A New Locus for Autosomal Recessive Nuclear Cataract Mapped to Chromosome 19Q13 in a Pakistani Family

http://www.paper.edu.cn A New Locus for Autosomal Recessive Nuclear Cataract Mapped to Chromosome 19q13 in a Pakistani Family

S. Amer Riazuddin,1,2,3 Afshan Yasmeen,2,3 Qingjiong Zhang,1 Wenliang Yao,1 Muhammad Farooq Sabar,2 Zahoor Ahmed,2 Sheikh Riazuddin,2,3 and J. Fielding Hejtmancik1,3

PURPOSE. To identify the disease locus of autosomal recessive the lens or, in severe cases, the entire lens, with a variety of congenital nuclear cataracts in a consanguineous Pakistani fam- types of opacity. Congenital cataracts can lead to permanent ily. blindness by interfering with the sharp focus of light on the METHODS. A large Pakistani family with multiple individuals retina during critical developmental intervals. In addition, con- affected by autosomal recessive congenital cataracts was ascer- genital cataracts can provide insight into the biology of the lens tained. Patients were examined, blood samples were collected, as well as age-related cataract, which affects large parts of the and DNA was isolated. A genome-wide scan was performed aging population. Approximately one third of congenital cataract cases are using 382 polymorphic microsatellite markers on genomic 3 DNA from affected and unaffected family members. Two-point familial. Although autosomal dominant congenital cataract appears to be the most common familial form in the Western lod scores were calculated, and haplotypes were formed by 4 inspection. world, autosomal recessive and X-linked cataract also occur. To date, 21 loci have been identified for autosomal dominant RESULTS. In the genome-wide scan, a maximum lod score of cataract, and mutations in 13 of these genes have been re- 2.89 was obtained for marker D19S414 on 19q13. Fine map- ported.2 Fewer autosomal recessive congenital cataract loci ping using D19S931, D19S433, D19S928, D19S225, and genes have been identified. Congenital recessive cataracts D19S416, D19S213, D19S425, and D19S220 markers from have been mapped to 6 loci residing on chromosomes 3p22- the Geńe´thon database showed that markers in a 14.3-cM 24.2, 6p23-24, 9q13-22, 16q21-22, 19q13.4, and 21q22.3.5–11 (12.66-Mb) interval flanked by D19S928 and D19S420 coseg- Of these, mutations in four genes: GCNT2, HSF4, LIM2, and regated with the cataract locus. Lack of homozygosity further CRYAA have been found.5,6,9,10,12,13 suggests that the cataract locus may lie in a 7-cM (4.3-Mb) Herein, we report a consanguineous Pakistani family with interval flanked by D19S928 proximally and D19S425 distally. multiple members affected by autosomal congenital recessive On fine mapping, a maximum lod score of 3.09 was obtained ␪ ϭ nuclear cataract. Initially, a genome-wide search including ex- with D19S416 at 0. clusion of known cataract loci was completed. Linkage analysis CONCLUSIONS. Linkage analysis identified a new locus for auto- provided evidence of a new locus for autosomal congenital somal recessive congenital nuclear cataracts on chromosome cataract on chromosome 19q13. The maximum lod score is ϭ ␪ ϭ 19q13 in a consanguineous Pakistani family. (Invest Ophthal- obtained with D19S416 (Zmax 3.09, at 0), and the mol Vis Sci. 2005;46:623–626) DOI:10.1167/iovs.04-0955 cataract locus cosegregates in a 14.3-cM (12.66-Mb) interval flanked by D19S928 and D19S420. Lack of homozygosity fur- ongenital cataracts are one of the major causes of vision ther suggests that the cataract locus may lie in a 7-cM (4.3 Mb) Closs in children worldwide1,2 and are responsible for ap- interval flanked by D19S928 proximally and D19S425 distally. proximately one third of blindness in infants. Congenital cataracts can occur in an isolated fashion or as one component of a syndrome affecting multiple tissues. Nonsyndromic congen- MATERIALS AND METHODS ital cataracts have an estimated frequency of 1 to 6 per 10,000 live births. They vary markedly in severity and morphology, Clinical Ascertainment affecting the nuclear, cortical, polar, or subcapsular parts of A four-generation consanguineous Pakistani family with nonsyndromic congenital cataract was recruited to participate in a collaborative study between the Center of Excellence in Molecular Biology (Lahore, Paki- 1 From the Ophthalmic Genetics and Visual Function Branch, Na- stan) and the National Eye Institute (Bethesda, MD), to identify new tional Eye Institute, National Institutes of Health, Bethesda, Maryland; 2 disease loci causing inherited visual diseases. Institutional review board and the National Centre of Excellence in Molecular Biology, Univer- approval was obtained for this study from both centers. The partici- sity of the Punjab, Lahore, Pakistan. 3Contributed equally to the work and therefore should be consid- pating subjects gave informed consent consistent with the tenets of the ered equivalent authors. Declaration of Helsinki. Supported in part by Higher Education Commission and Ministry The family described in this study is from a remote village in the of Science and Technology, Islamabad, Pakistan. Punjab province of Pakistan. A detailed medical history was obtained Submitted for publication August 6, 2004; revised October 25, by interviewing family members. Medical records of clinical examina- 2004; accepted November 1, 2004. tions previously conducted with slit lamp biomicroscopy reported Disclosure: S.A. Riazuddin, None; A. Yasmeen, None; Q. bilateral nuclear cataracts in all affected individuals for whom records Zhang, None; W. Yao, None; M.F. Sabar, None; Z. Ahmed, None; S. were available. Cataracts were either present at birth or developed in Riazuddin, None; J.F. Hejtmancik, None infancy. The cataracts showed constant morphology, but varied in size. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- All affected individuals had cataract surgery in the early years of their ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. lives, and hence no pictures of the lenses were available. Blood sam- Corresponding author: J. Fielding Hejtmancik, OGVFB/NEI/NIH, ples were collected from affected and unaffected family members. Building 10, Room 10B10, 10 Center Drive MSC 1860, Bethesda, MD DNA was extracted by a nonorganic method, as described by Grimberg 20892-1860; [email protected]. et al.14

624 Riazuddin et al. IOVS, February 2005, Vol. 46, No. 2

TABLE 1. Two-Point Lod Scores of Chromosome 19q Markers ␪ Marker cM Mb 0 0.01 0.05 0.1 0.2 0.3 0.4 Zmax max

D19S221* 35.5 12.57 Ϫϱ Ϫ2.86 Ϫ1.51 Ϫ0.55 0.21 0.29 0.24 0.29 0.30 D19S226* 41.7 14.49 Ϫϱ 0.58 0.98 1.18 1.21 1.08 0.81 1.24 0.18 D19S931 48.9 33.31 Ϫϱ 0.62 1.16 1.26 1.13 0.84 0.49 1.27 0.09 D19S433 50.82 35.11 Ϫϱ 0.58 1.09 1.14 0.94 0.63 0.27 1.15 0.08 D19S928 51.7 35.84 Ϫϱ Ϫ0.76 Ϫ0.15 0.04 0.13 0.1 0.05 0.15 0.21 D19S414* 53.2 36.60 2.93 2.87 2.67 2.39 1.84 1.24 0.96 2.93 0.00 D19S225 55.9 37.51 3.01 2.91 2.69 2.43 1.85 1.27 0.99 3.01 0.00 D19S416 58.1 38.76 3.09 2.94 2.72 2.45 1.87 1.26 0.97 3.09 0.00 D19S213 58.1 38.80 2.65 2.59 2.41 2.15 1.64 1.11 0.54 2.65 0.00 D19S425 58.7 40.18 Ϫ1.33 0.81 1.33 1.4 1.2 0.84 0.40 1.49 0.09 D19S220* 61.4 43.12 Ϫ2.27 Ϫ1.65 Ϫ1.18 Ϫ0.99 Ϫ0.56 Ϫ0.23 0.11 0.11 0.40 D19S420* 66.0 48.50 Ϫϱ Ϫ1.84 Ϫ0.61 Ϫ0.17 0.13 0.20 0.15 0.21 0.31 D19S902* 76.2 53.02 Ϫϱ Ϫ1.74 Ϫ0.47 Ϫ0.03 0.22 0.19 0.14 0.22 0.20 D19S571* 87.7 57.98 Ϫϱ Ϫ1.82 Ϫ0.52 Ϫ0.05 0.27 0.31 0.37 0.37 0.40

* Markers included in genome-wide scan.

Genotype Analysis greater than 1.0 only for markers D1S498, D9S164, D19S226, and D19S414. Of these, D1S498 and D9S164 had closely The initial genome scan was performed with 382 highly polymorphic flanking markers yielding large negative lod scores. D19S414 fluorescent markers (PRISM Linkage Mapping Set MD-10; Applied Bio- and D19S226 are adjacent markers in the MD-10 mapping set, systems, Inc. [ABI], Foster City, CA) that have an average spacing of 10 yielding lod scores of 2.93 at ␪ ϭ 0 and 1.24 at ␪ ϭ 0.18, cM. Multiplex polymerase chain reactions (PCR) were performed, as respectively. In addition, D19S221 on the proximal side of previously described.15 Briefly, each reaction was performed in a 5-␮L D19S414 supported linkage to this region with a maximum lod mixture containing 40 ng genomic DNA, various combinations of 10 score of 0.29 at ␪ ϭ 0.3. ␮M dye-labeled primers pairs, 0.5 ␮L10ϫ PCR buffer (GeneAmp Fine mapping using markers on chromosome 19 confirmed Buffer II; ABI) 0.5 ␮L 10 mM dNTP mix, 2.5 mM MgCl , and 0.2 U of 2 linkage to this region (Table 1). The maximum lod scores in the Taq DNA polymerase (AmpliTaq Gold Enzyme, ABI). Amplification region are 3.09 with D19S416 at ␪ ϭ 0, 3.01 with D19S225 at was performed in a PCR system (GeneAmp 9700; ABI). Initial denatur- ␪ ϭ 0, 2.93 with D19S414 at ␪ ϭ 0, and 2.65 with D19S213 at ation was performed for 5 minutes at 95°C, followed by 10 cycles of 15 ␪ ϭ 0. Obligate recombinants shown by lod scores of Ϫϱ at seconds at 94°C, 15 seconds at 55°C, and 30 seconds at 72°C and then ␪ ϭ 0 are obtained with the flanking markers D19S928 prox- 20 cycles of 15 seconds at 89°C, 15 seconds at 55°C, and 30 seconds imally and D19S420 distally. In addition, D19S425 and at 72°C. The final extension was performed for 10 minutes at 72°C and D19S220 yield a lod score of Ϫ1.33 at ␪ ϭ 0 and Ϫ2.27 at ␪ ϭ followed by a final hold at 4°C. PCR products from each DNA sample 0, respectively, strongly suggesting that the cataract locus lies were pooled and mixed with a loading cocktail containing size stan- in the D19S928 to D19S425 interval. dards (HD-400; ABI) and loading dye. The resultant PCR products were Visual inspection of the haplotypes of the markers used in separated on a 5% denaturing urea-polyacrylamide gel (Long Ranger; fine mapping supports the linkage analysis, localizing the cat- ABI) in a DNA sequencer (model 377; ABI) and analyzed by computer aract locus to this region and placing it on chromosome 19q13 (Genescan, ver. 3.1 and Genotyper, ver. 2.1 software packages; ABI). in the 14.3 cM (12.66 Mb) interval between D19S928 and Linkage Analysis D19S420 (Fig. 1). There is a proximal recombination event at D19S928 in affected individual 13 and a distal recombination Two-point linkage analyses were performed with the FASTLINK ver- event at D19S420 in affected individual 11. In addition, lack of sion of MLINK from the LINKAGE program package.16,17 Maximum lod homozygosity at markers D19S425, D19S220, and D19S420 in scores were calculated using ILINK (all LINKAGE packages are pro- affected individuals 11 to 14 of this consanguineous family vided in the public domain by the Human Genome Mapping Project suggests that the disease locus might be in the 7-cM (4.3-Mb) Resources Centre, Cambridge, UK; http:www.hgmp.mrc.ac.uk). Auto- region bounded by D19S928 and D19S425. This lack of ho- somal recessive nuclear cataracts were analyzed as a fully penetrant mozygosity is the source of the negative lod score for D19S425 trait with an affected allele frequency of 0.001. The marker order and and D19S220 on fine mapping (Table 1). distances between the markers were obtained from the Geńe´thon database (http://www.genethon.fr/ provided in the public domain by the French Association against Myopathies, Evry, France) and the DISCUSSION National Center for Biotechnology Information chromosome 19 se- quence maps (http://www.ncbi.nlm.nih.gov/mapview/ provided in We report linkage of autosomal recessive nuclear cataracts in a the public domain by National Center for Biotechnology, Bethesda, consanguineous Pakistani family to markers on 19q13. The MD). For the initial genome scan, equal allele frequencies were as- maximum lod score of 3.09 was obtained with D19S416 at ␪ ϭ sumed, whereas, for fine mapping, allele frequencies were estimated 0, and the cataract locus cosegregated with markers in a from 125 unrelated and unaffected individuals from the Punjab prov- 14.3-cM (12.66-Mb) region of chromosome 19q13 flanked by ince of Pakistan. D19S928 and D19S420. Lack of homozygosity further suggests that the cataract locus may lie in a 7-cM (4.3-Mb) D19S928 to RESULTS D19S425 interval. Although the maximum lod score of 3.09 is only slightly higher than the traditional limiting value of 3.0, it Linkage to five known autosomal recessive cataract loci— represents the maximum value obtainable with this family. In 3p23, 6p23-24, 9q13-p24, 19q14, and 21q22.3—was initially addition, the lack of any lod scores above 1.5, except for excluded by haplotype analysis using closely flanking markers marker D19S414, obtained in the remainder of the genome- (data not shown). A genome-wide scan yielded lod scores wide scan and the large negative lod scores of markers flanking 中国科技论文在线 http://www.paper.edu.cn

IOVS, February 2005, Vol. 46, No. 2 New Locus for AR Nuclear Cataract 625

To date, six loci for congenital recessive cataract have been reported, and mutations have been found in genes at four of these loci: GCNT2 on chromosome 6, HSF4 on chromosome 16, LIM2 on chromosome 19, and CRYAA on chromosome 21. With the exception of ␣A-crystallin, these genes represent a rather different set than those in which mutations have been associated with autosomal dominant cataract. Most genes associated with dominant cataracts encode structural genes such as the highly expressed lens crystallin,18–20 cytoskeletal proteins such as the beaded filament protein BFSP2,21 and mem- brane proteins such as aquaporin0.22 Recessive cataracts ap- pear more likely to be associated with enzymes such as GCNT2.10 Growth factors such as HSF423 or LIM26 have been shown to cause either autosomal dominant or recessive cataracts, depending on the type of mutation. The dual role of ␣-crystallin as both a structural crystallin and a chaperone may help to explain why mutations in this gene also can cause both autosomal dominant and recessive cataracts. Nuclear cataract is the most common form of congenital cataract. Congenital nuclear cataracts have been associated both with autosomal dominant and autosomal recessive inher- itance. To date, three autosomal dominant loci, 1pter-p36.13,24 2p12,25 and 12q13,26 and two autosomal recessive loci, 3p11 and 21q,5 have been identified in families with nuclear cataract. Mutations in CRYAA5 on chromosome 21 have been associated with nuclear cataracts. Moreover, mutations in ␤B1- crystallin (CRYBB1),27 ␤B2-crystallin (CRYBB2),18 ␥C-crystallin (CRYGC),19 and connexins Cx5028 and Cx4629 have been associated with nuclear pulverulent cataracts. Crystallins make up 90% of soluble lens proteins and have an essential role in maintaining lens transparency. Another characteristic feature of the lens is its extensive system of low-resistance gap junctions between lens fiber cells. Struc- tural proteins belonging to the connexin family make up the intercellular channels present in these gap junctions. Hence, together, crystallins and connexins are the most compelling candidates to screen for mutations for inherited cataract. No genes encoding crystallins or connexins are present in the 7-cM interval between D19S928 and D19S425. Other potential candidate genes present in this region are currently being screened for a possible role in the congenital nuclear cataract in this family. Identification of the specific mutation and gene associated with these cataracts will increase our understanding of lens biology and the nuclear cataract at a molecular level.

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