A Novel Nonsense Mutation in CRYGC Is Associated with Autosomal Dominant Congenital Nuclear Cataracts and Microcornea

Molecular Vision 2009; 15:276-282 <http://www.molvis.org/molvis/v15/a28> © 2009 Molecular Vision Received 28 October 2008 | Accepted 29 January 2009 | Published 6 February 2009 A novel nonsense mutation in CRYGC is associated with autosomal dominant congenital nuclear cataracts and microcornea Lu Zhang,1 Songbin Fu,2 Yangshan Ou,1 Tingting Zhao,1 Yunjuan Su,1 Ping Liu1 1Eye hospital, the First Affiliated Hospital, Harbin Medical University, Harbin, China; 2Laboratory of Medical Genetics, Harbin Medical University, Harbin, China Purpose: To report the identification of a novel nonsense mutation in CRYGC in a Chinese family with autosomal dominant congenital nuclear cataracts and microcornea. Methods: We investigated a four-generation Chinese family with six members affected with nuclear cataracts and microcornea. The family resides in a relatively isolated region of northern China. Genomic DNA was isolated from blood leucocytes, genotyping was performed using more than 100 microsatellite markers for the known cataract candidate gene loci, and LOD scores were calculated using the LINKAGE programs. Mutations were detected by DNA sequence analysis of the candidate genes. Results: Evidence for linkage was detected at marker D2S325 (LOD score [Z]=2.29, recombination fraction [θ]=0.0), which closely flanks the γ-crystallin gene cluster (CRYGA-CRYGD) on chromosome 2q32.3-q35. Direct sequencing of the candidate CRYGA-CRYGD gene cluster revealed a c.470G>A transversion in exon 3 of CRYGC, which cosegregated with cataracts in the family and was not observed in 100 normal controls. This single nucleotide change was predicted to introduce a translation stop codon at tryptophan 157 (W157X). Conclusions: The present study has identified a novel nonsense mutation in CRYGC associated with autosomal dominant cataracts and microcornea in a Chinese family. Our finding expands the spectrum of CRYGC mutations associated with congenital cataract and confirms the role of γ-crystallin in the pathogenesis of congenital nuclear cataracts. Congenital cataract is a clinically and genetically two genes that encode beaded filament proteins, BFSP1 [35] heterogeneous lens disorder that typically appears as a sight- and BFSP2 [36-38]; and (5) seven genes that encode growth threatening trait in childhood and accounts for one-tenth of and transcription factors, PITX3 [39], MAF [40,41], HSF4 the cases of childhood blindness [1]. The prevalence of [42], FOXE3 [43], EYA1 [44], CHX10 [45], and PAX6 [46]. congenital cataracts is estimated to vary from 0.6 to 6 per Crystallins are the dominant structural components of the 10,000 live births with an incidence of 2.2-2.49 per 10,000 vertebrate eye lens. At least 11 crystallin genes encode over live births [2]. Approximately half of all congenital cataract 95% of the water-soluble structural proteins present in the cases are inherited either in isolation or as part of a syndrome crystallin lens, representing more than 30% of its mass and of ocular or systemic abnormalities [3]. All three classical accounting for its optical transparency and high refractive forms of Mendelian inheritance have been associated with index [47]. Therefore, mutations in the crystallin genes are non-syndromic cataracts. However, the majority of families strong candidates for certain hereditary forms of congenital with a history of congenital cataracts show an autosomal cataracts. dominant transmission pattern. In this study, we performed linkage analysis of a four- There are currently 34 loci implicated in nonsyndromic generation Chinese family with autosomal dominant nuclear congenital cataract, and of these, over 20 have been associated cataract and microcornea, and we identified a novel nonsense with mutations in specific genes [4]. These genes can be mutation in CRYGC on 2q. considered in five functional groups: (1) 10 genes that encode crystallines, CRYAA [5,6], CRYAB [7,8], CRYBB1 [9,10], METHODS CRYBB2 [11-14], CRYBB3 [15], CRYBA1/A3 [16,17], Clinical evaluation and DNA specimens: We identified a four- CRYBA4 [18], CRYGC [19-23], CRYGD [19,21,24-26], and generation Chinese family with autosomal dominant nuclear CRYGS [27]; (2) two genes that encode the gap junction cataracts and microcornea in the absence of other ocular or proteins, GJA8 [28,29], and GJA3 [30,31]; (3) two genes that systemic abnormalities. The family resides in a relatively encode membrane proteins, MIP [32] and LIM2 [33,34]; (4) isolated region of northern China. Informed consent in accordance with the Declaration of Helsinki was obtained Correspondence to: Ping Liu, Eye Hospital, the First Affiliated from all participants. Thirteen members participated in the Hospital, Harbin Medical University, 23 Youzheng Road, Harbin, study, six affected and seven unaffected family members 150001, China; Phone: 0086-451-85553958; FAX: (Figure 1). Affected status was determined by a history of 0086-451-53650320; email: [email protected] cataract extraction or ophthalmologic examination, which Dr. Zhang's email: [email protected] 276 Molecular Vision 2009; 15:276-282 <http://www.molvis.org/molvis/v15/a28> © 2009 Molecular Vision included visual acuity testing, slit lamp examination, 10 min and a final hold at 4 °C. The PCR products were pooled intraocular pressure measurement, and fundus examination on the basis of size (Genescan-400HD ROX; Perkin Elmer, with dilated pupils. Phenotypes were documented using slit Foster City, CA), denatured at 95 °C for 1 min, and lamp photography. Peripheral blood was collected and electrophoresed in a 96 capillary automated DNA analysis genomic DNA was extracted from blood leukocytes using a system (MegaBACE 1000, Amersham, Freiburg, Germany). QIAampDNA Blood Mini Kit (Qiagen, Hilden, Germany). The results were analyzed by Genetic Profiler version 1.5 Genotyping and linkage analysis: We conducted a genome (Amersham). Two point LOD scores (Z) were calculated wide linkage scan based on a set of dinucleotide repeat using the LINKAGE software package (version 5.1). A gene microsatellite markers spaced at approximately 10 cM frequency of 0.0001 and a penetrance of 100% were assumed intervals using an ABI PRISM Linkage Mapping Set version for the cataract locus. LOD scores were calculated at 2.5 (Applied Biosystems, Foster City, CA). “Touchdown” recombination fractions (θ) of 0.00, 0.05, 0.1, 0.2, 0.3, and polymerase chain reaction (PCR) was performed in a 5 μl 0.4. reaction volume containing 20 ng of genomic DNA, 1 µl of Mutational analyses: Four functional candidate genes CRYGA CRYGD CRYGE CRYGF 10X PCR buffer, 7 mM MgCl2, 0.2 mM dNTPs, 0.3 U HotStar ( - ) and two pseudogenes ( - ) Taq DNA polymerase, and 0.05 µM microsatellite markers. with in-frame translation stop codons are clustered within the After an initial denaturation period of 12 min at 95 °C, 14 physical region defined for the cataract on 2q33–35. We cycles were performed at 95 °C for 30 s, 63–56 °C for 30 s systematically sequenced all four functional CRYG genes, (with a 0.5 °C decrease at each step), and 72 °C for 1 min. each comprising three exons and two introns, in two affected Thirty cycles were performed at 95 °C for 30 s, 56 °C for 30 and two unaffected members of the family using specific s, and 72 °C for 1 min followed by an extension at 72 °C for primers (Table 1). Genomic DNA was PCR amplified, purified, and sequenced directly using dye-terminator chemistry. The purified PCR products were sequenced on both DNA strands using an ABI 3100 sequencer (Applied Biosystems). After identifying a nonsense mutation in exon 3 of CRYGC, all of the family members (six affected and seven unaffected family members) and 100 unrelated normal individuals were screened. RESULTS Clinical findings: We report the identification of a four- generation Chinese family with a clear diagnosis of congenital nuclear cataracts and microcornea. Autosomal dominant inheritance of the cataract phenotype was supported by the presence of affected individuals in each of the four generations and male-to-male transmission. The affected individuals presented with bilateral congenital nuclear cataracts that consisted of a central nuclear opacity affecting the embryonic, fetal, and infantile nucleus of the lens (Figure 2) and with congenital small eyeballs and corneas with a diameter of approximately 9–10 mm. Every affected individual had the same poor visual acuity of hand movement in front of the eye, and they also had nystagmus and amblyopia. Linkage analysis: By following the exclusion of large chromosomal regions, we obtained suggestive evidence of linkage for marker D2S2321 (Z=1.88, θ=0) on 2q33.3. Further analysis of the marker showed a positive LOD score at the recombination fraction of 0.00 and strongly supported this Figure 1. Pedigree and haplotypes of the Chinese family with candidate region. The maximum two-point LOD score (Zmax) cataracts. A pedigree is shown with the haplotype analysis of the of 2.29 was obtained at marker D2S325 with recombination Chinese family with cataracts, which shows the segregation of 10 θ=0.00. The adjacent markers (D2S2321, D2S1385, and microsatellite markers on chromosome 2q in descending order from D2S1242) also showed LOD scores greater than 1.0. The the centromere. Squares and circles represent males and females, respectively. Black and white symbols denote affected and results of the two-point LOD scores are summarized (Table unaffected individuals, respectively. 2). 277 Molecular Vision 2009; 15:276-282 <http://www.molvis.org/molvis/v15/a28> © 2009 Molecular Vision

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