Phenotypic Mutation 'whisper3' (pdf version)
Allelewhisper3
Mutation Type missense
Chromosome11
Coordinate80,448,347 bp (GRCm39)
Base Change T ⇒ A (forward strand)
Gene Myo1d
Gene Name myosin ID
Synonym(s) D11Ertd9e, 9930104H07Rik
Chromosomal Location 80,372,952-80,670,851 bp (-) (GRCm39)
Accession Number

NCBI RefSeq: NM_177390; MGI:107728

MappedYes 
Amino Acid Change Aspartic acid changed to Valine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000037819]
AlphaFold Q5SYD0
SMART Domains Protein: ENSMUSP00000037819
Gene: ENSMUSG00000035441
AA Change: D926V

DomainStartEndE-ValueType
MYSc 3 696 N/A SMART
IQ 697 719 1.46e-3 SMART
Pfam:Myosin_TH1 803 1006 4.1e-49 PFAM
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000041065)
Meta Mutation Damage Score 0.9431 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All alleles(6) : Targeted(5) Gene trapped(1)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00432:Myo1d APN 11 80492566 missense probably benign
IGL01087:Myo1d APN 11 80573261 missense probably damaging 1.00
IGL01326:Myo1d APN 11 80575147 splice site probably benign
IGL01431:Myo1d APN 11 80565665 missense probably damaging 1.00
IGL01595:Myo1d APN 11 80566936 missense probably benign 0.00
IGL01811:Myo1d APN 11 80583823 missense probably damaging 0.96
IGL02301:Myo1d APN 11 80567679 missense probably benign 0.23
IGL02388:Myo1d APN 11 80528823 nonsense probably null
IGL02485:Myo1d APN 11 80557407 missense probably damaging 1.00
IGL03017:Myo1d APN 11 80492452 missense probably benign 0.26
horton UTSW 11 80565534 missense probably damaging 1.00
multifaceted UTSW 11 80583898 missense probably damaging 1.00
whisper UTSW 11 80375158 missense probably damaging 0.99
whisper2 UTSW 11 80557404 missense probably damaging 1.00
R0069:Myo1d UTSW 11 80528779 missense probably damaging 1.00
R0069:Myo1d UTSW 11 80528779 missense probably damaging 1.00
R0081:Myo1d UTSW 11 80448349 missense probably benign 0.00
R0096:Myo1d UTSW 11 80375158 missense probably damaging 0.99
R0096:Myo1d UTSW 11 80375158 missense probably damaging 0.99
R0244:Myo1d UTSW 11 80565534 missense probably damaging 1.00
R0711:Myo1d UTSW 11 80375158 missense probably damaging 0.99
R0746:Myo1d UTSW 11 80477705 missense possibly damaging 0.94
R1084:Myo1d UTSW 11 80575221 missense probably damaging 1.00
R1514:Myo1d UTSW 11 80576734 missense probably damaging 0.97
R1676:Myo1d UTSW 11 80575247 missense probably damaging 1.00
R1862:Myo1d UTSW 11 80553874 missense probably damaging 1.00
R2497:Myo1d UTSW 11 80565647 missense probably damaging 1.00
R2512:Myo1d UTSW 11 80670543 missense probably benign 0.00
R3425:Myo1d UTSW 11 80492464 missense probably benign
R3429:Myo1d UTSW 11 80573236 missense probably damaging 1.00
R3917:Myo1d UTSW 11 80557404 missense probably damaging 1.00
R3928:Myo1d UTSW 11 80375087 missense probably benign 0.09
R4706:Myo1d UTSW 11 80557467 missense probably damaging 0.96
R4723:Myo1d UTSW 11 80670667 utr 5 prime probably benign
R4924:Myo1d UTSW 11 80565504 missense probably damaging 1.00
R5042:Myo1d UTSW 11 80448347 missense probably damaging 1.00
R5320:Myo1d UTSW 11 80575149 critical splice donor site probably null
R5481:Myo1d UTSW 11 80553921 missense possibly damaging 0.79
R6214:Myo1d UTSW 11 80670617 start codon destroyed probably null 0.98
R6235:Myo1d UTSW 11 80583770 missense probably benign 0.23
R6282:Myo1d UTSW 11 80448338 missense probably damaging 0.99
R6468:Myo1d UTSW 11 80448300 missense probably benign 0.00
R6668:Myo1d UTSW 11 80474701 intron probably benign
R6954:Myo1d UTSW 11 80565783 missense probably benign 0.21
R7077:Myo1d UTSW 11 80565460 missense probably damaging 1.00
R7078:Myo1d UTSW 11 80565460 missense probably damaging 1.00
R7080:Myo1d UTSW 11 80565460 missense probably damaging 1.00
R7172:Myo1d UTSW 11 80483621 missense probably benign 0.16
R7276:Myo1d UTSW 11 80583898 missense probably damaging 1.00
R7467:Myo1d UTSW 11 80477743 missense probably damaging 1.00
R7650:Myo1d UTSW 11 80492510 missense probably benign
R7678:Myo1d UTSW 11 80567719 missense possibly damaging 0.80
R7859:Myo1d UTSW 11 80575203 missense probably damaging 1.00
R8324:Myo1d UTSW 11 80448347 missense probably damaging 1.00
R8329:Myo1d UTSW 11 80528900 missense probably benign 0.21
R8474:Myo1d UTSW 11 80561745 missense possibly damaging 0.93
R8799:Myo1d UTSW 11 80575205 missense probably damaging 1.00
R8810:Myo1d UTSW 11 80567758 missense probably benign 0.30
R8810:Myo1d UTSW 11 80565758 missense probably damaging 1.00
R8823:Myo1d UTSW 11 80492571 missense possibly damaging 0.91
R9221:Myo1d UTSW 11 80565744 missense probably damaging 1.00
R9494:Myo1d UTSW 11 80375093 missense probably benign 0.02
R9625:Myo1d UTSW 11 80448296 missense possibly damaging 0.95
R9626:Myo1d UTSW 11 80448296 missense possibly damaging 0.95
R9628:Myo1d UTSW 11 80448296 missense possibly damaging 0.95
Z1088:Myo1d UTSW 11 80565724 missense probably benign 0.01
Mode of Inheritance Autosomal Recessive
Local Stock
Repository
Last Updated 2019-09-04 9:40 PM by Anne Murray
Record Created 2017-03-27 10:43 AM
Record Posted 2018-10-11
Phenotypic Description

Figure 1. Whisper3 mice exhibited susceptibility to DSS-induced colitis at day 7 post-DSS treatment. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

Figure 2. Whisper3 mice exhibited susceptibility to DSS-induced colitis at day 10 post-DSS treatment. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The whisper3 phenotype was identified among G3 mice of the pedigree R5042, some of which showed susceptibility to dextran sulfate sodium (DSS)-induced colitis exhibiting diarrhea and rectal bleeding as well as weight loss by seven days after exposure to DSS (Figure 1); weight loss continued to progress through day 10 post-DSS treatment (Figure 2).

Nature of Mutation

Figure 3. Linkage mapping of the day 10 DSS susceptibility phenotype using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 53 mutations (X-axis) identified in the G1 male of pedigree R5042. 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 53 mutations. Both of the above anomalies were linked by continuous variable mapping to a mutation in Myo1d: an A to T transversion at base pair 80,557,521 (v38) on chromosome 11 corresponding to base pair 222,535 in the GenBank genomic region NC_000077 encoding Myo1d. The strongest association was found with a recessive model of linkage to the DSS-induced weight loss at day 10, wherein seven affected variant homozygotes departed phenotypically from 11 homozygous reference mice and 16 heterozygous mice with a P value of 8.026 x 10-5 (Figure 3).

The mutation corresponds to residue 3,010 in the mRNA sequence NM_177390 within exon 21 of 22 total exons.

2994 ACAAAAGACAACAAAGACCTCATTGTCTGCCTC

921  -T--K--D--N--K--D--L--I--V--C--L-


 

The mutated nucleotide is indicated in red.  The mutation results in an aspartic acid to valine substitution at position 926 (D926V) in the Myo1d protein, and is strongly predicted by Polyphen-2 to cause loss of function (probably damaging; score = 1.00).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 4. Domain structure of myosin 1D.  The head domain contains the actin-binding (not shown) and ATP-binding sites and generates force.  A central neck domain or light-chain binding domain contains two calmodulin binding IQ motifs.  The C-terminal half of myosin 1D consists of tail homology-1 (TH1) domain. The whisper3 mutation is an aspartic acid to valine substitution at position 926 (D926V) (red asterisk). This image is interactive. Click on the image to view another mutation found in Myo1d (red). Click on the mutations for more specific information.

Myo1d encodes myosin 1D (Myo1d; alternatively, Myr4), a member of the class I family of unconventional myosins (Figure 4). The class I myosins are molecular motors that control the mechanical properties of cell membranes by mediating membrane/cytoskeleton adhesion (1). The whisper3 mutation (D926V) is within the TH1 domain of Myo1d. The TH1 domain in class I myosins mediates myosin dimerization, targets each myosin to its subcellular location, binds directly to acidic phospholipids, and specifies the function(s) (e.g., cargo binding and enzymatic activities) of a myosin (2;3).

For more information on Myo1d, please see the record for horton.

Putative Mechanism

The apical brush border of the intestinal epithelial cells that line the small intestine is comprised of tightly packed microvilli (4). A core actin bundle and associated actin-binding proteins are essential to maintain the stability of each microvillus (4). Unconventional myosins in the intestine have been studied. Myosin-1a (Myo1a) connects the microvillar membrane to the actin bundle underneath (5-7). Myosins from classes I, II, V, VI, and VII also target to the actin-rich domain in the brush border (8;9). Myo1a has several functions within the enterocyte including the organization of apical membrane domains (10), controlling apical membrane tension (1), and the shedding of vesicles from the tips of the microvilli (11). Knockout of Myo1a (Myo1a-/-) in mice resulted in defects in the brush border membrane composition and apical membrane herniations in some enterocyte brush borders (12), but the knockout mice exhibited no noticeable physiological symptoms indicating that other myosins may compensate for Myo1a. In the wild-type microvillus, Myo1a is excluded from the distal tip compartment where Myo1d localized, indicating that Myo1a and Myo1d have different functions within the microvillus (4). In Myo1a-/- mice, the levels of Myo1d in the brush border are upregulated and redistributed along the length of the microvillus (4). The redistribution of Myo1d upon the loss of Myo1a expression indicates that Myo1d can compensate for the function of Myo1a in the brush border (4). Benesh et al. propose that Myo1d functions in the early stages of vesicle formation at the tips of the microvilli and that the redistribution of Myo1d upon loss of Myo1a expression indicates that Myo1d and Myo1a may compete for a shared binding site within the microvillus (4). The localization of Myo1d to the terminal web, a filamentous structure at the apical surface of epithelial cells that possess microvilli, indicates that Myo1d may function in the short-range transport, docking, and/or fusion of apically directed vesicles derived from the Golgi (13). The Myo1d at the tips of the microvilli may function to control actin dynamics or to transport components along the microvillar axis (4). Alternatively, Myo1d may also function in the formation and/or release of vesicles from the microvillar tips (14).

Primers PCR Primer
whisper3_pcr_F: GCCGGATGCTAATTTCACCTTC
whisper3_pcr_R: CTCCTTACTCCATGAAACTTGTTAAAG

Sequencing Primer
whisper3_seq_F: AAAGATGCTTTAATTTGTCCTTGTGG
whisper3_seq_R: GGTTAAAATCGTCACCATCGTTGC
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 433 nucleotides is amplified (chromosome 11, - strand):


1   ctccttactc catgaaactt gttaaaggtt aaaatcgtca ccatcgttgc tatggcctgc
61  tctaattcat tcttccagga gataccctaa cagcctctgt gtgttccctc cttgcagttg
121 actggtctga gtgtctccaa tggaaaggac caacttgtag tgttccatac aaaagacaac
181 aaagacctca ttgtctgcct cttcagcaaa cagccaaccc atgagagtcg tattggagaa
241 cttgttggag tcctggtgaa tcatttcaag aggtaaaatt ttatttttgt agataaaaga
301 caacttttaa atacacaaaa tgttgaatca tacttatcac taaccttggt ttgtgccaat
361 gtgaatatat tgttttcctc cacaaggaca aattaaagca tctttttcat agaaggtgaa
421 attagcatcc ggc


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

References
Science Writers Anne Murray
Illustrators Katherine Timer
AuthorsWilliam McAlpine, Emre Turer, and Bruce Beutler