Phenotypic Mutation 'ghost' (pdf version)
Alleleghost
Mutation Type missense
Chromosome7
Coordinate87,121,703 bp (GRCm39)
Base Change T ⇒ C (forward strand)
Gene Tyr
Gene Name tyrosinase
Synonym(s) skc35, Oca1
Chromosomal Location 87,073,979-87,142,637 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The enzyme encoded by this gene catalyzes the first 2 steps, and at least 1 subsequent step, in the conversion of tyrosine to melanin. The enzyme has both tyrosine hydroxylase and dopa oxidase catalytic activities, and requires copper for function. Mutations in this gene result in oculocutaneous albinism, and nonpathologic polymorphisms result in skin pigmentation variation. The human genome contains a pseudogene similar to the 3' half of this gene. [provided by RefSeq, Oct 2008]
PHENOTYPE: Numerous mutations at this locus result in albinism or hypopigmentation. Albinism is associated with reduced number of optic nerve fibers and mutants can have impaired vision. Some alleles are lethal. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_011661; Ensembl: ENSMUST00000004770; MGI: 98880

MappedYes 
Amino Acid Change Histidine changed to Arginine
Institutional SourceBeutler Lab
Gene Model not available
AlphaFold P11344
SMART Domains Protein: ENSMUSP00000004770
Gene: ENSMUSG00000004651
AA Change: H363R

DomainStartEndE-ValueType
signal peptide 1 18 N/A INTRINSIC
low complexity region 91 112 N/A INTRINSIC
Pfam:Tyrosinase 170 403 4.8e-45 PFAM
transmembrane domain 474 496 N/A INTRINSIC
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000004770)
Meta Mutation Damage Score Not available question?
Is this an essential gene? Probably nonessential (E-score: 0.194) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance 100% 
Alleles Listed at MGI

All alleles(104) : Spontaneous(26) Chemically induced(9) Radiation induced(69) Other(1)  

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01568:Tyr APN 7 87087156 missense probably damaging 1.00
IGL01594:Tyr APN 7 87133022 splice site probably benign
IGL02963:Tyr APN 7 87133205 missense probably benign
IGL03356:Tyr APN 7 87141922 missense possibly damaging 0.71
pale UTSW 7 87087175 missense probably damaging 1.00
pale_rider UTSW 7 87087231 missense probably damaging 1.00
rufus UTSW 7 87141914 missense probably damaging 1.00
shocked UTSW 7 87142330 missense probably damaging 1.00
siamese UTSW 7 87087252 missense probably damaging 0.99
Venusaur UTSW 7 87141914 missense probably damaging 1.00
waffle UTSW 7 87142429 missense possibly damaging 0.94
R0322:Tyr UTSW 7 87142125 missense probably benign 0.35
R0479:Tyr UTSW 7 87142429 missense possibly damaging 0.94
R1544:Tyr UTSW 7 87141914 missense probably damaging 1.00
R1546:Tyr UTSW 7 87087200 missense probably benign 0.02
R1606:Tyr UTSW 7 87087179 missense probably benign 0.01
R1666:Tyr UTSW 7 87142149 missense probably damaging 1.00
R2064:Tyr UTSW 7 87142051 missense probably benign 0.13
R2213:Tyr UTSW 7 87142086 missense probably damaging 1.00
R2420:Tyr UTSW 7 87078397 missense probably benign 0.17
R4013:Tyr UTSW 7 87087148 missense probably benign 0.00
R4014:Tyr UTSW 7 87087148 missense probably benign 0.00
R4015:Tyr UTSW 7 87087148 missense probably benign 0.00
R4016:Tyr UTSW 7 87087148 missense probably benign 0.00
R4202:Tyr UTSW 7 87078276 missense possibly damaging 0.92
R4205:Tyr UTSW 7 87078276 missense possibly damaging 0.92
R4206:Tyr UTSW 7 87078276 missense possibly damaging 0.92
R4361:Tyr UTSW 7 87078284 missense probably benign 0.01
R4738:Tyr UTSW 7 87141855 missense probably null 1.00
R5306:Tyr UTSW 7 87087222 missense probably damaging 1.00
R5378:Tyr UTSW 7 87121703 missense probably damaging 1.00
R5395:Tyr UTSW 7 87121698 missense probably damaging 0.98
R5782:Tyr UTSW 7 87142224 missense probably damaging 1.00
R7007:Tyr UTSW 7 87142548 missense probably benign 0.04
R7609:Tyr UTSW 7 87133092 missense probably benign 0.06
R7767:Tyr UTSW 7 87142218 missense probably benign 0.37
R7794:Tyr UTSW 7 87133028 critical splice donor site probably null
R8158:Tyr UTSW 7 87121724 missense probably damaging 0.99
R8383:Tyr UTSW 7 87133200 missense probably damaging 1.00
R8403:Tyr UTSW 7 87087175 missense probably damaging 1.00
R8544:Tyr UTSW 7 87142000 missense probably benign 0.05
R8822:Tyr UTSW 7 87142330 missense probably damaging 1.00
R8837:Tyr UTSW 7 87087223 missense probably damaging 1.00
R9492:Tyr UTSW 7 87121705 missense possibly damaging 0.63
R9492:Tyr UTSW 7 87121704 missense probably damaging 1.00
R9748:Tyr UTSW 7 87142072 missense possibly damaging 0.89
Mode of Inheritance Autosomal Recessive
Local Stock Sperm, gDNA
Repository

none

Last Updated 2016-05-13 3:09 PM by Peter Jurek
Record Created unknown
Record Posted 2008-10-10
Phenotypic Description

Ghost homozygotes exhibit albinism with white fur and red eyes.

Nature of Mutation
The ghost mutation was mapped to Chromosome 7, and results from an N-ethyl-N-nitrosourea (ENU)-induced A to G transition at position 1148 of the Tyr transcript in exon 3 of 5 total exons.
 
1133 TCTCAAAGTAGCATGCACAATGCCTTACATATC
358  -S--Q--S--S--M--H--N--A--L--H--I-
 
The mutated nucleotide is indicated in red lettering and causes a histidine to arginine substitution at residue 363 (H363R) of the tyrosinase protein.
Illustration of Mutations in
Gene & Protein
Protein Prediction
Mouse tyrosinase is a type I membrane glycoprotein with 533 amino acids. It shares 85% identity with its 529 residue human orthologue mapped to 11q14-q21 (1;2).  Tyrosinases, catechol oxidases, and hemocyanins all belong to the type 3 copper protein family.  Although they share a common active site, they exhibit different functions.  While tyrosinase and catechol oxidase are enzymes, hemocyanins are oxygen carrier proteins.  Tyrosinase is able to catalyze both the hydroxylation and oxidation steps in melanin biosynthesis (see Background), but catechol oxidases can catalyze only the oxidation of of o-diphenols to o-quinones (3;4).  Strongly related to tyrosinase are the melanogenic enzymes, the tyrosine-related protein 1 (Tyrp1) and tyrosine-related protein 2 (Tyrp2), also known as Dopachrome tautomerase (DCT) (5).
 
Figure 1. Domain structure of tyrosinase. The ghost mutation causes a histidine to arginine substitution at residue 363 of the tyrosinase protein. SP, signal peptide; EGF-like, epidermal growth factor-like laminin domain; TM, transmembrane; CT, cytoplasmic tail. Glycosylation and copper binding sites are indicated in light and dark pink, respectively. Click on the image to view other mutations found in TYR. Click on each mututation for more specific information.
The mouse and human tyrosinase-encoding genes have a similar structure, with five exons that encode a protein with an 18 amino acid N-terminal signal peptide followed by an epidermal growth factor (EGF)-like laminin domain, two copper-binding sites composing the catalytic domain, a 26 amino acid transmembrane domain near the C-terminus, and a cytoplasmic tail (Figure 1) (5-9).  The N-terminal portion of the processed protein, consisting of the EGF-like and active domains, has seven glycosylation sites, six of which are conserved between mouse and human, and 15 conserved cysteine residues (9).  The C-terminal domain has two intracellular targeting signals, a di-leucine motif and a tyrosine-based motif (10-13).  Mutations of the conserved glycosylation sites, the cysteines, or the intracellular signaling domains within the C-terminal domain cause loss of tyrosinase function by disrupting its maturation or cofactor binding (9;14-16).  Expression of the mouse gene yields predominantly a full-length mRNA, but alternative splicing produces multiple smaller mRNAs that do not encode active enzyme (6;17;18).
 
Figure 2. Crystal structure of bacterial tyrosinase in its copper-bound oxygenated form. Copper is shown as spheres. The ghost mutation is indicated. UCSF Chimera model is based on PDB 1WX4, Matoba et al. J. Biol. Chem. 281, 8981-8990 (2006). Click on the 3D structure to view other mutations found in TYR. Click again to view it rotate.

The tyrosinase active site is thought to be a hydrophobic pocket with a number of conserved aromatic residues near the copper-binding histidines (5).  At the active site of tyrosinases, one oxygen molecule binds in a side-on coordination between two copper ions, as has recently been shown for bacterial tyrosinase (19) (Figure 2; PDB ID 1WX2).  Each of the copper ions is coordinated by three histidines (His) in the protein matrix.  These His residues are located in two His-rich regions named CuA and CuB.  It is unclear whether CuA or CuB actually binds substrate, although models point to CuA, with CuB aiding in orientation and stabilization of the substrate in relation to CuA (3;4;19;20).  However, removal of only one of the copper-binding His residues results in loss of the copper ions, thereby abolishing dioxygen binding and enzyme activity (3;4;21-24).  The CuB domain is more highly conserved between tyrosinases than the CuA domain (5).  The entire active site is situated in a region of the protein that forms four α-helices (19;20).

 
The ghost mutation corresponds to an H363R substitution in tyrosinase.  This residue is highly conserved in all tyrosinases and is one of the three copper-binding histidines in CuB.  It is necessary for enzymatic activity (3;25).
Expression/Localization
The Tyr transcript is detected in a number of tissues, but the gene is expressed predominantly in skin and eyes.  The protein is localized to melanosomes, endolysosome-like organelles that store pigment. Melanosomes are found in melanocytes located in skin and hair follicles, and in the choroid layer, ciliary body, and iris of the eye. Melanosomes are also located in the pigmented retinal epithelium (9). In the melanosome, tyrosinase is located in the membrane with the C-terminus oriented toward the cytosol and the active domain located in the lumenal compartment (9;26).
Background
Figure 3. Biochemical pathway leading to the synthesis of Eumelanins.
Figure 4. Premelanosomes arise from the late secretory or endosomal pathway. Stage 1 premelanosomes (depicted here as "Early endosome/Stage I" for simplicity) lacking pigment are thought to correspond to the coated endosome, an intermediate between early and late endosomes densely coated on one face with clathrin. PMEL17, a structural component of the melanosome on which melanins are deposited (not depicted) accumulates in stage I and II; PMEL17 is masked by melanin in later stages. Melanin synthesis begins in Stage II premelanosomes that contain regular arrays of parallel fibers that give these organelles a striated appearance by electron microscopy. During Stage III, these fibers gradually darken and thicken (red arrows, inset) as eumelanin is deposited along them, such that by Stage IV no striations are visible and the melansome is filled with melanin; this action has been illustrated in the inset. All cargoes required for melanin synthesis, processing, and transport (OCA2, SLC45A2, Rab32, Rab38, DCT (alternatively, Tyrp2), Tyr, and Tyrp1) derive from the Golgi and traverse vacuolar and/or tubular elements (not shown) of early endosomes en route to the stage III melanosome. Adaptor protein-3 and -1 as well as biogeneisis of lysosome-related organelle complex-1 (BLOC-1) and BLOC-2 regulate the intracellular trafficking of Tyrp1. SLC45A2 and OCA2 function in the trafficking of Tyrp1 and DCT to melanosomes. OCA2 maintains the proper pH in the melanosomes and transports glutathione, a protein necessary for Tyr and Tyrp1 trafficking to melanosomes. Black arrows represent transport of vesicles; red arrows represent protein-mediated regulation at a specific transport step as indicated in the key. Tyr expression is regulated by the microphthalmia transcription factor (MITF). The image is interactive, click to reveal mutations in several of the proteins. Several mouse models with mutations in components of the pigment-producing pathway are shown below; the mutation name (black) and mutated gene (red) are indicated. The melanosome pathway has been adapted from several sources including: Raposo, G. and Marks, M.S. (2007), Nat. Rev. Mol. Cell Biol., 8:786 and Lakkaraju, A. et al. (2009), J. Cell Biol., 187:161.

Phenoloxidases, including tyrosinases, are found in almost all organisms and fulfill several essential biological functions.  They initiate the synthesis of melanin and are responsible for pigmentation of skin, hair and eyes, as well as browning of fruit and wound healing in plants and arthropods (Figure 3).  Due to the fungistatic and antibacterial properties of melanin and its intermediates, phenoloxidases are also key components of the primary immune response of arthropods (4;27-29).  Some in vitro studies have suggested that melanin has antiviral properties as well (30;31).
 
Melanin pigment is produced primarily in two different cell types, the neural crest-derived melanocytes found in skin, hair follicles, and the choroid, ciliary body, and iris of the eye, and the retinal pigment epithelium, a cell layer of the retina that is derived from the optic cup (32).  In the pigment cells, melanin is synthesized and deposited within melanosomes by a series of enzymatic and chemical reactions beginning with tyrosine as substrate (5).  Tyrosinase and the related enzymes, Tyrp1 and DCT, are involved in this process (4;25;33-36).  The melanogenic pathway starts with the tyrosinase-catalyzed conversion of L-tyrosine into L-dopaquinine (L-DQ).  The reaction involves two steps:  the rate-limiting hydroxylation of L-tyrosine to L-dopa (monophenolase activity of tyrosinase), and the oxidation of this intermediate o-diphenol to L-DQ (o-diphenoloxidase activity of tyrosinase).  Tyrosinase catalyzes the direct transformation of L-tyrosine into L-DQ without releasing L-dopa (37;38).  Tyrosinase is activated by its own substrates and products, tyrosine and L-dopa (9).  Other products of the melanin pathway also affect the process (9;39).  Tyrp1 (see the record for chi) and DCT are involved in later stages of the melanogenic pathway (Figure 4).  DCT catalyzes the non-decarboxylative rearrangement of dopachrome to 5,6-dihydroxyindole-2 carboxylic acid (DHICA).  In vitro, mouse Tyrp1 shows DHICA oxidase activity, catalyzing the oxidation of DHICA to the corresponding quinine (4;25;33;35;36).  There are differences in the catalytic activities between human and mouse proteins involved in this pathway.  For example, human Tyrp1 does not display DHICA oxidase activity in vitro (40), but human tyrosinase does (35).  Conversely, mouse Tyrp1 has DHICA oxidase activity, but mouse tyrosinase does not (5;33;41).  The melanin product is deposited within the melanosome as eumelanic (brown or black) or pheomelanic (yellow/red) pigment (42). 
 
Mice with tyrosinase mutations display a variety of pigmentation phenotypes that deviate from the wild-type black fur and eyes due to a reduction or a lack of melanin, (1;32;43-45).  The BALB/c mouse used extensively in laboratory research is a Tyr mutant (1;32;43;44).  The genetic defect in the tyrosinase gene affects the quantity of pigment produced within the melanosome; melanin is absent or reduced, but melanocytes (and melanosomes) are present in the skin and hair follicles.  Albino mice with tyrosinase mutations have defects in the visual projections at the optic chiasm (46), decreased numbers of rod photoreceptors (47;48), and spatiotemporal defects in neuronal production (48).  Furthermore, a role for tyrosinase in the occurrence of glaucoma by a mechanism apparently unlinked to melanin production has recently been suggested.  It is thought that the tyrosinase product L-dopa may be involved in this phenotype (49).  Often the effects of tyrosinase mutations can differ strikingly in eyes and fur.  This might be due to transfer of melanosomes from neural crest-derived melanocytes in skin and hair follicles, whereas they are retained in the retinal pigment epithelial cells and the choroidal melanocytes.  Alternatively, there might be differential regulation distinguishing optic cup-derived and neural crest-derived pigment cells (32;50-53)
 
Oculocutaneous albinism in humans is a recessive genetically heterogeneous congenital disorder characterized by decreased or absent pigmentation in the hair, skin, and eyes.  The reduction of melanin pigment in the skin and eyes results in an increased sensitivity to ultraviolet radiation and a predisposition to skin cancer (54).  The reduction of melanin pigment in the eye during development leads to foveal hypoplasia and abnormal routing of the nerve fibers from the eye to the brain, resulting in nystagmus (rapid movements of the eyes), strabismus (lazy eye), reduced visual acuity, and loss of binocular vision (54;55).  OCA is caused by mutations in several genes including tyrosinase (OCA1A, OMIM #203100 and OCA1B, OMIM #606952), Pink-eyed dilution or P (OCA2, OMIM #203200, mutated in snowflake, whitemouse, charbon, faded, and quicksilver), Tyrp1 (OCA3, OMIM #203290 or red OCA, OMIM #278400), and the solute carrier family 45, member 2 gene or Slc45a2 (OCA4, OMIM #606574, mutated in cardigan, grey goose, galak, and sweater) (54;56-59).  Tyrp1, aside from its direct role in melanin synthesis, also stabilizes tyrosinase (9;60;61), while the putative melanocyte membrane transporters P and SLC45A2 are important for proper maturation, processing and trafficking of tyrosinase to post-Golgi melanosomes (9;57;63;64).  OCA, amongst other phenotypes, is also characteristic of Hermansky-Pudlak syndrome (HPS; OMIM #203300), and Chediak-Higashi syndrome (CHS; OMIM #214500).  Mutations in genes associated with these diseases cause a more generalized defect in protein trafficking resulting in defects in lysosome-related organelles including melanosomes (please see toffee, dorian gray, pam gray, stamper-coat, minnie, bullet gray, sooty, souris, and grey wolf) (64;65).  For more information on the genes that cause these syndromes as well as non-syndromic albinism, please see the Hermansky-Pudlak Syndrome Database
 
OCA1A is characterized by a complete lack of tyrosinase activity (and pigment), while individuals with OCA1B usually have some residual pigment due to reduced activity of the enzyme. A subtype of OCA1B is caused by temperature-sensitive tyrosinase activity and is equivalent to the Himalayan mouse mutant (32;45;66).  The mutation is at amino acid 420 for mouse (a histidine) and amino acid 422 (an arginine) for human and allows pigment production in cooler areas of the body, such as the extremities.  Polymorphisms in the tyrosine gene are associated with skin and eye color variation in certain populations (67;68).  Loss of tyrosinase activity is also associated with Menkes disease (OMIM #309400), an X-linked recessive copper deficiency disorder caused by mutations in the ATP7A gene (mutated in Tigrou and Tigrou-like) (69;70).  ATP7A is a copper-transporting P-type ATPase that is localized predominantly in the trans-Golgi network, but relocates to the plasma membrane in cells exposed to elevated copper where it functions in copper efflux.  ATP7A transports copper into the secretory pathway of cells to activate copper-dependent enzymes such as tyrosinase (71).  More recently, the subcellular localization of ATP7A to melanosomes was found to be critical for tyrosinase activity, and was dependent on proteins involved in HPS (72).  Patients suffering Menkes disease have a wide variety of defects including whitish hair and skin.
Putative Mechanism
The ghost phenotype results from substitution of a metal-coordinating histidine with a basic arginine residue.  It is probable that this mutation completely abolishes tyrosinase activity due to a disruption in Cu2+ binding at the active site, as previous studies have shown that disruption of any of the copper-binding histidines in human tyrosinase leads to inactivation (25).  Indeed, most human tyrosinase mutations affecting the active site result in OCA1, which is characterized by a complete lack of pigment and enzyme activity (54).
Primers Primers cannot be located by automatic search.
Genotyping
Ghost genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transition. This protocol has not been tested.
 
Primers
ghost (F): 5’- CATATTGGCCCAGCCTATCTCACTG -3’
ghost (R): 5’- GTCCCAAGACATGGCTGTATTTGACTC -3’
 
PCR program
1) 95°C             2:00
2) 95°C             0:30
3) 56°C             0:30
4) 72°C             1:00
5) repeat steps (2-4) 29X
6) 72°C             7:00
7) 4°C              ∞
 
Primers for sequencing
ghost_seq(F): 5’- ACTGCTTAACCTATCTCACAGTG -3’
ghost_seq(R): 5’- GACATGGCTGTATTTGACTCCTAAAC -3’
 
The following sequence of 668 nucleotides (from Genbank genomic region NC_000073 for linear genomic sequence of Tyr) is amplified:
 
20621                                             catattggcc cagcctatct
20641 cactgcttaa cctatctcac agtgtacaaa acctaatatc tttcagttct gaagagatct
20701 aaatgaatat tcagaatccc aatatcaaat gagttttctt attgattttc tttacttgaa
20761 atcatgattt ttcctctgga gttgtagatt agatgagata attagtgaga ttttcaatca
20821 tttagaaaaa ctaatttttt tttaaatttc cctttatttc aacaggattt gccagtccac
20881 tcacagggat agcagatcct tctcaaagta gcatgcacaa tgccttacat atctttatga
20941 atggaacaat gtcccaagta cagggatcgg ccaacgatcc catttttctt cttcaccatg
21001 cttttgtgga caggttggtt gacatttcct cataaattat gcatttattg catttaaatg
21061 tattatcaac gtgtgtttca aatgtgactt tatcatacca attcttataa attatggtgg
21121 tttgctattt tgagagaacc taaattaatc ctaaaatttc aaatgattta ttaaagtagt
21181 atttactgaa gagaaaccag taaggtgtaa gctatgataa ggaaatagaa attttcatgt
21241 taagctacaa taaaagttta ggagtcaaat acagccatgt cttgggac
 
Primer binding sites are underlined; sequencing primers are highlighted in gray; the mutated A is indicated in red.
References
Science Writers Nora G. Smart
Illustrators Diantha La Vine
AuthorsCeline Eidenschenk, and Bruce Beutler
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