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|Coordinate||94,341,087 bp (GRCm38)|
|Base Change||C ⇒ T (forward strand)|
|Gene Name||a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 20|
|Chromosomal Location||94,270,163-94,465,418 bp (-)|
|MGI Phenotype||Homozygous mutants have a white belt across the back in the midtrunk region and a white belly patch that coalesces to form a white belt.|
|Amino Acid Change||Serine changed to Asparagine|
|Institutional Source||Beutler Lab|
|Gene Model||not available|
AA Change: S750N
|Predicted Effect||probably damaging
PolyPhen 2 Score 0.999 (Sensitivity: 0.14; Specificity: 0.99)
|Phenotypic Category||pigmentation, skin/coat/nails|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Live Mice, Sperm, gDNA|
|Last Updated||09/16/2016 1:07 PM by Katherine Timer|
|Record Created||03/03/2015 5:16 PM by Jeff SoRelle|
The buckeye phenotype was identified among N-ethyl-N-nitrosourea (ENU)-induced G3 mice of the pedigree R1809, some of which exhibit a variable belted phenotype (Figure 1).
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 112 mutations. Among these, only one affected a gene with known effects on pigmentation, Adamts20. The mutation in Adamts20 was presumed to be causative because the buckeye piebald spotting phenotype mimics other known alleles of Adamts20 (see MGI for a list of Adamts20 alleles). The Adamts20 mutation is a G to A transition at base pair 94,341,087 (v38) on chromosome 15, or base pair 124,341 in the GenBank genomic region NC_000081. The mutation corresponds to residue 2,510 in the mRNA sequence NM_177431 (isoform 1) within exon 16 of 39 total exons and residue 2,510 in the mRNA sequence NM_00164785 (isoform 2) within exon 16 of 28 total exons.
Genomic numbering corresponds to NC_000081. The mutated nucleotide is indicated in red. The mutation results in a serine (S) to asparagine (N) substitution at position 750 (S750N) in both Adamts20 protein variants, and is strongly predicted by Polyphen-2 to cause loss of function (score = 0.999).
ADAMTS-20 is a 1906 amino acid protein that is a member of a large family of secreted zinc-dependent metalloproteases known as the ADAMTS (a disintegrin and metalloprotease domain, with thrombospondin type-1 repeats) family (Figure 2). ADAMTS-20 has an N-terminal signal peptide and a propeptide domain which contains three conserved Cys residues and ends in a basic region (aa 246-259). Following the prodomain, ADAMTS-20 contains the zinc-coordinating catalytic sequence HELGHVFNVPHD (aa 399-410) that is closely related to sequences found in metalloproteases. The catalytic domain of ADAMTS20 is followed by a disintegrin-like domain similar in size and structure amongst all ADAMTS proteins (76 residues) and includes eight conserved Cys residues. The disintegrin domain precedes a single thrombospondin type I repeat (TSP1) which is well-conserved and similar to a domain in Thrombospondin I. The first TSP1 repeat is followed by a cysteine (Cys)-rich domain that has ten conserved Cys residues as well as a spacer characteristic of ADAMTS family members. The spacer domain contains a putative sequence for glycosaminoglycan attachment and may be important in substrate recognition and ECM association (1;2). C-terminal to the spacer region, ADAMTS proteins have a highly variable number of TSP1 repeats. The full-length ADAMTS-20 protein contains fourteen of these motifs with linker sequences between the fifth and sixth repeat and the seventh and eighth repeat. The smaller splice variant for mouse ADAMTS-20 contains 9 instead of 14 TSP1 motifs at the C-terminal domain while the human splice variant contains 11 instead of 14 TSP1 domains (1;3-5). The buckeye mutation (S750N) is within the spacer domain of both splice variants.
Please see the record for splotch2 for more information about Adamts20.
The ADAMTS proteins are predicted to interact with the extracellular matrix (ECM) and play a wide role in normal and pathological processes (1;3). White-spotting mutants are often the result of improper melanoblast development or survival. The lack of pigment in the adult reflects the absence of mature melanocytes in that area due to defects at various stages of melanocyte development including proliferation, survival, migration, invasion of the integument, hair follicle entry and melanocyte stem cell renewal (6;7). Adamts20 is the gene mutated in belted and splotch2 mutant mice. Belted animals exhibit a mostly pigmented coat except for a region near the hindlimbs that resembles a belt (8). At least fifteen different belted alleles are in existence and four of these, including splotch2, have been sequenced. Two of the three previously described mutations result in single amino acid changes either in the Cys-rich domain or one of the C-terminal TSP1 repeats. The third mutation generates a premature stop codon, resulting in truncation of the protein and loss of eight C-terminal TSP1 domains. Deficient ADAMTS-20 activity may cause loss of melanoblast entry into the hair follicles (3). ADAMTS-20 may degrade specific peptide substrates, either ECM components or signaling molecules, and this degradation could be necessary for melanoblast migration to the proper areas. Examination of ADAMTS-20 function during melanoblast development determined that mice with mutations in Adamts20 had a normal distribution of melanoblasts during embryogenesis suggesting that melanoblast migration is normal in these animals. The phenotype of the buckeye mice resembles those of other Adamts20 alleles indicating that the mutation results in impaired ADAMTS-20 function.
buckeye(F):5'- GCCAAAGAGTACTACCATGAACATTAG -3'
buckeye(R):5'- GAGCTTTGTGCAATAGTGAGC -3'
buckeye_seq(F):5'- AACTAAAAATTCTCCCCGTTATTCC -3'
buckeye_seq(R):5'- GTGCAATAGTGAGCTTATTGCAATAG -3'
1. Tang, B. L. (2001) ADAMTS: A Novel Family of Extracellular Matrix Proteases. Int J Biochem Cell Biol. 33, 33-44.
2. Somerville, R. P., Longpre, J. M., Jungers, K. A., Engle, J. M., Ross, M., Evanko, S., Wight, T. N., Leduc, R., and Apte, S. S. (2003) Characterization of ADAMTS-9 and ADAMTS-20 as a Distinct ADAMTS Subfamily Related to Caenorhabditis Elegans GON-1. J Biol Chem. 278, 9503-9513.
3. Rao, C., Foernzler, D., Loftus, S. K., Liu, S., McPherson, J. D., Jungers, K. A., Apte, S. S., Pavan, W. J., and Beier, D. R. (2003) A Defect in a Novel ADAMTS Family Member is the Cause of the Belted White-Spotting Mutation 1. Development. 130, 4665-4672.
4. Llamazares, M., Cal, S., Quesada, V., and Lopez-Otin, C. (2003) Identification and Characterization of ADAMTS-20 Defines a Novel Subfamily of Metalloproteinases-Disintegrins with Multiple Thrombospondin-1 Repeats and a Unique GON Domain 1. J Biol Chem. 278, 13382-13389.
5. Cal, S., Obaya, A. J., Llamazares, M., Garabaya, C., Quesada, V., and Lopez-Otin, C. (2002) Cloning, Expression Analysis, and Structural Characterization of Seven Novel Human ADAMTSs, a Family of Metalloproteinases with Disintegrin and Thrombospondin-1 Domains. Gene. 283, 49-62.
6. Baxter, L. L., Hou, L., Loftus, S. K., and Pavan, W. J. (2004) Spotlight on Spotted Mice: A Review of White Spotting Mouse Mutants and Associated Human Pigmentation Disorders 1. Pigment Cell Res. 17, 215-224.
7. Bennett, D. C., and Lamoreux, M. L. (2003) The Color Loci of Mice--a Genetic Century. Pigment Cell Res. 16, 333-344.
8. Murray, J. M., and Snell, G. D. (1945) Belted, A New 6Th Chromosome Mutation in the Mouse. J Hered. 36, 266-268.
|Science Writers||Anne Murray|
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