Phenotypic Mutation 'Typhoon'

• Allele: Typhoon
• Mutation Type: critical splice donor site
• Chromosome: 11
• Coordinate: 60,487,425 bp (GRCm38)
• Base Change: G ⇒ A (forward strand)
• Gene: Myo15
• Gene Name: myosin XV
• Synonym(s): sh2; sh-2; Myo15a
• Chromosomal Location: 60,469,339-60,528,369 bp (+)
• MGI Phenotype: FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes an unconventional myosin. This protein differs from other myosins in that it has a long N-terminal extension preceding the conserved motor domain. Studies in mice suggest that this protein is necessary for actin organization in the hair cells of the cochlea. Mutations in this gene have been associated with profound, congenital, neurosensory, nonsyndromal deafness. This gene is located within the Smith-Magenis syndrome region on chromosome 17. Read-through transcripts containing an upstream gene and this gene have been identified, but they are not thought to encode a fusion protein. Several alternatively spliced transcript variants have been described, but their full length sequences have not been determined. [provided by RefSeq, Jul 2008] ; PHENOTYPE: Mutations in this gene result in profound deafness and neurological behavior. [provided by MGI curators]
• Mapped: Yes
• Institutional Source: Beutler Lab
• Gene Model: predicted gene model for protein(s): [ENSMUSP00000071777 ^†] [ENSMUSP00000080507 ^†] [ENSMUSP00000091686 ^†] † probably from a misspliced transcript
• AlphaFold: no structure available at present
• SMART Domains: Protein: ENSMUSP00000071777; Gene: ENSMUSG00000042678

Domain	Start	End	E-Value	Type
low complexity region	4	24	N/A	INTRINSIC
low complexity region	87	100	N/A	INTRINSIC
low complexity region	107	120	N/A	INTRINSIC
low complexity region	269	292	N/A	INTRINSIC
low complexity region	295	306	N/A	INTRINSIC
low complexity region	311	325	N/A	INTRINSIC
low complexity region	349	384	N/A	INTRINSIC
low complexity region	425	435	N/A	INTRINSIC
low complexity region	487	498	N/A	INTRINSIC
low complexity region	502	509	N/A	INTRINSIC
low complexity region	653	681	N/A	INTRINSIC
low complexity region	692	705	N/A	INTRINSIC
low complexity region	737	747	N/A	INTRINSIC
low complexity region	758	775	N/A	INTRINSIC
low complexity region	781	792	N/A	INTRINSIC
low complexity region	796	809	N/A	INTRINSIC
low complexity region	825	849	N/A	INTRINSIC
low complexity region	883	897	N/A	INTRINSIC
low complexity region	1067	1082	N/A	INTRINSIC
low complexity region	1115	1130	N/A	INTRINSIC
MYSc	1200	1884	N/A	SMART
IQ	1885	1907	1.63e-1	SMART
IQ	1908	1930	1.77e-2	SMART
IQ	1931	1953	2.97e2	SMART
low complexity region	1955	1974	N/A	INTRINSIC
low complexity region	1992	2006	N/A	INTRINSIC
MyTH4	2049	2195	1.8e-42	SMART
low complexity region	2396	2405	N/A	INTRINSIC
low complexity region	2451	2461	N/A	INTRINSIC
Blast:MYSc	2665	2848	2e-14	BLAST
SH3	2851	2933	1.55e-4	SMART
low complexity region	2949	2962	N/A	INTRINSIC
MyTH4	3031	3185	5.59e-48	SMART
B41	3188	3400	6.94e-3	SMART

• Predicted Effect: probably null
• SMART Domains: Protein: ENSMUSP00000080507; Gene: ENSMUSG00000042678

Domain	Start	End	E-Value	Type
MYSc	13	697	N/A	SMART
IQ	698	720	1.63e-1	SMART
IQ	721	743	1.77e-2	SMART
IQ	744	766	2.97e2	SMART
low complexity region	787	801	N/A	INTRINSIC
MyTH4	844	990	1.8e-42	SMART
low complexity region	1191	1200	N/A	INTRINSIC
low complexity region	1246	1256	N/A	INTRINSIC
Blast:MYSc	1460	1643	7e-15	BLAST
SH3	1646	1728	1.55e-4	SMART
low complexity region	1744	1757	N/A	INTRINSIC
MyTH4	1826	1980	5.59e-48	SMART
B41	1983	2195	6.94e-3	SMART

• Predicted Effect: probably null
• SMART Domains: Protein: ENSMUSP00000091686; Gene: ENSMUSG00000042678

Domain	Start	End	E-Value	Type
low complexity region	4	24	N/A	INTRINSIC
low complexity region	87	100	N/A	INTRINSIC
low complexity region	107	120	N/A	INTRINSIC
low complexity region	269	292	N/A	INTRINSIC
low complexity region	295	306	N/A	INTRINSIC
low complexity region	311	325	N/A	INTRINSIC
low complexity region	349	384	N/A	INTRINSIC
low complexity region	425	435	N/A	INTRINSIC
low complexity region	487	498	N/A	INTRINSIC
low complexity region	502	509	N/A	INTRINSIC
low complexity region	653	681	N/A	INTRINSIC
low complexity region	692	705	N/A	INTRINSIC
low complexity region	737	747	N/A	INTRINSIC
low complexity region	758	775	N/A	INTRINSIC
low complexity region	781	792	N/A	INTRINSIC
low complexity region	796	809	N/A	INTRINSIC
low complexity region	825	849	N/A	INTRINSIC
low complexity region	883	897	N/A	INTRINSIC
low complexity region	1067	1082	N/A	INTRINSIC
low complexity region	1115	1130	N/A	INTRINSIC
MYSc	1200	1884	N/A	SMART
IQ	1885	1907	1.63e-1	SMART
IQ	1908	1930	1.77e-2	SMART
IQ	1931	1953	2.97e2	SMART
low complexity region	1974	1988	N/A	INTRINSIC
MyTH4	2031	2177	1.8e-42	SMART
low complexity region	2378	2387	N/A	INTRINSIC
low complexity region	2433	2443	N/A	INTRINSIC
Blast:MYSc	2647	2830	2e-14	BLAST
SH3	2833	2915	1.55e-4	SMART
low complexity region	2931	2944	N/A	INTRINSIC
MyTH4	3013	3167	5.59e-48	SMART
B41	3170	3382	6.94e-3	SMART

• Predicted Effect: probably null
• SMART Domains: Protein: ENSMUSP00000120839; Gene: ENSMUSG00000042678

Domain	Start	End	E-Value	Type
MYSc	34	716	N/A	SMART
IQ	717	739	1.63e-1	SMART
IQ	740	762	1.77e-2	SMART
IQ	763	785	2.97e2	SMART
low complexity region	806	820	N/A	INTRINSIC
MyTH4	863	1009	1.8e-42	SMART
low complexity region	1210	1219	N/A	INTRINSIC
low complexity region	1265	1275	N/A	INTRINSIC
Blast:MYSc	1479	1662	5e-15	BLAST
SH3	1665	1747	1.55e-4	SMART
low complexity region	1763	1776	N/A	INTRINSIC

• Predicted Effect: probably null
• Meta Mutation Damage Score: 0.9496
• Is this an essential gene?: Non Essential (E-score: 0.000)
• Phenotypic Category: Autosomal Recessive
• Mode of Inheritance: Autosomal Recessive
• Local Stock: Live Mice
• Last Updated: 2019-10-23 1:57 PM by Anne Murray
• Record Created: 2017-02-16 8:48 AM by Jamie Russell
• Record Posted: 2017-03-03
• Science Writers: Anne Murray
• Illustrators: Katherine Timer
• Authors: Lauren Prince, Jamie Russell, and Bruce Beutler

Accession Number

NCBI RefSeq: NM_010862, NM_182698, NM_001103171; MGI:1261811

Alleles Listed at MGI

All alleles(13) : Targeted(6) Spontaneous(2) Chemically induced (ENU)(3) Chemically induced (other) (1) Radiation induced(1)

Phenotypic Description

Figure 1. The typhoon mice exhibit ataxia and head tilt.

The typhoon phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R5051, some of which showed ataxia and a head tilt (Figure 1). Some mice also ran in circles.

Nature of Mutation

Figure 2. Linkage mapping of the behavioral phenotypes using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 54 mutations (X-axis) identified in the G1 male of pedigree R5051. 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 54 mutations. The behavioral phenotypes were linked to two genes: Nlrp3 and Myo15. However, the mutation in Myo15 was presumed to be causative because the typhoon behavior phenotypes mimic other known alleles of Myo15 (see MGI for a list of Myo15 alleles as well as the entry for parker). The mutation in Myo15 a G to A transition at base pair 60,487,425(v38) on chromosome 11, or base pair 113,685 in the GenBank genomic region NC_000077 encoding Myo15; the mutation is within the donor splice site of intron 10.  Linkage was found with a recessive model of inheritance (P = 1.981 x 10^-5), wherein four variant homozygotes departed phenotypically from 20 homozygous reference mice and 18 heterozygous mice with (Figure 2).  A substantial semidominant effect was also observed (P = 4.274 x 10^-5). 

 

The effect of the mutation at the cDNA and protein level have not examined, but the mutation is predicted to result in the use of a cryptic site in intron 10, resulting in a transcript that has an 8-base pair insertion on intron 10. This predicts a frame shifted protein product beginning after amino acid 1,386 of the protein, which is normally 3,511 amino acids in length, and terminating after the inclusion of 23 aberrant amino acids.

<--exon 9  <--exon 10 intron 10--> <--exon 11--> 17906 ……TTTCTAGAAGG ……ATTGTGTTCCAG gtaggcaggtcagag…… GCCAAAAATGAAA……TCCCTGCTCAACTAA…… 18927 1362 ……-F--L--E--G ……-I--V--F--Q-                   --P--K--M--K-……-S--L--L--N--*-                  correct                                       aberrant

The donor splice site of intron 10, which is destroyed by the typhoon mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red.

Protein Prediction

Figure 3. Domain structure of myosin XV. At its N-terminus, myosin XV has an N-terminal extension (NTE); isoform 3 does not have the NTE. Within the motor domain, myosin XV has an ATP- and actin-binding site. Two IQ motifs are located within the neck of myosin XV. The C-terminal tail contains two myosin tail homology 4 (MyTH4) domains, a Src homology 3 (SH3) domain, and a 4.1/ezrin/radixin/moesin (FERM) domain. A FERM-like domain is predicted based on sequence similarity to one in human myosin VIIa, however, the precise location in mouse myosin XV has not been documented. At the C-terminus, myosin XV has a PDZ-ligand motif (ITLL*). The typhoon mutation (red asterisk) is located in the donor splice site of intron 10. See the text for more details.

Myo15 encodes myosin XV (alternatively, Myosin XVa), a 3,511 amino acid member of the unconventional myosin family. Myosin XV has a proline-rich N-terminal extension that does not have sequence similarity to reported proteins and the function is unknown [Figure 3; amino acids 1-1200; SMART; (1-3)]. Myosin XV has a highly conserved motor domain (amino acids 1200-1884; NM_010862; SMART) following the N-terminal extension (2). The motor domain contains an adenosine triphosphate (ATP)- and an actin-binding site (amino acids 1299-1306 and 1776-1783, respectively; Uniprot) (4-6). The myosin neck region contains a variable number of light-chain binding (IQ) motifs (IQxxxRGxxxRK) and is linked to the motor domain by a converter region [(7); reviewed in (8;9)]. Myosin XV has two IQ motifs (amino acids 1909-1920; LQRCLRGFFIKR and amino acids 1932-1943; LQSRARGYLARQ) (5;8;9). The IQ motif is an α-helical structure that often mediates the binding of myosins to calmodulin, members of the EF-hand family of calcium-binding proteins, or myosin light chains [reviewed in (8;9)]. The myosin XV tail region is 1584 amino acids in length and has two myosin tail homology 4 (MyTH4) domains (amino acids 2049-2195 and 3031-3185; SMART), a band 4.1/ezrin/radixin/moesin (FERM)-like domain (amino acids 2687-2867, human myosin XVa), a Src homology 3 (SH3) domain (amino acids 2851-2933), and a FERM domain (alternatively, talin-like domain; amino acids 3188-3400) (1;2;10;11). The MyTH4 domain is proposed to function in microtubule binding as well as in actin binding to the plasma membrane (12). The FERM domain of myosin XV is proposed to be involved in anchoring myosin XV to the cell membrane (1). The function of the myosin XV SH3 domain is unknown, but it is proposed to mediate an intramolecular interaction with a region in the proline-rich N-terminal extension to regulate the activity of myosin XV (2). Myosin XV is unique among the myosins in that it has a predicted class I PDZ-ligand motif (ITLL*) at the C-terminus (11;13;14). The PDZ-ligand motif of myosin XV is required for association of myosin XV with whirlin as well as the localization of whirlin to stereocilia tips (13;15).

 

The typhoon mutation is predicted to result in a frame-shift and coding of a premature stop codon within exon 10. Premature truncation of myosin XV within exon 10 would result in loss in a portion of the motor domain and the domains following the motor domain.

Putative Mechanism

Myosin XV functions in the assembly and maintenance of actin organization in hair cells of the inner ear by acting as a motor and carrier along the length of the actin filament within the hair cells (16). Myosin XV senses the tension between the plasma membrane and the actin filaments, a function that is necessary in the growth of the steocilia (11). Myosin XV contributes to the elongation of stereocilia by delivering whirlin, a multi-PDZ domain-containing scaffold protein, to the stereocilia tips (13). Both myosin XV and whirlin are required for the elongation and staircase formation of the stereocilia bundle and myosin XV and whirlin may function as part of a complex that modulates the growth of actin bundles in the stereocilia (11;13). Another proposed function of myosin XV is the maintenance of the hair cell mechanotransduction apparatus at the tips of the stereocilia (4;13). Two myosin XV mutant mouse models, shaker 2 (Myo15^sh2; MGI:1857036) and shaker 2^J (Myo15^sh2J; MGI:1889795) have been characterized. Both the shaker 2 and shaker 2^J mice have abnormally short stereocilia bundles on the inner ear hair cells compared to wild-type mice; the bundles are correctly positioned (1;4). As a result, the shaker 2 and shaker 2^J homozygous mice are congenitally deaf and exhibit vestibular defects that cause head-tossing and circling behavior (2-4). Similar to the shaker 2^J mice, the typhoon mice exhibit head-tossing and circling behavior. Anderson et al. propose that the myosin XV^{shaker 2J} protein is unable to exert force on the actin cytoskeleton because the truncated protein is improperly anchored, resulting in a failure to form the scaffolding to form the normal stereocilia structure (1).  

Primers

PCR Primer
Typhoon_pcr_F: AGGAATGACAACTCCAGCCG
Typhoon_pcr_R: AAGTGACAGGTTGCAGGCTC

Sequencing Primer
Typhoon_seq_F: GAATGACAACTCCAGCCGCTTTG
Typhoon_seq_R: TTGCAGGCTCAGGAACAC

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 11, + strand):

1   aggaatgaca actccagccg ctttgggaag tttgtggaaa tctttctaga agggtgagtg
61  ggactgtggc agattctcag aggccctgca gagcccttgg tggcccacag cagcatccaa
121 ggccacctca gtctccaaga tctgactgtt ctttcccagg ggtgtgatct gtggtgccat
181 aacctctcag tacctgctgg agaagtcaag gattgtgttc caggtaggca ggtcagaggc
241 ctgagtggca ccaacactca tgtgtataca tgtggatgga tggatggata gatggataga
301 tagatagata gatagataga tagatagata gatagataga tagacagaca gactctgtga
361 atgcatgctg tctgtgttcc tgagcctgca acctgtcact t

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

References

  1. Anderson, D. W., Probst, F. J., Belyantseva, I. A., Fridell, R. A., Beyer, L., Martin, D. M., Wu, D., Kachar, B., Friedman, T. B., Raphael, Y., and Camper, S. A. (2000) The Motor and Tail Regions of Myosin XV are Critical for Normal Structure and Function of Auditory and Vestibular Hair Cells. Hum Mol Genet. 9, 1729-1738.

  2. Liang, Y., Wang, A., Belyantseva, I. A., Anderson, D. W., Probst, F. J., Barber, T. D., Miller, W., Touchman, J. W., Jin, L., Sullivan, S. L., Sellers, J. R., Camper, S. A., Lloyd, R. V., Kachar, B., Friedman, T. B., and Fridell, R. A. (1999) Characterization of the Human and Mouse Unconventional Myosin XV Genes Responsible for Hereditary Deafness DFNB3 and Shaker 2. Genomics. 61, 243-258.

  3. Nal, N., Ahmed, Z. M., Erkal, E., Alper, O. M., Luleci, G., Dinc, O., Waryah, A. M., Ain, Q., Tasneem, S., Husnain, T., Chattaraj, P., Riazuddin, S., Boger, E., Ghosh, M., Kabra, M., Riazuddin, S., Morell, R. J., and Friedman, T. B. (2007) Mutational Spectrum of MYO15A: The Large N-Terminal Extension of Myosin XVA is Required for Hearing. Hum Mutat. 28, 1014-1019.

  4. Probst, F. J., Fridell, R. A., Raphael, Y., Saunders, T. L., Wang, A., Liang, Y., Morell, R. J., Touchman, J. W., Lyons, R. H., Noben-Trauth, K., Friedman, T. B., and Camper, S. A. (1998) Correction of Deafness in Shaker-2 Mice by an Unconventional Myosin in a BAC Transgene. Science. 280, 1444-1447.

  5. Wang, A., Liang, Y., Fridell, R. A., Probst, F. J., Wilcox, E. R., Touchman, J. W., Morton, C. C., Morell, R. J., Noben-Trauth, K., Camper, S. A., and Friedman, T. B. (1998) Association of Unconventional Myosin MYO15 Mutations with Human Nonsyndromic Deafness DFNB3. Science. 280, 1447-1451.

  6. Cope, M. J., Whisstock, J., Rayment, I., and Kendrick-Jones, J. (1996) Conservation within the Myosin Motor Domain: Implications for Structure and Function. Structure. 4, 969-987.

  7. Karolyi, I. J., Probst, F. J., Beyer, L., Odeh, H., Dootz, G., Cha, K. B., Martin, D. M., Avraham, K. B., Kohrman, D., Dolan, D. F., Raphael, Y., and Camper, S. A. (2003) Myo15 Function is Distinct from Myo6, Myo7a and Pirouette Genes in Development of Cochlear Stereocilia. Hum Mol Genet. 12, 2797-2805.

  8. Friedman, T. B., Sellers, J. R., and Avraham, K. B. (1999) Unconventional Myosins and the Genetics of Hearing Loss. Am J Med Genet. 89, 147-157.

  9. Mooseker, M. S., and Cheney, R. E. (1995) Unconventional Myosins. Annu Rev Cell Dev Biol. 11, 633-675.

  10. Chen, Z. Y., Hasson, T., Kelley, P. M., Schwender, B. J., Schwartz, M. F., Ramakrishnan, M., Kimberling, W. J., Mooseker, M. S., and Corey, D. P. (1996) Molecular Cloning and Domain Structure of Human Myosin-VIIa, the Gene Product Defective in Usher Syndrome 1B. Genomics. 36, 440-448.

  11. Delprat, B., Michel, V., Goodyear, R., Yamasaki, Y., Michalski, N., El-Amraoui, A., Perfettini, I., Legrain, P., Richardson, G., Hardelin, J. P., and Petit, C. (2005) Myosin XVa and Whirlin, Two Deafness Gene Products Required for Hair Bundle Growth, are Located at the Stereocilia Tips and Interact Directly. Hum Mol Genet. 14, 401-410.

  12. Weber, K. L., Sokac, A. M., Berg, J. S., Cheney, R. E., and Bement, W. M. (2004) A Microtubule-Binding Myosin Required for Nuclear Anchoring and Spindle Assembly. Nature. 431, 325-329.

  13. Belyantseva, I. A., Boger, E. T., Naz, S., Frolenkov, G. I., Sellers, J. R., Ahmed, Z. M., Griffith, A. J., and Friedman, T. B. (2005) Myosin-XVa is Required for Tip Localization of Whirlin and Differential Elongation of Hair-Cell Stereocilia. Nat Cell Biol. 7, 148-156.

  14. Sheng, M., and Sala, C. (2001) PDZ Domains and the Organization of Supramolecular Complexes. Annu Rev Neurosci. 24, 1-29.

  15. Shearer, A. E., Hildebrand, M. S., Webster, J. A., Kahrizi, K., Meyer, N. C., Jalalvand, K., Arzhanginy, S., Kimberling, W. J., Stephan, D., Bahlo, M., Smith, R. J., and Najmabadi, H. (2009) Mutations in the First MyTH4 Domain of MYO15A are a Common Cause of DFNB3 Hearing Loss. Laryngoscope. 119, 727-733.

  16. Held, N., Smits, B. M., Gockeln, R., Schubert, S., Nave, H., Northrup, E., Cuppen, E., Hedrich, H. J., and Wedekind, D. (2011) A Mutation in Myo15 Leads to Usher-Like Symptoms in LEW/Ztm-ci2 Rats. PLoS One. 6, e15669.