Allele | L10 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Mutation Type | missense | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Chromosome | 1 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Coordinate | 65,121,316 bp (GRCm39) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Base Change | T ⇒ A (forward strand) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gene | Crygb | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gene Name | crystallin, gamma B | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Synonym(s) | Cryg-3, DGcry-3 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Chromosomal Location | 65,119,381-65,121,449 bp (-) (GRCm39) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MGI Phenotype |
FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] Crystallins are separated into two classes: taxon-specific, or enzyme, and ubiquitous. The latter class constitutes the major proteins of vertebrate eye lens and maintains the transparency and refractive index of the lens. Since lens central fiber cells lose their nuclei during development, these crystallins are made and then retained throughout life, making them extremely stable proteins. Mammalian lens crystallins are divided into alpha, beta, and gamma families; beta and gamma crystallins are also considered as a superfamily. Alpha and beta families are further divided into acidic and basic groups. Seven protein regions exist in crystallins: four homologous motifs, a connecting peptide, and N- and C-terminal extensions. Gamma-crystallins are a homogeneous group of highly symmetrical, monomeric proteins typically lacking connecting peptides and terminal extensions. They are differentially regulated after early development. Four gamma-crystallin genes (gamma-A through gamma-D) and three pseudogenes (gamma-E, gamma-F, gamma-G) are tandemly organized in a genomic segment as a gene cluster. Whether due to aging or mutations in specific genes, gamma-crystallins have been involved in cataract formation. [provided by RefSeq, Jul 2008] PHENOTYPE: Homozygotes and heterozygotes for a spontaneous mutation exhibit cataracts characterized by nuclear and polar opacity with vacuoles and a reduction in lens weight. [provided by MGI curators] |
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Accession Number | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Mapped | Yes | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Amino Acid Change | Isoleucine changed to Phenylalanine | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Institutional Source | Beutler Lab | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gene Model | not available | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AlphaFold | P04344 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SMART Domains |
Protein: ENSMUSP00000027090 Gene: ENSMUSG00000073658 AA Change: I4F
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Predicted Effect | probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00) |
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Meta Mutation Damage Score | Not available | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Is this an essential gene? | Non Essential (E-score: 0.000) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Phenotypic Category | Autosomal Semidominant | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Candidate Explorer Status | loading ... | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Single pedigree Linkage Analysis Data |
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Penetrance | 100% | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Alleles Listed at MGI | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Lab Alleles |
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Mode of Inheritance | Autosomal Semidominant | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Local Stock | None | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MMRRC Submission | 030337-UCD | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Last Updated | 2017-03-30 4:50 PM by Katherine Timer | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Record Created | unknown | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Record Posted | 2012-01-24 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Phenotypic Description | The Clapper (L10) mutant was identified by slit-lamp examination (which detects cataracts) among G1 mice born to ENU-mutagenized sires (1). Clapper mice develop cataracts similar to human hereditary lamellar cataracts; they are observed in both heterozygous and homozygous mutant mice at the age of 3 weeks. The lenses of homozygotes show a much denser opacity than the lenses of heterozygotes, and in the former, roughly 75% of the lens is opacified with the lens periphery remaining clear. The cataracts in homozygous animals decrease in opacity with age (Figure 1).
Closer examination of Clapper heterozygous and homozygous lenses revealed that peripheral cortical fibers appear normal for both genotypes, but that aggregates are observed in the deep cortex of the lens. Disorganized, irregularly shaped, and smaller fibers as well as interfiber spaces are observed in the inner regions of both heterozygous and homozygous lenses. These defects are more severe in the homozygous mice (1). |
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Nature of Mutation | The Clapper mutation was mapped to Chromosome 1, and corresponds to an A to T transversion at position 22 of the Crygb transcript, in exon 2 of 3 total exons.
13 ATGGGAAAGATCACCTTCTTCGAGGAC
1 -M--G--K--I--T--F--F--E--D-
The mutated nucleotide is indicated in red lettering, and corresponds to the missense error I4F in the polypeptide chain (Figure 2).
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Illustration of Mutations in Gene & Protein |
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Protein Prediction |
Crygb encodes mouse γB-crystallin, a 175 amino acid protein that is a member of the β/γ superfamily of crystallin proteins that are primarily lens structural proteins (Figure 3, see L23) (2). Seven proteins make up the γ-crystallins. Human γB-crystallin also has 175 residues and is 83% identical to the mouse protein.
Unlike β-crystallins which form multimeric complexes, γ-crystallins are a homogeneous group of highly symmetrical, monomeric proteins with molecular masses around 21 kDa and typically lacking N- and C-terminal extensions. Along with the β-crystallins, the γ-crystallins contain four highly conserved homologous regions known as Greek key motifs. The Greek key motifs are able to tightly fold up, allowing these crystallins to be densely packed in the eye lens. The Cryg genes in all mammals consist of three exons: the first exon codes for three amino acids, and the subsequent two each encode two Greek key motifs (2;3).
A common tertiary structure is predicted among γ-crystallins with most of the hydrophobic residues buried in the center and charged residues located mainly on the surface (Figure 4) (1). Protein structures of the γ-crystallins are based on X-ray crystallographic studies of several γ-crystallins as well as on molecular modeling programs (2-4). Each Greek key motif contains about 40 amino acids that form four antiparallel β-strands: a,b,c, and d. Strands a, b, and d align to form three strands of a β-pleated sheet, with a fourth strand provided by the short c strand of the adjacent Greek key motif. The most characteristic feature of this structure is an unusual fold-over on the β hairpin formed by strands a and b and also involving the beginning of strand d. This structure requires certain conserved amino acids, particularly a glycine, which is the only residue able to adopt dihedral angles. The Greek key motifs are highly conserved with some conservative amino acid variability that is more pronounced in the third motif of all γ-crystallins. The fourth Greek key motif contains several predicted sites that are at the same location in most γ-crystallins: a tyrosine phosphorylation site (amino acids 147-154), an N-myristoylation site (amino acids 158-163) and a PKC phosphorylation site (amino acids 166-168) (3). The N- and C-terminal domains are covalently linked by a six- to eight-amino-acid connecting peptide, with the domain orientation determined by a patch of hydrophobic residues located at the interface between the domains (4;5). The small size and structure of γ-crystallins make them highly soluble and stable proteins. The L10 (Clapper) mutation results in the replacement of the fourth amino acid residue, isoleucine, with a phenylalanine in the first β-strand of the first Greek key motif. This amino acid substitution does not appear to alter the secondary and tertiary structure of the protein, but does alter its stability (1). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Expression/Localization | The ocular lens contains high levels of all crystallin proteins mostly in the cytoplasm of lens fibers, but the expression patterns of specific crystallin genes vary in the lens throughout embryonic development and adult life. The transcripts of α and β-crystallins (see L1N) are first detected at the early elongation stage of primary fibers during embryonic development; γ-crystallin transcripts do not appear until the late elongation phase (mid-late gestation). All areas of the lens contain crystallin mRNA until the beginning of secondary fiber formation at embryonic day 18 (E18). At that time, α- and β-crystallin mRNA is found in the equatorial part of the lens, unlike γ-crystallin transcripts which are localized in the lens core by postnatal day 1 (6). After birth, the concentration of αA-crystallin transcripts remains high until 6 months of age, while the concentration of γ-crystallin transcripts decreases gradually (6;7). Closer examination of γ-crystallin expression revealed lower levels of Crygb and Crygc mRNA and higher levels of Cryga, Crygd, Cryge, and Crygf during early stages of lens development. After 30-40 days of postnatal development, the levels of Cryga, Cryge, and Crygf mRNAs decline rapidly, and the Crygb, Crygc, and Crygd mRNAs became the major γ-crystallin transcripts in the lens (7). γB-crystallin, as assessed by RT-PCR and antibody staining, is expressed in the lens beginning on E14.5 (8).
Both mRNA and protein expression of all the Cryg genes have been detected at low levels in the retina. γ-crystallins are localized particularly to the inner retina, outer plexiform layer, and the photoreceptors during postnatal development (9).
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Background |
The ocular lens is a unique organ, able to focus light on the retina as a result of physiological properties and architecture that eliminates light scattering. Normal development of the mouse lens begins by E9.5-10 when the developing optic vesicles have approached the surface ectoderm, which begins to thicken and form the lens placode (Figure 5). At E10.5, the lens placode begins to invaginate to form the lens vesicle. At E11-13, the lens is characterized by elongation of cells from the posterior wall of the lens vesicle to form primary lens fibers, leading to obliteration of the vesicle cavity. These primary fibers are maintained throughout life and constitute the nucleus of the adult lens. The lens of the adult eye is an avascular tissue surrounded by the lens capsule, an extracellular matrix (ECM) secreted by lens cells. Beneath the capsule, a single layer of cuboidal epithelial cells covers the anterior surface and is bathed in aqueous humour. Further enlargement of the lens continues by proliferation and elongation of the epithelial cells at the lens equator to form secondary fibers, a process that continues throughout adult life. Lens fibers face the posterior chamber of the eye and are bathed in vitreous humour. Bioactive molecules such as fibroblast growth factor (FGF), present in the vitreous humour, initiate the change from lens epithelial cell to lens fiber [reviewed in (10)]. The fiber cells adopt a flattened hexagonal profile that facilitates packing into an ordered array that promotes lens transparency. Additionally, the fiber cells lose their intracellular organelles and undergo dramatic changes in the expression of cytoplasmic and membrane proteins (Figure 6). The high expression of soluble and stable cytoplasmic crystallins creates a high index of refraction. The crystallin concentration is highest in the lens nucleus and creates a radial gradient in refractive index that corrects for the curved shape of the lens (10-13). The avascular lens is maintained through a flow of ionic current that is directed inwards at the poles and outwards at the equator and allows for nutrient and waste transport and maintenance of appropriate membrane potential. Na+-K+ pumps, water channels, glucose transporters and gap junctions are all critical components of the lens microcirculatory system (13-15). Loss of transparency of the lens due to disruption of the microcirculatory system or the lens architecture is manifested macroscopically in cataracts, the most common cause of blindness in humans. Clinical descriptions of cataracts are based on the physical appearance and location of opacities within the lens. Whole cataracts affect the entire lens while nuclear cataracts affect the center of the lens and the primary lens fibers. Cortical cataracts originate in the lens cortex and lamellar cataracts are present in only one layer of the lens. The term “zonular” refers to opacities that are confined to one or more discrete zones of the lens other than the poles. The term “pulverulent” refers to powdery dustlike opacities that can be either zonular or widely dispersed throughout the lens. Other terms have also been used to describe specific cataract phenotypes including cerulean, which describes a bluish coloring, and aculeiform, which refers to needle-like crystals in the lens. For a full list of clinical cataract phenotypes, please refer to (16). Cataracts are often caused by mutations in crystallins (please see L1N, L23), channel proteins involved in the transport of water and metabolites, and connexins, the components of gap junction channels (please see L1) (2;10;14). The crystallin proteins in the lens are divided into three main families, α-, β-, and γ-crystallins with the β/γ-crystallins forming a superfamily. Crystallins appear to have evolved from preexisting proteins that were recruited to new structural roles in the lens. The α-crystallins, αA and αB, resemble heat-shock proteins and have chaperone functions, maintaining the stability and solubility of other proteins in the lens fibers (17). Theβ/γ-crystallins show similarities to bacterial spore coat protein and to Physarum species stress-induced proteins and act solely as structural protein in the lens. Additional crystallins, known as the taxon-specific crystallins, also exist. These are often structurally related to metabolic enzymes such as lactate dehydrogenase and aldose reductase (2).
The γ-crystallin family consists of seven genes. Six of these genes (Cryga--Crygf) are located in a cluster on mouse chromosome 1 or the long arm of human chromosome 2, q33-35 (18). The seventh Cryg gene (Crygs) maps to mouse chromosome 16 and human chromosome 3. In mice, mutations in all of the Cryg genes have been shown to cause mostly dominant cataracts of varying phenotypes and severity depending on the nature of the mutation (3;19). Ten of these mutations can be found in the Cryge locus and six in the Crygd gene suggesting that these two genes are hot spots for cataract mutations (3;20). Only two other cataract-causing mutations in Crygb are known aside from CrygbClapper (1;3;21). The CrygbNop mutation results in a truncated 144 amino acid γB-crystallin with six aberrant C-terminal amino acids (19). Although the truncated protein is stable, it has lost the fourth Greek key domain resulting in the formation of amyloid fibers in the nuclei of primary lens fiber cells. This process is associated with retention of nuclei in these cells (8). Very recently, the CrygbNm3062 mutation was described (21). This mutation replaces a serine at position 11 of γB-crystallin with an arginine. CrygbNm3062 animals display nuclear cataracts associated with disrupted inner lens fiber cells along with γ-crystallin aggregates and increased degradation of other crystallin proteins. The increased degradation of crystallin proteins results from an elevated calcium concentration in the mutant lenses leading to activation of calcium-dependent proteases.
Mutations in CRYG genes also result in inherited cataracts in humans. Missense mutations of CRYGC and CRYGD cause dominant cataracts (OMIM #604219) (22). Recently, another missense mutation of CRYGD was found to cause cataract-microcornea syndrome (CCMC, OMIM #116150), which is also caused by mutations in CRYAA and GJA8 (23). However, no disease-causing human mutation in the CRYGB gene has been reported.
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Putative Mechanism | The substitution of the fourth isoleucine with phenylalanine in the γB-crystallin protein lowers the stability of the protein with increasing temperature; the tertiary structure of the protein is completely disrupted at 50 oC in vitro. Clapper lenses, especially in homozygous mice, exhibit an increase in insoluble crystallins, particularly the γ-crystallins. In vivo, the mutant γB-crystallin associates with α-crystallins to form soluble complexes, an interaction that is not normally observed in wild type mice. It is hypothesized that α-crystallins recognize the inherent instability of the mutant protein and act to maintain its solubility and stability. Cataracts in the Clapper mutants are probably caused by the presence of insoluble lens structural proteins and also by light scattering caused by protein aggregates consisting of mutant γB-crystallin and α-crystallins. These animals also display a 15% reduction in the gap junction channel protein connexin 50. The distribution of this protein appears to be slightly less uniform in the lens of homozygous mutants, making it possible that the homozygous Clapper phenotypes could be caused, in part, by abnormal functioning of gap junction channels (please refer to L1) (1).
It is not understood why homozygous Clapper mutants exhibit reduced lens opacity with age, because abnormal protein aggregates and modifications that cause cataracts usually increase with age due to an accumulation of defects caused by oxidative reactions and other mechanisms (24).
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Primers | Primers cannot be located by automatic search. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Genotyping | Clapper genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.
Primers for PCR amplification
L10(F): 5’- ATCCCCTTACTCACCGAAATGGGC -3’
L10(R): 5’- CCACGTTCCTGAGAACTGAGATTCC -3’
PCR program
1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 35X
6) 72°C 3:00
7) 4°C ∞
Primers for sequencing
L10_seq(F): 5’- ATTTCCTGTGGAGGCAGC -3'
L10_seq(R): 5’- GCTCTAGTTGAAACGACTTGC -3’
The following sequence of 633 nucleotides (from Genbank genomic region NC_000067 for linear genomic sequence of Crygb plus 118 additional nucleotides from the 5’UTR of Crygb taken from NCBI m37 mouse assembly Chromosome 1: 65126801:65128959) is amplified:
-118 atcccctt actcaccgaa atgggcccct ttgtgtgatt tcctgtggag gcagcagtca
-61 tgacagctat atataccagg ggagctcccc tagagtctca cagctcccag ggcatctctt
1 actctcagcg agatgggaaa ggtaagtcct ggaaccctga cctttgcccg caagcagcat
61 ccttgctggc agaaatcact tatttgtctg gtccctttct gcgcttacag atcaccttct
121 tcgaggaccg cagcttccag ggccgctgct atgagtgcag cagcgactgc cccaacctgc
181 agacctactt cagccgctgc aattctgtcc gcgtggacag tggctgctgg atgctctatg
241 agcgccccaa ctaccagggc caccagtact tcctgagacg tggagagtac cctgactacc
301 agcagtggat gggtttcagc gactccattc gttcctgctg cctcatcccc caagtgagtt
361 tggctgtctt tattattgat ctctgggaac acaaattatc ctaaaagatc atcttaaagc
421 aagtcgtttc aactagagca aagagtgggt gtaataccta gtatgtgtat ccagtaggat
481 acctgagaga ggaatctcag ttctcaggaa cgtgg PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated A is shown in red text.
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References | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Science Writers | Nora G. Smart | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Illustrators | Victoria Webster | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Authors | Haiquan Liu, Xin Du, Xiaohua Gong, Bruce Beutler |