I:1 were subcloned, anda probable cis position of the rare SNP variants was confirmedby sequencing of the subclones. Sequence analysis of the SNPsin CRYBB2 exon 5 for 100 normal unrelated individuals ofmatching ethnic background only detected the wild-type allelec.[433C; 440A; 449C] (data not shown) and none of the SNPswas found to be polymorphic in any of the other sequencedcataract family members. The SNP rs2330992 (p.Gln147Arg) isnonpolymorphic and represented only by the A-allele(p.Gln147) in the HapMap program (dbSNP, ss3282510, Build129, NCBI, http://www.ncbi.nlm.nih.gov/). This strongly sup-ports the unusual character of the G-allele for rs2330992 andfrom the above mentioned observations we consider it reason-able to assume that the occurrence of the three mutations in cisare pathogenic, possibly by transforming the secondary struc-ture of the -crystallin protein (Fig. 3B). A gene conversion

    between wild-type CRYBB2 and the pseudogene CRYBB2P1has been shown to be the most likely mechanism for thep.Gln155X mutation.25 A similar mechanism is the most likelycause for the complex allele, as shown by alignment of thehomologous DNA sequences for the wild-type CRYBB2 andthe CRYBB2P1 pseudogene with the sequence of individualII:1-CC00133 (Fig. 2D).

    The mutation found in CRYBB3 (c.224GA, p.Arg75His;Table 3) is the first report of a cataract-associated CRYBB3mutation with a dominant effect. The mutant genotype was notdetected in 238 normal individuals of matching ethnic back-ground or among the other cataract families. The mutation is inthe second Greek key motif (Fig. 3B) and destroys a highlyconserved amino acid (Fig. 3D) and is therefore most likelypathogenic. Unfortunately, only a single affected family mem-

    ber, individual I:1 (Fig. 3A) with a microcornea cataract wasavailable for investigation. One other CRYBB3 mutation(p.Gly165Arg) has been reported in a consanguineous Paki-stani family with recessively inherited cataract.28

    Mutations in the Gap Junction Proteins

    The six mutations, among which two were novel (Table 3),represent 22% of the families in our sample (Fig. 4). Three ofthe mutations were located in the first extracellular loop of thetwo gap junction proteins, and all three amino acid positionsare highly conserved in humans (Fig. 4E). This finding impliesthat the primary structure of transmembrane regions and theextracellular loops are crucial for the assembly of gap junction

    proteins into connexons. The GJA8 mutation affecting aminoacid p.Ser259 in the carboxyl terminus was novel. The muta-

    FIGURE 4. Pedigrees and restriction digests of four families with mutations in the gap junction proteins. (A ) Pedigree of family CC00145 showsthe haplotypes for all analyzed persons. The STS marker D1SGJA5-GJA8 was located between two genes, GJA5 and GJA8. The EarII restrictionenzyme digest showed cosegregation of the mutation GJA8c.218CT with the disease trait in the family. Wild-type allele: 199 bp; mutant allele:165 bp. (B) Pedigree of family CC00162. The AciI restriction enzyme digest illustrated the segregation of the mutation GJA3 c.227GA. Note thehealthy carrier V:2a. Wild-type allele: 151, 91, and 79 bp; mutant allele: 191, 91, and 79 bp; M: 50 bp DNA ladder. ( C) Pedigree of family CC00110.The BseRI restriction enzyme digest showed segregation of the mutation GJA8 c.836C A in both affected individuals. Wild-type allele (C): 114,135, 154, and 225 bp; mutant allele: 114, 225, and 289 bp. (D) The pedigree of family CC00129. The AluI restriction enzyme digest illustrates themutation in individual II:1. Wild-type allele (C): 129 and 328 bp; mutant allele: 129, 160, and 168 bp; U: undigested PCR products; M: 50 bp DNAladder. (E) Graphic representation of the two lens-specific gap junction proteins and the mutations found in the Danish cohort. CP, cytoplasmicdomain; TM, transmembrane domain; EC, extracellular domain. Alignment of the group of human gap junction proteins demonstratedconservation of the mutant positions except for the C-terminal mutation. Protein Ref. Seq.: Gja1, NP_000156; Gja3, NP_068773; Gja4, NP_002051;Gja5, NP_005257; Gja7, NP_005488; Gja8, P_005258; Gja10, NP_115991; and Gja12, NP_065168.

    IOVS, July 2009, Vol. 50, No. 7 Hereditary Congenital Cataract Mutations 3299

  • 8/3/2019 Catarata Congenita Hereditaria

    10/13

    3300 Hansen et al. IOVS, July 2009, Vol. 50, No. 7

  • 8/3/2019 Catarata Congenita Hereditaria

    11/13

    tion segregated with the phenotype in all members of the smallfamily (Fig. 4C), which supports the interpretation as a patho-genic mutation, although the amino acid position is less con-served among the human gap junction proteins. All GJA3mutations have been reported previously in association withisolated congenital cataract (Table 3). Of interest, the mutationp.Arg76His is characterized by incomplete penetrance in fam-ily CC00162 which also was observed in the first report of themutation.14 A third mutation affecting the same arginine resi-due (p.Arg76Gly) has been described in association with a fullypenetrant total cataract.17

    Mutations inHSF4

    Both HSF4 mutations have previously been described by Bu etal.15 The mutation p.Leu114Pro was reported in a large Chi-nese family with cataract and p.Arg119Cys in the large Danishfamily with cataract first described by Marner in 1949 (Table 3,Fig. 5, MIM 116800).2931 The repetition of the latter mutationin another Danish family suggests a common founder, whichcould not be documented by genealogical studies. The nomen-clature for the two mutations has been corrected (Fig. 5C)according to the recommendation from the Human Genome

    Variation Society (http://www.hgvs.org/).16

    MAF Mutations

    Previously, two MAF mutations have been published in asso-ciation with microcornea cataract.32,33 The recurrent mutationp.Arg299Ser was ascertained in family CCMC0112 (Table 3)and is predicted to modify the conserved DNA-binding region(Fig. 5D).7 The novel mutation p.Lys320Glu detected in familyCCMC0113 affects the leucine zipper region and is the firstpathogenic cataract MAF mutation outside the DNA bindingdomain. Of note, an apparent case of incomplete expressionwas observed in individual III:2 who had microcornea, but nocataract. This shows that isolated microcornea may be causedby a mutation in a cataract-associated transcription factor gene.

    This observation points toward a common regulatory mecha-nism of corneal and lens crystallins in humans. Experimentalevidence in mice seems to support the presence of such amechanism. Recently Davis et al.34 showed activation of thespecific mouse corneal crystallin Aldh3a1 by different tran-scription factors as Pax6, Oct1, and p300. Confirmation of apossible dual mechanism involving corneal and lens develop-ment induced by the novel MAF zipper domain mutationawaits experimental elucidation.

    Polymorphisms

    Several DNA variations of the major cataract-associated genes were found by the sequence analyses. Among eight novelpolymorphisms, we encountered one synonymous base-ex-change, five missense mutations, one intronic substitution, andone promoter deletion (Table 4). All variations except twowere present in normal control samples. The nonsynonymousHSF4change p.Met212Ile in the C-terminal part of the protein(Table 4) was initially considered to be causal in familyCC00109, because of the absence among 170 normal unrelatedindividual of Danish origin. The variation, however, was alsoabsent in two affected relatives and therefore was classified asa rare polymorphism. A CRYGD promoter deletion was de-tected in one affected individual of family CC00805 (Table 4). Analyses of 170 unrelated normal individuals of same ethnicbackground failed to identify the deletion, but it was not foundin an affected daughter of the proband and therefore consid-ered to be a rare DNA variant without pathogenic effects. Anonsynonymous GJA8mutation was found in family CC00159(Table 4) in which no pathogenic mutation has been identifiedso far (Table 3). The mutation, p.Asn220Asp involves an aminoacid position that is highly conserved among human and mam-malian gap junction proteins (data not shown). Surprisingly,the mutation was found in one allele among 170 normal per-sons with same ethnic background, which led to a classifica-tion as nonpathogenic.

    FIGURE 5. Pedigrees and restriction digests of families withHSF4and MAFmutations. (A) Pedigree with haplotypes for family CC00128. The BsrIrestriction enzyme digests showed that the mutation HSF4c.341TC cosegregates with the disease. Wild-type allele: 49, 57, 60, 74, and 252 bp;mutant allele: 49, 57, 74, and 312 bp; U: uncut PCR products; M: 50 bp DNA ladder. (B) Pedigree of family CC00171. The mutation HSF4c.355CT

    was confirmed byHpyCH4V restriction enzyme digest in both affected individuals. Wild-type allele: 492 bp; mutant allele: 48 bp and 444 bp; U:uncut PCR products; M: 100 bp DNA ladder. (C) The mutations are identical with two mutations described in a Chinese and a Danish family. Buet al.15 named the mutations c.348TC L115P and c.362CT, R120C, respectively, according to the GenBank cDNA clone, accession numberD87673, that starts at the4 position from the first ATG codon. The translation of the D87673 results in a HSF4protein that includes an additional

    valine residue at position 2. This results in discrepancies between the mutation names published by Bu et al. and the nomenclature used herein(Fig. 4C). The HSF4 isoform A (NM_001538) has been used for the systematic nomenclature (http://www.hgvs.org/mutnomen/).16 (D) Pedigreeof family CCMC0113. The half-filled symbolof individual III:3 refers to a case of no cataract with microcornea. The restriction enzyme MboII digestshowed segregation of the mutation in the family. Wild-type allele: 84, 92 (seen as one band), and 231 bp; mutant allele: 92 and 315 bp; U: uncutPCR products; M: 100-bp DNA ladder. The graphic representation of the MAF protein shows the known mutations6,32,33 and the novel cataractmutation.

    TABLE 4. Novel Polymorphisms Identified in the Danish Cataract Study

    Gene Exon Variation Amino Acid Change Allele Frequency* Family (dbLaH)

    CRYBA1 Ex2 c.74CT p.Pro25Leu 6/110 (1/38) CC00159 (391)CRYBA1 Ivs3 c.21516CT (2/30) CC00805 (395)CRYGD Promoter c.16_37del 0/340 (1/34) CC00805 (395)CRYGD Ex3 c.376G A p.Val126Met 2/240 (1/34) CC00171 (393)

    HSF4 Ex7 c.636GT p.Met212Ile 0/340 (1/34) CC00109 (387)GJA8 Ex2 c.658A G p.Asn220Asp 1/340 (1/38) CC00159 (391)

    MIP Ex1 c.141A G p.Ala45Ala (4/30) Several familiesMIP Ex1 c.319G A p.Val107Ile 5/76 (1/30) CC00110 (388)

    * Allele frequencies are given for number of chromosomes analyzed in the normal persons and in brackets the number chromosomesrepresenting persons from the cataract cohort.

    IOVS, July 2009, Vol. 50, No. 7 Hereditary Congenital Cataract Mutations 3301

  • 8/3/2019 Catarata Congenita Hereditaria

    12/13

    Phenotypes

    Most of our patients showed a composite morphology withregard to size, density, and localization of the lens opacitiesshowing mixtures of nuclear, cortical, and polar cataracts.

    This finding was further accentuated by considerable intrafa-milial differences in phenotypes and additional congenitaldysmorphology (microcornea and coloboma). In addition,some cataracts were progressive, leading to changing mor-phology during infancy. The highly heterogenic phenotypespreclude sound genotypephenotype predictions based onthis study. It should be kept in mind, however, that ourphenotype data were historical. The descriptive terminologyprobably differed among clinicians, and centers and routineexamination before surgery may have been cursory. A qual-ified phenotype description should rely on photographicdocumentation and be based on a descriptive standard forinfantile cataracts.

    CONCLUSION

    A mutational analysis strategy involving direct sequencing of 17cataract genes identified nearly three fourths of the mutationsin a cohort of hereditary congenital cataracts of NorthernEuropean descent. We propose the application of the strategyto investigate cases of isolated congenital cataract and un-known etiology.

    Acknowledgments

    The authors thank the families for their participation, Jeanette An-

    dreasen and Maria Jrgensen for excellent technical assistance, and

    Erik Kann for the genealogical studies.

    References

    1. Shiels A, Hejtmancik JF. Genetic origins of cataract. Arch Ophthal-mol. 2008;125:165173.

    2. Hejtmancik JF. Congenital cataracts and their molecular genetics.Semin Cell Dev Biol. 2007;19:134149.

    3. Bonneau D, Winter-Fuseau I, Loiseau MN, et al. Bilateral cataractand high serum ferritin: a new dominant genetic disorder? J MedGenet. 1995;32:778779.

    4. Beaumont C, Leneuve P, Devaux I, et al. Mutation in the ironresponsive element of the L ferritin mRNA in a family with domi-nant hyperferritinaemia and cataract. Nat Genet. 1995;11:444446.

    5. Hansen L, Yao W, Eiberg H, et al. The congenital ant-egg cataractphenotype is caused by a missense mutation in connexin46. MolVis. 2006;12:10331039.

    6. Hansen L, Yao W, Eiberg H, et al. Genetic heterogeneity inmicrocornea-cataract: five novel mutations in CRYAA, CRYGD,and GJA8. Invest Ophthalmol Vis Sci. 2007;48:39373944.

    7. Hansen L, Eiberg H, Rosenberg T. Novel MAF mutation in a familywith congenital cataract-microcornea syndrome. Mol Vis. 2007;13:20192022.

    8. Grnskov K, Rosenberg T, Sand A, Brndum-Nielsen K. Mutationalanalysis of PAX6: 16 novel mutations including 5 missense muta-

    tions with a mild aniridia phenotype. Eur J Hum Genet. 1999;7:274286.

    9. Eiberg H, Nielsen LS, Klausen J, et al. Linkage between serumcholinesterase 2 (CHE2) and crystalline gene cluster (CRYG): as-signment to chromosome 2. Clin Genet. 1989;35:313321.

    10. Ott J. A computer program for linkage analysis of general humanpedigrees. Am J Hum Genet. 1976;28:528529.

    11. Kent WJ, Sugnet CW, Furey TS, et al. The human browser at UCSC.Genome Res. 2002;12:9961006.

    12. Mackay DS, Andley UP, Shiels A. Cell death triggered by a novelmutation in the alphaA-crystallin gene underlies autosomal domi-nant cataract linked to chromosome 21q. Eur J Hum Genet.2003;11:784793.

    13. Bennett TM, Mackay DS, Knopf HL, Shiels A. A novel missensemutation in the gene for gap-junction protein alpha3 (GJA3) asso-

    ciated with autosomal dominant nuclear punctate cataractslinked to chromosome 13q. Mol Vis. 2004;10:376382.

    14. Burdon KP, Wirth MG, Mackey DA, et al. A novel mutation in theConnexin 46 gene causes autosomal dominant congenital cataract

    with incomplete penetrance. J Med Genet. 2004;41:e106.

    15. Bu L, Jin Y, Shi Y, et al. Mutant DNA-binding domain of HSF4 isassociated with autosomal dominant lamellar and Marner cataract.

    Nat Genet. 2002;31:276278.

    16. den Dunnen JT, Antonarakis SE. Mutation nomenclature exten-sions and suggestions to describe complex mutations: a discus-sion. Hum Mutat. 2000;15:712.

    17. Devi RR, Reena C, Vijayalakshmi P. Novel mutations in GJA3associated with autosomal dominant congenital cataract in theIndian population. Mol Vis. 2005;11:846852.

    18. Devi RR, Vijayalakshmi P. Novel mutations in GJA8 associated with

    autosomal dominant congenital cataract and microcornea. Mol Vis.2006;12:190195.

    19. Devi RR, Yao W, Vijayalakshmi P, Sergeev YV, Sundaresan P,Hejtmancik JF. Crystallin gene mutations in Indian families withinherited pediatric cataract. Mol Vis. 2008;14:11571170.

    20. Burdon KP, Wirth MG, Mackey DA, et al. Investigation of crystallingenes in familial cataract, and report of two disease associatedmutations. Br J Ophthalmol. 2004;88:7983.

    21. Shiels A, Bennett TM, Knopf HL, et al. The EPHA2 gene is associ-ated with cataracts linked to chromosome 1p. Mol Vis. 2008;14:20422055.

    22. Graw J, Klopp N, Illig T, Preising MN, Lorenz B. Congenitalcataract and macular hypoplasia in humans associated with a denovo mutation in CRYAA and compound heterozygous mutationsin P. Graefes Arch Clin Exp Ophthalmol. 2006;244:912919.

    FIGURE 6. Photograph of the right eye of an individual with nuclearcataract. The 26 year-old patient III:5 belonged to family CCMC0113

    with microcornea cataract and a MAFp.Lys320Glu mutation (Table 3).The corneal diameter was not measured but she had steep corneas(K-readings 6.7 6.85 mm of curvature), which indirectly indicates areduced overall corneal size. The cataract consisted of a circular densenuclear opacity with condensations in a triangular configuration ac-cording to the fetal Y-suture. Faintly seen triangular extensions outsidethe central opacity were present. The cortex and the anterior polarzones were clear. The left eye had identical findings. The patientunderwent surgery with insertion of artificial lenses at the age of 43

    years.

    3302 Hansen et al. IOVS, July 2009, Vol. 50, No. 7

  • 8/3/2019 Catarata Congenita Hereditaria

    13/13

    23. Litt M, Carrero-Valenzuela R, LaMorticella DM, et al. Autosomaldominant cerulean cataract is associated with a chain terminationmutation in the human beta-crystallin gene CRYBB2. Hum MolGenet. 1997;6:665668.

    24. Gill D, Klose R, Munier FL, et al. Genetic heterogeneity of theCoppock-like cataract: a mutation in CRYBB2 on chromosome22q11.2. Invest Ophthalmol Vis Sci. 2000;41:159165.

    25. Vanita V, Sarhadi V, Reis A, et al. A unique form of autosomaldominant cataract explained by gene conversion between beta-

    crystallin B2 and its pseudogene. J Med Genet. 2001;38:392396.26. Yao K, Tang X, Shentu X, Wang K, Rao H, Xia K. Progressivepolymorphic congenital cataract caused by a CRYBB2 mutation ina Chinese family. Mol Vis. 2005;11:758763.

    27. Bateman JB, von-Bischhoffshaunsen FR, Richter L, Flodman P,Burch D, Spence MA. Gene conversion mutation in crystallin,beta-B2 (CRYBB2) in a Chilean family with autosomal dominantcataract. Ophthalmology. 2007;114:425432.

    28. Riazuddin SA, Yasmeen A, Yao W, et al. Mutations in betaB3-crystallin associated with autosomal recessive cataract in two Pa-kistani families. Invest Ophthalmol Vis Sci. 2005;46:21002106.

    29. Marner E. A family with eight generations of hereditary cataract.Acta Ophthalmol. 1949;27:537551.

    30. Eiberg H, Marner E, Rosenberg T, Mohr J. Marners cataract (CAM)assigned to chromosome 16: linkage to haptoglobin. Clin Genet.1988;34:272275.

    31. Marner E, Rosenberg T, Eiberg H. Autosomal dominant congenitalcataract: morphology and genetic mapping. Acta Ophthalmol.1989;67:151158.

    32. Jamieson RV, Munier F, Balmer A, Farrar N, Perveen R, Black GC.

    Pulverulent cataract with variably associated microcornea and iriscoloboma in a MAF mutation family. Br J Ophthalmol. 2003;87:411412.

    33. Vanita V, Singh D, Robinson PN, Sperling K, Singh JR. A novelmutation in the DNA-binding domain of MAF at 16q23.1 associated

    with autosomal dominant cerulean cataract in an Indian family.Am J Med Genet A. 2006;140:558666.

    34. Davis J, Davis D, Norman B, Platigorsky J. Gene expression of themouse corneal crystalline Aldh3a1: activation by Pax6, Oct1, andp300. Invest Ophthalmol Vis Sci. 2008;49:18141826.

    IOVS, July 2009, Vol. 50, No. 7 Hereditary Congenital Cataract Mutations 3303