|Molecular Vision 1999;
Received 29 April 1999 | Accepted 3 September 1999 | Published 10 September 1999
Identification of a missense mutation in the [alpha]A-crystallin gene of the lop18 mouse
N. L. Hawes,1 T. H.
Roderick,1 R. S. Smith,1
J. R. Heckenlively,2,3
M. T. Davisson1
1The Jackson Laboratory, Bar Harbor, ME; 2Jules Stein Eye Institute, Harbor-UCLA Medical Center, Torrance, CA; 3Jules Stein Eye Institute, University of California Los Angeles School of Medicine, Los Angeles, CA
Correspondence to: Bo Chang, M.D., The Jackson Laboratory, Bar Harbor, ME, 04609; Phone: (207) 288-6394; FAX: (207) 288-6149; email: email@example.com
Purpose: The mouse lop18 (lens opacity 18) mutation causes a white cataract obvious at weaning age. It soon progresses to a large white nuclear cataract with mild cortical changes. The mutation maps to mouse Chromosome 17 in close linkage to the [alpha]A-crystallin (Crya) gene, which encodes one of the major vertebrate eye lens proteins. Here we report the identification of a missense mutation in the [alpha]A-crystallin gene of lop18/lop18 mutant mice.
Methods: PCR primers were designed based on the [alpha]A-crystallin gene sequence from GenBank and PCR products were sequenced.
Results: We have analysed the sequence of the [alpha]A-crystallin gene from the lop18/lop18 mouse and identified a missense mutation. This mutation is tightly associated with the cataract phenotype, as no recombination was detected in 112 meioses.
Conclusions: Our results suggest that a missense mutation in the [alpha]A-crystallin gene is responsible for the lop18/lop18 phenotype and Cryalop18 should be used as a gene symbol for the lop18 mutation.
The lens contains a set of highly expressed proteins known as the crystallins whose structural function is to provide the proper refractive index for the focusing of light .
[alpha]-crystallin is one of the major vertebrate eye lens proteins. It is usually isolated as a large water-soluble aggregate with an average molecular mass of 800 kDa. The aggregate consists of two types of subunits, [alpha]A- and [alpha]B-crystallin, comprising 173 and 175 amino acid residues, respectively. [alpha]-crystallin is the first crystallin to appear during lens differentiation in the mouse . The [alpha]A-crystallin gene codes for two polypeptides ([alpha]A2 and [alpha]ins) that are produced by alternative RNA splicing [3,4]. Expression of [alpha]A is primarily lens-specific with only small amounts of the protein detectable in other tissues such as spleen, thymus, and retina [5-7]. [alpha]B on the other hand, while most abundant in the lens, is present at significant levels in a variety of tissues such as the heart, muscle, kidney, and in most epithelial cells [8,9]. [alpha]-crystallin is a member of the small heat shock protein (sHSP) family and possesses molecular chaperone activity . A missense mutation in the [alpha]B-crystallin chaperone gene causes a desmin-related myopathy .
In mice homozygous for a targeted null mutation of the [alpha]A-crystallin gene, lenses are initially normal in structure, though smaller than wild type. They become opaque with age, with dense inclusion bodies in the central lens fiber cells that react to antibodies against [alpha]B. [alpha]B shifts into an insoluble fraction in these mutant mice, suggesting that the action of [alpha]A is to maintain solubility of [alpha]B in vivo . Further studies using this targeted null mutation show a critical role for [alpha]A in lens cell growth and a photoprotective phenotype can be conferred on lens epithelial cells in culture by the expression of [alpha]A-crystallin .
To understand the role of crystallins in lens development, we have been studying a mutant mouse carrying the spontaneous mutation lop18 (lens opacity 18) . This mutation, which is recessive and fully penetrant, causes prominent degeneration of the cortex, posterior migration of the lens epithelial nuclei, and formation of abnormal lens fibers at the posterior pole resulting in a large white nuclear cataract with mild cortical changes. Since lop18 was genetically linked to the [alpha]A-crystallin gene (Crya) on mouse Chromosome 17 [14-18], the mutant phenotype was postulated to be caused by a mutation in the [alpha]A-crystallin gene.
In this report, we describe our characterization of the [alpha]A-crystallin gene in lop18/lop18 mice and show that there is a single-nucleotide substitution in the coding region of this gene creating a missense mutation. The absence of this change in any of several wild type strains of mice and other strains with cataracts strongly suggests that this alteration is responsible for the lop18 phenotype.
Isolation of Genomic DNA
Genomic DNA from CBA/CaGnLe-lop18/lop18, CBA/CaGnLe +/+, C57BL/6J, 112 backcross mice produced by mating CBA/CaGnLe-lop18/lop18 with (CBA/CaGnLe-lop18/lop18 X C57BL/6J)F1, other inbred strains and strains with cataracts (Table 1) was isolated from tail tips by a standard phenol/chloroform extraction procedure. The DNAs were dissolved in Tris-HCl/EDTA, pH 8.0, and used directly for PCR.
The following oligonucleotides were used for amplification or direct sequencing (Crya-# indicates the base pair location in the [alpha]A-crystallin gene):
Crya-29 Forward (5' GAT GGC CTG CTA ATC TGT GC 3') and Crya-1133 Reverse (5' AGG GAA GCT AGA AAG GAG CC 3') were used to amplify a 1.1 kb DNA segment which covered 5' flanking sequence, exon 1, intron A, insert exon and partial intron B. Crya-889 Forward (5' CAG CTC ATG ACC CAT ATG TGG 3') and Crya-2308 Reverse (5' ATC CCA CTT AAT CCT GCT GC 3') were used to amplify a 1.4 kb DNA segment which covered partial intron A, insert exon, intron B, exon 2 and partial intron C. Crya-315 Forward (5' ATT CCT CCA TTC TGT GCA GG 3') and Crya-466 Reverse (5' GAA CCA AGG ATG CTG AAT GG 3') were used to detect the mutation in exon 1.
PCR was performed on the GeneAmp PCR System 9600 and was carried out in a 10 µl reaction volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% (w/v) gelatin, dATP, dCTP, dGTP, and dTTP each at 0.2 mM, 0.13 µM of each primer, 100 ng genomic DNA, and 0.25 units of AmpliTaq DNA polymerase. The PCR conditions were 94 °C for 3 min, then 35 cycles of 94 °C for 15 s, 55 °C for 2 min and 72 °C for 2 min followed by a final incubation of 72 °C for 7 min. The PCR products were electrophoresed on 3% agarose gel (FMC corporation, Rockland, ME). For Dra III digestion, genomic DNA was amplified with Primers Crya-315 and Crya-645. Dra III digestion was carried out directly in a 10-µl volume by adding 8 µl of PCR products, 1 µl of Dra III 10x buffer (Buffer H), and 2-5 units of Dra III (Boehringer Mannheim).
For direct sequencing, the PCR reaction was scaled up to 30 µl. The products were purified from 1% agarose gels using a Qiagen kit and sequenced on an ABI 373A automated sequencer. Both strands were sequenced using either the forward or reverse primers for PCR.
The [alpha]A-crystallin mutation
To determine the sequence of the [alpha]A-crystallin gene in the lop18/lop18 mouse, we first amplified the whole gene by polymerase chain reaction (PCR), with the primers Crya-29, Crya-1133, Crya-889 and Crya-2308 (Figure 1), for direct sequencing. Genomic DNAs from two lop18/lop18 mutants and a normal CBA/CaGnLe +/+ control were used as templates. Three differences were observed in the sequence obtained from the lop18/lop18 mutant (Figure 1) and two differences were observed in the sequence obtained from the +/+ control as compared to that published for the [alpha]A-crystallin gene [3,4]. The two base substitutions at nucleotide positions 264 and 273 in exon 2 were silent sequence polymorphisms that did not change the encoded amino acids and were present in both mutant and +/+ DNA (Figure 1). The important difference in exon 1 (a G to A transition at position 161) produces a missense mutation converting codon 54 arginine (CGC) to histidine (CAC), thereby creating a new Dra III restriction site (Figure 1). Digestion of amplified products of normal and lop18/lop18 DNA produces the expected Dra III restriction fragment length polymorphism (RFLP), Figure 2. No sequence difference was found in the insert exon.
To confirm the presence of the missense codon in the lop18 [alpha]A-crystallin gene, we re-examined the 112 DNAs from a previous linkage analysis  for the Dra III RFLP. We amplified with primers Crya315/Crya645 a 331-bp genomic fragment that contains one Dra III site in the lop18 [alpha]A-crystallin allele and none in the normal allele (Figure 1). Digestion of the PCR amplified products with Dra III from wild type, heterozygous and homozygous lop18 DNA revealed the predicted RFLP pattern (Figure 2). This analysis showed that there was an absolute concordance between the lop18/lop18 phenotype and the missense mutation ([chi]2 = 112, p << 0.0001) (Table 2). The RFLP pattern thus provides a tool for verification of the presence or absence of the lop18 allele in genetic analysis. We also typed 5 additional mutant mice from the lop18 strain and found the Dra III site caused by the mutation. Although 0/112 gives an estimated recombination value of 0 with upper 95% confidence limit of 0.026393, these data taken together with the creation of a missense mutation and the absence of this mutation in any of the other strains examined is strong evidence that lop18 is the mutation in the Crya gene.
Identification and distribution of the missense mutation in other inbred strains with or without other types of cataract
To determine whether the sequence alteration at position 161 was specific for the [alpha]A-crystallin gene of the lop18/lop18 mice, we analysed the corresponding genomic DNA region in 18 different inbred mouse strains and 16 other mouse strains with cataracts by RFLP analysis. No sequence alteration was found in the 18 inbred strains and 16 other strains with cataracts examined (Table 1), further arguing that the G161 substitution is the mutation underlying lop18 rather than a sequence polymorphism.
Our sequence and genetic data show that the cataract development observed in the lop18/lop18 mice is due to a missense mutation in the first exon of the [alpha]A-crystallin gene. [alpha]A-crystallin is an abundant eye lens protein in vertebrates. It is usually found as large aggregates, consisting of two types of subunits, [alpha]A and [alpha]B [1,25]. It has a structural function in the lens, contributing to the proper refractive properties and transparency of the lens . Normal visual function depends on the transparency of the lens, which must retain this optical characteristic or severe loss of acuity results.
The missense mutation caused by a G to A transition at position 161 of the [alpha]A-crystallin gene in lop18/lop18 mice converts codon 54 from CGC encoding arginine to CAC encoding histidine. Arginine (arg) and histidine (his) are basic amino acids, but their molecular weights (MW) and isoelectric points (pH) are different (arg - MW 174.20, pH 11.15; his - MW 155.16, pH 7.47). Arginine is positively charged at neutral pH while histidine can be uncharged or positively charged, depending upon its local environment. The arg residue is highly conserved, found in all [alpha]A-crystallin from all mammalian species examined as well as in chicken and frog [3,27-29]. The replacement of arg by his in [alpha]A-crystallin may change the refractive properties and transparency of this lens protein, which could explain the lens opacity in the lop18/lop18 mice.
Comparison of the eye phenotypes of the lop18/lop18 mutant and mice with the targeted disruption of the mouse [alpha]A-crystallin gene shows similarities in cataract formation. Lenses of mice that are homozygous for the disrupted [alpha]A allele develop a progressive lens opacification that becomes apparent several weeks after birth . The lop18/lop18 mouse has a white cataract obvious at weaning age that soon progresses to a large white nuclear cataract with mild cortical changes. However, although eyes of both mutants have normal gross structure, [alpha]A-/- mouse eyes and lenses are smaller than wild type controls , while the lop18/lop18 mouse has a normal sized lens and eye. The structural changes in [alpha]A-crystallin caused by the arg to his amino acid change in the lop18 mutant appear to cause only lens opacification, while lack of [alpha]A-crystallin in the mouse with the targeted disruption of the [alpha]A-crystallin gene affects lens development (smaller lens and eye) as well as causing lens opacification. Decreased lens and eye size could result from the lens actually lacking one of the crystallins, while opacification could result from failure of [alpha]A and [alpha]B to form complex aggregation due to either protein structural/conformational abnormalities (lop18) or missing protein ([alpha]A-/-). Another explanation would be that the molecular chaperone function of [alpha]A-crystallin in lens cell growth is affected in [alpha]A(-/-) mice  causing smaller lens and eye, while this molecular chaperone function is not affected in lop18 mice.
[alpha]-crystallin and small heat-shock protein (sHSP) have a conserved similar sequence of 80-100 amino acids in the C-terminal region of the sequence and numerous studies have been undertaken to determine the functional elements involved in the chaperone-like function of sHSP and [alpha]-crystallin [25,30,31]. The point mutation in codon 54 from CGC encoding arginine to CAC encoding histidine found in the lop18 mouse is not in the region identified in the structure-function analyses of [alpha]A-crystallin.
A family with autosomal dominant congenital cataract, described as congenital zonular central nuclear opacities, was identified as having a missense mutation, arginine 116 to cysteine (R116C), in the CRYAA (human homologue of [alpha]A) gene in affected individuals . Arg116 is invariant in 28 mammalian species, as well as in chicken and frog. The net charge of alpha-crystallins is strongly conserved throughout evolution. The lop18 spontaneous mutation is the first identified in the [alpha]A-crystallin gene in the mouse and provides an important mouse model for studying cataract formation.
This work was supported by the National Institutes of Health grant R01-EY07758 and The Foundation Fighting Blindness. Institutional shared services are supported by National Cancer Institute Cancer Center grant, CA-34196.
1. Wistow GJ, Piatigorsky J. Lens crystallins: the evolution and expression of proteins for a highly specialized tissue. Annu Rev Biochem 1988; 57:479-504.
2. Zwann J. The appearance of alpha-crystallin in relation to cell cycle phase in the embryonic mouse lens. Dev Biol 1983; 96:173-81.
3. King CR, Piatigorsky J. Alternative RNA splicing of the murine alpha A-crystallin gene: protein-coding information within an intron. Cell 1983; 32:707-12.
4. King CR, Piatigorsky J. Alternative splicing of alpha A-crystallin RNA. Structural and quantitative analyses of the mRNAs for the alpha A2- and alpha Ains-crystallin polypeptides. J Biol Chem 1984; 259:1822-6.
5. Kato K, Shinohara H, Goto S, Inaguma Y, Morishita R, Asano T. Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle. J Biol Chem 1992; 267:7718-25.
6. Srinivasan AN, Nagineni CN, Bhat SP. alpha A-crystallin is expressed in non-ocular tissues. J Biol Chem 1992; 267:23337-41.
7. Deretic D, Aebersold RH, Morrison HD, Papermaster DS. Alpha A- and alpha B-crystallin in the retina. Association with the post-Golgi compartment of frog retinal photoreceptors. J Biol Chem 1994; 269:16853-61.
8. Bhat SP, Nagineni CN. alpha B subunit of lens-specific protein alpha-crystallin is present in other ocular and non-ocular tissues. Biochem Biophys Res Commun 1989; 158:319-25.
9. Dubin RA, Wawrousek EF, Piatigorsky J. Expression of the murine alpha B-crystallin is not restricted to the lens. Mol Cell Biol 1989; 9:1083-91.
10. Graw J. The crystallins: proteins and diseases. Biol Chem 1997; 378:1331-48.
11. Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D, Fardeau M. A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy. Nat Genet 1998; 20:92-5.
12. Brady JP, Garland D, Duglas-Tabor Y, Robison WG Jr, Groome A, Wawrousek EF. Targeted disruption of the mouse alpha A-crystallin gene induces cataract and cytoplasmic inclusion bodies containing the small heat shock protein alpha B-crystallin. Proc Natl Acad Sci U S A 1997; 94:884-9.
13. Andley UP, Song Z, Wawrousek EF, Bassnett S. The molecular chaperone alphaA-crystallin enhances lens epithelial cell growth and resistance to UVA stress. J Biol Chem 1998; 273:31252-61.
14. Chang B, Hawes NL, Smith RS, Heckenlively JR, Davisson MT, Roderick TH. Chromosomal localization of a new mouse lens opacity gene. Genomics 1996; 36:171-3.
15. Kaye NW, Church RL, Piatigorsky J, Petrash JM, Lalley PA. Assignment of the mouse alpha A-crystallin structural gene to chromosome 17. Curr Eye Res 1985; 4:1263-8.
16. Kaye NW, Lalley PA, Petrash JM, Church RL. Regional assignment of the mouse alpha A2-crystallin gene (Crya-1) to chromosome 17A3----B by in situ hybridization. Cytogenet Cell Genet 1990; 53:95-6.
17. Skow LC, Donner ME. The locus encoding alpha A-crystallin is closely linked to H-2K on mouse chromosome 17. Genetics 1985; 110:723-32.
18. Skow LC, Nadeau JN, Ahn JC, Shin HS, Artzt K, Bennett D. Polymorphism and linkage of the alpha A-crystallin gene in t-haplotypes of the mouse. Genetics 1987; 116:107-11.
19. Taylor BA, Rowe L. Genes for serum amyloid A proteins map to Chromosome 7 in the mouse. Mol Gen Genet 1984; 195:491-9.
20. Hawes NL, Roderick TH, Chang B, Heckenlively JR. Mouse cataract models; characterization and linkage data. Invest Ophthalmol Vis Sci 1996; 37:S988.
21. Runge PE, Hawes NL, Heckenlively JR, Langley SH, Roderick TH. Autosomal dominant mouse cataract (Lop-10). Consistent differences of expression in heterozygotes. Invest Ophthalmol Vis Sci 1992; 33:3202-8.
22. Smith RS, Hawes NL, Kuhlmann SD, Heckenlively JR, Chang B, Roderick TH, Sundberg JP. Corn1: a mouse model for corneal surface disease and neovascularization. Invest Ophthalmol Vis Sci 1996; 37:397-404.
23. Kuck JF, Kuwabara T, Kuck KD. The Emory mouse cataract: an animal model for human senile cataract. Curr Eye Res 1981-82; 1:643-9.
24. Kuck JF. Late onset hereditary cataract of the emory mouse. A model for human senile cataract. Exp Eye Res 1990; 50:659-64.
25. Groenen PJ, Merck KB, de Jong WW, Bloemendal H. Structure and modifications of the junior chaperone alpha-crystallin. From lens transparency to molecular pathology. Eur J Biochem 1994; 225:1-19.
26. Tardieu A. Eye lens proteins and transparency: from light transmission theory to solution X-ray structural analysis. Annu Rev Biophys Biophys Chem 1988; 17:47-70.
27. de Jong WW, Zweers A, Versteeg M, Nuy-Terwindt EC. Primary structures of the alpha-crystallin A chains of twenty-eight mammalian species, chicken and frog. Eur J Biochem 1984; 141:131-40.
28. de Jong WW, van der Ouderaa J, Versteeg M, Groenewoud G, van Amelsvoort JM, Bloemendal H. Primary structures of the alpha-crystallin A chains of seven mammalian species. Eur J Biochem 1975; 53:237-42.
29. King CR, Shinohara T, Piatigorsky J. alpha A-crystallin messenger RNA of the mouse lens: more noncoding than coding sequences. Science 1982; 215:985-7.
30. Koteiche HA, Berengian AR, Mchaourab HS. Identification of protein folding patterns using site-directed spin labeling. Structural characterization of a beta-sheet and putative substrate binding regions in the conserved domain of alpha A-crystallin. Biochemistry 1998; 37:12681-8.
31. Berengian AR, Bova MP, Mchaourab HS. Structure and function of the conserved domain in alphaA-crystallin. Site-directed spin labeling identifies a beta-strand located near a subunit interface. Biochemistry 1997; 36:9951-7.
32. Litt M, Kramer P, LaMorticella DM, Murphey W, Lovrien EW, Weleber RG. Autosomal dominant congenital cataract associated with a missense mutation in the human alpha crystallin gene CRYAA. Hum Mol Genet 1998; 7:471-4.