A Novel Locus for Autosomal Dominant Nuclear Cataract Mapped to Chromosome 2P12 in a Pakistani Family

A Novel Locus for Autosomal Dominant Nuclear Cataract Mapped to Chromosome 2p12 in a Pakistani Family

Shagufta Khaliq, Abdul Hameed, Muhammad Ismail, Khalid Anwar, and S. Qasim Mehdi

PURPOSE. To map the disease locus in a four-generation, con- dominant congenital cataract, at least 16 loci have been iden- sanguineous Pakistani family affected by autosomal dominant tified by linkage analysis on chromosomes 1q21-q25,3 1pter- congenital nuclear cataract (adNCat). All affected individuals p36.13,4 2q33-35,5 3q21.2-22.3, 6 11q22-q22.3,7 12q13,8 had early onset of bilateral nuclear cataract. 13q11-q13,9 14q24-qter,10 15q21-22,11 16q22,12 17q24,13 14 15 16 17 18 METHODS. Genomic DNA from family members was typed for 17q11-q12, 17p13, 20p12-q12, 21q22.3, and 22q alleles at more than 300 known polymorphic genetic markers (Eye Research Network, provided in the public domain by the by polymerase chain reaction. The lod scores were calculated Eye Research Institute, Oakland University, Rochester, MI; by using two-point linkage analysis of the genotyping data. Kenneth P, Mitton, editor; available at http://ken.mitton.com/ ern/lensbase.html). RESULTS. The maximum lod score, 4.05, was obtained for the Mutations in several genes are known to cause cataracts. marker D2S2333. Proximal and distal crossovers were ob- These include the genes encoding lens connexins (CX50,18,19 served with markers D2S286 and D2S1790, respectively. These and CX46,20), crystallins (CRYAA,21,22 CRYAB,23 CRYBA,24 crossovers define the critical disease locus to an interval of CRYBB,25 CRYGC,26 and CRYGD27), lens-developmental pro- approximately 9 centimorgans (cM). tein PITX3,28 the major intrinsic protein of lens fiber (MIP),29 CONCLUSIONS. Linkage analysis identified a novel locus for and cytoskeletal protein CP49.30 The connexins comprise a adNCat on chromosome 2p12 in a Pakistani family. A genome family of structural proteins that are important in the formation database analysis of the target interval is being undertaken to of gap junctions and play a vital role in the structure and identify candidate gene(s) for the disease. (Invest Ophthalmol function of the lens. Mutations in the crystallin genes that Vis Sci. 2002;43:2083–2087) encode more than 90% of the lens cytoplasmic proteins have been identified as a cause of dominant cataracts. MIP is a utosomal dominant congenital cataract (adCat) is a clini- member of the aquaporin 0 (AQP0) family of membrane-bound Acally and genetically heterogeneous lens disorder. Cataract water channels.30 may occur as an isolated anomaly or as a component of a In this study we describe a large, four-generation, consan- multisystem syndrome. As an isolated anomaly, congenital cat- guineous Pakistani family affected by adCat (Fig. 1). Morpho- aracts can occur sporadically, but many are familial. Although logically the cataract in all the affected members of this family X-linked and autosomal recessive transmission of hereditary was nuclear with a phenotype typical of early onset of disease. cataracts has been observed, the most frequent mode of inher- After exclusion of all the candidate genes that are known to itance is autosomal dominant with a high degree of pen- be involved in the structure and function of the lens and all the etrance.1 Cataract is one of the most common major disorders known loci for adCat, a whole-genome search was performed. of the eye in infants and accounts for 10% to 30% of legal Linkage analysis provided evidence for a novel locus for auto- blindness in children. Cataract shows a wide variety of pheno- somal dominant nuclear cataract (adNCat) on chromosome typic and genotypic heterogeneity.2 Although there is no 2p12. agreed nomenclature for the morphologic patterns of cataract, depending on the location, opacity, and appearance the fol- lowing types of cataracts are generally described in the litera- MATERIALS AND METHODS ture: anterior polar, posterior polar, nuclear, lamellar (zonular), coralliform blue dot, cortical, pulverulent, and total (available Clinical Details at http://www.ncbi.nlm.nih.gov/omim, provided in the public A four-generation consanguineous Pakistani family with cataract was domain by the National Center for Biotechnology Information, ascertained. Based on family history and clinical diagnosis, the type of Bethesda, MD; accessed February 2002). For nonsyndromic cataract was classified as congenital embryonic nuclear, with an autosomal dominant mode of inheritance. In each generation, either one or both parents were affected (Fig. 1). Previous records showed that From the Dr. A. Q. Khan Research Laboratories, Biomedical and cataract was either present at birth, or developed in infancy and that Genetic Engineering Division, Islamabad, Pakistan. there was no family history of any other ocular or systemic abnormal- Supported by The Wellcome Trust Grant 063406/Z/2000/Z ities. Two individuals, III:3 and IV:4, had had bilateral cataract surgery (SQM). before the age of 40 years. Detailed ophthalmic examination was Submitted for publication September 19, 2001; revised February performed on 20 family members. Analysis included slit lamp biomi- 19, 2002; accepted March 6, 2002. croscopy and photography of the lens to record the cataract type. Commercial relationships policy: N. Twelve members of the family were found to be affected with bilateral The publication costs of this article were defrayed in part by page cataract, whereas the remaining eight were unaffected. After correc- charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. tion of astigmatism, all affected individuals had adequate vision. For Corresponding author: Shagufta Khaliq, Biomedical and Genetic example, individual IV:5 achieved a corrected vision of 6/15 and 6/18 Engineering Division, Dr. A. Q. Khan Research Laboratories, PO Box in the left and right eyes, respectively. Figure 2 shows photographs and 2891, 24 Mauve Area, Islamabad, Pakistan; schematic views of lens opacities in individuals III:4 (Figs. 2a, 2b) and [email protected]. IV:5 (Figs. 2c, 2d), aged 65 and 28 years, respectively. Slit lamp

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FIGURE 1. Pedigree of an adCat family with genotypic data for the microsatellite markers analyzed. Circles: females; squares: males; ﬁlled symbols: affected individuals; unﬁlled symbols: unaffected individuals; diagonal line through a symbol: deceased family member; double line between individuals: consanguinity; crosshatching: parental crossover.

biomicroscopy of the lens showed that the opacity consisted of a large obtained from 100 unrelated, normal Pakistani individuals with no number of minute shiny particles with a distinct bluish hue in the fetal symptoms of cataract or any other disease of the eye. part of the nucleus. The cataracts were identical in appearance in all patients but were of various sizes in different subjects. Cataract was Microsatellite and Linkage Analysis also progressive in nature and, as shown in Figures 2c and 2d, the changes were more prominent in the offspring(s) (IV:5) of a ﬁrst- For linkage analysis polymorphic microsatellite markers (Human Map- cousin marriage (Fig. 1: III:3 and III:4). ping Set, ver. 8; Research Genetics, Inchinnan, Scotland, UK) were For genetic analysis, blood samples were collected from the whole ampliﬁed by polymerase chain reaction (PCR). PCR reactions were ␮ family with voluntarily signed informed consent. The protocol of the each performed in a 10 L volume, containing 1.5 mM MgCl2, 0.4 mM study conformed with the tenets of the Declaration of Helsinki. of each primer, 200 ␮M dNTPs, 1 U Taq DNA polymerase, and PCR

Genomic DNA was extracted from whole blood using a commercially buffer containing 16 mM (NH4)2SO4, 67 mM Tris-HCl [pH 8.8], and available extraction kit (Nucleon II: Scotlab Bioscience, Strathclyde, 0.01% of the nonionic detergent Tween-20 (Bio-Line, London, UK). Scotland, UK). To calculate the allele frequencies, samples were also Ampliﬁcation was performed with an initial denaturation for 3 minutes

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FIGURE 2. Photographs of the lens opacities of individuals containing a single disease chromosome (a) and two disease chromosomes (c) and their respective schematic diagrams (b) and (d).

at 95°C, followed by 30 cycles of denaturation at 95°C for 1 minute, RESULTS annealing at 55°C for 1 minute, extension at 72°C for 1 minute, and a final extension at 72°C for 3 minutes. The PCR products were sepa- Known loci for adCat were first excluded by linkage analysis rated on 8% to 10% nondenaturing polyacrylamide gels (Protogel; using locus-specific microsatellite markers. Subsequently, a ge- National Diagnostics, Edinburgh, Scotland, UK). The gel was stained nome-wide search was performed, using a set of 296 polymor- with ethidium bromide and photographed under UV illumination. phic markers spanning the entire human genome at approxi- Alleles were assigned to individuals, and haplotypes of all family mem- mately 15- to 20-cM intervals (Human Mapping Set; Research bers were constructed. On the first indication of linkage to chromo- Genetics). Linkage was obtained for markers located on chromosome 2p12. More markers in this region were analyzed to some 2p12, the family was further genotyped for more markers across obtain proximal and distal crossovers to localize the disease this region (Fig. 1). The genotypic data were used to calculate the lod region. Haplotypes for these markers are shown in Figure 1. scores using the MLINK software program (ver. 5.2, ftp://linkage. Two-point lod scores between adNCat and the markers in this rockefeller.edu/software/linkage/, provided in the public domain by region are summarized in Table 2. The maximum lod score of Rockefeller University, New York, NY). Allele frequencies were also 4.05 was obtained for the marker D2S2333 with no crossover. calculated of the normal, ethnically matched population. The pheno- A proximal crossover was obtained for individual IV:8 with type was analyzed as an autosomal dominant trait with nearly complete marker D2S286, and a distal crossover was obtained with penetrance (0.99) at a frequency of 0.0001 for the disease allele. marker D2S1790 in individuals III:5 and IV:11. These crossovers localized the disease gene within approximately 9 cM on Mutation Screening chromosome 2p12. Individuals III:3 and III:4 were cousins, and because the To examine the probability of the existence of the disease-associated mode of inheritance of the disease is autosomal dominant, both mutations, exon-specific intronic primers were designed for the can- appeared to carry the same disease chromosomes in a het- didate gene for mammalian cytochrome c oxidase subunit Vb (COX5B, erozygous state. Individual III:3 was assumed to have a parental GenBank accession number M59250; provided in the public domain by

the National Center for Biotechnology Information, Bethesda, MD, and TABLE 1. COX5B Gene-Sequencing Primers and Their Product Sizes available at http://www.ncbi.nlm.nih.gov/Genbank). The gene con- tains four coding exons spanning a region of approximately 2.4 kb.31 PCR Product Each exon was amplified at an annealing temperature of 55°C under Exon Primer Sequence (bp) standard PCR conditions. Primer sequences and their PCR product F5؅-CTGCAGCTTGTTCCCGGAAG-3؅ 261 1 sizes are given in Table 1. The amplified exons were analyzed by R5؅-CGTAAAGCGGCCACTGGGACC-3؅ mutation-detection electrophoresis (MDE) at 180 V on a commercial 2 F5؅-GCCCGCTAAAACTTATACAGAAG-3؅ 296 apparatus (model 600S; Hoefer, San Francisco, CA). The products of R5؅-CATCCTTCCAGAAGCTCAGGGGAC-3؅ PCR amplification were purified and sequenced using a commercially 3 F5؅-GATACAGGAAACTTGGCTTGTAG-3؅ 349 available kit (Big Dye Terminator; PE Biosystems, Foster City, CA) and R5؅-GCTTGAGAGTGTCAAGACCCTTAG-3؅ ؅ ؅ the products were analyzed on an automated DNA sequencer (model 4 F5-GGAGGGCTTGAACTGATCTCTTAAG-3 547 R5؅-CACTATACACTGGTTATACACTTTC-3؅ 377; PE Biosystems). All PCR products were sequenced in both forward and reverse directions. F, forward; R, reverse.

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TABLE 2. Two-Point lod Scores between Congenital Nuclear Cataract Phenotype and Chromosome 2p12 Microsatellite Markers, with Zmax and ␪ max values

Recombination Fraction ␪ Marker ␪ Location Marker 0.0 0.01 0.05 0.1 0.2 0.3 0.4 Zmax max

80.82 D2S1394 1.97 1.93 1.78 1.58 1.15 0.70 0.28 1.97 0.00 94.05 D2S286 Ϫ1.10 0.85 1.33 1.35 1.03 0.60 0.19 1.35 0.10 99.41 D2S1777 2.59 2.54 2.33 2.08 1.56 1.06 0.55 2.59 0.00 103.16 D2S2333* 4.05 3.98 3.66 3.25 2.39 1.49 0.64 4.05 0.00 103.16 D2S1790 Ϫ9.75 Ϫ0.47 0.67 0.97 0.98 0.76 0.43 0.98 0.20 111.21 D2S2216 Ϫ0.68 0.55 1.05 1.12 0.93 0.59 0.22 1.12 0.10 114.42 D2S2972 Ϫ0.78 Ϫ1.78 0.05 0.64 0.87 0.66 0.29 0.87 0.20 125.18 D2S410 Ϫ3.14 Ϫ3.48 Ϫ0.93 Ϫ0.03 0.54 0.55 0.30 0.55 0.30

* Marker with maximum lod score. Two-point lod score was calculated at nearly complete penetrance (0.99) at a frequency of 0.0001 for the disease allele. Map locations are based on data obtained from Marshmed genetic maps; Marshﬁeld Laboratories (Marshﬁeld, WI).

crossover (Fig. 1). All their affected children inherited the 4. Eiberg H, Lund AM, Warburg M, Rosenberg T. Assignment of disease chromosomes from both parents—that is, they were congenital cataract Volkmann type (CCV) to chromosome 1p36. homozygous in the disease region. Further, there was a marked Hum Genet. 1995;96:33–38. difference in the size and severity of the cataract between 5. Lubsen NH, Renwick JH, Tsui LC, Breitman ML, Schoenmakers JG. individuals that inherited a single disease chromosome (Figs. A locus for a human hereditary cataract is closely linked to the 2a, 2b) compared with those with two disease chromosomes gamma-crystallin gene family. Proc Natl Acad Sci USA. 1987;84: (Figs. 2c, 2d). 489–492. 6. Kramer PL, LaMorticella D, Schilling K, Billingslea AM, Weleber RG, Litt M. A new locus for autosomal dominant congenital cataracts maps to chromosome 3. Invest Ophthalmol Vis Sci. 2000;41: DISCUSSION 36–39. 7. Berry V, Francis P, Reddy MA, et al. Alpha-B crystallin G (CRYAB) This novel locus, close to the centromeric region of chromo- mutation causes dominant congenital posterior polar cataract in some 2, is the third genetic locus reported to date for adNCat. humans. Am J Hum Genet. 2001;69:1141–1145. The other two loci for nuclear cataracts have been reported on 4 8. Bateman JB, Johannes M, Flodman P, et al. A new locus for chromosome 1pter-1p36 in a large Danish family and on autosomal dominant cataract on chromosome 12q13. Invest Oph- chromosome 12q13 in a white family of European descent in thalmol Vis Sci. 2000;41:2665–2670. 8 the United States. 9. Mackay D, Ionides A, Berry V, Moore A, Bhattacharya S, Shiels A. A Similar to retinal disorders, these results show the genetic new locus for dominant “zonular pulverulent” cataract, on chro- heterogeneity of cataract, and many genes appear to be in- mosome 13. Am J Hum Genet. 1997;60:1474–1478. volved for a particular phenotype. The ␥-crystallin gene that 10. Miller BA, Jaafar MS, Capo H. Chromosome 14-terminal deletion has been associated with Coppock-like cataract is also present and cataracts. Arch Ophthalmol. 1992;110:1053. on chromosome 2q33-35.26 However, it is far from this new 11. Vanita SJR, Sarhadi VK, Singh D, et al. A novel form of “central cataract locus, and the distal crossover with marker D2S1790 pouchlike” cataract, with sutural opacities, maps to chromosome excludes the possibility of this gene’s being responsible for the 15q21-22. Am J Hum Genet. 2001;68:509–514. disease haplotype in this family. The candidate genes in this 12. Marner E, Rosenberg T, Eiberg H. Autosomal dominant congenital disease region were searched from the genomic database (pro- cataract: morphology and genetic mapping. Acta Ophthalmol vided in the public domain by the Sanger Centre, Hinxton Hall, (Copenh). 1989;67:151–158. UK, and available at http://www.ensembl.org/). A candidate 13. Armitage MM, Kivlin JD, Ferrell RE. A progressive early onset gene of mammalian cytochrome c oxidase subunit Vb (COX5B; cataract gene maps to human chromosome 17q24. Nat Genet. GenBank accession number M59250) which is located on chro- 1995;9:37–40. mosome 2-region cen-q13 was screened for mutations.31 No 14. Padma T, Ayyagari R, Murty JS, et al. Autosomal dominant zonular disease-associated mutation was found, and thus the disease- cataract with sutural opacities localized to chromosome 17q11-12. Am J Hum Genet. 1995;57:840–845. causing gene for this novel locus is yet to be identiﬁed. 15. Berry V, Ionides ACW, Moore AT, Plant C, Bhattacharya SS, Shiels A. A locus for autosomal dominant anterior polar cataract on Acknowledgments chromosome 17p. Hum Mol Genet. 1996;5:415–419. 16. Yamada K, Tomita HA, Kanazawa S, Mera A, Amemiya T, Niikawa The authors thank the family members for taking part in this study and N. Genetically distinct autosomal dominant posterior polar cata- Shomi S. Bhattacharya and Qasim Ayub for valuable help. ract in a four-generation Japanese family. Am J Ophthalmol. 2000; 129:159–165. References 17. Kramer P, Yount J, Mitchell T, et al. A second gene for cerulean cataracts maps to the beta crystallin region on chromosome 22. 1. Merin S, Inherited cataracts. In: Merin S, ed. Inherited Eye Disease: Genomics. 1996;35:539–542. Diagnosis and Clinical Management. New York: Marcel Dekker; 18. Shiels A, Mackay D, Ionides A, Berry V, Moore A, Bhattacharya SS. 1991:86–120. A missense mutation in the human Connexin 50 gene (GJA8) 2. Francois J. Genetics of cataract. Ophthalmologica. 1982;184:61– underlies autosomal dominant “zonular pulverulent” cataract, on 71. chromosome 1q. Am J Hum Genet. 1998;62:526–532. 3. Renwick JH, Lawler SD. Probable linkage between a congenital 19. Berry V, Mackay D, Khaliq S, et al. Connexin 50 mutation in a cataract locus and the Duffy blood group locus. Ann Hum Genet. family with congenital zonular nuclear pulverulent cataract of 1963;27:67–84. Pakistani origin. Hum Genet. 1999;105:168–170.

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20. Mackay D, Ionides A, Kibiar Z, et al. Connexin 46 mutations in 26. Heon E, Priston M, Schorderet DF, Billingsley GD, Girard PO, autosomal dominant congenital cataract. Am J Hum Genet. 1999; Lubsen N, Munier FL. The gamma-crystallins and human cataracts: 64:1357–1364. a puzzle made clearer. Am J Hum Genet. 1999;65:1261–1267. 21. Litt M, Kramer P, LaMorticella DM, Murphey W, Lovrien EW, 27. Heon E, Liu S, Billingsley G, et al. Gene localization for a cuneiform Weleber RG. Autosomal dominant congenital cataract associated cataract, on chromosome 2q33-35. Am J Hum Genet. 1998;63: with a missense mutation in the human alpha crystallin gene 921–926. CRYA. Hum Mo Genet. 1998;7:471–474. 28. Semina EV, Ferrell RE, Mintz-Hittner HA, et al. A novel homeobox 22. Pras E, Frydman M, Levy-Nissenbaun E, et al. A nonsense mutation gene PITX3 is mutated in families with autosomal-dominant cata- (W9X) in CRYAA causes autosomal recessive cataract in an inbred racts and ASMD. Nat Genet. 1998;19:167–170. Jewish Persian family. Invest Ophthalmol Vis Sci. 2000;41:3511– 3515. 29. Berry V, Francis P, Kaushal S, Moore A, Bhattacharya SS. Missense mutations in MIP underlie autosomal dominant “polymorphic” and 23. Berry V, Francis P, Reddy MA, et al. Alpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar lamellar cataracts linked to 12q. Nat Genet. 2000;25:15–17. cataract in humans. Am J Hum Genet. 2001;69:1141–1145. 30. Jakobs PM, Hess JF, FitzGerald PG, Kramer P, Weleber RG, Litt M. 24. Kannabiran C, Rogan PK, Olmos L, et al. Autosomal dominant Autosomal dominant congenital cataract associated with a deletion zonular cataract with sutural opacities is associated with a splice mutation in the human beaded ﬁlament protein gene BFSP2. Am J mutation in the beta A3/A1-crystallin gene. Mol Vis. 1998;4:18. Hum Genet. 2000;66:1432–1436. 25. Litt N, Carrero-Valenzuela R, LaMorticella DM, et al. Autosomal 31. Lomax MI, Hsieh CL, Darras BT, Francke U. Structure of the human dominant cerulean cataract is associated with a chain termination cytochrome c oxidase subunit Vb gene and chromosome mapping mutation in the human beta-crystallin gene CRYBB2. Hum Mol of the coding gene and of seven pseudogenes. Genomics. 1991; Genet. 1997;6:665–668. 10:1–9.

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