|Molecular Vision 2005;
Received 15 March 2005 | Accepted 11 July 2005 | Published 8 August 2005
CRYBB1 mutation associated with congenital cataract and microcornea
Willoughby,1,2,3 Ayad Shafiq,4 Walter Ferrini,1,2
Louie Loh Yen Chan,2 Gail Billingsley,2 Megan
Priston,2 Calvin Mok,2 Arvind Chandna,5 Stephen
Kaye,6 Elise Héon1,2
Departments of 1Ophthalmology and Vision Sciences and 2Genetics and Genomic Biology, The Hospital for Sick Children, Toronto, Canada; 3Department of Ophthalmology and Vision Science, Queens University Belfast, Belfast, UK; 4Royal Victoria Infirmary, Newcastle-upon-Tyne, UK; 5Royal Liverpool Children's Hospital and 6Royal Liverpool University Hospital, Liverpool, UK
Correspondence to: Dr Elise Héon, Department of Ophthalmology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada; FAX: (416) 813-8266; email: firstname.lastname@example.org
Purpose: The molecular characterization of a UK family with an autosomal dominant congenital cataract associated with microcornea is reported.
Methods: Family history and clinical data were recorded. This phenotype was linked to a 7.6 cM region of chromosome 22q11.2-q12.2, spanning the β-crystallin gene cluster (ZMax of 3.91 for marker D22S1114 at θ=0). Candidate genes were PCR amplified and screened for mutations on both strands using direct sequencing.
Results: Sequencing of the coding regions and flanking intronic sequences of CRYBB2 and CRYBB1 showed the presence of a novel, heterozygous X253R change in exon 6 of CRYBB1. SSCP analysis confirmed that this sequence change segregated with the disease phenotype in all available family members and was not found in 109 ethnically matched controls.
Conclusions: X253R is predicted to elongate the COOH-terminal extension of the protein and would be expected to disrupt β-crystallin interactions. This is the first documented involvement of CRYBB1 in ocular development beyond cataractogenesis.
The vast majority of world blindness results from untreated cataract , with the number of people blind from cataract increasing by approximately 1 million per year . The development of novel treatments to slow down the progression of cataract is hindered by a limited understanding of cataractogenesis. The refractive properties of the lens are dependent on the accumulation of high concentrations of water soluble structural proteins known collectively as crystallins [3,4]. Crystallins account for about 90% of total lens proteins, and form complex protein-protein interactions with each other [4,5]. They fall into two classes; the α-crystallin family and the β/γ-crystallin superfamily . The four Greek key motifs of β-crystallin genes are mostly encoded by exons 3 to 6. The NH2-terminal extensions and the intramolecular domain interfaces are thought to play a role in the ability of β-crystallin to self associate to form homo- or heterodimers with other β-crystallins. The NH2- and COOH-terminal extensions of β-crystallins have extended conformations which have been suggested to modulate protein association . Post-translational modifications, which accumulate during aging, may disturb the normal interactions between these proteins and may eventually lead to cataract formation [7,8]. Several autosomal dominant human cataracts resulting of heterozygous mutations in the β-crystallins have been identified [9-20].
Microcornea, defined by a horizontal corneal diameter of less than 11.00 mm [21,22], can be seen as an isolated anomaly or with microphthalmia (small eye). The association of cataract and microcornea only has been described in rare autosomal dominant pedigrees [23-29]. The molecular basis of cataract with microcornea in the absence of microphthalmia, anterior segment dysgenesis, or coloboma has not been elucidated (OMIM 116150). More complex ocular phenotypes such as Peters' anomaly, sclerocornea, aniridia, iris coloboma, and ectopia pupillae are also associated with cataract and microcornea [24,28-30]. Some of these complex anomalies have an identified molecular basis. The combination of cataract and microphthalmia with or without microcornea was associated with a missense mutation in CRYAA in one case  and a homozygous mutation in CRYBB2 in another [11,31]. Mutation in the DNA binding domain of the bZIP transcription factor (MAF; OMIM 177075) results in pulverulent cataract alone or cataract associated with microcornea or iris coloboma . PAX6 (OMIM 607108) mutations have also been identified in patients with cataract, microcornea, and anterior segment dysgenesis [32,33].
We report here the linkage of cataract with microcornea to a region of chromosome 22q11.2-q12.2, for which a novel mutation X253R in the CRYBB1 gene was identified. These findings link ocular development and cataractogenesis.
Respecting the Tenets of the Declaration of Helsinki, a three generation family from the UK with autosomal dominant congenital cataract and microcornea was recruited. Seventeen family members (11 affected and 4 unaffected) had a full ocular assessment to document the phenotype.
This included slit-lamp biomicroscopy, measurement of the horizontal corneal diameter, and measurement of the axial length using A-scan ultrasonography. Following informed consent, genomic DNA was extracted from peripheral blood leukocytes using standard protocols.
Genotyping and linkage analysis
Linkage analysis of a panel of 14 candidate loci included those with genes involved in cataract and anterior segment malformations. These included PAX6 (OMIM 607108), PITX2 (OMIM 601542), PITX3 (OMIM 602669), FOXC1 (OMIM 601090), CHX10 (OMIM 142993), MAF (OMIM 177075), CRYAA (OMIM 123580), CRYAB (OMIM 123590), CRYGC (OMIM 123680), CRYGD (OMIM 123690), CRYBB2 (OMIM 123620), CRYBB1 (OMIM 600929), LIM2 (OMIM 154045), and NHS (OMIM 300457). Details of the markers and protocols used are available upon request. The position of genes in relation to markers was determined using the UCSC Human Genome Browser (July 2003 build; Figure 1B). Two-point linkage analysis was performed using the MLINK program of the LINKAGE software package version 5.2.
Microsatellite markers (D22S1174, D22S258, and D22S1144) were selected from the Marshfield Genetic Database. The polymorphic CRYB2-CA dinucleotide repeat (X62390) was also used as a marker [14,34].
Genomic DNA samples from affected and unaffected members of the family and from 109 ethnically matched control individuals were screened for mutations with a combination of direct cycle sequencing, single strand conformation polymorphism (SSCP) analysis, and restriction endonuclease digestion. The coding region (exons 2-6) and flanking intronic sequences of CRYBB2 (NM_000496; BAC clone accession number Z99916) were directly sequenced as previously described [9,11]. A NlaIV restriction endonuclease digestion NEBcutter was used to screen for the previously published mutation G220X in CRYBB1 . The coding region (exons 2-6) and flanking intronic sequences of CRYBB1 (NM_001887; BAC clone accession number Z95115) were also directly sequenced as previously described . A population of patients with senile (n=50) and congenital cataracts (n=50) were screened by SSCP for sequence variations in exon 6 of CRYBB1.
cDNA sequences for CRYBB2 (NM_000496), and CRYBB1 (NM_001887) were retrieved from GenBank and aligned with the fully annotated chromosome 22 sequence (Z95115). Multiple sequence alignments of nucleotide sequences of CRYBB1 from various species were performed using the sequence alignment program ClustalX (version 1.8).
The three generation family studied included 11 affected and 4 unaffected with autosomal dominant cataract and microcornea (Figure 1A). The clinical phenotype of affected individuals is summarized in Table 1. Briefly, all affected family members had congenital cataracts. All 11 affected family members had congenital cataracts, and microcornea was present in 8/10 individuals (where data was available) with an average horizontal corneal diameter of 10.2 mm (range 9-11 mm; Figure 1C). The average horizontal corneal diameter was 12 mm in the 3/4 unaffected family members available for full clinical examination (III:3, III:6, and III:7). Individual II:1 was not available for study but was a member of the Royal Air Force (UK) and so had passed a full ophthalmological assessment as part of his employment. A-scan ultrasonography measurements showed normal axial length of the eyes for age [35,36]. The mean axial length in affected individuals (excluding III-8) was 22.04 mm, and 22.20 mm in unaffected individuals. The axial lengths of the four-week-old patient III-8 (18.11 mm right eye and 17.91 mm left eye) were within the normal range for a neonate (normal: 16.7-17.6 mm) . The morphology of the cataract was assessed in one individual at birth (III-8). The cataract was dense and nuclear but included cortical fibers (riders) and anterior and posterior polar opacities. Previous medical records showed that a similar cataract morphology was present at birth in all affected family members. No photographic documentation was available. Patient III-5 also had a single fine irido-lenticular strand in each eye. The majority of affected family members underwent surgery in infancy. Surgery was delayed in some cases, despite the early diagnosis, either because of initial post-operative complication (II-10) or for family reasons (II-4). The presence of nystagmus and level of postoperative visual impairment suggests that visual deprivation was significant in the critical period of visual development. Younger affected individuals had improved visual outcomes. Five affected individuals developed glaucoma postoperatively at different ages, three of which (II-10, III-1, and III-2) required glaucoma filtration surgery to control their intraocular pressure.
Molecular genetic data
Thirteen candidate genes were excluded by haplotype and linkage analysis (data not shown). The family could not be excluded from the β-crystallin cluster on chromosome 22q11.2-12.1 (Figure 1A). This region includes the β-crystallin genes CRYBB1, CRYBB2, CRYBB3, and CRYBA4 (Figure 1B), and the CRYBB2 pseudogene (CRYBB2P1). Linkage analysis using the MLINK program of the LINKAGE package version 5.2 gave a maximum two-point LOD score (ZMax) of 3.91 for marker D22S1114 at θ=0 (Table 2). A homozygous mutation in CRYBB2 was previously associated with cataract and microphthalmia, but no pathological sequence variations were detected in CRYBB2 (exons 2-6) in this family. The previously reported nonsense heterozygous mutation (G220X) in exon 6 of CRYBB1 was excluded using NlaIV restriction endonuclease digestion. Direct sequencing of the complete coding sequence of CRYBB1 (exons 2-6) and flanking intronic regions identified a novel c.827T>C change (nucleotide change based on sequence NM_001887; gi:21536279; Figure 2B), changing the stop codon (TGA) to an arginine (CGA). SSCP analysis confirmed that this sequence alteration, X253R, segregated with the phenotype in the family (Figure 2A). X253R is predicted to result in translational read through at the stop codon causing elongation of the COOH-terminal chain until the next in-frame stop codon in the downstream 3'-UTR. This would result in elongation of the COOH-terminal chain by an additional 26 amino acid residues. Multiple sequence alignments of 4 mammalian CRYBB1 genes demonstrates that the coding region of exon 6 and the stop codon are highly conserved (data not shown). Analysis of 109 ethnically matched control individuals failed to detect this sequence variation and was not detected in 50 patients with age related cataracts or 50 patients with congenital cataracts.
This study identified a novel mutation in the CRYBB1 gene and provides the first molecular basis for cataract with microcornea (OMIM 116150) in the absence of microphthalmia, anterior segment dysgenesis, or coloboma. This work demonstrates that CRYBB1 plays a role not only in cataractogenesis but also in ocular development. The mutational mechanism resulting in cataract with microcornea is also unique, as the previous mutations reported in the β-crystallins were nonsense chain terminating mutations in exon 6 of CRYBB1 and CRYBB2 [9,11,14,16].
βB1-crystallin is a major subunit of the β-crystallins and comprises 9% of the total soluble crystallins in the human lens . The major sequence difference between oligomeric β-crystallin and monomeric γ-crystallin is that β-crystallin have long terminal extensions. There is interest in the structural role played by sequence extensions which have been suggested to modulate protein association . Removal of residues at the COOH-terminus does not appear to have a major effect on dimer formation or protein folding of bovine βB2-crystallin . The novel mutation, X253R detected in this family is predicted to elongate the COOH-terminal extension and would be expected to disrupt β-crystallin interactions and truncation. As previous mutations in β-crystallins associated with cataract are chain terminating mutations, this work is the first association of COOH-terminal extending mutations with cataract, offering new insight into the development of the lens is intrinsically linked to the development of the anterior segment and this family highlights the complexity present in these early developmental cataractogenesis and crystallin biology.
The development of the lens is intrinsically linked to the development of the anterior segment and this family highlights the molecular complexity involved in these early developmental processes. A transcriptional cascade is involved in early lens development through Pax6 expression, followed by the expression of Mafs, Soxs, and Prox1, resulting in the initiation of lens differentiation and crystallin expression [41-46]. Mutations in these genes result in a variery of ocular phenotypes combining cataract, anterior segment dysgenesis, and specific iris defects [29,32,33], while mutations in crystallins mainly have a phenotype restricted to the lens [9-20]. In this family the mutation, X253R in CRYBB1 results in congenital cataract with microcornea. Individual III-5 had a single iridolenticular strand in each eye suggesting a slight element of anterior segment dysgenesis but not as severe as that associated with PAX6 and MAF mutations [29,32,33]. This is in keeping with experimental work which shows that βB1-crystallin expression occurs downstream of PAX6 and MAF [41,42,47].
The novel X253R mutation in the CRYBB1 gene is predicted to elongate the COOH-terminal extension and would be expected to disrupt β-crystallin interactions and as such, offers new insight into cataractogenesis and anterior segment development.
We are grateful to the enthusiastic participation of the family in this study and acknowledge the support of the Canadian Institutes of Health Research number CIHR94282 (EH), the Canadian Genetic Diseases Network (EH), and the BUPA Foundation, Royal College of Ophthalmologists and the Research Training Centre, The Hospital for Sick Children, Toronto (CW). We are grateful to Alan Shiels for providing the CRYBB1 PCR primer sequences, Francis Munier for DNA from cataract patients and Roger Mountford for DNA banking. Grant support: This work was funded by a grant from the Canadian Institutes of Health Research number CIHR94282 (EH).
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