Phenotypic Mutation 'wash' (pdf version)
Allelewash
Mutation Type nonsense
Chromosome15
Coordinate94,245,551 bp (GRCm39)
Base Change C ⇒ A (forward strand)
Gene Adamts20
Gene Name ADAM metallopeptidase with thrombospondin type 1 motif 20
Synonym(s) ADAMTS-20, bt
Chromosomal Location 94,166,177-94,329,966 bp (-) (GRCm39)
MGI Phenotype FUNCTION: This gene encodes a member of "a disintegrin and metalloproteinase with thrombospondin motifs" (ADAMTS) family of multi-domain matrix-associated metalloendopeptidases that have diverse roles in tissue morphogenesis and pathophysiological remodeling, in inflammation and in vascular biology. The encoded preproprotein undergoes proteolytic processing to generate an active protease. Certain mutations in this gene cause defective development of neural crest-derived melanoblasts resulting in a "belted" phenotype that is characterized by white spots in the lumbar region. [provided by RefSeq, Jul 2016]
PHENOTYPE: Mice homozygous for spontaneous or ENU-induced mutations exhibit abnormal coat/hair pigmentation, including a typical white belt phenotype. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_177431, NM_00164785, NM_00164786; MGI:2660628

MappedYes 
Amino Acid Change Glutamic Acid changed to Stop codon
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000036330] [ENSMUSP00000121696]
AlphaFold P59511
SMART Domains Protein: ENSMUSP00000036330
Gene: ENSMUSG00000022449
AA Change: E584*

DomainStartEndE-ValueType
signal peptide 1 19 N/A INTRINSIC
Pfam:Pep_M12B_propep 41 186 1.4e-30 PFAM
Pfam:Reprolysin_5 253 445 3.6e-13 PFAM
Pfam:Reprolysin_4 253 460 1.1e-7 PFAM
Pfam:Reprolysin 255 464 1.5e-26 PFAM
Pfam:Reprolysin_2 272 454 1.8e-10 PFAM
Pfam:Reprolysin_3 276 410 5.8e-10 PFAM
TSP1 556 608 7.73e-11 SMART
Pfam:ADAM_spacer1 718 836 2.6e-34 PFAM
TSP1 846 901 1.47e-1 SMART
TSP1 904 958 2.83e0 SMART
TSP1 965 1019 4.28e-4 SMART
TSP1 1020 1074 1.89e-5 SMART
TSP1 1075 1131 4.87e-8 SMART
TSP1 1152 1201 6.05e-4 SMART
TSP1 1204 1260 1.22e-8 SMART
TSP1 1304 1356 1.37e-2 SMART
TSP1 1357 1411 6e-8 SMART
TSP1 1416 1470 1.69e-2 SMART
TSP1 1471 1526 2.3e0 SMART
TSP1 1530 1579 1.23e0 SMART
TSP1 1653 1706 5.27e-4 SMART
Pfam:GON 1708 1905 5.8e-80 PFAM
Predicted Effect probably null
SMART Domains Protein: ENSMUSP00000121696
Gene: ENSMUSG00000022449
AA Change: E584*

DomainStartEndE-ValueType
signal peptide 1 19 N/A INTRINSIC
Pfam:Pep_M12B_propep 40 186 1e-31 PFAM
Pfam:Reprolysin_5 253 445 2.7e-13 PFAM
Pfam:Reprolysin_4 253 460 7.2e-8 PFAM
Pfam:Reprolysin 255 464 2.4e-28 PFAM
Pfam:Reprolysin_2 272 454 4e-10 PFAM
Pfam:Reprolysin_3 276 410 1.1e-10 PFAM
TSP1 556 608 7.73e-11 SMART
Pfam:ADAM_spacer1 718 836 2e-34 PFAM
TSP1 846 901 1.47e-1 SMART
TSP1 904 958 2.83e0 SMART
TSP1 965 1019 4.28e-4 SMART
TSP1 1020 1074 1.89e-5 SMART
TSP1 1075 1131 4.87e-8 SMART
TSP1 1152 1201 6.05e-4 SMART
TSP1 1204 1260 1.22e-8 SMART
TSP1 1304 1356 1.37e-2 SMART
TSP1 1357 1411 6e-8 SMART
Predicted Effect probably null
Meta Mutation Damage Score 0.9755 question?
Is this an essential gene? Probably nonessential (E-score: 0.233) question?
Phenotypic Category Unknown
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(26) : Chemically induced (ENU)(10) Chemically induced (other)(1) Radiation induced(2) Spontaneous(12) Targeted(1)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00433:Adamts20 APN 15 94292522 missense probably benign
IGL00491:Adamts20 APN 15 94171113 missense possibly damaging 0.89
IGL00502:Adamts20 APN 15 94301278 missense probably damaging 0.99
IGL00672:Adamts20 APN 15 94238986 missense probably damaging 0.99
IGL00840:Adamts20 APN 15 94180363 missense probably damaging 1.00
IGL00909:Adamts20 APN 15 94277694 missense probably damaging 1.00
IGL01101:Adamts20 APN 15 94241923 missense probably damaging 1.00
IGL01137:Adamts20 APN 15 94292492 critical splice donor site probably null
IGL01457:Adamts20 APN 15 94229329 missense probably damaging 0.97
IGL01685:Adamts20 APN 15 94301327 missense possibly damaging 0.81
IGL01949:Adamts20 APN 15 94223987 missense probably benign 0.08
IGL02525:Adamts20 APN 15 94180959 splice site probably null
IGL03088:Adamts20 APN 15 94227795 critical splice donor site probably null
IGL03175:Adamts20 APN 15 94171136 nonsense probably null
belt UTSW 15 94243871 missense probably damaging 1.00
buckeye UTSW 15 94238968 missense probably damaging 1.00
jack_white UTSW 15 unclassified
meowth UTSW 15 94229339 missense probably damaging 1.00
nidoking UTSW 15 94301326 missense probably damaging 1.00
panda UTSW 15 94224580 intron probably benign
pikachu UTSW 15 94243871 missense probably damaging 1.00
poliwag UTSW 15 94292503 nonsense probably null
splotch2 UTSW 15 94233442 intron probably benign
whitebelly UTSW 15 unclassified
whopper UTSW 15 94245691 missense probably damaging 1.00
R0483:Adamts20 UTSW 15 94251452 missense probably benign 0.00
R0514:Adamts20 UTSW 15 94168257 missense probably damaging 1.00
R0568:Adamts20 UTSW 15 94189594 splice site probably benign
R0730:Adamts20 UTSW 15 94245571 missense probably benign 0.00
R0973:Adamts20 UTSW 15 94184252 missense probably benign 0.00
R1339:Adamts20 UTSW 15 94220777 missense probably benign 0.19
R1721:Adamts20 UTSW 15 94236340 missense probably benign 0.44
R1809:Adamts20 UTSW 15 94238968 missense probably damaging 1.00
R1832:Adamts20 UTSW 15 94184225 missense probably benign 0.00
R1846:Adamts20 UTSW 15 94243871 missense probably damaging 1.00
R1867:Adamts20 UTSW 15 94236340 missense probably benign 0.44
R1875:Adamts20 UTSW 15 94229277 missense probably benign 0.01
R1930:Adamts20 UTSW 15 94301891 missense probably benign 0.03
R1931:Adamts20 UTSW 15 94301891 missense probably benign 0.03
R1932:Adamts20 UTSW 15 94301891 missense probably benign 0.03
R2001:Adamts20 UTSW 15 94245599 missense possibly damaging 0.96
R2116:Adamts20 UTSW 15 94253243 missense probably damaging 1.00
R2162:Adamts20 UTSW 15 94229339 missense probably damaging 1.00
R2350:Adamts20 UTSW 15 94181797 missense probably damaging 1.00
R2887:Adamts20 UTSW 15 94228459 missense probably benign 0.00
R2889:Adamts20 UTSW 15 94228459 missense probably benign 0.00
R2890:Adamts20 UTSW 15 94228459 missense probably benign 0.00
R3109:Adamts20 UTSW 15 94243785 splice site probably benign
R3719:Adamts20 UTSW 15 94259719 missense probably damaging 0.99
R3832:Adamts20 UTSW 15 94229339 missense probably damaging 1.00
R3901:Adamts20 UTSW 15 94226726 missense possibly damaging 0.81
R4398:Adamts20 UTSW 15 94231576 missense possibly damaging 0.93
R4402:Adamts20 UTSW 15 94277827 missense probably benign
R4431:Adamts20 UTSW 15 94241924 missense probably damaging 1.00
R4479:Adamts20 UTSW 15 94301326 missense probably damaging 1.00
R4482:Adamts20 UTSW 15 94243801 missense probably damaging 1.00
R4503:Adamts20 UTSW 15 94277631 missense probably damaging 0.99
R4671:Adamts20 UTSW 15 94301206 missense possibly damaging 0.48
R4700:Adamts20 UTSW 15 94292503 nonsense probably null
R4707:Adamts20 UTSW 15 94231528 missense possibly damaging 0.53
R4725:Adamts20 UTSW 15 94249643 missense probably damaging 0.99
R4771:Adamts20 UTSW 15 94249516 splice site probably null
R4829:Adamts20 UTSW 15 94224277 missense probably benign 0.01
R4937:Adamts20 UTSW 15 94277656 missense probably benign
R4960:Adamts20 UTSW 15 94277655 missense probably benign
R5270:Adamts20 UTSW 15 94180400 missense probably benign 0.00
R5388:Adamts20 UTSW 15 94243659 missense possibly damaging 0.81
R5410:Adamts20 UTSW 15 94179838 missense possibly damaging 0.94
R5453:Adamts20 UTSW 15 94223969 missense possibly damaging 0.69
R5611:Adamts20 UTSW 15 94171161 missense possibly damaging 0.65
R5687:Adamts20 UTSW 15 94223852 missense probably benign 0.36
R5758:Adamts20 UTSW 15 94292531 missense probably benign 0.00
R5801:Adamts20 UTSW 15 94245551 nonsense probably null
R5834:Adamts20 UTSW 15 94251465 missense probably damaging 0.99
R5993:Adamts20 UTSW 15 94236604 missense probably damaging 0.99
R5997:Adamts20 UTSW 15 94277628 missense probably damaging 1.00
R6044:Adamts20 UTSW 15 94180364 missense probably damaging 1.00
R6058:Adamts20 UTSW 15 94227928 nonsense probably null
R6217:Adamts20 UTSW 15 94236596 missense probably benign 0.00
R6283:Adamts20 UTSW 15 94249602 missense probably benign
R6354:Adamts20 UTSW 15 94245691 missense probably damaging 1.00
R6415:Adamts20 UTSW 15 94222540 critical splice donor site probably null
R6419:Adamts20 UTSW 15 94231556 missense possibly damaging 0.84
R6476:Adamts20 UTSW 15 94259691 missense probably benign 0.22
R6485:Adamts20 UTSW 15 94241852 missense probably benign 0.17
R6517:Adamts20 UTSW 15 94180985 splice site probably null
R6675:Adamts20 UTSW 15 94229197 critical splice donor site probably null
R6863:Adamts20 UTSW 15 94277627 nonsense probably null
R7186:Adamts20 UTSW 15 94220689 missense possibly damaging 0.76
R7263:Adamts20 UTSW 15 94220772 missense possibly damaging 0.52
R7441:Adamts20 UTSW 15 94251554 missense probably damaging 1.00
R7519:Adamts20 UTSW 15 94223869 missense possibly damaging 0.64
R7747:Adamts20 UTSW 15 94189468 nonsense probably null
R7770:Adamts20 UTSW 15 94231579 missense probably benign 0.02
R7816:Adamts20 UTSW 15 94220725 missense probably benign 0.00
R7827:Adamts20 UTSW 15 94223814 missense probably damaging 1.00
R7853:Adamts20 UTSW 15 94243871 missense probably damaging 1.00
R7894:Adamts20 UTSW 15 94249641 missense probably damaging 1.00
R7951:Adamts20 UTSW 15 94238947 missense probably damaging 1.00
R8233:Adamts20 UTSW 15 94189533 missense probably benign 0.19
R8458:Adamts20 UTSW 15 94251521 missense probably benign 0.02
R8709:Adamts20 UTSW 15 94238947 missense probably damaging 1.00
R8719:Adamts20 UTSW 15 94241903 missense probably damaging 0.99
R8728:Adamts20 UTSW 15 94229281 missense probably benign 0.00
R8787:Adamts20 UTSW 15 94184294 missense possibly damaging 0.83
R8801:Adamts20 UTSW 15 94258490 missense probably damaging 1.00
R9055:Adamts20 UTSW 15 94181867 missense probably damaging 0.98
R9069:Adamts20 UTSW 15 94236349 missense probably benign 0.00
R9297:Adamts20 UTSW 15 94301321 missense possibly damaging 0.88
R9318:Adamts20 UTSW 15 94301321 missense possibly damaging 0.88
R9362:Adamts20 UTSW 15 94236626 missense possibly damaging 0.86
R9658:Adamts20 UTSW 15 94249626 missense probably damaging 1.00
R9747:Adamts20 UTSW 15 94180943 missense probably damaging 1.00
R9769:Adamts20 UTSW 15 94251459 missense probably damaging 1.00
R9795:Adamts20 UTSW 15 94301180 missense possibly damaging 0.78
Mode of Inheritance Unknown
Local Stock Live Mice
Repository
Last Updated 2021-10-21 7:36 AM by Diantha La Vine
Record Created 2017-10-24 7:01 AM by Carlos Reyna
Record Posted 2018-03-13
Phenotypic Description
Figure 1. The wash mice exhibit a variable belted phenotype.

The wash phenotype was identified among N-ethyl-N-nitrosourea (ENU)-induced G3 mice of the pedigree R5801, some of which exhibit a variable belted phenotype (Figure 1).

Nature of Mutation

Figure 2. Linkage mapping of the pigmentation phenotype using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 61 mutations (X-axis) identified in the G1 male of pedigree R5801. Normalized phenotype data are shown for single locus linkage analysis without consideration of G2 dam identity. Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 61 mutations. The pigmentation phenotype was linked to a mutation in Adamts20: a G to T transversion at base pair 94,347,670 (v38) on chromosome 15, or base pair 84,405 in the GenBank genomic region NC_000081. Linkage was found with a recessive model of inheritance (P = 9.848 x 10-7), wherein 6 affected mice were homozygous for the variant allele, and 27 unaffected mice were either heterozygous (N = 17) or homozygous for the reference allele (N = 10) (Figure 2).

The mutation corresponds to residue 2,011 in the mRNA sequence NM_177431 (isoform 1) within exon 12 of 39 total exons.

1996 CTCTGTGACCGGCCCGAGCCACGAAACGGAGGA
579  -L--C--D--R--P--E--P--R--N--G--G-
 

The mutated nucleotide is indicated in red. The mutation results in substitution of glutamic acid 584 for a premature stop codon (E584*) in both Adamts20 protein variants.

Illustration of Mutations in
Gene & Protein
Protein Prediction

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 3). 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 wash mutation results in substitution of glutamic acid 584 for a premature stop codon (E584*); amino acid 584 is within the TSP1 domain.

Please see the record for splotch2 for more information about Adamts20.

Putative Mechanism

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 wash mice resembles those of other Adamts20 alleles indicating that the mutation results in impaired ADAMTS-20 function.

Primers PCR Primer
wash_pcr_F: AGCTGCCTCCGTTCTAACTG
wash_pcr_R: GTGACCGTGATGTTTTAACTACAG

Sequencing Primer
wash_seq_F: GCCTCCGTTCTAACTGAGAAATTTAG
wash_seq_R: GAGACACAAGCCATTCCTGTGTG
Genotyping

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) 40x
6) 72°C 10:00
7) 4°C hold

The following sequence of 401 nucleotides is amplified (chromosome 15, - strand):

1   gtgaccgtga tgttttaact acagagacac aagccattcc tgtgtgtgtg cttcttctgt
61  tcctagcact gccaccgtgg gctgtgtgtg accagggaca tggagacccg tcctgtggat
121 ggcgagtggg gaccatgggg accctacagc tcgtgttcaa ggacatgcgg aggtggaatc
181 aagagcacag cccggctctg tgaccggccc gagtacgtct tcttattcac tgtaaattaa
241 gattgccccc aagcaccaga aagtcaaaat cgcttccgct cacttccgga gctgggggtc
301 agtctgcgag tttcagagat tcggaatccg cctggaagaa tttcatttgt aggttcagac
361 tttttaaaaa tctaaatttc tcagttagaa cggaggcagc t

Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.

References
1. Tang, B. L. (2001) ADAMTS: A Novel Family of Extracellular Matrix Proteases. Int J Biochem Cell Biol. 33, 33-44.
  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
Illustrators Diantha La Vine
AuthorsCarlos Reyna, Jamie Russell, and Bruce Beutler