Crygf ^Rop : The First Mutation in the Crygf Gene Causing a Unique Radial Lens Opacity

Abstract

purpose. The Rop (radial opacity) mutation, which was recovered in a mutagenicity screen after paternal treatment with procarbazine, was analyzed to determine phenotype, chromosomal localization, candidate genes, and molecular lesion.

methods. Native lenses were photographed under a dissecting microscope. Histologic sections of the eye were made according to standard procedures. Fine mapping of the mutation in relation to microsatellite markers for mouse chromosome 1 was performed. Candidate genes were amplified by PCR from cDNA or genomic DNA and sequenced.

results. The nuclear opacity of the heterozygous mutants showed radial structures, whereas the opacity of the homozygotes was homogenous. The histologic analysis revealed changes in the lens nucleus, which corresponds to the pronounced opacification in lenses of homozygous mutants. The allelism of Rop to the Cat2 group of dominant cataracts on mouse chromosome 1 was confirmed by linkage to microsatellite markers D1Mit156 and D1Mit181. The cluster of the Cryg genes and the closely linked Cryba2 gene were tested as candidates. A T→A exchange in exon 2 of the Crygf gene leads to a Val→Glu exchange in codon 38 and was considered to be causative for the cataract phenotype; therefore, Crygf ^Rop has been suggested as the designation for the mutation.

conclusions. Crygf ^Rop is the first mutation affecting the Crygf gene. Dominant cataract mutations for all six Cryg genes on mouse chromosome 1 have now been characterized, demonstrating the importance of this gene cluster in lens transparency.

The murine α-, β-, and γ-crystallins are a major protein component of the lens and are considered essential for lens transparency. The family of γ-crystallin encoding genes (Cryg) consists of seven highly homologous genes: Six (Cryga– Crygf) are located in a tight cluster on mouse chromosome 1 (human chromosome 2q33-35) and the seventh (Crygs) is on mouse chromosome 16 (human chromosome 3). ^{1 2 3}  

Crystallographic analyses have shown that the γ-crystallins are composed of two domains, each consisting of two Greek key motifs. The Cryg genes in all mammals consist of three exons: exon 1 codes only for three amino acids, and exons 2 and 3 each encode two Greek key motifs. Biochemically, the γ-crystallins are monomers with a molecular mass of 21 kDa. ^{2 3 4}  

Up to now, several types of hereditary cataracts in humans have been shown to be caused by mutations in CRYG genes. ^{5 6 7 8 9} In humans two of the six CRYG genes are pseudogenes (CRYGE and CRYGF) that are not expressed, because of several mutations in their promoters. ^{1 5 10} In the mouse, all seven Cryg genes are expressed in the lens. 

Several mouse mutations in the Cryg genes leading to cataracts have been identified: Cryga ^ENU436 , Crygb ^Nop , ¹¹ Crygc ^Chl3 , ¹² Crygd ^Lop12 , ¹³ and Crygd ^Aey4 . ¹⁴ For the Cryge gene, five cataract alleles have been reported so far: Cryge ^Elo , ¹⁵ Cryge ^t , ¹¹ Cryge ^ns , ¹⁶ Cryge ^nz , ¹⁷ and Cryge ^Aey1 . ¹⁸ Just recently, a temperature-sensitive mutation of the Crygs gene, Crygs ^Opj , was characterized in the mouse. ¹⁹ However, to date, no mutation has been detected in the Crygf gene. 

A mutation, previously designated as Rop (radial opacity), was found by ophthalmic screening of mice after paternal treatment with procarbazine. ²⁰ Rop has been shown to be allelic or tightly linked with a group of cataract mutations on mouse chromosome 1, ²¹ and some of these mutations have been identified as mutant alleles of different Cryg genes. ^{11 17} Based on chromosome location and lens phenotype, the six genes (Cryga– Crygf) of the Cryg gene cluster, as well as the closely linked Cryba2 gene, were considered to be good candidates for Rop mutation analysis. Results revealed that the Rop mutation is associated with a sequence alteration of the Crygf gene. This report describes the molecular lesion and provides morphologic data on Rop mutants. 

Materials and Methods

Animals

The Rop mutant was detected among offspring of procarbazine-treated male (102/ElxC3H/El)F₁ mice. ²⁰ The screening for the lens anomalies was performed in 3-week-old mice with a slit lamp (SLM30; Carl Zeiss, Oberkochen, Germany). Before these studies were initiated, the Rop mutation was backcrossed more than 10 generations to a C3H background. Homozygous mutant lines have been maintained by brother x sister matings. All breeding was performed in our animal facility according to the German Law on the Protection of Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Morphologic Analysis

For gross observation, mice at different ages (ranging from 2 weeks to 3 months) were killed and the eyes dissected. The lenses were enucleated under a dissecting microscope (MZ APO; Leica, Bensheim, Germany) and photographed at ×20 magnification. For histologic analysis, eyes from 4-day-old mice were fixed for 24 hours in Carnoy solution, dehydrated, and embedded in plastic medium (JB-4Plus; Polysciences Inc., Eppelheim, Germany), according to the manufacturer’s instructions. Sectioning was performed with an ultramicrotome (Ultratom OMU3; Reichert, Walldorf, Germany). Serial transverse 2-μm sections were cut with a dry glass knife and stained with methylene blue and basic fuchsin. The sections were evaluated by light microscope (Axioplan; Carl Zeiss, Hallbergmoos, Germany). Images were acquired by means of a scanning camera equipped with a screen-capture program (Axiocam and Axiovision; Carl Zeiss) and imported into an image-processing program (Photoshop, ver. 6.0; Adobe Illustrator 9.0, Adobe Systems, Unterschleissheim, Germany). 

Fine Mapping of the Rop Mutation

For fine mapping of the Rop mutation, linkage was tested in relation to the markers D1Mit156 and D1Mit181 in the cross (C3H ^Rop x C57BL/6)F₁ x C57BL/6. 

Isolation of RNA, DNA, and PCR Conditions

Genomic DNA was prepared from spleen or tail tips of 3-week-old mice according to standard procedures. RNA was isolated from lenses (stored at –80°C) of newborn mice. cDNA synthesis and PCR using genomic DNA or cDNA as a template were performed essentially as reported previously. ¹¹ Because Crygf cDNA is difficult to amplify because of its similarity to and the predominance of Cryge, it was amplified from genomic DNA using a primer pair for exons 1 and 2, together with their flanking regions and the connecting intron A (forward: 5′-GTT ATT CAA ATT CTC TTA GTG TGA GAA TTA TAA ACC-3′; reverse: 5′-ACA AAG AAG GTA GCA GAT ATC CTA ACC-3′; annealing temperature 58°C) and for exon 3 (forward 5′-AAA CAC ACA GGA AAT ATT TTA CTG TCC-3′ and reverse 5′-GAT GTC CCC TTG TCT GCT GTT C-3′; annealing temperature 48–49°C). Besides the Cryg genes, the closely linked Cryba2 (GenBank/EMBL accession number NM_021541; GenBank is provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD, and is available at http://www.ncbi.nlm.nih.gov/Genbank; EMBL is provided in the public domain by the European Molecular Biology Laboratory, Heidelberg, Germany, and is available at http://www.embl-heidelberg.de) was tested as a candidate as described recently. ¹⁸ PCR products were sequenced commercially (SequiServe, Vaterstetten, Germany) either after cloning into the pCR2.1 vector (Invitrogen, Leek, The Netherlands) or directly after elution from the agarose gel, with kits from Qiagen (Hilden, Germany) or Bio-Rad (Munich, Germany) and subsequent precipitation by ethanol and glycogen. MnlI digested PCR fragments were analyzed in a 10% polyacrylamide gel. 

General

Chemicals were from Merck (Darmstadt, Germany) or Sigma Chemical Co. (Deisenhofen, Germany). The enzymes used for cloning and reverse transcription were from Roche Diagnostics (Mannheim, Germany), and restriction enzymes were from MBI Fermentas (St. Leon-Rot, Germany), if not otherwise mentioned. 

Result

Phenotype and Lens Morphology

The only defect of the Rop mutants is the altered lens transparency. The size of the lens is not affected, and also the content of water-soluble protein remains similar in the mutants compared with the wild type. The mutation is semidominant with full penetrance and does not influence the viability and fertility of the mutants. No variation in expressivity was observed in outcrosses to C3H, 102/El, C57BL/6 strains. The cataracts are stationary and observable even macroscopically from the time, when the mice first open the eyes. The microscopic examination revealed multiple concentric hazy layers in the heterozygous mutants (Fig. 1A) and pronounced milky opacity in the homozygous mutants (Fig. 1B) . The lens cortex remains transparent. 

The histologic sections of homozygous mutants at postnatal day (P)4 revealed a dark-stained lens nucleus, which is shifted anteriorly (Fig. 2B) . In the sections of the heterozygous mutants; however, no aberrant structures were found (not shown). The dark staining in the histologic sections corresponds to the strong opacification observed in the core of the native homozygous lenses. The relatively slight hazy radial opacity of the heterozygous mutants has no counterpart in the histologic sections and may not change the morphology of the lens fibers. 

Genomic Analysis

Because allelism test of Rop revealed a close linkage to other Cat2/Cryg mutations on mouse chromosome 1, ²¹ fine mapping of the mutation was performed in 107 backcross animals using the markers D1Mit156 (position, 32.8 centimorgans [cM] from the centromere) and D1Mit181 (position, 42 cM from the centromere). We observed no recombination to the marker D1Mit156, but two recombinations to the marker D1Mit181 (calculated distance 1.9 ± 1.3 cM). Based on these data, the members of the Cryg gene cluster (position, 32 cM from the centromere) as well as Cryba2 (position, 40.8 cM from the centromere) were considered to be candidate genes for the Rop mutation. 

The Cryba2 gene and all six members of the Cryg gene cluster were amplified and sequenced. No difference was found between the wild types and the Rop mutants in the Cryga, Crygb, Crygc, Crygd, Cryge, and Cryba2 cDNA or genomic DNA. In the Cryg genes, some polymorphic sites were discovered in the Rop mutant and the wild types, as described previously. ¹⁸  

The only DNA sequence difference between wild-type C3H and mutant Rop, which leads to an alteration of the amino acid sequence, was identified in the Crygf gene (GenBank accession number, M11039) as a T→A exchange at position 113 of the cDNA (Fig. 3) . The mutation creates a new MnlI restriction site. This new MnlI restriction site was only present in the genomic DNA of the mutants (as demonstrated for four different homozygotes), but it was always absent in wild-type mice, as demonstrated for four different strains (Fig. 4) . Therefore, we concluded that this point mutation in the Crygf gene is responsible for the cataractous phenotype; the suggested new allele symbol is Crygf ^Rop . 

The deduced amino acid sequence demonstrates an alteration from Val to Glu at codon 38. A Prosite scan (provided in the public domain by the Swiss Institute of Bioinformatics, Geneva, Switzerland and available at http://www.expasy.ch/tools/scanprosite) of the mutant protein sequence revealed that an additional casein kinase II site is formed at the amino acid positions 35 to 38. The Val at position 38 is conserved among all mammalian γ-crystallins analyzed (human, mouse, rat, bovine). Moreover, the exchange of this hydrophobic, neutral Val residue at position 38 by the charged, acidic Glu leads to a clear decrease of the isoelectric point (pI) from pH 7.1 in the wild type to pH 6.7 in the mutants. If the pI is calculated only for the short decapeptide demonstrated in Figure 3 , the decrease of the pI in this microenvironment is more than 1 pH unit (from pH 5.8 to 4.4). However, the highly conserved Val residue, which occupies a buried environment in the wild type, is changed to a bulkier, charged Glu residue, suggesting the protein to be in an unfolded, precipitated stage that affects in particular the first Greek key motif, which is defined between amino acid positions 3 and 39. ²²  

Discussion

We have presented a morphologic description and a molecular characterization of a cataract mutation in the mouse. We identified a base-pair substitution within the second exon of the Crygf gene associated with the Rop mutation, representing the first mutation in this gene. Thus, mutations have now been detected in all seven Cryg genes in the mouse. The finding of a base-pair substitution after paternal procarbazine treatment is in agreement with previous characterizations of procarbazine-induced mutations in Neurospora demonstrating solely gene/point mutations that result, predominantly or exclusively, from base-pair substitutions. ²³  

The Crygf ^Rop point mutation causes an amino acid exchange of Val to Glu in the first Greek key motif. This Val is highly conserved in all mammalian γ-crystallins, and its alteration may disturb the folding characteristic of the γF-crystallin. Unfortunately, it is not possible to test specifically whether the altered protein is expressed in the lens. However, for all other mouse Cryg cataract mutations, for which the mutant gene product could be specifically assayed by Western blot analysis, there was stable expression of the corresponding protein. ^{11 17 18} Dramatic changes in the biophysical properties of the altered protein are indicated by the decrease of the overall pI of the mutant protein by approximately a half pH unit or by the decrease by 1.5 pH units in the microenvironment of the altered amino acid. 

The Crygf ^Rop mutation is the first mutation in the mouse Crygf gene, but the 10th mutation identified in the Cryg gene cluster. Thus, we have characterized the Cryga ^1Neu , Crygb ^nop , Crygc ^Chl3 , Crygd ^Aey4 , Cryge ^Aey1 , Cryge ^ns , Cryge ^nz , and Cryge ^{t 11 12 14 16 17 18} ; the Crygd ^Lop12 and the Cryge ^elo have been reported by others. ^{13 15} The Crygf ^Rop heterozygotes express a radial opacity. This resulting phenotype is unique among the numerous mutants maintained in the collection of the genetic institutes at Neuherberg. 

Currently, also in humans, an increasing number of mutations in the CRYG genes has been found to lead to cataract formation: the Coppock-like cataract and a variable zonular pulverulent cataract in the presence of the CRYGC gene and an aculeiform cataract, a punctate cataract, and a crystal cataract in the presence of CRYGD. ^{5 6 7 8 9} Moreover, a polymorphic congenital cataract has been mapped very close to the CRYGB gene. ²⁴  

From the high frequency of cataracts with different clinical phenotypes associated with mutations affecting one of the Cryg genes, it can be concluded that the Cryg gene cluster is very important for the maintenance of lens transparency. All Cryg mutations reported so far are dominant or semidominant, and all lead to structural alterations of the proteins. Neither gene deletions nor recessive mutations in the γ-crystallin encoding genes have yet been described. 

In the context of the first mouse Crygf mutation, the CRYGE and CRYGF genes in humans are pseudogenes. Among the 10 mouse mutations in the Cryg genes, a relatively high number (50%) affect the Cryge gene and one affects the Crygf gene. Therefore, specific gene functions must be elaborated for the individual members of the Cryg gene cluster. Such studies may explain the specific differences in the frequencies of the gene mutations and the loss of the CRYGE and CRYGF gene expression in humans compared with the mouse. Nevertheless, the existing data suggest that the consequences of expressed altered proteins may be more complicated (“dominant negative”) than the loss of the corresponding gene function, as indicated by the two CRYG pseudogenes in humans. 

²  Present affiliations: GSF, Institute of Epidemiology, Neuherberg, Germany; and

³  GSF, Institute of Human Genetics, Neuherberg, Germany.

 Submitted for publication March 1, 2002; revised April 29, 2002; accepted May 29, 2002.

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Figure 1.

 

Dissected lenses of 3-month-old mice. (A) Multiple concentric spheres of hazy opacities were observed in the lens core of the heterozygous Rop mutants. (B) The homogenous, relatively dense opacity in the lens core of the homozygous mutants was surrounded by a slightly opaque zone.

Figure 2.

 

Histologic sections of eyes at postnatal day 4. The eyes of 4-day-old wild-type and homozygous Rop mutants were embedded in plastic medium, sectioned, and stained with methylene blue and basic fuchsin. (A) The lens of a wild type was regularly organized. (B) In the lens of a homozygous Rop mutant, the lens core was shifted anteriorly and abnormally stained (arrows). Lens fiber cells were swollen, and there were clefts in the core of the lens. These features are considered to be responsible for the nuclear cataract. Other ocular tissues outside the lens are normal. C, cornea; L, lens; LB, lens bow; LE, lens epithelium; R, retina.

Figure 3.

 

Sequence analysis of the Rop mutant. The Crygf DNA sequence from wild-type mice was compared with that from homozygous Rop mutants. The deduced amino acid composition is demonstrated above or below the DNA sequence. The T→A exchange in exon 2 at position 113 (box) leads to a change from Val→Glu in codon 38. The MnlI recognition site is created by the mutation and is not present in the wild-type mouse.

Figure 4.

 

Crygf digest by MnlI. (A) A simplified restriction map of the 800-bp fragment covering exons 1 and 2 and their flanking regions indicates schematically the sizes of nine MnlI fragments in the wild-type mouse. Because the mutation creates a new MnlI restriction site, the fragment of 235 bp is cleaved into two smaller fragments of 90 and 145 bp. (B) An 800-bp fragment covering the promoter, exons 1 and 2, and part of intron B was amplified from genomic DNA and analyzed by polyacrylamide gel electrophoresis (10%) after digestion by MnlI. The presence of the mutation is monitored in four homozygous Crygf ^Rop mutants by the appearance of the 145-bp band (arrow). This particular band is not present in four wild-type animals from different strains (C3H/El, C57BL/6; T-Stock, or JF1). However, because of the fragments of similar sizes after MnlI digest of the Crygf PCR product, the loss of the 235-bp fragment and the appearance of the 90-bp one cannot be identified in the figure. The numbers at the left side indicate the size of the fragments in base pairs. M, marker.

The authors thank Carmen Arnhold and Erika Bürkle for expert technical assistance and Utz Linzner (National Research Center for Environment and Health [GSF], Bioinformatics Group, Institute of Experimental Genetics) for the oligonucleotides.