New Phenotype Associated with an Arg116cys Mutation in the CRYAA Gene Nuclear Cataract, Iris Coloboma, and Microphthalmia

OPHTHALMIC MOLECULAR GENETICS

SECTION EDITOR: JANEY L. WIGGS, MD, PhD New Phenotype Associated With an Arg116Cys Mutation in the CRYAA Gene Nuclear Cataract, Iris Coloboma, and Microphthalmia

Francis Beby, MD; Claire Commeaux; Muriel Bozon; Philippe Denis, MD, PhD; Patrick Edery, MD, PhD; Laurette Morle´, PhD

Objective: To describe a new phenotype with an argi- Conclusion: To our knowledge, this is the first case to nine-to-cysteine mutation at position 116 (Arg116Cys) date in which an Arg116Cys mutation in the CRYAA gene in the CRYAA gene. was associated with nuclear cataract and iris coloboma.

Methods: We investigated a 4-generation French fam- Clinical Relevance: This study indicates that an ily with autosomal dominant cataract and performed a Arg116Cys mutation in the CRYAA gene could be asso- genetic linkage analysis using microsatellite DNA mark- ciated with an unusual phenotype in affected individu- ers encompassing 15 known cataract loci. Exons 1, 2, and als. In this family, the clinical observation of iris colo- 3 and flanking intronic sequences of the CRYAA gene were boma allows for the possibility of identifying individuals amplified and analyzed using direct sequencing. carrying the mutation. Iris coloboma is particularly im- portant in terms of perinatal diagnosis because its detec- Results: All of the affected individuals had nuclear cata- tion in the newborn requires a careful and regular ex- ract and iris coloboma. Genetic analysis revealed the pre- amination of the lens. viously described Arg116Cys mutation in the CRYAA gene in the heterozygous state in all of the affected members of the family but not in unaffected individuals. Arch Ophthalmol. 2007;125:213-216

ONGENITAL CATARACTS less than 20 mm. All of the affected patients are a common cause of vi- had a bilateral early-onset cataract. The lens sual impairment in chil- opacity appeared at birth or developed during dren.1,2 Approximately the first years of life. The cataract consisted of 30% of nonsyndromic a single dense axial opacity of 3 mm confined congenital cataracts have a genetic ori- to the embryonic and fetal nuclei of the lens. C3 In all of the affected individuals, the bilateral gin. To date, 22 independent autosomal nuclear cataract was also associated with bi- dominant cataract loci have been mapped lateral iris coloboma (Figure 1). Two af- and 14 genes have been identified, includ- fected individuals (II:7 and III:5) (Figure 2) 1-5 ing 8 genes coding for crystallins. Here had congenital microphthalmia in conjunc- we report the first case in which an argi- tion with cataract. Physical examination of the Author Affiliations: nine-to-cysteine mutation at position 116 affected individuals did not reveal any dysmor- Department of Ophthalmology (Arg116Cys) in the CRYAA gene was as- phic facial features, mental retardation, or de- (Drs Beby and Denis) and sociated with autosomal dominant nuclear velopmental malformations that could indi- Service de Cytoge´ne´tique cataract and iris coloboma. cate that the cataract was syndromic. The power Constitutionnelle (Dr Edery), to detect a linkage in this family was esti- 6 Edouard Herriot Hospital, Place mated using the SLINK simulation program METHODS d’Arsonval, and Equipe (Rockefeller University, New York, NY) and as- d’Acceuil 3739, Universite´ suming an autosomal dominant model of in- Claude Bernard Lyon 1 A full ophthalmological examination was per- heritance, a 95% penetrance, and a gene fre- (Dr Edery), Lyon, France; and formed in 16 members of a 4-generation French quency of 0.00015. We performed a genetic Molecular and Cellular Genetic family with autosomal dominant cataract. linkage analysis using microsatellite DNA mark- Center, Centre National de la Twelve affected and 4 unaffected members were ers7 encompassing 15 known cataract loci: Recherche Scientifique Unite´ studied. The onset of the cataract was deter- 1q21-q25 (CX50); 2q33-q35 (CRYGB); 3p22.1- Mixte de Recherche 5534, mined by medical documentation and surgi- 3p21; 3q21.3-22.3 (BFPS2); 9q13-q22; 11p13 University of Lyon, cal records of cataract extraction. For each in- (PAX6); 11q22.3-q23.1 (CRYAB); 12q13-q14 Villeurbanne, France dividual, ocular axial length was determined (MIP); 13q11-q13 (CX46); 14q24 (CHX10); (Mss Commeaux and Bozon by A-scan ultrasonography. A diagnosis of mi- 15q21-q22; 16q22 (HSF4); 17q11.2-q12 and Dr Morle´). crophthalmia was based on a total axial length (CRYBA1); 20q12-p12; and 21q22.3 (CRYAA).

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 Logarithm of odds (LOD) scores were calculated using the with markers D21S1912 (LOD score=2.6 at ␪=0) and LINKAGE version 5.2 package8 (Universite´ Paris 7, Paris, France) D21S1890 (LOD score=1.9 at ␪=0). Haplotype analysis and the parameters described for the SLINK program. Exons indicated that a specific haplotype segregates with the dis- 1, 2, and 3 and flanking intronic sequences of the CRYAA gene 9 ease (Figure 2). The CRYAA gene, which lies within the were amplified as previously described and analyzed using di- D21S1260-D21S1890 interval and encodes a crystallin pro- rect sequencing. tein, was considered to be a good candidate. Sequence analysis of the 3 CRYAA exons revealed the previously RESULTS described Arg116Cys mutation9 in the heterozygous state in all of the affected members of this family but not in All of the affected individuals had an autosomal domi- unaffected individuals. In addition, a synonymous change nant bilateral nuclear cataract with bilateral iris colo- at the third base of codon 2 (GAC→GAT) (single nucleo- boma. A microphthalmia also appeared in 2 of 14 af- tide polymorphism rs872331, http://www.ncbi.nih.gov fected members. A SLINK simulation calculation yielded /SNP/) was observed on the CRYAA mutated allele. This expected LOD scores of 3.5 at ␪=0, 3.2 at ␪=0.05, and polymorphism was found in the homozygous state in the 2.9 at ␪=0.01, indicating a high probability of finding a 2 affected individuals (II:7 and III:5) who have both cata- linkage in this family. Although we were able to exclude ract and microphthalmia and in 5 of 12 other affected 14 of 15 candidate loci, we detected positive LOD scores patients who have normal eye size. This frequent polymorphism was also observed in the heterozygous state in 5 of 12 patients with cataract and in 3 unaffected members of the family. Thus, its occurrence in the homozygous state did not correlate with microphthalmia and iris coloboma.

COMMENT

An Arg116Cys mutation of the CRYAA gene was identified in all of the affected members from the family with autosomal dominant cataract described here. This mutation was previously reported in 1 family9 with isolated autosomal dominant cataract but no iris coloboma. The preservation of a positive charge at position 116 was found Figure 1. Slitlamp photograph of the left eye of an affected individual (III:9) showing an iris coloboma (arrows) and a nuclear cataract. The lens opacity to be critical for the structural and functional integrity is located in the embryonal and fetal nucleus of the lens (dilated pupil). of the ␣A-crystallin.10 Indeed, Bera et al10 have demon-

D21S1260 6.1 cM CRYAA D21S1890 0.6 cM D21S1912

1 2 I

∗ 1 2 3 4 5 6 7 8 9 10 11 II 1 3 1 3 1 3 1 3 1 3 3 3 3 5 3 5 3 5 3 5 5 1 2 5 4 2 4 2 4 2 4 2 4 1 4 2

∗ 1 2 3 4 5 6 7 8 9 10 III 2 3 1 3 1 3 3 3 1 1 1 3 1 3 2 5 5 1 3 1 5 1 3 5 5 5 4 4 3 2 2 1 4 1 2 1 4 4 4 2 6 6

1 2 3 4 IV 1 3 1 3 1 3 1 4 5 4 5 4 5 V 6 2 6 2 6 2 1 3 4 6 6 5

Figure 2. Pedigree of the family. cM indicates centimorgans; open circles, unaffected females; filled circles, females with both a nuclear cataract and an iris coloboma; open squares, unaffected males; filled squares, males with both a nuclear cataract and an iris coloboma; asterisks, individuals with microphthalmia. The numbers below the symbols indicate the genotypes for 3 markers, and the disease-associated haplotypes appear in bold numbers.

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 strated that the Arg116 residue can be replaced by an- microphthalmia or coloboma. Thus, CRYBB1 also plays other positively charged amino acid, Lys, without any a role in early ocular development. Altogether, these re- effect on protein structure and function. Moreover, mu- sults provide evidence for an early expression of MAF, tation of Arg116 to Cys or to another neutral amino acid, CRYBB1, and CRYAA in the embryogenesis of the eye and Gly, showed very similar changes in structure, oligomer- confirm that mutations in each of these genes can result ization, and chaperone function. This suggests that an not only in a phenotype restricted to the lens but also in extra Cys residue per se is not the cause of the changes. a complex variety of ocular phenotypes combining cata- In contrast, the replacement of the Arg116 residue by a ract, microphthalmia, and anterior segment dysgenesis negatively charged amino acid, Asp, has a devastating such as microcornea or iris coloboma. effect on the secondary and tertiary protein structures.10 Mouse mutants with the Val142Glu CRYAA muta- The ␣A-crystallin Arg116Cys mutant forms larger oligo- tion25 or with a CRYAA gene homozygous invalidation26 merized heteroaggregates with ␣B-crystallin11 whereas its have both microphthalmia and cataract. In the family de- interaction with the ␤B2- and ␥C-crystallins is described here, we suggest the possibility that the Arg116Cys creased.12 In addition, the mutant Arg116Cys protein has mutation acting in combination with a trans genetic modi- reduced chaperone activity.13,14 fier such as a polymorphic allele of a gene involved in In this study, an iris coloboma was observed in all of eye development may be responsible for the occurrence the patients with cataract, and 2 individuals have mi- of microphthalmia in 2 individuals. crophthalmia in addition to cataract and iris coloboma. Autosomal dominant cataract is phenotypically and Two families with different CRYAA mutations have been genetically highly heterogeneous, making it a major ob- described.15,16 An Arg49Cys mutation was previously re- stacle to the phenotype-genotype relationship and di- ported in a family with isolated autosomal dominant cata- rect linkage of congenital cataract genes.1-5 We describe ract15 whereas a Trp9Xaa mutation was identified in a fam- in this study a new phenotype associated with an ily with autosomal recessive cataract.16 No iris coloboma Arg116Cys mutation of the CRYAA gene in a French fam- was described in these 2 families. Litt et al9 noted that 5 ily. In this family, all of the affected individuals had nuclear of 13 individuals with the Arg116Cys mutation had mi- cataract and iris coloboma. crophthalmia in addition to their cataract. In contrast, no ocular abnormality aside from cataract was found either in the family with autosomal dominant cataract with a Submitted for Publication: March 13, 2006; final revi- missense Arg49Cys mutation of the human CRYAA gene15 sion received June 14, 2006; accepted June 22, 2006. or in the family with autosomal recessive cataract with a Correspondence: Francis Beby, MD, 30 rue du Professeur nonsense Trp9Xaa mutation of the CRYAA gene.16 Florence, 69003 Lyon, France ([email protected]). Coloboma may be caused by a defect in the closure of Author Contributions: Dr Beby had full access to all of the optic fissure or by an abnormal development of the iris the data in the study and takes responsibility for the in- stroma and epithelium.17 Although some CRYAA tran- tegrity of the data and the accuracy of the data analysis. scripts have been detected in an iris complementary DNA Financial Disclosure: None reported. library (National Eye Institute NEIBank library NbLib0016, Funding/Support: This work was supported by contracts http://neibank.nei.nih.gov), a direct implication of CRYAA HCL 1999 and HCL 2001 from the Hospices Civils de Lyon, in iris development remains to be demonstrated. Never- by the Centre National de la Recherche Scientifique (Unite´ theless, investigators18-20 have previously demonstrated that Mixte de Recherche 5534), and by the Claude Bernard Lyon the lens produces growth factors and influences the devel- 1 University. Dr Beby is the recipient of a grant from the opment of the ciliary body and iris. Based on the features Fondation pour la Recherche Me´dicale. found, our results also indicate that CRYAA protein is prob- Acknowledgment: We are grateful to the family mem- ably involved in choroidal development. In the family we bers in this study. We thank the De´veloppements Tech- have described, the occurrence of an iris coloboma could niques pour l’Analyse Mole´culaire de la Biodiversite´ for be related to a higher expression level of the Arg116Cys their technical support. ␣A-crystallin mutant allele compared with the previously described family.9 Indeed, polymorphisms in the CRYAA REFERENCES promoter region appear to influence transcriptional activ- ity21 and may be responsible for a different expression level 1. Reddy MA, Francis PJ, Berry V, Bhattacharya SS, Moore AT. Molecular genetic of the mutant protein. basis of inherited cataract and associated phenotypes. Surv Ophthalmol. 2004; 49:300-315. 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