|Molecular Vision 2007;
Received 21 February 2007 | Accepted 24 August 2007 | Published 10 September 2007
Localization of autosomal recessive congenital cataracts in consanguineous Pakistani families to a new locus on chromosome 1p
Wenliang Yao,2 Haiba Kaul,1
Xiaodong Jiao,2 Libe Gradstein,2
Yan Zhang,2 Tayyab Husnain,1
J. Fielding Hejtmancik,2
S. Amer Riazuddin,1
Drs. Butt, Yao, S. Riazuddin, Hejtmancik, and S.A. Riazuddin contributed equally to this publication.
1National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore Pakistan, 2Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, MD
Correspondence to: S. Amer Riazuddin, Ph.D., National Centre of Excellence in Molecular Biology, 87 West Canal Bank Road, Lahore, Pakistan 53700; Phone: 92-42-542-1235; FAX: 92-42-542-1316; email: firstname.lastname@example.org
Purpose: To identify the disease locus for autosomal recessive congenital cataracts in two consanguineous Pakistani families.
Methods: Two Pakistani families were ascertained, ophthalmologic examination including slit lamp biomicroscopy was performed on all members, blood samples were collected and DNA was extracted. A genome-wide scan was performed using 382 polymorphic microsatellite markers on genomic DNA from affected and unaffected family members. Two-point logarithm of odds (LOD) scores were calculated using the LINKAGE program package.
Results: All the affected individuals of family PKCC009 show bilateral membranous cataract, whereas the affected individuals of family PKCC039 show bilateral posterior sub-capsular cataract. Other ocular abnormalities include corneal opacities, microcornea and nystagmus in the affected individuals of PKCC009. Maximum two point LOD scores were obtained with D1S186 (4.14 at θ= 0), D1S432 (4.01 at θ= 0), D1S2892 (4.11 at θ= 0), and D1S2797 (4.07 at θ= 0) for family PKCC009 and with D1S496 (4.73 at θ= 0), D1S2892 (4.34 at θ= 0), D1S3721 (4.83 at θ= 0), and D1S2797 (4.32 at θ= 0) for family PKCC039. The common linked region, 20.76 cM (20.80 Mb), is flanked by markers D1S2729 and D1S2890 and co-segregates with the disease in both families, placing the disease locus on chromosome 1p34.3-p32.2.
Conclusions: Linkage analysis of autosomal recessive cataracts in two consanguineous Pakistani families localizes a novel locus for autosomal recessive congenital cataract on chromosome 1p.
Congenital cataracts are one of the major causes of vision loss in children world-wide and are responsible for about one-third of blindness in infants [1,2]. Congenital cataracts can occur in an isolated fashion or as one component of a syndrome affecting multiple tissues. Non-syndromic congenital cataracts have an estimated frequency of 1-6 per 10,000 live births . They vary markedly in severity and morphology affecting the nuclear, cortical, polar, or subcapsular parts of the lens or in severe cases, the entire lens with various types of opacity. Congenital cataracts can lead to permanent blindness by interfering with the sharp focus of light on the retina during critical developmental intervals.
Approximately one-third of congenital cataract cases are familial . To date, 23 independent autosomal dominant cataract loci have been reported. Genes that have been previously reported to be associated with autosomal dominant congenital cataract include crystallins, connexins, beaded fiber specific proteins, aquaporin 0, and developmental and transcription factors. Conversely, fewer autosomal recessive cataract loci have been mapped. Thus far, eleven loci residing on chromosomes 1q21.1, 3p22-24.2, 6p23-24, 9q13-22, 16q21-22, 19q13.3, 19q13.4, 20p12.1, 21q22.3, 22q11.2, and 22q11.2 have been mapped. Of these loci, mutations in eight genes: gap junction protein alpha 8 (GJA8), glucosaminyl (N-acetyl) transferase 2 (GCNT2), heat shock transcription factor 4 (HSF4), lens intrinsic membrane protein 2 (LIM2), beaded filament structural protein 1 (BFSP1), αA-crystallin (CRYAA), βB1-crystallin (CRYβB1), and βB3-crystallin (CRYβB3) have been found with three of these also causing autosomal dominant cataracts [5-15].
Here we report two consanguineous Pakistani families with multiple members affected by autosomal recessive congenital cataract. Initially, a genome-wide search including exclusion of known cataract loci was completed. Linkage analysis provided evidence of a locus for autosomal recessive congenital cataract on chromosome 1p34.3-p32.2. In the genome wide scan, a maximum two point lod scores of 3.81 and 4.07 at θ=0 were obtained with markers D1S255 and D1S2797 for family PKCC009, respectively. Similarly, a maximum two point lod scores of 2.36 and 4.32 at θ=0 were obtained with markers D1S255 and D1S2797 for family PKCC039, respectively. Fine mapping using markers from the Marshfield database localizes the diseased locus to a 20.76 cM (20.80 Mb) interval flanked by markers D1S2729 and D1S2890. This region is distinct from the autosomal dominant cataract loci described by Eiberg et al.  and Mackay et al.  as well as the cataract region reported by Ionides et al., which overlaps the first two loci. Thus, there are at least three distinct cataract loci on chromosome 1p.
Seventy-five Pakistani families with nonsyndromic congenital cataract were recruited to participate in a collaborative study between the National Centre of Excellence in Molecular Biology (NCEMB), Lahore, Pakistan and the National Eye Institute, Bethesda, MD to identify new disease loci that causes inherited visual diseases. This study was approved by Institutional Review Board (IRB) of the National Centre of Excellence in Molecular Biology and the National Eye Institute. The participating subjects gave informed consent consistent with the tenets of the Declaration of Helsinki. The ophthalmologic examination was performed at the Laytton Rahmatullah Benevolent Trust Hospital, Lahore, Pakistan.
The families described in this study, PKCC009 (60009) and PKCC039 (60039) are from the North West Frontier Province and the Punjab Province of Pakistan, respectively. A detailed medical history was obtained by interviewing family members. Cataracts in affected individuals were either present at birth or developed during infancy. Medical records of clinical exams conducted with slit lamp biomicroscopy reported different phenotypes of cataract in affected individuals of PKCC009 and PKCC039. Affected individuals in family PKCC009 had membranous cataracts whereas clinical examinations suggested that affected individuals in family PKCC039 had posterior subcapsular cataracts.
Blood samples were collected from affected and unaffected family members. Genomic DNA was extracted from white blood cells as described by Grimberg et al. . Genotyping for all participating family members was performed using 5' fluorescently labeled microsatellite markers. A genome wide scan was conducted using the Applied Biosystems PRISM Linkage Mapping Set MD-10 Version 2.5 (Applied Biosystems, Foster City, CA) with markers spaced at average intervals of about 10 cM. PCR was conducted with an initial denaturing step of 94 °C for 8 min followed by 10 cycles of amplification at 94 °C for 15 s, at 55 °C for 15 s, and at 72 °C for 30 s; then 20 cycles at 89 °C of 15 s, at 55 °C for 15 s, and at 72 °C for 30 s. The final extension step was performed at 72 °C for 10 min followed by a final hold at 4 °C. PCR products were denatured at 95 °C for five min and then immediately placed on ice for 5 min. Amplified DNA fragments were analyzed on ABI 3100 DNA Genetic analyzer(s) and alleles were assigned using GeneScan (version 3.7; Applied Biosystems) and Genotyper Software (version 3.7; Applied Biosystems).
Two-point linkage analyses were performed with the FASTLINK version of MLINK from the LINKAGE program package [20,21]. Maximum LOD scores were calculated using ILINK. Autosomal recessive cataracts were analyzed as a fully penetrant trait with an affected allele frequency of 0.001. The marker order and distances between the markers were obtained from the Marshfield database and the National Center for Biotechnology Information chromosome 1 sequence maps (National Center for Biotechnology, Bethesda, MD provided the information in the public domain). For the initial genome scan, equal allele frequencies were assumed whereas for fine mapping, allele frequencies were estimated from 100 unrelated and unaffected individuals from the North West Frontier province and the Punjab province of Pakistan.
The families described in this study, PKCC009 and PKCC039, are from the North West Frontier province, bordering Afghanistan and the Punjab Province of Pakistan, respectively. Cataracts in affected individuals were either present at birth or developed during infancy. Clinical examination conducted with slit lamp biomicroscopy reveals congenital cataract. Affected individuals of family PKCC009 exhibit bilateral membranous cataract (Figure 1A,B) accompanied by other ocular abnormalities including corneal opacity, microcornea, and nystagmus. Affected individuals of PKCC039 reveal posterior sub capsular cataract without other accompanying abnormalities (Figure 1C).
Linkage to known autosomal recessive cataract loci was initially excluded by haplotype analysis using closely flanking markers (data not shown). For family PKCC009, a genome wide scan yields lod scores greater than 1.0 for markers D1S450, D1S255, D1S2797, and D9S1776. Of these markers, only D1S450 and D9S1776 had closely flanking markers yielding large negative LOD scores thus excluding the linkage regions. D1S255 and D1S2797 are adjacent markers in the MD-10 mapping set, yielding lod scores of 3.81 and 4.07 at θ=0, respectively. For family PKCC039 LOD scores of 2.36 and 4.32 at θ=0 were obtained with markers D1S255 and D1S2797, respectively, during the genome wide scan. No LOD scores greater than 1.0 were obtained for family PKCC039 other than with chromosome 1p markers.
Fine mapping with closely spaced markers was carried out in both linked families in order to refine the region further. Maximum lod scores were obtained without recombination with D1S255 (3.81), D1S186 (4.14), D1S432 (4.01), D1S2892 (4.11), D1S3721 (3.73), D1S2713 (2.34), D1S2797 (4.07), D1S197 (3.98), and D1S2652 (1.61) for family PKCC009 and with D1S496 (4.73), D1S2729 (2.36), D1S255 (2.36), D1S186 (3.99), D1S432 (3.99), D1S2892 (4.34), D1S3721 (4.83), D1S2713 (3.99), D1S2797 (4.32), D1S197 (4.51) and D1S2652 (2.40) for family PKCC039 (Table 1).
Visual inspection of the haplotypes of both families supports the results of linkage analysis. As shown in Figure 2A,B, there is a proximal recombination in affected individual 16 of family PKCC009 at D1S199, individual 18 of family PKCC039 at D1S234, individual 19 of family PKCC039 at D1S513, and individual 14 of family PKCC009 at D1S2729. Similarly, there is distal recombination in individual 10 of family PKCC009 and individuals 14 and 19 of family PKCC039 at D1S2890. Taken together, these results strongly suggest that the disease locus cosegregates in a 20.76 cM (20.80 Mb) interval flanked by markers D1S2729 and D1S2890.
Here we report linkage of congenital cataract inherited as an autosomal recessive trait in two consanguineous Pakistani families to markers on chromosome 1p34.3-p32.2. The lack of significant lod scores other than chromosome 1p region during the genome scan in both families and homozygous alleles for all the affected individuals of both families for markers of 1p34.3-p32.2 along with lack of homozygosity in all the unaffected individuals strongly suggest that the cataract locus maps to the linked region, 20.76 cM (20.80 Mb), flanked by markers D1S2729 and D1S2890 on chromosome 1p34.3-p32.2.
Previously, the autosomal dominant Volkmann cataract (CCV) locus described by Eiberg et al.  and a second locus for autosomal dominant cataracts described by Mackay et al.  have been mapped to chromosome 1p. In addition, the cataract region reported by Ionides et al.  overlaps the first two with the highest lod score in the Volkmann region. In addition, an autosomal dominant zonular pulverulent cataract locus (CAE1) has been mapped to chromosome 1q21-25 and shown to result from mutations in the gene for connexin 50, GJA8 . During the genome wide scan, we found no evidence of linkage to these regions or other known cataract regions.
The affected individuals of family PKCC039 have posterior subcapsular cataract, whereas affected individuals of PKCC009 exhibit bilateral membranous cataract, which can be an end stage of several forms of severe cataract, usually associated with rupture of the lens capsule or absorption of the lens. This leads to the question: why families linked to the same locus would have different cataract phenotypes. One possible explanation is that distinct cataract phenotype results from modifying genetic and epistemic factors in families with the same mutation. Previously, it has been shown that a single mutation, i.e. Q155X mutation in CRYβB2, could result in cerulean cataract, Coppock-like cataract, and sutural cataract with punctate and cerulean opacities [23-25]. Another plausible explanation is that different alterations in the same gene can result in distinct phenotypes. Mutations reported for CRYβB1 strongly suggest that mutations in a single gene can not only result in different phenotypes but also can lead to different modes of inheritance [15,26]. Lastly, we can not rule out the possibility that both of these families have mutations in two different closely linked genes residing on the same locus.
Transparency and precise shape are distinctive features of the lens that are critical for proper light refraction. Elucidating the molecular mechanisms that maintain or disrupt lens transparency is a fundamental precursor for preventing cataract. The search for genes responsible for these phenotypically distinct congenital cataracts is in progress. As other ocular abnormalities such as corneal opacity, microcornea, and nystagmus were reported by the affected individuals of PKCC009 along with congenital cataract, the disease-causing gene may not be lens specific, although they are all consistent with early severe disruption of the lens. Identification of the mutations causing these cataracts is likely to lead to a better understanding of the mechanisms involved in the loss of transparency in the lens and to better conventional treatments for preventing this loss.
The authors are grateful to the families for their participation in this study. We sincerely thank the staff of Lyton Rehmatullah Benevolent Trust (LRBT) Hospital, especially Dr. Saleem Akhtar, for the identification of the families and expert clinical evaluation of affected individuals. We also thank Mr. Farooq Sahbir, Mr. Muhammad Assad, and Mr. Muhammad Awais for their technical help. This study was supported in part by Higher Education Commission and Ministry of Science and Technology, Islamabad, Pakistan.
1. Robinson GC, Jan JE, Kinnis C. Congenital ocular blindness in children, 1945 to 1984. Am J Dis Child 1987; 141:1321-4.
2. Hejtmancik JF, Smaoui N. Molecular genetics of cataract. Dev Ophthalmol 2003; 37:67-82.
3. Francis PJ, Berry V, Bhattacharya SS, Moore AT. The genetics of childhood cataract. J Med Genet 2000; 37:481-8.
4. Foster A, Johnson GJ. Magnitude and causes of blindness in the developing world. Int Ophthalmol 1990; 14:135-40.
5. Pras E, Pras E, Bakhan T, Levy-Nissenbaum E, Lahat H, Assia EI, Garzozi HJ, Kastner DL, Goldman B, Frydman M. A gene causing autosomal recessive cataract maps to the short arm of chromosome 3. Isr Med Assoc J 2001; 3:559-62.
6. Pras E, Raz J, Yahalom V, Frydman M, Garzozi HJ, Pras E, Hejtmancik JF. A nonsense mutation in the glucosaminyl (N-acetyl) transferase 2 gene (GCNT2): association with autosomal recessive congenital cataracts. Invest Ophthalmol Vis Sci 2004; 45:1940-5.
7. Heon E, Paterson AD, Fraser M, Billingsley G, Priston M, Balmer A, Schorderet DF, Verner A, Hudson TJ, Munier FL. A progressive autosomal recessive cataract locus maps to chromosome 9q13-q22. Am J Hum Genet 2001; 68:772-7.
8. Smaoui N, Beltaief O, BenHamed S, M'Rad R, Maazoul F, Ouertani A, Chaabouni H, Hejtmancik JF. A homozygous splice mutation in the HSF4 gene is associated with an autosomal recessive congenital cataract. Invest Ophthalmol Vis Sci 2004; 45:2716-21.
9. Riazuddin SA, Yasmeen A, Zhang Q, Yao W, Sabar MF, Ahmed Z, Riazuddin S, Hejtmancik JF. A new locus for autosomal recessive nuclear cataract mapped to chromosome 19q13 in a Pakistani family. Invest Ophthalmol Vis Sci 2005; 46:623-6.
10. Pras E, Levy-Nissenbaum E, Bakhan T, Lahat H, Assia E, Geffen-Carmi N, Frydman M, Goldman B, Pras E. A missense mutation in the LIM2 gene is associated with autosomal recessive presenile cataract in an inbred Iraqi Jewish family. Am J Hum Genet 2002; 70:1363-7.
11. Pras E, Frydman M, Levy-Nissenbaum E, Bakhan T, Raz J, Assia EI, Goldman B, Pras E. A nonsense mutation (W9X) in CRYAA causes autosomal recessive cataract in an inbred Jewish Persian family. Invest Ophthalmol Vis Sci 2000; 41:3511-5.
12. Riazuddin SA, Yasmeen A, Yao W, Sergeev YV, Zhang Q, Zulfiqar F, Riaz A, Riazuddin S, Hejtmancik JF. Mutations in betaB3-crystallin associated with autosomal recessive cataract in two Pakistani families. Invest Ophthalmol Vis Sci 2005; 46:2100-6.
13. Ponnam SP, Ramesha K, Tejwani S, Ramamurthy B, Kannabiran C. Mutation of the gap junction protein alpha 8 (GJA8) gene causes autosomal recessive cataract. J Med Genet 2007; 44:e85.
14. Ramachandran RD, Perumalsamy V, Hejtmancik JF. Autosomal recessive juvenile onset cataract associated with mutation in BFSP1. Hum Genet 2007; 121:475-82.
15. Cohen D, Bar-Yosef U, Levy J, Gradstein L, Belfair N, Ofir R, Joshua S, Lifshitz T, Carmi R, Birk OS. Homozygous CRYBB1 deletion mutation underlies autosomal recessive congenital cataract. Invest Ophthalmol Vis Sci 2007; 48:2208-13.
16. Eiberg H, Lund AM, Warburg M, Rosenberg T. Assignment of congenital cataract Volkmann type (CCV) to chromosome 1p36. Hum Genet 1995; 96:33-8.
17. McKay JD, Patterson B, Craig JE, Russell-Eggitt IM, Wirth MG, Burdon KP, Hewitt AW, Cohn AC, Kerdraon Y, Mackey DA. The telomere of human chromosome 1p contains at least two independent autosomal dominant congenital cataract genes. Br J Ophthalmol 2005; 89:831-4.
18. Ionides AC, Berry V, Mackay DS, Moore AT, Bhattacharya SS, Shiels A. A locus for autosomal dominant posterior polar cataract on chromosome 1p. Hum Mol Genet 1997; 6:47-51.
19. Grimberg J, Nawoschik S, Belluscio L, McKee R, Turck A, Eisenberg A. A simple and efficient non-organic procedure for the isolation of genomic DNA from blood. Nucleic Acids Res 1989; 17:8390.
20. Schaffer AA, Gupta SK, Shriram K, Cottingham RW Jr. Avoiding recomputation in linkage analysis. Hum Hered 1994; 44:225-37.
21. Lathrop GM, Lalouel JM. Easy calculations of lod scores and genetic risks on small computers. Am J Hum Genet 1984; 36:460-5.
22. Shiels A, Mackay D, Ionides A, Berry V, Moore A, Bhattacharya S. A missense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant "zonular pulverulent" cataract, on chromosome 1q. Am J Hum Genet 1998; 62:526-32.
23. Litt M, Carrero-Valenzuela R, LaMorticella DM, Schultz DW, Mitchell TN, Kramer P, Maumenee IH. Autosomal dominant cerulean cataract is associated with a chain termination mutation in the human beta-crystallin gene CRYBB2. Hum Mol Genet 1997; 6:665-8.
24. Gill D, Klose R, Munier FL, McFadden M, Priston M, Billingsley G, Ducrey N, Schorderet DF, Heon E. Genetic heterogeneity of the Coppock-like cataract: a mutation in CRYBB2 on chromosome 22q11.2. Invest Ophthalmol Vis Sci 2000; 41:159-65.
25. Vanita, Sarhadi V, Reis A, Jung M, Singh D, Sperling K, Singh JR, Burger J. A unique form of autosomal dominant cataract explained by gene conversion between beta-crystallin B2 and its pseudogene. J Med Genet 2001; 38:392-6.
26. Mackay DS, Boskovska OB, Knopf HL, Lampi KJ, Shiels A. A nonsense mutation in CRYBB1 associated with autosomal dominant cataract linked to human chromosome 22q. Am J Hum Genet 2002; 71:1216-21.