A1-Crystallin Encoding Gene Cryba1 Causes a Dominant Cataract in the Mouse

Genomics 62, 67–73 (1999) Article ID geno.1999.5974, available online at http://www.idealibrary.com on

Mutation in the bA3/A1-Crystallin Encoding Gene Cryba1 Causes a Dominant Cataract in the Mouse Jochen Graw, 1 Martin Jung,* ,2 Jana Lo¨ster, Norman Klopp, Dian Soewarto, Christiane Fella, Helmut Fuchs, Andre´ Reis,* Eckhard Wolf,† Rudi Balling, and Martin Hrabe´ de Angelis Institute of Mammalian Genetics, GSF-Research Center for Environment and Health, D-85764 Neuherberg, Germany; *Institute of Molecular Genetics, Max-Delbru¨ck-Center for Molecular Medicine, D-13122 Berlin, Germany; and †Lehrstuhl fu¨r Molekulare Tierzucht und Haustiergenetik, Ludwig-Maximilians-Universita¨t Mu¨nchen, D-81377 Munich, Germany Received July 9, 1999; accepted August 27, 1999

detected only in the eye and mainly in the ocular lens. The common characteristic of all b- and g-crystallins is the so-called Greek key motif. Crystallography has shown that each of the b- and g-crystallins is composed of two domains, each built up by two Greek key motifs. It is widely accepted that b/g-crystallins evolved in two duplication steps from an ancestral protein folded like a Greek key. In the b-crystallins, individual Greek key motifs are encoded by separate exons. The Cryb genes consist of six exons: the first exon is not translated, the second encodes the N-terminal extension, whereas the subsequent four are responsible for one Greek key motif each. Biochemically, the b-crystallins are characterized as oligomers (the molecular masses of the monomers are between 22 and 28 kDa) with native molecular masses ranging up to 200 kDa for octameric forms. The N-termini are blocked by acetylation (Wistow, 1995; Lampi et al., 1997). The family of b-crystallins can be divided into more acidic (bA-) and more basic (bB-) crystallins. Each subgroup is encoded by three genes (Cryba1, -2, -4; Crybb1, -2, -3); however, Cryba1 encodes two proteins (bA1and bA3-crystallin). This feature is conserved among all mammals, birds, and frogs. In mouse and human, the Cryb genes are distributed among three chromosomes (mouse: 1, 5, and 11; human: 2, 17, and 22; for a recent review see Graw, 1997, and references therein). In the course of analysis of mouse mutants obtained by a large-scale ENU mutagenesis program (Hrabe´ de Angelis and Balling, 1998), we identified several mutants with dominant cataracts. Here we report one of them, which has been mapped to mouse chromosome 11 and identified as the first murine mutation in the Cryba1 gene.

During the mouse ENU mutagenesis screen, mice were tested for the occurrence of dominant cataracts. One particular mutant was discovered as a progressive opacity (Po). Heterozygotes show opacification of a superficial layer of the fetal nucleus, which progresses and finally forms a nuclear opacity. Since the homozygotes have already developed the total cataract at eye opening, the mode of inheritance is semidominant. Linkage analysis was performed using a set of genome-wide microsatellite markers. The mutation was mapped to chromosome 11 distal of the marker D11Mit242 (9.3 6 4.4 cM) and proximal to D11Mit36 (2.3 6 2.3 cM). This position makes the bA3/A1-crystallin encoding gene Cryba1 an excellent candidate gene. Mouse Cryba1 was amplified from lens mRNA. Sequence analysis revealed a mutation of a T to an A at the second base of exon 6, leading to an exchange of Trp by Arg. Computer analysis predicts that the fourth Greek key motif of the affected bA3/A1-crystallin will not be formed. Moreover, the mutation leads also to an additional splicing signal, to the skipping of the first 3 bp of exon 6, and finally to the deletion of the Trp residue. Both types of mRNA are present in the homozygous mutant lenses. The mutation will be referred to as Cryba1 po1. This particular mouse mutation provides an excellent animal model for a human congenital zonular cataract with suture opacities, which is caused by a mutation in the homologous gene. © 1999 Academic Press

INTRODUCTION

The b- and g-crystallins are recognized as members of a related b/g-crystallin superfamily, which can be Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. AJ239052. 1 To whom correspondence should be addressed at GSF-Research Center for Environment and Health, Institute of Mammalian Genetics, Ingolsta¨dter Landstrasse 1, D-85764 Neuherberg, Germany. Telephone: 0049-89/3187-2610. Fax: 0049-89/3187-2210. E-mail: [email protected]; Internet, http://www.gsf.de/isg/groups/eye_devel.html. 2 Present address: Ingenium Pharmaceuticals AG, D-82152 Martinsried, Germany.

MATERIALS AND METHODS Mice. Male C3HeB/FeJ mice were treated with ethylnitrosourea (ENU; 160 mg/kg) at the age of 10 weeks according to Ehling et al. (1985) and mated to untreated female C3HeB/FeJ mice. Mice were kept under specific pathogen-free conditions at the GSF and monitored within the ENU mouse mutagenesis screen project (Hrabe´ de Angelis and Balling, 1998). 67

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FIG. 1. Isolated eyes of the progressive opacity (Po) mutant. Gross appearance of unfixed eyes under the dissecting microscope at the age of 5 weeks. On the left, eye of a wildtype with a clear lens; in the middle, microphthalmic eye of a homozygous Po mutant with a pronounced lens opacity; and on the right, eye of a heterozygous Po mutant showing a zonular opacity and a faint opacity of the lens nucleus. Phenotypic characterization. F 1 offspring of the ENU-treated mice were screened at the age of 4 – 6 months with the aid of a slit lamp for the presence of cataracts (Kratochvilova and Ehling, 1979). Mice with lens opacities were tested for a dominant mode of inheritance. Homozygotes were obtained by brother 3 sister mating. For documentation, lenses were enucleated under a dissecting microscope (Leica MZ APO) and photographed. Mapping. Heterozygous carriers (first generation) were mated to wildtype C57BL/6J mice; offspring (second generation) with cataracts were backcrossed to the wildtype C57BL/6J mice. DNA was prepared from tail tips of 48 cataractous offspring of the third generation (G3) according to standard procedures; DNA was adjusted to a concentration of 50 ng/ml. For a genome-wide linkage analysis, several microsatellite markers were used for each chromosome (Table 1). PCR and sequencing. For mutation analysis, RNA was isolated from lenses of wildtype C3HeB/FeJ or C57BL/6J mice or homozygous mutant mice at the age of 4 weeks; for expression studies, RNA was isolated from C3Heb/FeJ mouse embryos at embryonic days 13.5 (head), embryonic days 14.5 and 15.5 (eye), or postnatal days 1, 14, or 21 (lens). The age of the embryos was timed from the morning of detection of the vaginal plug; this day was considered as embryonic day 0.5. RNA was transcribed to cDNA using the Ready-to-go kit from Pharmacia Biotech (Freiburg, Germany); genomic DNA was isolated from tail tips or spleen of wildtype or homozygous mutants according to standard procedures. For amplification of Cryba1 from cDNA, primers were selected from reported sequences (forward primer, 59-GACTATAAAGAGGGGATCCGGAGGC-39 according to Peterson and Piatigorsky, 1986; reverse primer, 59-AATTCTAGAGTGCTTAGCAAGATGTCATGC39; GenBank Accession No J00378). For amplification of the 39terminal part of the Cryba1 gene from genomic DNA (39 part of exon 5, intron 5, and exon 6), the primer Cryba1-Ex5L (59-GTTGGTTCAACAATGAAGTTGGTTCC-39) was combined with the reverse primer mentioned above. Cryga primers were described previously (Klopp et al., 1998), and Gapdh primers were 59-GTCACCAGGGCTGCCATT-

TGC-39 (forward) and 59-GAGATGATGACCCGTTTGGCTCCACC-39 (reverse) according to GenBank Accession No. M32599. PCR was performed using a Hybaid OmniGene Thermocycler (MWG BioTech, Ebersberg, Germany). For Cryba1 amplification, 35– 40 cycles of 30 s at 95°C, 30 s at 58°C for annealing, and 30 s at 72°C for polymerase action were performed. For Cryga and Gapdh, the cDNA was diluted 1:10, and the annealing temperature was raised to 60°C for 35 cycles only. PCR products were analyzed on a

FIG. 2. Haplotype analysis of the Po mutation localized at chromosome 11. Heterozygous mutant mice were outcrossed to wildtype C57BL/6J mice; the heterozygous carriers were backcrossed to wildtype C57BL/6J mice. Among the offspring, only the cataractous mice were analyzed for their parental genotypes with respect to a variety of microsatellite markers; results are given for those at chromosome 11. The total number of progeny scored for each locus is given on the right of the boxes, including the calculated distances between the loci (in cM). The number of progeny inherited each haplotype is given below the boxes.

CrybA1 MUTATION IN THE MOUSE

TABLE 1 Genome-wide Linkage Analysis for progressive opacity Marker

Animals tested

% of homozygotes

Linkage

D1Mit211 D1Mit216 D2Mit148 D2Mit206 D3Mit307 D3Mit44 D3Mit77 D4Mit203 D5Mit138 D6Mit102 D7Mit31 D8Mit121 D8Mit242 D9Mit12 D9Mit95 D10Mit42 D10Mit86 D11Mit36 D11Mit224 D11Mit242 D11Mit263 D11Mit271 D12Mit221 D12Mit259 D13Mit14 D13Mit53 D13Mit64 D13Mit67 D15Mit171 D15Mit85 D16Mit146 D16Mit189 D17Mit185 D18Mit60 D19Mit10

46 46 46 43 46 46 46 46 46 46 46 45 46 45 45 46 41 46 39 45 46 44 46 46 46 45 46 46 44 46 45 44 46 42 46

37 50 57 63 50 41 39 47 39 48 57 53 83 58 60 63 66 2 21 11 9 20 57 52 54 47 54 57 50 57 44 41 54 62 52

No No No No No No No No No No No No No No No No No Yes Yes Yes Yes Yes No No No No No No No No No No No No No

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ysis. The results for several markers are given in Table 1. Linkage was found with markers at mouse chromosome 11. The gene order is D11Mit271–(14.0 6 5.3 cM)–D11Mit242–(9.3 6 4.4 cM)–Po–(2.3 6 2.3 cM)– D11Mit36–(4.7 6 3.2 cM)–D11Mit263. These data are in good agreement with the current report from the 1999 report of the Chromosome Committee for Mouse Chromosome 11 (http://www.informatics.jax.org/bin/ ccr/). A detailed haplotype analysis is given in Fig. 2, and the partial map of mouse chromosome 11 is depicted in Fig. 3. Based upon this mapping information and its lenspreferred expression, the Cryba1 gene (44.7 cM from the centromere) was considered as a candidate gene. From lenses of 4-week-old mice, corresponding PCR products could be amplified both in the wildtypes and in the homozygous mutants using cDNA as template. The full-length coding sequence including some flanking information on both sides is given in Fig. 4 (EMBL Accession No. AJ239052). Sequencing revealed two al-

Note. Linkage is considered, if the number of homozygous animals for the C57BL/6J marker is lower than 25%. 1% agarose gel. Sequencing from both ends was performed commercially (SequiServe, Vaterstetten, Germany) directly after isolation of the DNA from the gel using the QiaQuick extraction kit (Qiagen, Hilden, Germany). A part of the PCR product was digested for 2 h at 37°C with 5 U restriction endonuclease DraII (5Eco01091; MBI Fermentas, St. Leon-Rot, Germany); the reaction products were analyzed on a 4% agarose gel.

RESULTS

Among the F 1 offspring of ENU-treated male mice, a presumptive mutant was detected because of its lens opacity, which could be observed by slit lamp screening. The mutation is semidominant without effects on viability and penetrance. In the heterozygous mutants, a cortical opacity can be observed with a slit lamp, when the mice open their eyes (i.e., 12 days after birth). The perinuclear zonular opacification (Fig. 1) progresses to total cataract at 8 weeks of age. The total cataract is already developed in the homozygotes at eye opening. For mapping of the mutation, 48 heterozygous G3 mutants were screened by a genome-wide linkage anal-

FIG. 3. Partial map of mouse chromosome 11. A partial chromosome map shows the location of the mutation progressive opacity in relation to relevant markers and to the candidate gene Cryba1. Numbers to the left of the chromosome indicate the genetic distance in cM from the centromere as given by the 1999 Chromosome Committee report. On the right, the actual linkage data are summarized.

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FIG. 4. Characterization of the progressive opacity mutation within the Cryba1 gene. The cDNA sequence of the mouse Cryba1 gene is given (EMBL Accession No. AJ239052). The exon boundaries were deduced from the corresponding human gene and are indicated by vertical lines below the DNA sequence. The amino acid sequence is given above the DNA, and the regions coding for the four Greek key motifs are marked by arrows. The mutation is indicated in boldface type and shaded in gray; the exchanged amino acid is mentioned below the sequence. The polymorphic site at position 395 is underlined.

terations compared to the partial sequences available from the databases. First, the wildtype derived from C3HeB/FeJ or C57BL/6J mice as well as the homozygous mutants revealed at position 395 a T instead of an A as reported in the database (GenBank Accession No J00378), predicting a substitution from Asn to Ile. This altered sequence is obviously not responsible for the phenotype. Therefore, the sequence reported in the database might be considered strain specific, since it was determined from male mice of a National Institutes of

Health stock (Inana et al., 1982). Ile is usually encoded at this position in rat, bovine, and human, and as the chicken sequence demonstrates a Val at the corresponding position at the beginning of motif III (aa 96), the change at this position is most likely a polymorphism without genetic significance. The second sequence alteration was found in mutants only and led to a mixture of two sequences in the PCR products derived from cDNA (Fig. 5c: position 169; since Fig. 5c shows the reverse sequence, it corresponds to position 513 in Fig. 4). The analysis of the

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CrybA1 MUTATION IN THE MOUSE

FIG. 5. Cryba1 sequence analysis. Cryba1 sequences were amplified from genomic DNA of one wildtype (a) or homozygous Po mutant (b). Additionally, cDNA from the lenses of one homozygous Po mutant was amplified (c). All PCR products were directly sequenced using the Cryba1 reverse primer. The only difference between the genomic sequences is the T 3 A exchange in the coding strand (Fig. 4). The arrows point to the corresponding sites at position 172 in the reverse sequence. The bases 173–148 correspond to the 59-part of exon 6; bases 202 to 174 correspond to the 39-end of intron 5 (reverse sequence). In all the cDNAs (c), a mixture of two sequences was observed from position 169 onward (arrow). The relative concentration of the two types of cDNA might be roughly estimated by the peak areas. Bases 202 to 172 of the main sequence correspond to the 39-end of exon 5; exon 6 starts at position 171 and is given here till position 146 (reverse sequence). The bases CTG (169–171 in the main sequence) are deleted in the sequence of lower concentration.

sequences revealed for the main sequence a substitution of T to A at the second base of exon 6 (at position 170, corresponding to position 514 in Fig. 4). The second (lower) sequence indicated the loss of bases 169 – 171 (corresponding to position 513 to 515 in Fig. 4). Both alterations led to the loss of a DraII restriction site, which is present in the wildtype. The presence of the mutation was proven by the absence of the DraII restriction site in the homozygous mutants. The appearance of two slightly different cDNA sequences at the beginning of exon 6 indicated alterations in the splicing signals. Analysis of the genomic DNA supported this interpretation since in the

genomic DNA of homozygous mutants, only the exchange of T 3 A at position 514 was observed (Figs. 5a and 5b; reverse strand). Obviously, this point mutation creates a new splice signal at the transition of intron 5 to exon 6 as outlined in Fig. 6. A rough estimation from the parallel analysis of the independent cDNAs from three different homozygous mutants and the corresponding genomic DNA from the same animals indicated that about 30% of the mRNA from the mutant lenses is represented by the 3-bp deletion and about 70% is represented by the T 3 A exchange. Since no other differences in comparison to the corresponding wildtype sequences were found, this mutation is considered to be causative for the cataractous phenotype. The onset of Cryba1 expression was investigated by RT-PCR at various stages of head, eye, and lens development (Fig. 7). Cryba1 transcripts were not found prior to E14.5; however, the main expression starts at birth. Compared to Cryga, coding for one of the major structural proteins of the lens, the Cryba1 expression starts later and remains at a lower level. The finding of the onset of the main Cryba1 expression at birth is in good agreement with the onset of cataract formation. At the protein level, two consequences are deduced by computer-assisted translation: At first, the substitution of the A to T at the beginning of the sixth exon will lead to a substitution of Trp by Arg. Computerassisted prediction of the quaternary structure (PCGENE: Prosite) suggests that this particular alteration will prohibit the formation of the fourth Greek key motif in the mutants. The Trp residue is conserved among all mammalian bA3/A1 crystallins characterized so far and is also found in chicken. Second, the loss of the 3 bp at the beginning of the sixth exon will lead to the loss of the Trp residue. Computer-assisted prediction revealed in this particular case the existence of a shorter fourth Greek key motif. However, final interpretations must use physicochemical data from the corresponding recombinant proteins combined with a more sophisticated computer analysis. DISCUSSION

A new cataract mutation was observed among the F 1 offspring of ENU-treated male mice. Po was demonstrated to be caused by a mutation within the Cryba1 gene; the mutant allele was, therefore, designated Cryba1 po1. The mutation is predicted to affect the formation of the fourth Greek key motif. Since the correct folding of the b-crystallins as well as that of the g-crystallins is essential for the functional integrity of the lens, this finding is consistent with the phenotype of the mutants. Additionally, the progressive character of the cataract parallels the expression profile of Cryba1 (this study) and of b-crystallins in general (van Leen et al., 1987). The observation of the progressive cataract formation and the characterization of the molecular lesion within the bA3/A1-crystallin encoding gene demonstrates some similarities to the Philly cataract in the

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FIG. 6. Alternative splicing of Cryba1 transcripts in Po mutant lenses. (a) Consensus splice sites are shown. (b) The boundaries of intron 5 with exon 5 and exon 6 are given. The mutation at the second base of exon 6 forms a new consensus splice site, which allows also an alternative start for exon 6 three bases downstream (underlined). (c). The deduced sequences for the mRNA and the protein are demonstrated.

mouse. This mutation was also reported to lead to a dominant cataract (Kador et al., 1980), which was caused by an in-frame deletion of 12 bp in the 39-end of the Crybb2 gene (Chambers and Russell, 1991). The mutation affects a region of the protein close to the carboxy terminus, which is considered to be essential for the correct formation of the tertiary structure of the bB2-crystallin. The phenotype of the Philly mouse cataract was also reported as a progressive cataract, which develops after the first week of postnatal life. At

FIG. 7. Expression of Cryba1. Expression of Cryba1 was investigated by RT-PCR during embryonic development starting at E13.5 and during the first days after birth (P1 to P21). Weak expression of Cryba1 was observed only from E14.5 onward; the main expression, however, starts at birth. For comparison, the expression of Cryga and Gapdh is given. The cDNA template for the Cryga and Gapdh was diluted 1:10 before PCR was allowed.

that time, particles appear in the anterior cortex that extend by the 10th day in the anterior subcapsular area. A loss of the normal lens denucleation process and swelling of the lens fibers follow. The characteristic bow configuration of the nuclei is replaced by a fan-shaped configuration (Uga et al., 1980). Faint anterior opacities seen at postnatal day 15 are followed by sutural opacities at day 25, nuclear cataract at 30 days, lamellar perinuclear opacities at 35 days, and total nuclear cataracts at 45 days. During cataractogenesis, an intralenticular increase in water, sodium, and calcium and a decrease in potassium, reduced glutathione, and ATP were reported. An altered membrane permeability is the cause of the increased outward leak of potassium (Kador et al., 1980). The increasing severity of the phenotype is temporally correlated with the expression of the Crybb2 gene. A corresponding human counterpart to the Philly mouse mutant is the cerulean blue cataract detected in a large family as a dominantly inherited disorder. This disease was mapped to the region of human chromosome 22 that includes two b-crystallin encoding genes (CRYBB2 and 3) and a pseudogene (CRYBB2c1) (Kramer et al., 1996). Recently, a G 3 A transition was reported at the position of the first base of the codon normally coding for Glu residue 155 in CRYBB2. This

CrybA1 MUTATION IN THE MOUSE

mutation creates a stop codon that truncates the bB2crystallin by 51 amino acids (Litt et al., 1997). The human homologue phenotypically similar to the actual mouse Cryba1 pol mutation described in this communication is a semidominant, zonular cataract with sutural opacities. Padma et al. (1995) reported a linkage of the corresponding disease gene to human chromosome 17q11– q12 in a three-generation family. Since the CRYBA1 gene is localized in this region, it was considered a good candidate gene. Recently, Kannabiran et al. (1999) reported that this particular form of cataract is caused by a lack of exons 3 and 4 in the corresponding mRNA (resulting in a protein with only the C-terminal globular domains). The present paper describes the first mutation within the mouse Cryba1 gene, providing an excellent animal model for the homologous disease in human. Moreover, together with recent reports in the literature concerning mutations affecting b- and g-crystallin encoding genes in human and mouse, it demonstrates the importance of the b/g-crystallin superfamily defined by the presence of the four Greek key motifs for the functional integrity of the eye lens (Cartier et al., 1992; He´on et al., 1999; Klopp et al., 1998; Litt et al., 1997; Stephan et al., 1999; Wistow et al., 1998). ACKNOWLEDGMENTS The expert technical assistance of Erika Bu¨rkle, Monika Stadler, Gerlinde Bergter, Andreas Mayer, Nicole Hirsch, Sabine Manz, and Sylvia Prettin is gratefully acknowledged. Oligonucleotides were obtained from Utz Linzner (GSF-AG BIODV). Part of this work was supported by a grant from the German Human Genome Project (DHGP) to R.B., E.W., and M.H.d.A. (01KW9610/1).

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