Aey2, a New Mutation in the ßb2-Crystallin–Encoding Gene Of

Aey2, a New Mutation in the ␤B2-Crystallin–Encoding Gene of the Mouse

Jochen Graw,1 Jana Lo¨ster,1 Dian Soewarto,2 Helmut Fuchs,2 Andre´ Reis,3,4 Eckhard Wolf,5 Rudi Balling,1,6 and Martin Hrabe´ de Angelis2

PURPOSE. During an ethylnitrosourea (ENU) mutagenesis he ␤- and ␥-crystallins form the major part of the water- screen, mice were tested for the occurrence of dominant Tsoluble proteins of the eye lens. The ␤- and ␥-crystallins are cataracts. One particular mutant was found that caused pro- recognized as members of a superfamily and have been con- gressive opacity and was referred to as Aey2. The purpose of sidered for a long time to be present only in the eye and mainly the study was to provide a morphologic description, to map in the ocular lens.1,2 However, just recently, expression of the the mutant gene, and to characterize the underlying molecular ␤B2-crystallin mRNA and protein was reported also in brain lesion. and testis.3 The common characteristic of all ␤- and ␥-crystal- METHODS. Isolated lenses were photographed, and histologic lins is the so-called Greek key motif. Crystallography of bovine sections of the eye were analyzed according to standard pro- ␤B2- and ␥B-crystallins has shown that each of the ␤- and cedures. Linkage analysis was performed using a set of micro- ␥-crystallins is composed of two domains, each of them built satellite markers covering all autosomal chromosomes. cDNA up by two Greek key motifs.4,5 from candidate genes was amplified after reverse transcription It is widely accepted that ␤- and ␥-crystallins evolved in two of lens mRNA. duplication steps from an ancestral protein folded like a Greek 6 RESULTS. The cortical opacification visible at eye opening pro- key. Greek key motifs also have been reported for a few other gressed to an anterior suture cataract and reached its final proteins, such as a yeast killer toxin from Williopsis mrakii 7 phenotype as total opacity at 8 weeks of age. There was no (WmKT; 1 Greek key motif) the protein S from Myxococcus obvious difference between heterozygous and homozygous xanthus (4 Greek key motifs), spherulin 3c from Physarum mutants. The mutation was mapped to chromosome 5 proxi- polycephalon (2 Greek key motifs), the epidermis-specific pro- mal to the marker D5Mit138 (8.7 Ϯ 4.2 centimorgan [cM]) and tein from Cynops pyrrhogaster (EDSP or ED37; 4 Greek key distal to D5Mit15 (12.8 Ϯ 5.4 cM). No recombinations were motifs),6 and the putative human tumor suppressor protein observed to the markers D5Mit10 and D5Mit25. This position AIM1 (12 Greek key motifs).8 makes the genes within the ␤A4/␤B-crystallin gene cluster The function of the Greek key motifs has not been elabo- excellent candidate genes. Sequence analysis revealed a muta- rated in detail; however, computer-based analysis suggests that tion of T3A at position 553 in the Crybb2 gene, leading to an it may be responsible for particular protein–protein interac- exchange of Val for Glu. It affects the same region of the tions similar to those seen with immunoglobulins.9 Similar to Crybb2 gene as in the Philly mouse. Correspondingly, the loss the immunoglobulins, the ␤- and ␥-crystallins are folded in an of the fourth Greek key motif is to be expected. all-␤ structure. Undoubtedly, the accumulation of hydrogen CONCLUSIONS. The Aey2 mutant represents the second allele of bonds in the symmetrical, twisted, antiparallel, ␤-sheet struc- Crybb2 in mice. Because an increasing number of ␤- and ture of each domain, and the hydrophobic interactions be- ␥-crystallin mutations have been reported, a detailed pheno- tween them, contribute significantly to their stability. This type–genotype correlation will allow a clearer functional un- overall stability can be understood as the sum of the contribu- derstanding of ␤- and ␥-crystallins. (Invest Ophthalmol Vis Sci. tion of independent folding units.10,11 2001;42:1574–1580) Mutations in the ␤- and ␥-crystallin–encoding genes (gene symbols are Cryb and Cryg, respectively) have been demonstrated to lead to lens opacification in mice12–17 and humans.18–24 In most cases, the formation of at least one of the 1 From Forschungszentrum fu¨r Umwelt und Gesundheit (GSF; Na- Greek key motifs is affected by the mutation. In total, 13 genes tional Research Center for Environment and Health), Institute of Mam- belong to this superfamily in mammals. malian Genetics, Neuherberg; the 2Institute of Experimental Genetics, 3 In the ␤-crystallins, individual Greek key motifs are encoded Neuherberg; the Institute of Molecular Genetics, Max Delbru¨ck Cen- 25 ter for Molecular Medicine, Berlin; and the 5Lehrstuhl fu¨r Molekulare by separate exons. The Cryb genes consist of six exons: The Tierzucht und Haustiergenetik, Ludwig Maximilians Universita¨t, Mu- first exon is not translated, the second exon encodes the nich, Germany. N-terminal extension, and the subsequent four exons are re- 4Present affiliation: Institute of Human Genetics, University of sponsible for one Greek key motif each. Biochemically, the Erlangen-Nu¨rnberg, Germany. 6 ␤-crystallins are characterized as oligomers (the molecular Present affiliation: German Research Center for Biotechnology masses of the monomers ranges between 22 and 28 kDa) with (GBF), Braunschweig, Germany. native molecular masses ranging up to 200 kDa for octomeric Supported by Grant 01KW9610/1 from the German Human Ge- 6,26 nome Project (RB, EW, MHdA). forms. The N termini are blocked by acetylation. Submitted for publication November 28, 2000; revised January 29, The family of ␤-crystallins can be divided into more acidic 2001; accepted February 7, 2001. (␤A-) and more basic (␤B-) crystallins. Each subgroup is en- Commercial relationships policy: I, C (RB, MHdA); N (all others). coded by three genes (respectively, Cryba1,-2, and -4; and The publication costs of this article were defrayed in part by page Crybb1,-2, and -3), however, Cryba1 encodes two proteins: charge payment. This article must therefore be marked “advertise- ␤A1- and ␤A3-crystallins. This feature is conserved among all ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: Jochen Graw, GSF–National Research Cen- vertebrates. In mammals, the Cryb genes are distributed among ter for Environment and Health, Institute of Mammalian Genetics, three chromosomes (mouse: 1, 5, and 11; man: 2, 17, and 22; Ingolsta¨dter Landstrasse 1, D-85764 Neuherberg, Germany. for recent review see Ref. 15 and references therein). Among [email protected] the Cryb genes, the nucleotide sequence of Crybb2 was char-

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TABLE 1. PCR Primers with Annealing Temperatures and Length of the Products

Accession Annealing Temperature (Designation Lab Number Sequence (5؅ 3 3؅) Number (°C); Fragment Size (bp

Cryba4-L1 25026 GGC CCT TCT TGG ACC GTG C AJ272228 53–60; 747 Cryba4-R2 26526 TGG CGG GGC ATG GGC AJ272228 Crybb1-L1 25028 CCA ATA CAG CAC CAG GAA CCA TGT C AF106853 60; 517 Crybb1-R1 25029 CTC TTG GAT CTC CAT GGT GTT GCC AF106853 Crybb2-L1 24468 CTC GAC GCC AGA GAG TCC ACC M60559 55; 728 Crybb2-R1 24469 GGC ACG AGC CAC ACT TTA TTC TTC M60559 Crybb2-L2 27037 GGC TAC CGT GGG CTG CAG TAC M60559 58; 215 Crybb2-R1 24469 GGC ACG AGC CAC ACT TTA TTC TTC M60559 Crybb3-L1 25030 CTG GTT CTC CTC CAG TTT GAG GC AJ272229 53–60; 738 Crybb3-R2 25031 GGC TGG GCA CAT CGT CAT CTA C AJ272229

acterized at first because of the high abundance of this basic, PCR and Sequencing principal ␤-crystallin.27 In the course of analysis of mouse mutants obtained by a For the molecular analysis, RNA was isolated from lenses of C3HeB/FeJ, large-scale ethylnitrosourea (ENU) mutagenesis program,28,29 T-stock, 129, JF-1, and C57BL/6J wild-type mice and from homozygous we identified several cataract mutations. Herein, we report one mutant mice at the age of 4 weeks. RNA from lens, brain, and testis of them, which was mapped to mouse chromosome 5 and were transcribed to cDNA by using a kit (Ready-to-Go; Pharmacia identified as the second allele in the mouse Crybb2 gene. Biotech, Freiburg, Germany). Genomic DNA was isolated from tail tips or spleen of wild-type C3HeB/FeJ and C57BL/6J mice or homozygous mutants, according to standard procedures. For amplification of cDNA MATERIALS AND METHODS from Cryba4 and the three Crybb genes, primers were selected from the EMBL GenBank databases (provided in the public domain by the Mice European Bioinformatics Institute at Hinxton, Cambridge, UK, at Mice were kept under specific pathogen-free conditions at the National http//:www.ebi.ac.uk/ and the National Center for Biotechnology In- Research Center for Environment and Health (GSF) and monitored formation at http//:www.ncbi.nlm.nih.gov/, respectively; Table 1). within the ENU mouse mutagenesis screen project.28,29 The use of PCR was performed using a thermocycler (Hybaid OmniGene; animals was in accordance with the ARVO Statement for the Use of MWG BioTech, Ebersberg, Germany). The annealing temperatures are Animals in Ophthalmic and Vision Research and with the German Law included in Table 1. PCR products were analyzed on a 1% agarose gel. on Animal Protection. Sequencing was performed commercially (SequiServe, Vaterstetten, Male C3HeB/FeJ mice were treated with ENU (160 mg/kg) at the Germany) immediately after isolation of the DNA from the gel using an age of 10 weeks according to Ehling et al.30 and mated to untreated extraction kit (Qia-Quick; Qiagen, Hilden, Germany). Some of the PCR female C3HeB/FeJ mice. The offspring of the ENU-treated mice were products, using the primers Crybb2-L2 and Crybb2-R1, were digested screened at the ages of 4 to 6 months for the presence of cataracts, for 2 hours at 37°C by 5 U restriction endonuclease DdeI (MBI Fer- with the aid of a slit lamp (SLM30; Carl Zeiss, Oberkochen, Ger- mentas, St. Leon-Rot, Germany), and the reaction products were ana- many).31 Mice with lens opacities were tested for a dominant mode of lyzed on a 4% agarose gel. inheritance. Homozygotes were obtained by brother ϫ sister mating. Phenotypic Characterization RESULTS The eyes of the mutants were examined continuously during postnatal The Aey2 mutant was detected by slit lamp screening of the life with the slit lamp. For documentation, lenses were enucleated offspring of ENU-treated male mice. The mutation has a com- under a dissecting microscope (MZ APO; Leica, Heidelberg, Germany) plete penetrance. The litter size in the matings of heterozy- and photographed. gotes and of homozygotes indicated normal fertility and viabil- Histologic analysis was performed at the second day after birth (P2) ity of the mutants. and at 4 and 11 weeks of age. The eye globes were fixed for 3 hours The Aey2 mutant displays a progressive cataract. The cata- in Carnoy’s solution and embedded in JB-4 plastic medium (Poly- ractous changes were observed as early as at eye opening (i.e., sciences, Inc., Eppelheim, Germany) according to the manufacturer’s 12 days after birth) as a diffuse opacity in the cortex and protocol. Sectioning was performed with an ultramicrotome (OMU3; abnormally branched anterior suture (Fig. 1a). This type of Reichert-Jung, Walldorf, Germany). Serial transverse 3-␮m sections opacity remained stationary until 8 to 11 weeks of age, after were cut with a glass knife and stained with methylene blue and basic which a total opacity developed (Fig. 1b). fuchsin. The sections were evaluated with a light microscope (Axio- The histologic sections of mutant eyes at 4 and 11 weeks of plan; Zeiss). Images were acquired by means of a scanning camera age demonstrated striking irregularities in the anterior suture (Progress 3008; Jenoptik, Jena, Germany) and imported into an image- and a dark subcortical zone (Fig. 2a). A higher power view processing program (Photoshop V5.5, Adobe Illustrator 8.0; Adobe, showed swollen and liquefied fibers in the anterior suture with Unterschleissheim, Germany). dark particles (Fig. 2b). Abnormal dark bodies and dustlike particles were also present in the subcortical zone (Fig. 2c). Mapping The first 46 cataractous mice from the backcross (G3) were Homozygous carriers (first generation) were mated to wild-type taken for genome-wide mapping. The results indicate linkage C57BL/6J mice, and the offspring (second generation) were back- with markers on mouse chromosome 5. The following gene crossed to the wild-type C57BL/6J mice. DNA was prepared from tail order was observed: D5Mit15—(12.8 Ϯ 5.4 cM)—Aey2, tips of cataractous offspring of the third generation (G3), according to D5Mit10, D5Mit25—(8.7 Ϯ 4.2 cM)—D5Mit138. These re- standard procedures. DNA was adjusted to a concentration of 50 ng/␮l. sults are in good agreement with the current report of the For a genome-wide linkage analysis, several microsatellite markers Chromosome Committee for mouse chromosome 5 (available were used for each autosome.15 at http://www.informatics.jax.org/bin/ccr/). A detailed haplo-

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FIGURE 1. Gross appearance of lenses of the Aey2 mutant mouse. (a) Lenses of wild-type (left) and homozygous Aey2 mutant (right) mice at the age of 3 weeks. Both genotypes showed the same cortical and anterior suture opacity. The anterior suture was abnormally branched. Original size of the lenses was approximately 2 mm. (b) The lens of a heterozygous Aey2 mutant at the age of 11 weeks was completely opaque (right). For comparison, an age- matched wild-type (left) is shown. Original size of the wild-type lens is approximately 3 mm.

type analysis is given in Figure 3a and a partial map of mouse mouse ␤-crystallins. Only the acidic ␤A1/A3- and ␤A2-crystal- chromosome 5 in Figure 3b. lins had an Ile at this position. It is expected that this exchange Based on this mapping information, the cluster encoding prohibits the formation of the fourth Greek key motif in the the four Cryb genes (Cryba4, Crybb1, Crybb2, and Crybb3; mutants. However, final interpretations should include physi- 59–60 cM from the centromere) was considered to contain cochemical data from corresponding recombinant proteins, good candidate genes for progressive cataract formation. The together with a more sophisticated computer analysis. corresponding cDNAs were amplified both in the wild-type Because Crybb2 expression also has been reported recently mice and in the homozygous mutants, by using cDNA tem- in the brain and testis,3 we tested this expression also in our plates derived by reverse transcription of RNA from lenses of wild-type C3H and cataractous mice. We observed conflicting 4-week-old mice. results with different sets of primers. The primer pair The only sequence alteration cosegregating with the mutant Crybb2-L1 and -R1 amplified specifically the full-length cDNA phenotype was a T3A exchange at position 553 (exon 6) of of Crybb2 in the lens only. No transcripts were detected, either the Crybb2 gene (Fig. 4). The mutation lead also to the appear- in brain or testis (Fig. 6a). The detection limit was determined ance of a new DdeI restriction site in the mutant mice that was for 10Ϫ18 moles (Fig. 6b). However, using another 5Ј primer, not present in the wild-type sequence of C3H and several other Crybb2-L2, located inside the Crybb2 transcript, a transcript mouse strains. The presence of the mutation was validated by was observed in brain and testis of the same size as in the lens the presence of the DdeI restriction site in five homozygous (Fig. 6c). The sequence analysis confirmed this short amplifi- mutants (Fig. 5). Therefore, the new allele should be referred cation product as part of Crybb2. The absence of the entire to as Crybb2Aey2. Crybb2 transcript but presence of a shorter 3Ј end indicates The mutation is predicted to lead to a Val3Glu exchange at the presence of a novel splice product of Crybb2 in brain and codon 187. At this position Val occurred in four of the six testis that is missing the N-terminal part. This is in line with the

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FIGURE 3. Linkage analysis of the Aey2 mutation localized on chromosome 5. (a) Heterozygous mutant mice were outcrossed to wild- type C57BL/6J mice, and the heterozygous carriers were backcrossed to wild-type 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 on chromosome 5. The total number of progeny scored for each locus is to the right of the boxes, including the calculated distances between the loci (in cM). The number of progeny that inherited each haplotype is below the boxes. (b) A partial chromosome map shows the location of the Aey2 mutation in relation to relevant markers and to the candidate genes Cryba4 and Crybb1, -2, and -3. Numbers to the left of the chromosome indicate the genetic distance (in cM) from the centromere, as given by the Chromosome Committee report. At right, the actual linkage data are summarized.

observation by Mugabo et al.3 who presented just the C-terminal part of ␤B2-crystallin, but not the entire protein. The presence of the mutation in brain and testis was conﬁrmed by

Aey2 mutants. They were caused by a disturbed denucleation process in those fibers, which were completely degraded in the normal inner lens fiber, suggesting impaired chromatin degradation. (b) Higher magnification of the central anterior region of the lens demonstrates the irregular fiber cells in the outer cortical fiber cells and shows a distinct zone of discontinuity between the outer and inner fiber cells. The cellular structure of the inner fiber cells was markedly destroyed. The epithelial cells remained unaffected. (c) The remnants of the fiber FIGURE 2. Histologic analysis of lenses of the Aey2 mutant at the age cell nuclei were still present in the lens bow of the Aey2 lens. Partially of 11 weeks. The section through a lens of an 11-week-old mouse is degraded nuclei are obvious behind the zone of discontinuity from the shown. Arrows: Irregular remnants of fiber cell nuclei. (a) Striking outer cortex to the cataractous inner cortex. c, cornea; i, iris; lb, lens abnormality of the anterior suture, a layer of dustlike particles in the bow; le, lens epithelium; r, retina; s, anterior suture. Staining, methyl- anterior cortex and intercellular clefts in the lens nucleus appeared in ene blue and basic fuchsin. Bar, 100 ␮m.

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FIGURE 4. Characterization of the Aey2 mutation within the Crybb2 gene. The cDNA sequence of the mouse Crybb2 gene is shown (EMBL accession number, M60559). The amino acid sequence is above the DNA sequence. The mutation is indicated in bold; the exchanged amino acid is noted below the sequence. The Dde1 restriction site at position 553 is underlined.

the presence of the DdeI restriction site as in the lens cDNA sponding alteration at the amino acid level (Val187Glu) is (Fig. 6c). thought to affect the formation of the fourth Greek key motif of the ␤B2-crystallin. Additionally, the progressive character of the cataract is in line with the expression proﬁle of Crybb2.32 DISCUSSION

A new cataract mutation was observed among the F1 offspring of ENU-treated male mice. This progressive opacity, prelimi- narily referred to as Aey2, was mapped to mouse chromosome 5, close to the cluster of the ␤A4-, ␤B1-, ␤B2-, and ␤B3- crystallin–encoding genes. By molecular analysis of all four genes, the mutation was ﬁnally demonstrated to be caused by an A3T exchange within exon 6 of the Crybb2 gene. The mutant allele was therefore designated Crybb2Aey2. The corre-

FIGURE 6. RT-PCR for Crybb2 expression in lens, testis, and brain. cDNA from lenses, testis, and brain from 3-week-old wild-type male mice were analyzed for Crybb2 expression by RT-PCR. Ϫ, negative control; M, marker. (a) Amplification of full-length Crybb2 revealed amplification only in the lens, not in the testis or brain. (b) One femtomole of cloned entire Crybb2 cDNA was used as a template for PCR reactions (lane 1). Stepwise 1:10 dilution of the template DNA (lanes 2–7) revealed a detection limit of 10Ϫ18 moles for the PCR FIGURE 5. Crybb2 digest by DdeI. The cDNA of the Crybb2 gene was reaction conditions reported in (a). (c) Amplification of the 215-bp amplified, and the PCR fragment was analyzed by agarose gel electro- fragment of the 3Ј end of Crybb2 cDNA detected corresponding phoresis with (ϩ) and without (Ϫ) subsequent digest by DdeI. The transcripts in lens, brain, and testis of wild-type C3H and mutant Aey2 fragment from the wild-type C3H, C57BL/6J (C57BL), T-stock (T), 129, mice. The amplification product of the Aey2 mutants were digested in and JF-1 (JF) mice were not digested (a), but the cDNA from lenses of all tissues, indicating that it represented the 3Ј end of the Crybb2 five Aey2 mutants elicited the expected digest pattern (b). transcripts.

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The observation of the progressive cataract formation and mutation creates a stop codon and truncates the ␤B2-crystallin the characterization of the molecular lesion within the ␤B2- by 51 amino acids.19 crystallin–encoding gene demonstrates very strong similarities Another human cataract mutation affecting a CRYB locus to the Philly cataract in the mouse. This mutation leads also to was described as a semidominant zonular cataract with sutural 33,34 a progressive dominant cataract, which is caused by an opacities. Padma et al.18 reported a linkage of the correspond- 12 in-frame deletion of 12 bp in the 3Ј end of the Crybb2 gene. ing disease gene to human chromosome 17q11-12 in a three- The Philly mouse cataract develops after the first week of generation family. Because the CRYBA1 gene is localized in postnatal life. At that time, abnormal particles appear in the this region, it was considered to be a good candidate gene. anterior cortex that extend by the 10th day in the anterior 21 34 Recently, Kannabiran et al. reported that this particular form subcapsular area. Two weeks after birth, enlargement of the of a cataract is caused by the absence of exons 3 and 4 in the persisting bow nuclei becomes prominent. During the fourth corresponding mRNA (resulting in a protein with only the postnatal week, posterior lens fibers are swollen, and degen- C-terminal globular domains). erating cells and large intercellular spaces are present in the superficial cortex. Between 5 and 7 weeks, epithelial cells at Also in this human mutation, a homologous semidominant mutation in the mouse has been reported recently.2 The het- the equator become tall, and the number of their mitotic po1 figures are markedly reduced. The lens cells in the posterior erozygous Cryba1 mutants exhibit a progressive cataract, cortex degenerate, causing widening of the posterior suture. reaching its final stage at 8 weeks of age, whereas in the At this final stage of Philly mouse cataractogenesis, the lentic- homozygotes the total cataract has already developed at eye ular nucleus becomes markedly opaque.34 The increasing se- opening. The mutation affects the splice site at the beginning verity of the phenotype is temporally correlated with the of exon 6 and leads to two different cDNA forms and also to expression of the Crybb2 gene.32 two different proteins. One of them is thought to affect the Biochemical studies indicate that the cataractous process in formation of the fourth Greek key motif.2 the Philly mouse is associated with a variety of osmotic The ␤-crystallins form a superfamily of proteins together with changes. At 20 days of age, there is an increase in lens water the ␥-crystallins, because of their common structural Greek key along with an alteration in electrolyte levels. Lenticular sodium motifs.1,7 They are distinct from the ␣-crystallins in sequence, rapidly increases, and potassium levels decrease. Concomitant structure, and function. Physicochemical data point out that the with cataract formation is an increase in total lenticular cal- monomeric ␥-crystallins and the oligomeric ␤-crystallins can build cium and a decrease in lens dry weight, in reduced glutathione, similar folding structures that allow a dense package of these 33 and in adenosine triphosphate. Associated with this altered proteins without loss of transparency.5,41 Also in the ␥-crystallin– membrane permeability in the Philly mouse lens, a changed 35 36 encoding genes, a broad variety of mutations are currently being pattern of membrane glycoproteins and membrane lipids characterized in mice13,14,16,17,42 and humans.20,22–24,43 Most of has been reported. them also affect the Greek key motifs, by the loss of the corre- The cause of these biochemical changes may be the differ- sponding sequence, by predicted changes of their folding prop- ent biophysical properties of the altered ␤B2-crystallin, as out- erties, or by changes of their steric coordinations. lined by the absence of heat-stable characteristics.37 Moreover, Changes of the structural characteristics of lens proteins the altered form of the ␤B2-crystallin in the Philly mouse lens can be rescued, at least in part, by the chaperone activity of the is present primarily in the heavy-molecular-weight fraction, 44 indicating that the altered ␤B2-crystallin in the Philly lens can ␣-crystallins, which are also abundant in the lens. However, interact with other ␤-crystallins in the lens.38 It is a matter for the prevention of denaturation and/or renaturation is restricted speculation whether the interactions of the altered ␤B2-crys- to physicochemical effects on the already-formed proteins, tallin with other lens proteins cause a rapid aggregation of the such as thermal or oxidative stress. The ␣-crystallins are not cellular proteins, leading to the formation of the heavy-weight able to bring a protein with an altered primary structure (i.e., material and resulting finally in the cataract. altered amino acid sequence) to its “regular” folding. This The Philly mutation affects the same region of the protein inability was demonstrated recently in a mutated form of a (close to the carboxyl-terminus) as does the Crybb2Aey2 muta- ␨-crystallin.45 tion. This region is considered to be essential for the correct From the morphologic observations reported in the current formation of the tertiary structure of the ␤B2-crystallin, even if study and in the references cited herein, it becomes evident previous structural examination of the C-terminal region fo- that inherited congenital cataracts have some similar features, cused on the amino acids from 173 to 185, but did not include even if they are distinct in their details. The most common 39 Val187. Therefore, it is very likely that the mechanisms in- aspect is the presence of the fiber cell nuclei throughout the volved in both cataractogenic processes are very similar. How- entire lens. They may be shifted to the anterior or posterior ever, the lenses of the Aey2 mutant showed a slower progres- pole, but usually they are not correctly degraded and are still sion of the opacification, which was terminated between 8 and present, at least in a pyknotic form. This feature reflects a 11 weeks of age. As in the Philly mouse, dustlike particles were disturbance of the differentiation program of the lens fiber present in the anterior cortex. They seemed to be breakdown cells. The onset of the first cataractogenic characteristics usu- products of the cell nuclei, which do not undergo the regular ally correlates with the expression profile of the gene, which is degradation process that occurs in the normal lens fiber cells. affected by the deleterious mutation. However, it would be This slower progress in cataractogenesis may be due to a smaller molecular lesion in the Aey2 mice than that in the interesting to elaborate the underlying molecular and biochem- Philly mouse. ical mechanisms in detail. A corresponding human counterpart to the mouse mutants In conclusion, the present article describes a second allele at the Crybb2 gene locus is the cerulean cataract detected in a of the mouse Crybb2 gene, providing a further excellent ani- large family as a dominantly inherited disorder. This disease mal model for the homologous disease in humans. Moreover, was mapped to the region of human chromosome 22 that together with recent reports in the literature concerning mu- includes two ␤-crystallin–encoding genes (CRYBB2 and -3) tations affecting ␤- and ␥-crystallin–encoding genes in human and a pseudogene (CRYBB2␺1).40 Recently, a G3A transition and mouse, it demonstrates the importance of the ␤- and has been reported at position 155 in CRYBB2. It affects the first ␥-crystallin superfamily in the functional integrity of the eye base of a codon, usually coding for a Glu residue, but the lens.

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