Genetic and Allelic Heterogeneity of Cryg in Eight Distinct Forms of Dominant in the Mouse

Jochen Graw,1 Angelika Neuha¨user-Klaus,2,3 Norman Klopp,4 Paul B. Selby,5 Jana Lo¨ster,1 and Jack Favor 2,3

PURPOSE. The purpose of this study was the characterization of exons: The first one codes for three amino acids, and the eight new dominant cataract mutations. subsequent two are responsible for two Greek key motifs each. ␥ METHODS. Lenses of mutant mice were described morphologi- Biochemically, the - are characterized as monomers cally and histologically. Each was mapped by linkage with a molecular mass of 21 kDa (for reviews see Refs. 6 and 7). 3 studies. The candidate (the Cryg cluster and the Six members of the Cryg family (Cryga Crygf) are located closely linked Cryba2 gene) were sequenced. in a cluster on mouse 1 or the long arm of human , region 33-35, whereas the seventh Cryg gene RESULTS. Molecular analysis confirmed all mutations in Cryg (Crygs) maps to mouse chromosome 16 and human chromo- genes. Five mutations lead to amino acid exchanges, two some 3. The Cryba2 gene encoding the ␤A2- is lo- are due to premature stop codons, and one is a 10-bp dele- cated approximately 8 cM distal to the mouse Cryg gene tion in the Cryge gene. Morphologically, mutant carriers cluster. In humans, the relative map positions of the CRYG expressed nonsyndromic , ranging from diffuse len- ENU910 ENU449 and the CRYBA2 gene are similar to the CRYBA2 ticular opacities (Crygd and Cryge ), to dense nu- 8 K10 MNU8 Z2 located on the long arm of chromosome 2, region 34-36. clear and subcortical opacity (Crygd , Crygc , Cryge , In mice, mutations in all six genes of the Cryg gene clus- CrygdENU4011, and CrygeADD15306), to dense nuclear opacity ENU469 ter have been identified and shown to lead to dominant, and ruptured lenses (Cryga ). Results of histologic anal- congenital cataracts: CrygaENU-436, CrygbNop,9 CrygcChl3,10 yses correlate well with the severity of opacity, ranging CrygdLop12,11 and CrygdAey4.12 Five cataract-causing alleles of from alterations in the process of secondary fiber nucleus Cryge have been reported so far in the mouse: CrygeElo,13 degradation to lens vacuoles, fiber degeneration, and disrup- Cryget,9 Crygenz,14 CrygeAey1,15 and CrygeENU418.16 Moreover, tion of the lens capsule. one dominant cataract was reported recently in the sixth gene CONCLUSIONS. In total, 20 mutations have been described that of the Cryg cluster, Crygf.17 In addition, mutations in the affect the Cryg gene cluster: Nine mutations affect the Cryge mouse Crygs gene were demonstrated to be causative of a gene, but only one affects the Crygb or Crygf genes. No dominant18 and a recessive cataract.19 Several hereditary cata- mutation was observed in the closely linked Cryba2. Two racts in humans have also been shown to be caused by muta- mutations occur at the same site in the Crygd and Cryge genes tions in CRYG genes.20–24 (Leu453Pro). The unequal distribution of mutations suggests In the course of a large-scale mouse mutagenesis pro- hot spots in the Cryg genes. The overall high number of gram,25,26 we identified more than 200 mutants with different mutations in these genes demonstrates their central role in the dominant cataracts. As reported previously,26 the largest sub- maintenance of lens transparency. (Invest Ophthalmol Vis Sci. group among our collection is located on mouse chromosome 2004;45:1202–1213) DOI:10.1167/iovs.03-0811 1 close to the Cryg gene cluster and the Cryba2 gene. Looking at this unequal distribution of mutations in the genome, the he ␤- and ␥-crystallins were first characterized by Mo¨rner1 fundamental question comes up of why this particular region Tmore than 100 years ago. Based on their unique folding on mouse chromosome 1 is more affected than others. There- structure, they are now recognized as members of one ␤/␥- fore, it is necessary to characterize, as a first step, the under- crystallin superfamily. The corresponding genes are expressed lying mutations to enable future experiments to provide more preferentially in the eye and mainly in the ocular lens, and low detailed analysis of the mechanisms leading to the different expression is found in the retina,2,3 brain, and testes.4,5 The types of cataracts. Herein, we report eight novel dominant common characteristic of all ␤- and ␥-crystallins is the Greek cataract mutations: Seven come from our own mutagenesis key motif, which allows a dense packing of in the screening and an additional one from a similar study at the Oak ocular lens. The Cryg genes in all mammals consist of three Ridge National Laboratory (ORNL, Oak Ridge, TN). Molecular analyses show that all eight mutations affect genes in the Cryg gene cluster on mouse chromosome 1, but none of them affect the closely linked Cryba2 gene. From the 1Institute of Developmental Genetics, the 2Clinical Oph- thalmogenetics Cooperation Group, the 3Institute of Human Genetics, 4 and the Institute of Epidemiology, GSF-National Research Center for MATERIALS AND METHODS Environment and Health, Neuherberg, Germany; and 5RiskMuTox, Oak Ridge, Tennessee. Submitted for publication July 30, 2003; revised September 11, Animals 2003; accepted October 5, 2003. For the mutation screens, chemically treated male mice, either (102/ Disclosure: J. Graw, None; A. Neuha¨user-Klaus, None; N. ElxC3H/El)F1 hybrids or the strain DBA/2, were mated with untreated Klopp, None; P.B. Selby, None; J. Lo¨ster, None; J. Favor, None female T-stock mice. In the study at ORNL, mutagenized C3Hf/Sl males The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- were mated with T-stock females. Offspring were ophthalmically ex- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. amined for eye abnormalities at weaning, with a slit lamp microscope Corresponding author: Jochen Graw, GSF-National Research Cen- (model SLM30; Carl Zeiss Meditec, Oberkochen, Germany). Presumed ter for Environment and Health, Institute of Developmental Genetics, mutations from the treatment or control groups or even from the D-85764 Neuherberg, Germany; [email protected]. breeding colony were genetically confirmed and further outcrossed to

Investigative Ophthalmology & Visual Science, April 2004, Vol. 45, No. 4 1202 Copyright © Association for Research in Vision and Ophthalmology

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27 either strain 102/El or (102/ElxC3H/El)F1 hybrid mice. All mutant sequence notation, the A of the ATG start codon in the cDNA was lines were subsequently backcrossed to strain C3H. Homozygous mu- assigned nucleotide position 1. Correspondingly, at the level, tant lines were established and have been maintained by brother x the first Met was assigned position 1 of the amino acid sequence. sister matings. All breeding was performed in the National Center for Environment and Health (GSF, Neuherberg, Germany) animal facility, Computer-Assisted Prediction of the Biochemical according to the German Law on the Protection of Animals and the Properties of the Mutated Proteins ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The analyses were performed using the Proteomics tools of the ExPASy Molecular Biology server (http://www.expasy.ch; provided in the pub- Mutations lic domain by the Swiss Institute of Bioinformatics, Geneva, Switzer- land). In particular, we used Kyte-Doolittle algorithms for hydropho- Of the mutations characterized in the present study, seven were bicity,32 the TMpred and TopPred2 programs to detect transmembrane recovered in Neuherberg. Four were detected in the offspring of mice domains, GOR433 for secondary structures, and the ScanProsite pro- exposed to ethylnitrosourea (ENU). The founder mutant ENU369 ex- gram for additional biochemical features. Protein models were calcu- 28 pressed lens vacuolization and total opacity ; ENU449, diffuse total lated using the ExPASy first-view program and RasMol 2.6. opacity28; ENU910, total cloudy opacity29; and ENU4011, total cloudy opacity (Favor J, unpublished data, 1990). One mutation (MNU8) was General recovered in the offspring of mice exposed to methylnitrosourea (MNU), and the founder mutant expressed lens vacuolization and total Chemicals were from Merck (Darmstadt, Germany) or Sigma-Aldrich opacity (Favor J, unpublished data, 1989). The mutant K10 was recov- (Deisenhofen, Germany). If not indicated otherwise, the used ered in the offspring of untreated parental mice and expressed total for cloning and reverse transcription were from Roche Diagnostics opacity, whereas the mutant Z2 was discovered in breeding stocks and (Mannheim, Germany), and restriction enzymes were from MBI Fer- expressed nuclear and zonular opacity (Favor J, both unpublished, mentas (St. Leon-Rot, Germany). 1985 and 1997). One mutation was recovered at ORNL in the offspring of an ENU- ESULTS treated male. Heterozygous ADD15306 mutants expressed a nuclear R and zonular opacity (Selby PB, unpublished data, 1997). Morphology and Histology Mapping of the Mutations The lens opacities expressed by eight heterozygous and ho- The mutations were mapped relative to microsatellite markers accord- mozygous mutants are documented in Figure 1. Compared ing to methods previously described.30,31 The mutation ADD15306 with the homozygous wild-type (Fig. 1A), there was a wide was mapped commercially (i.puma, Bad Homburg, Germany). Chro- spectrum of lens opacities expressed by carriers of the differ- mosomal positions of genes or markers were taken from the MGI ent mutants. Most extreme is the phenotype observed in het- database (http://www.informatics.jax.org; provided in the public do- erozygotes and homozygotes of the mutation ENU369 (Figs. main by Jackson Laboratories, Bar Harbor, ME). Mice were kept under 1B, 1C, respectively), in which dense nuclear opacity was specific pathogen-free conditions at the GSF, according to the German observed, and lenses were ruptured at 3 weeks of age. Het- Law on the Protection of Animals. erozygous and homozygous mutants K10 (Figs. 1D, 1E), MNU8 (Figs. 1F, 1G), Z2 (Figs. 1H, 1I), ENU4011 (Fig. 1J, 1K), and Morphologic and Histologic Analysis ADD15306 (Figs. 1L, 1M) all expressed cataracts: a dense nuclear and subcortical cataract in heterozygotes and the more For gross documentation, lenses were prepared under a dissecting severe opacity in homozygotes. The phenotypes associated microscope (MZ APO; Leica, Heidelberg, Germany) and photographed with the mutations ENU910 (Figs. 1N, 1O) and ENU449 (Figs. ϫ at 25 magnification. For histologic analysis, eyes of 3-week-old ho- 1P, 1Q) were, by comparison, much milder and were charac- mozygous mutant mice were fixed for 24 hours in Carnoy solution, terized as diffuse lenticular opacities. dehydrated, and embedded in plastic medium (JB-4Plus; Polysciences Histologic analysis demonstrated also a broad spectrum of Inc., Eppelheim, Germany) according to the manufacturer’s instruc- severity (Fig. 2). Compared with the wild-type C3H lenses (Fig. tions. Sectioning was performed with an ultramicrotome (Ultratom 2A), the mutants ENU910 (Fig. 2H) and ENU449 (Fig. 2I) ␮ OMU3; Reichert, Walldorf, Germany). Serial transverse 2- m sections showed only subtle changes as a darkly stained homogenous were cut with a dry glass knife and stained with methylene blue and mass in the core of the lens. Because methylene blue can also basic fuchsin. The sections were evaluated by light microscope (Axio- be used as a stain for cell nuclei, the darkly stained areas in plan; Carl Zeiss Meditec, Halbergmoos, Germany). Images were im- these two mutants could be interpreted as homogenously dis- ported into image-processing programs (Photoshop 6.0, Illustrator 9.0; persed nuclei acids, liberated from imperfectly degraded cell Adobe, Unterschleissheim, Germany). All wild-type controls were nuclei. These minor changes correlate with the weak pheno- strain C3H/El. type observed in the slit lamp as well as in isolated lenses. The mutant ADD15306 (Fig. 2G) also showed sections without Isolation of RNA, DNA, and PCR Conditions major abnormalities. In addition to the darkly stained lens core, Genomic DNA was prepared from tail tip, liver, or spleen, and RNA tiny black spots were present inside the fiber cells, most likely was isolated from lenses (stored at Ϫ80°C) of newborn mice, accord- representing fiber cell nuclei, which are only partly degraded. ing to standard procedures. cDNA synthesis and PCR for mouse Cryg Correspondingly, the wave of the cell nuclei in these three or Cryba2 genes using genomic DNA or cDNA as template were mutants was not as well ordered as in the wild type, indicating performed, as described previously.9,15 For amplification of a smaller also a disturbance of the lens fiber cell differentiation. genomic fragment of Crygc, exon 3, we used an additional internal The mutants MNU8 (Fig. 2D), Z2 (Fig. 2E), and ENU4011 left-side primer (CCTCAGTGAGGTGCGCTCGC) together with the al- (Fig. 2F) exhibited an intermediate phenotype. Small vacuoles ready-described right-side primer, and the resultant PCR fragment of and swollen fiber cells were present in various parts of the lens 279 bp was digested by the restriction SduI. as were alterations in the lens core (anteriorly shifted lens core PCR products were sequenced commercially (SequiServe, Vater- in ENU4011 and Z2), which are consistent with the nuclear stetten, Germany) after cloning into the pCR II vector (Invitrogen, and subcortical cataracts observed in the corresponding lenses. Leek, The Netherlands) or directly after elution from the agarose gel, In the MNU8 and Z2 mutants, the lens nucleus seemed to be using kits from Qiagen (Hilden, Germany) or Bio-Rad (Munich, Ger- separated from the outer cortex. In particular, in the Z2 mu- many) and subsequent precipitation by ethanol and glycogen. For tants, a plaque of epithelial cells was present at the anterior

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FIGURE 1. Lens opacities expressed in 3-week-old heterozygous and ho- mozygous mutants: (A) C3H wild type; ENU369 (B) heterozygote and (C) homozygote; K10 (D) heterozy- gote and (E) homozygote; MNU8 (F) heterozygote and (G) homozygote; Z2 (H) heterozygote and (I) homozy- gote; ENU4011 (J) heterozygote and (K) homozygote; ADD15306 (L) het- erozygote and (M) homozygote; ENU910 (N) heterozygote and (O) homozygote; and ENU449 (P) het- erozygote and (Q) homozygote.

pole. In general, the denucleation process was abnormal, as sequenced either at the cDNA and/or at the genomic DNA reported several times in other cataract mutants.34–35 level. Because the Cryba2 gene is very close to the Cryg gene The most severe phenotypes were observed in the sections cluster (map distance just 8.8 cM according to the Chromo- from the ENU369 (Fig. 2B) and K10 (Fig. 2C) mutant mice. In some Committee Report, 2000; available online at http:// particular, the lens nucleus of the K10 mutant was completely www.informatics.jax.org/ccr/searches/index.cgi?yearϭ2000), shifted to the anterior epithelium and highly vacuolated. The it was also sequenced. primary lens fiber cells had degenerated. The secondary fiber cells did not enlarge, leading to a very small lens. In the Sequence Analysis and Molecular Modeling ENU369 mutant, the posterior lens capsule was ruptured, and In the present study, we identified causative mutations in the lens material was, therefore, also present in the vitreous. Other Cryga, Crygc, Crygd, and Cryge genes, but none in the Crygb, consequences of the rupture of the posterior lens capsule and Crygf,orCryba2 genes. A schematic overview of the general the loss of lens material are the high numbers of large vacuoles features of the Cryg genes and the encoded Greek key motifs in the remaining lens. However, in all these cases, the cell is given in Figure 3A; the three-dimensional (3-D) structure of nuclei were not fully degraded and could be stained by corre- a typical wild-type ␥-crystallin is based on crystallography data sponding techniques. In contrast to the lens, the retinas, iris, of the ␥E-crystallin from the rat and of the bovine ␥F-crystallin cornea were not affected. (Fig. 4A). The mutations will be discussed in alphabetical order (Cryga–Cryge). Mapping Sequencing of the mutation ENU369 revealed a specific The mutation ENU369 has been mapped to chromosome 1 T3C transition at position 127 in exon 2 of Cryga (Fig. 3B). relative to the visible markers fz and ln, and the results sug- The mutation creates a new HpaII restriction site that was not gested that it is located on chromosome 1 at cM 28.36 We found in six different wild-type strains (BALB/c, C57BL/6, JF1, mapped the remaining seven mutations to approximately the 129, DBA, or C3H), but was present in all four homozygous same chromosomal region (Table 1). Therefore, for all muta- mutants that were analyzed (Fig. 5A). The mutant allele was tions, the Cryg genes are considered candidate genes and were accordingly designated CrygaENU369.

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FIGURE 2. Histologic analysis of cat- aractous lenses from homozygous mice. A histologic overview of the entire lens is presented in the top rows of (A)to(H) and a higher mag- nification of the boxed region is shown in the bottom rows. For the weak phenotypes ENU910 and ENU449, only the overview is dem- onstrated. (A) C3H, (B) ENU369,(C) K10,(D) MNU8,(E) Z2,(F) ENU4011,(G) ADD15306,(H) ENU910, and (I) ENU449. Bars, 100 ␮m.

At the protein level, it leads to an amino acid change from the lower band represents the 125-bp fragment. Obviously, the an aromatic Trp to a basic Arg at position 43 (W43R) in the mixture of both fragments results in a slower velocity during second Greek key motif (Fig. 4B). The Trp occurs at this electrophoresis most likely due to hyperstructures. The se- position in all ␥-crystallins from mouse, rat, bovine, and human quence analysis of the band in the mutants revealed a mixture and is essential for the formation of the second Greek key of the 146- and 133-bp fragments, consistent with the destruc- motif. The program ScanProsite predicts that the second Greek tion of the first SduI restriction site. Therefore, we propose the key motif will not be formed in the mutant ␥A-crystallin. allele symbol CrygcMNU8. Moreover, it predicts that the hydrophobicity of the corre- The ScanProsite program suggests that two biochemically sponding region will be changed as well as the isoelectric point active sites are missing in the truncated ␥C-crystallin, namely ␥ (pI) of the entire protein (from pH 7.5 in the wild-type A- an N-myristoylation site (at amino acid position 158-163) and a crystallin to pH 8.5 in the mutated form). In addition, Scan- PKC phosphorylation site (at amino acid position 166-168); the Prosite suggests that a novel tyrosine kinase phosphorylation pI of the protein is calculated to be at pH 6.9 instead of pH 7.6, site may be created at amino acid position 43-51. as in the wild-type allele. Three-dimensional modeling (Fig. 4C) A mutation in the Crygc gene is causative of the cataractous demonstrates that an essential part of the fourth Greek key phenotype in the MNU8 mutant line. The exchange of the ␣ regular G with an A at position 471 of the Crygc gene leads to motif (the third antiparallel strand and a part of the helix) is a stop codon and a truncation of the protein at position 157 missing; however, ScanProsite suggests that the fourth Greek (Trp157Stop; Fig. 3B). It destroys one (of two very close) SduI key motif will form. restriction site, which was confirmed by the analysis of four Three of the new mutants evaluated in this study are asso- wild-type mice from different genetic backgrounds and four ciated with mutations in the Crygd gene: In the ENU4011 mutants (Fig. 5B). The observed pattern of bands in the PAGE mutant line, a T3C exchange was observed at position 134 of unexpectedly did not fit the predicted sizes of the DNA frag- the Crygd gene. Because the mutation did not affect a restric- ments. In wild type, the 125- and 133-bp fragments should be tion site, it was confirmed at the genomic DNA level by se- separated electrophoretically. Likewise, in mutants, the 146- quencing exons 1 and 2 and their flanking regions in five and 133-bp fragments should be separable. However, sequence mutants and by comparison to the database (NM_007776) and analysis of the two wild-type fragments revealed that the upper to four different wild-type strains (C57BL/6, JF1, DBA, and band is a mixture of the 125- and 133-bp fragments, whereas T-stock). The C at cDNA position 134 was found only in the

TABLE 1. Mapping Results from Seven Dominant-Eye Mutants of the Mouse, Relative to Chromosome 1 Microsatellite Markers

ENU449 - (0/102) - D1Mit156 - (2/102) - D1Mit181 D1Mit231 - (31/100) - ENU910 - (1/100) - D1Mit156 - (3/100) - D1Mit181 ENU4011 - (0/113) - D1Mit156 - (3/113) - D1Mit181 D1Mit213 - (9/109) - MNU8 - (0/109) - D1Mit156 - (5/109) - D1Mit181 - (6/109) D1Mit334 Z2 - (1/115) - D1Mit156 - (4/115) - D1Mit181 K10 - (0/107) - D1Mit156 - (6/107) - D1Mit181 D1Mit211 - (5/38) - ADD15306 - (2/38) - D1Mit216 - (14/38) - D1Mit206

Numbers in parentheses indicate the number of recombinants observed (numerator) in the total number of offspring genotyped (denominator) between the loci.

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FIGURE 3. A schematic overview of the Cryg genes, the encoded mutations, and the ␥-crystallin structure. (A) A typical Cryg gene consisting of 522 bp is shown. The four Greek key motifs are shown, including their N- and C-terminal amino acids and in relation to the coding exons. The A of the ATG initiation codon of the cDNA is counted as nucleotide 1; the first Met of the deduced amino acid sequence as amino acid 1. The Crygb gene of all mammals codes for an additional amino acid at the end of exon 2; the corresponding mRNA is 3 bp longer. The designation of the four Greek key motifs is according to Reference 37. (B) Alignments of the wild-type and mutated sequences: The mutations and the resultant changes in the amino acid sequence are shaded with white letters; the corresponding wild-type sequences are shaded with bold black letters. The altered restriction sites are underlined.

five homozygous cataractous mutants. Therefore, this new Two polymorphic sites in this mutant line ENU4011 are Crygd allele is referred to as CrygdENU4011. shared with the Test stock, one of which is close to the The mutation leads to a replacement of Leu at position 45 characterized mutation site. Therefore, it might be speculated by Pro (Fig. 3B). The Leu residue is present in most of the that this mutation was not induced by ENU, but occurred ␥-crystallins analyzed so far. It is replaced by Ile in the human spontaneously in the germ cells of the untreated T stock mouse ␥B- and the bovine ␥D-crystallin and by Val in the bovine (in our standard protocol, all parental mice were ophthalmo- ␥A-crystallin. Codon 45 is located in the second Greek key logically examined for the presence of pre-existing muta- motif (Fig. 5B). The ScanProsite program suggests that the tions28). folding properties of the ␥D-crystallin are strongly affected by The second mutation affecting the Crygd gene was recov- the substitution of Leu by Pro and that the second Greek key ered in the mutant line ENU910 as an A3T substitution at motif will be prevented from forming. position 268 (Fig. 3B). The mutation destroys a Bsp143I restric-

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FIGURE 4. Protein models of mu- tated ␥-crystallins. Based on the crys- tal structure of the rat ␥E- and bovine ␥D-crystallin, 3-D models of the mu- tated ␥-crystallins were calculated. The antiparallel ␤ sheets are yellow and the ␣ helices red; blue sections are looping regions. Amino acid sub- stitutions are green. N, N terminus; C, C terminus. (A) The consensus ␥-crystallin based on the rat ␥E- and the bovine ␥D-crystallin; the Greek key motifs are indicated by Roman numerals; (B) the novel point muta- tions (Arg43, CrygaENU369; Pro45, CrygdENU4011, and CrygeADD15306; Phe90, CrygdENU910; Met126, CrygeENU449); (C) C-terminal dele- tion in CrygcMNU8;(D) C-terminal de- letion in CrygdK10.

tion site in the Crygd gene. A digest of the corresponding puter-based analysis of the mutant protein predicts two trans- genomic fragment confirmed the absence of the Bsp143I re- membrane domains from amino acids 23 or 28 to position 44 striction site in all five homozygous mutant mice, but demon- and from position 46 or 50 to 68 or 70, and the N and C termini strated its presence in all four wild-type strains tested (Fig. 5C). are likely to be at the cytosolic site. ScanProsite suggests also Therefore, this allele was named CrygdENU910. the presence of a threonine-rich region. However, this predic- At the protein level, this mutation changes the Ile at tion is below the accepted threshold level and may be spuri- position 90 to a Phe. Comparing all available data on ␥D- ous. crystallin amino acid sequences in mammals, only Lys or Met The mutation ADD15306 is characterized by an exchange is present at this position without effects on the ␥-crystallin ofaTforaCatposition 134 (Fig. 3B). Because this mutation functions. Three-dimensional modeling (Fig. 4B) shows that does not affect a restriction site, it was confirmed by sequenc- this position is at the beginning of the third Greek key motif, ing in three additional mutant animals using genomic DNA as which will form correctly. The pI is slightly higher for the substrate, but was never found in wild-type mice of different mutated ␥D-crystallin (pH 7.0 versus pH 6.6 for the wild- genetic origins (C57BL/6, T-stock, BALB/c, 129, and JF1). type protein). Therefore, this Cryge allele is referred to as CrygeADD15306. The third mutation was confirmed in the K10 mutant line as The mutation is predicted to lead to a Leu3Pro exchange a unique 432C3G base-pair substitution (Fig. 3B) that destroys at codon 45 in the second Greek key motif, the same position an ScaI restriction site. A digest of the corresponding genomic as in the mutant CrygdENU4011 (Fig. 4B), and, similarly, Scan- fragment confirmed the absence or presence of the ScaI re- Prosite predicts the absence of this particular motif. The pI is striction site within the mutant lines or wild-type strains, re- reduced in the mutant ␥E-crystallin (pH 7.1) compared with spectively (Fig. 5D). Therefore, this allele was referred to as the wild type (pH 7.7); other alterations are not predicted. CrygdK10. The third mutation in the Cryge gene was found in the At the protein level, this mutation creates a stop codon in ENU449 mutant line. The mutation is characterized by a G3A the linker region between the third and fourth Greek key motif exchange at position 376 in exon 3 (Fig. 3B). It destroys an and leads to a truncation of the protein after 143 amino acids. Eco72I restriction site, which was confirmed in the genomic The 3-D model of the truncated protein (Fig. 4D) as well as DNA of five mutants and five wild-type mice of different ge- ScanProsite show that the mutant gene product lacks the last netic origin (Fig. 5F). Therefore, the mutation was named Greek key motif. The tyrosine kinase phosphorylation site CrygeENU449. (amino acids 147-154), the N-myristoylation site (amino acid It is predicted that the amino acid sequence is affected at position 158-163) and the PKC phosphorylation site (amino position 126 (Val3Met). ScanProsite does not suggest any acid position 166-168) are not present. The pI of the truncated effect on the Greek key motif formation, because the changed ␥D-crystallin is lower (pH 5.9) than the pI of the wild-type amino acid is considered to be localized just at the beginning of protein (pH 6.6). the fourth motif (Fig. 4B). However, comparing all available Three mutations were detected in the Cryge gene: The protein sequences revealed that the Val residue is highly con- spontaneous mutation Z2 exhibits a 10-bp deletion in the very served at this position and present in all ␥-crystallins analyzed beginning of the Cryge cDNA in exon 2 (del12-21; Fig. 3B). The so far. As for the ADD15306 mutation, the pI of the ␥E- mutation creates a new HphI restriction site that was con- crystallin affected by the ENU449 mutation is lowered also to firmed by analysis of four homozygous mutants and wild-type pH 7.1. mice of four different strains (Fig. 5E). Therefore, the mutation is called CrygeZ2. The deletion of 10 bp creates a new open Polymorphisms reading frame consisting of the first three N-terminal amino Several polymorphic sites were identified in the Cryg gene acids of the Cryge followed by 119 novel amino acids. Com- cluster (Table 2). Most of them occurred in the T-stock mice,

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TABLE 2. Polymorphisms in Mouse Cryg Genes

Gene Reference Acc. No (Strain) cDNA Protein Strain*

Cryga NM_007774 (DBA/2) 201 A3G No effect T-stock; all mutant lines Crygb Z22573 (102/ElxC3H/El)F1 238 C3T R80C T-stock 524 A3T Y175F T-stock Crygc Z22574 (102/ElxC3H/El)F1 111 A3C R37R T-stock; ENU4011 384 G3A E128E T-stock 513 A3C V171V ENU910 Crygd NM_007776† 95A3G H32R T-stock; all mutant lines 154G3A; 156A3G A52T T-stock 303/304 AG3GA QV101/102QM T-stock; all mutant lines 321A3G E107E 488A3G K163R T-stock; all mutant lines AJ224342 (102 ϫ C3H)F1 Intron A: ⌬GCCTT No effect JF1, C3H Cryge X57855 (101/ElxC3H/El)F1 139 C3G Q47E T-stock; all mutant lines 154 A3G T52A T-stock 189 T3C Y63Y T-stock; ENU4011 Intron A: ϩ11 A3G No effect 129, ADD15306 Intron B: reduced number No effect T-stock of CTCAG repeats Crygf Unpublished C3H 111 C3A R37R NM_027010 (B) 234 T3C S78S NM_027010 (B) 239 G3A R80H NM_027010 (B) 468 C3T D156D T-stock; NM_027010 (B) 484–486 GCC3TCA A162S T-stock, NM_027010 (B) 501–503 GAG3TGA LR167/168LE T-stock, NM_027010 (B)

A of the ATG start codon is counted as nt 1; Met encoded by the start codon is counted as amino acid 1. *Bϭ C57BL/6J. † No specific strain is known (NIH general purpose mice).

but some also in the mutant lines. They will be presented in 154/156 the commonly used sequence GCA (Ala) is replaced alphabetical order from Cryga to Crygf. only in the T-stock by ACG (Thr; nucleotide 52). This position In Cryga, all mutant lines tested as well as the wild-type does not seem to be highly conserved among mammals (hu- strain T-stock demonstrated a deviation from the database man, Ser; rat, Thr; bovine, Leu). At position 303/304, the sequence (accession no. NM_007774) at position 201 without database reports an AG, but all our lines have a GA, which leads effect on the amino acid composition. to a Val3Met exchange at amino acid position 102. The Met In the Crygb gene, two polymorphic sites were observed. residue is present in most of the other ␥-crystallins at this At position 238, the T-stock mice exhibit a T instead of a C, position; a Val was reported for the human ␥D-crystallin as well which leads to an exchange of an Arg for a Cys at codon 80. as for mouse and rat ␥A-crystallin. In the bovine ␥C- and This Cys is also present in human ␥B- and ␥C-crystallins and in ␥E-crystallins, an Ile occurs at this position. At nucleotide 488, bovine ␥C- and ␥E-crystallins. In the rat ␥F-crystallin, a His is the database A is replaced in all cases by a G; therefore, the present at this position. At position 524, a T was present in the DNA polymorphism causes an exchange from Lys to Arg at T-stock mice, changing the Tyr at codon 175 into a Phe, which codon 163. The Arg has been reported for human and rat is present in rat Crygf at the corresponding position. All other ␥D-crystallin, too. Cryg genes exhibit either Tyr or Leu. During this study, we also identified a small deletion in In the Crygc gene, we identified three polymorphic sites. In intron A of the JF1 wild-type mice. The sequence GCCTT is all cases, the encoded amino acids are not changed. present three times in three wild-type strains (C57BL/6; DBA/2, All four polymorphic sites identified in the Crygd gene are T-stock), but only twice in the JF1 strain and in the database predicted to cause changes in the amino acid composition. At entry based on C3H mice (accession no. AJ224342). That in the position 95, the database (accession no. NM_007776) reported JF1 mice the correct cDNA for Crygd was also observed sug- an A, which is replaced in all our sequences by a G, leading to gests that this deletion of one of the repeated GCCTT elements an exchange of His for Arg at codon 32. His at this position is does not affect the splicing mechanism in the small first intron reported also in all other species. In contrast, at position of the JF1 Crygd gene.

Š

FIGURE 5. Alterations in restriction sites associated with Cryg gene mutations and cataract formation. Genomic DNA from homozygous mutant or wild-type mice was amplified by PCR and digested by the corresponding restriction enzymes. Restriction maps of the PCR fragments are shown and the restriction sites indicated. The PCR fragments were analyzed by agarose gel electrophoresis with (ϩ) or without (Ϫ) digestion by the indicated restriction enzyme (see the Results section for interpretation of gels). (A) Cryga fragment digested by HpaII for CrygaENU369 mutation: The restriction site is present in the PCR-fragment of exons 1 and 2 in the four mutants only. (B) Crygc fragment digested by SduI for CrygcMNU8 mutation: The first SduI restriction site is missing in the 3Ј part of Crygc, exon 3 of the mutants, resulting in two fragments of apparently the same size, which were not electrophoretically separated. (C) Crygd fragment digested by Bsp143I for the CrygdENU910 mutation: The first Bsp143I restriction site in the PCR fragment containing exons 1 and 2 (Crygd) is missing only in the mutants, leading to two fragments of 226 and 237 bp, respectively. (D) Crygd fragment digested by ScaI for CrygdK10 mutation: The loss of the ScaI restriction site in exon 3 (Crygd) leads to an undigested fragment of 463 bp in all five mutants tested. (E) Cryge fragment digested by HphI for CrygeZ2 mutation: The 10-bp deletion results in loss of the second HphI restriction site in the PCR fragment containing exons 1 and 2 (Cryge), leading to two fragments of 198 and 356 bp. (F) Cryge fragment digested by Eco72 for the CrygeENU449 mutation: The loss of the second Eco72 restriction site results in a fragment of 490 bp in the mutants. The slightly larger size of the C3H fragment compared with the other wild-type strains is due to a larger repeat in intron 1. M, marker; BL6, mouse strain C57BL/6; T, test stock mice; JF1, Japanese fancy mouse.

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Next to the Crygd gene, the Cryge gene showed the most A second factor is the degree to which the mutated gene is polymorphic sites in the mouse strains investigated. There altered. In addition to these 8 Cryg gene mutations reported in were several polymorphisms that are present only in a few of this study, 12 other mouse mutations have been reported to the mutant lines or the T-stock strain. Some of them have no affect the Cryg gene cluster and to lead to cataracts (Table 3). effect on the amino acid composition. At position 47, Glu is the Most of these 20 Cryg mutations lead to an amino-acid ex- predominant form in all other ␥-crystallins. At codon 52, a change in an important region of the corresponding ␥-crystal- T52A polymorphism is observed in the T-stock mice. It should lin, or they express a truncated form of the ␥-crystallin with or be noted that, in the ␥D-crystallin, the common amino acid at without new amino acids. For the mutations with extreme this position is also Ala. cataract phenotype, the mutant proteins were predicted to be During this study, a further polymorphic site was observed altered drastically. Moreover, there are four frameshift muta- in intron A at position 11:aGispresent in the ADD15306 tions (CrygeAey1, CrygeENU418, CrygeNz, and CrygeZ2) leading mutant line and in the wild-type strain 129, but an A in all other to chimeric proteins, which include novel amino acid se- wild-type strains analyzed including the database (accession quences from the point of the frame shift that have no similar- no. X57855). Moreover, the frequency of the CTCAG repeat at ity to the ␥-crystallins. the 3Ј-end of intron B is lower in the T-stock mice as reported Among the biophysical data available, the changes of the previously for the (101xC3H)F1 hybrids (GenBank accession pIs were interesting. Recently, Ueda et al.39 reported that dur- no. X57855). ing ageing, an acidification of the mouse ␥-crystallins appears The specific amplification of the Crygf cDNA is not always (on average 0.47 pH units). The acidified forms were almost successful because of its extensive similarity to the Cryge exclusively found in the insoluble fraction. Among the novel cDNA, which is usually preferentially expressed. Therefore, in Cryg mutations reported in the present study, the CrygaENU369 all mutant lines the Crygf gene was analyzed at the genomic mutation has the most severe phenotype. However, the af- level by amplifying the coding (exon) regions together with fected ␥A-crystallin is changed into a basic form (pI changed flanking (intron) regions. Three polymorphic sites were recov- from pH 7.5 to 8.5). Among all the Cryg mutants listed in Table ered in the T-stock mice, two of which have no effect on the 3, the most extreme acidic alteration in the pI occurs in the amino acid composition. The 493 A3G exchange in the T- CrygbNop mutant (pH 7.6 to 5.5), and the most extreme basic stock and the ENU449 mutant line leads to Gly, which is alteration occurs in the CrygeENU418 (pH 7.7 to 10.1). How- present in all ␥-crystallins at this position; in contrast, the Ser in ever, in both cases the phenotype is not as severe as in the all other mutant lines seems to be the exception. CrygaENU369 or the Cryget, suggesting that the pI is not a In addition, we checked the expressed sequence tag (EST) valuable parameter for genotype–phenotype correlation. database entries sharing the key word ␥-crystallin for polymor- Posttranslational modifications of the mutated proteins phic sites and a surprisingly high number of additional poly- might occur (e.g., phosphorylation) depending on the site and morphic sites were found. Unfortunately, only a few of them the nature of the mutation. Such modifications have been could be attributed to strain-specific (mainly C57BL/6J) poly- predicted for Cryga ENU369, Crygc MNU8 and CrygdK10. How- morphisms, because most of the entries do not give informa- ever, biochemical analyses are required to determine if such tion on the particular strain characterized. However, because posttranslational modifications occur. in some cases, contradictory results were found, it may be Another approach might address the question of how the necessary to confirm these sequences before making conclu- mutations and the altered proteins interfere with the ongoing sions based on rough data. process of terminal differentiation in the lens fiber cells. We could show recently that Cryg-mediated cataractogenesis (in Crygbnop, Cryge Elo, and Cryget ) is associated with the forma- DISCUSSION tion of amyloid fibrils in the nuclei of primary lens fiber cells. This interpretation was supported by the observation of recom- In the present paper, we describe the phenotypes and results binant mutant ␥-crystallin forming amyloid fibrils also in cell of molecular characterizations of eight mouse cataract muta- culture, whereas native ␥-crystallins are soluble and nonfila- 38 tions affecting genes in the Cryg gene cluster. Because the mentous. This process is associated with an inhibition of the mutations segregate with the phenotypes, the evidence is degradation of the central lens fiber cell nuclei and of the strong that these mutations are responsible for the cataract chromosomal DNA. This is most likely caused by an impaired 2ϩ phenotypes. action of a Mg -dependent DNase, which was shown to correlate well with the severity of three types of cataract investigated (Cryge Ns Ͼ CrygbNop Ͼ Cryge t).40 This interpre- Genotype–Phenotype Considerations tation is supported by recent findings that mice deficient in the The eight novel Cryg alleles lead to different phenotypes of DLAD gene (coding for a DNase II–like acid DNase or DNase congenital lens opacifications, ranging from nuclear, to total, to II␤) are incapable of degrading DNA during lens cell differen- 41 lamellar cataracts. The cataractous eyes are in most cases tiation and develop nuclear cataract. However, the biochem- smaller, and the mutations show a semidominant mode of ical mechanisms during cataractogenesis remain to be elabo- inheritance. The most severe phenotype among the eight mu- rated. tants described herein is represented by the CrygaENU369 mutant, which is caused by a mutation affecting the Cryga Unequal Distribution of Mutations among the gene. In the mouse, it was shown recently that the Cryga Cryg Gene Cluster gene is expressed very early (at embryonic day [E] 12.5) and at a higher level than the Cryge/f genes.38 The other Cryg The mutations are not evenly divided among the six Cryg genes genes are expressed only from E14.5 onward. With the excep- in the cluster: nine in the Cryge gene, four in the Crygd gene, tion of the very mild phenotypes caused by the CrygdENU910 three in the Crygc gene, two in the Cryga gene, and only one and CrygeENU449 mutations, the other phenotypes are consis- mutation each in the Crygb and Crygf genes. Surprisingly, not tent with the hypothesis that the severity of the cataracts a single mutation or polymorphic site was found in the closely increases, if the affected Cryg gene is expressed earlier. Thus, linked Cryba2 gene. The six ␤-crystallin encoding genes are in one factor to be considered important in the degree of severity general less affected than the Cryg genes; there has been one of congenital cataract is the time of onset of mutation reported in Cryba142 and two in Crybb2.5,43 In this during embryonic development. context, it might be interesting to note that two mouse muta-

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TABLE 3. List of Characterized Mutations at the Cryg Gene Cluster on Mouse Chromosome 1

Consequence for Protein Phenotype Previous (Graded Paternal aa Greek Key Name Symbol Severity) Treatment Molecular Lesion Alterations pI Motif Reference

CrygaENU369 ENU369; Total cataract ENU Cryga: T127C Trp433Arg pH 7.5–8.5 2nd not This study Cat2tol with vacuoles formed (severe) Cryga1Neu ENU-436 Diffuse nuclear ENU Cryga: A222G Asp743Gly pH 7.5–8.0 No effect 9 opacity (medium) CrygbNop Nop Nuclear opacity Spontaneous Crygb: ⌬417–427/Ins4 Ser139: 6 pH 7.6–5.5 4th not 9 (medium) new aa; formed stop CrygcChl3 Chl3 Total and Chlorambucil Crygc: ⌬420–425 ⌬G141, R142 pH 7.6–6.9 4th not 10 lamellar formed opacity (medium) CrygcMNU8 MNU-8 Total opacity MNU Crygc: G471A Trp1573stop pH 7.6–6.9 No effect This study with vacuoles 15 aa (medium) missing CrygdENU4011 ENU4011 Cloudy nuclear ENU Crygd: T134C Leu453Pro pH 6.6–7.0 2nd not This study opacity formed (medium) CrygdAey4 Aey4 Nuclear and ENU Crygd: T227A Val763Asp pH 6.6–6.6 No effect 12 cortical cataract (medium) CrygdENU910 ENU910 Diffuse total ENU Crygd: A275T Ile903Phe pH 6.6–7.0 No effect This study cataract (mild) CrygdK10 K-10 Total cataract Spontaneous Crygd: C432G Tyr1443stop pH 6.6–5.9 4th not This study (medium) 28 aa formed missing CrygdLop12 Lop12 Irregular nuclear Spontaneous Crygd: G470A Trp1573stop pH 6.6–6.2 No effect 11 lens opacity 18 aa (medium) missing CrygeAey1 Aey1 Nuclear cataract ENU Cryge: A1T new protein pH 7.7–9.5 No motif 15 (medium) (13 kDa) CrygeENU418 ENU418 Nuclear and ENU Cryge: Intron A, Ile4: 153 new pH 7.7–10.1 No motif 16 lamellar A66G, lariat aa cataract formation and (medium) splicing inhibited CrygeZ2 Z2 Total lamellar Spontaneous Cryge: ⌬12–21 Ile4: 119 new pH 7.7–9.8 No motif This study cataract aa (medium) CrygeNz Nzc; 116 Nuclear and ␥-ray Cryge: ⌬89T Phe30: 96 pH 7.7–8.4 Motifs 2–4 not 14 zonular new aa formed cataract (medium) CrygeADD15306 ADD15306 Zonular cataract ENU Cryge: T134C; Leu453Pro pH 7.7–7.1 2nd not This study (medium) formed CrygeENU449 ENU449 Capsular opacity ENU Cryge: G376A Val1263Met pH 7.7–7.1 No effect This study (mild) CrygeElo Elo Eye lens Spontaneous Cryge: ⌬403 Glu135: 11 pH 7.7–6.4 4th not 13 obsolescence new aa and formed (medium) stop Cryget R324; Total opacity X-ray Cryge: C432G Tyr1443stop pH 7.7–6.4 4th not 9 Cat2t (severe) formed CrygeNs K134; Scat Suture cataract Spontaneous Cryge: Del Ͼ2kb Hybrid ? 4th motif 50 (mild) protein? affected? CrygfRop Rop Radial opacity Procarbazine Crygf: T113A Val383Glu pH 7.1–6.7 4th not 17 (medium) formed

A of the ATG start codon is counted as nt 1; Met encoded by the start codon is counted as amino acid 1.

tions affect also the Crygs gene at mouse chromosome 1618,19; genes. The T134C mutation was found in both Crygd one of them is inherited in a recessive mode even if the (ENU4011) and Cryge (ADD15306). Both were induced by mutation leads to a late truncation of the protein ENU, and it is also of interest that their phenotypes are differ- (Trp163Stop).19 The relatively high rates of mutation induction ent. Moreover, the G470A mutation was found both in the compared with other genes that cause cataracts, the high mouse Crygd and human CRYGD.11,24 number of polymorphic sites, and the unequal distribution Corresponding to the increasing number of characterized among the Cryg gene cluster were unexpected. Also unex- cataract mutants in mice, mutations in human CRYG genes pected were the findings of identical mutations in two different have been shown to be associated with cataract formation: the

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Coppock-like cataract22 and the variable zonular pulverulent 13. Cartier M, Breitman ML, Tsui L-C. A frameshift mutation in the cataract21 with the CRYGC gene and the aculeiform cataract,22 ␥E-crystallin gene of the Elo mouse. Nat Genet. 1992;2:42–45. a punctate cataract,23 and a crystal-deposition cataract20 with 14. Klopp N, Lo¨ster J, Graw J. Characterization of a 1-bp deletion in mutations in the CRYGD gene. Three other hereditary congen- the ␥E-crystallin gene leading to a nuclear and zonular cataract in ital cataracts with lamellar or nuclear opacity are also associ- the mouse. Invest Ophthalmol Vis Sci. 2001;42:183–187. ated with mutations and affect the CRYGC or CRYGD genes.24 15. Graw J, Klopp N, Lo¨ster J, et al. Ethylnitrosourea-induced mutation No mutation has been reported to date in the CRYGA or in mice leads to the expression of a novel protein in the eye and CRYGB genes or in the closely linked CRYBA2 gene. to dominant cataracts. Genetics. 2001;157:1313–1320. The only described change in the human CRYGA is an 16. Graw J, Neuha¨user-Klaus A, Lo¨ster J, Klopp N, Favor J. Ethylnitro- sourea-induced substitution affects splicing of the mouse insertion at pos. 43 leading to a frame shift and a premature ␥E-crystallin encoding gene leading to the expression of a hybrid stop codon after 7 novel amino acids; it is not known whether protein and to a cataract. Genetics. 2002;161:1633–1640. the corresponding 20-amino acid peptide is stable. In the 17. Graw J, Klopp N, Neuha¨user-Klaus A, Favor J, Lo¨ster J. CrygfRop: heterozygous situation this insertion is without pathological the first mutation in the Crygf gene causing a unique radial lens consequences; it is not known whether it might lead to cata- opacity. Invest Ophthalmol Vis Sci. 2002;43:2998–3002. 44 racts in homozygotes. Among the ␤-crystallin encoding 18. Sinha D, Wyatt MK, Sarra R, et al. A temperature-sensitive mutation genes, the CRYBB2 is most often affected because of possible of Crygs in the murine Opj cataract. J Biol Chem. 2001;276:9308– recombinations with its closely linked pseudogene45–47; fur- 9315. ther mutations have been reported only in CRYBA148 and 19. Bu L, Yan S, Jin M, et al. The ␥S-crystallin gene is mutated in CRYBB1.49 autosomal recessive cataract in mouse. Genomics. 2002;80:38– In conclusion, it is suggested even from the relatively small 44. number of characterized mutations that the 7 Cryg genes are 20. Kmoch S, Brynda J, Asfaw B, et al. Link between a novel human more frequently affected than the 6 Cryb genes. Moreover, ␥D-crystallin allele and a unique cataract phenotype explained by even among the Cryg genes there is an unequal distribution of protein crystallography. Hum Mol Genet. 2000;9:1779–1786. mutations suggesting a sub-group of Cryg genes, which are a 21. Ren Z, Li A, Shastry BS, et al. 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