Phenotypic Mutation 'Nidoqueen' (pdf version)
AlleleNidoqueen
Mutation Type missense
Chromosome1
Coordinate78,108,869 bp (GRCm39)
Base Change A ⇒ G (forward strand)
Gene Pax3
Gene Name paired box 3
Synonym(s) Splchl2, Pax-3
Chromosomal Location 78,077,904-78,173,771 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene is a member of the paired box (PAX) family of transcription factors. Members of the PAX family typically contain a paired box domain and a paired-type homeodomain. These genes play critical roles during fetal development. Mutations in paired box gene 3 are associated with Waardenburg syndrome, craniofacial-deafness-hand syndrome, and alveolar rhabdomyosarcoma. The translocation t(2;13)(q35;q14), which represents a fusion between PAX3 and the forkhead gene, is a frequent finding in alveolar rhabdomyosarcoma. Alternative splicing results in transcripts encoding isoforms with different C-termini. [provided by RefSeq, Jul 2008]
PHENOTYPE: Effects on homozygotes for mutations in this gene vary in severity and include embryonic to perinatal death, malformations of neural tube, spinal ganglia, heart, vertebral column, hindbrain and limb musculature. Heterozygotes have white belly spots and variable spotting on the back and extremeties. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_008781, NM_001159520; MGI:97487

MappedYes 
Amino Acid Change Valine changed to Alanine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000004994] [ENSMUSP00000084320]
AlphaFold P24610
SMART Domains Protein: ENSMUSP00000004994
Gene: ENSMUSG00000004872
AA Change: V263A

DomainStartEndE-ValueType
PAX 34 159 1.99e-91 SMART
low complexity region 164 185 N/A INTRINSIC
HOX 219 281 6.6e-27 SMART
Pfam:Pax7 347 391 5.9e-29 PFAM
Predicted Effect possibly damaging

PolyPhen 2 Score 0.956 (Sensitivity: 0.79; Specificity: 0.95)
(Using ENSMUST00000004994)
SMART Domains Protein: ENSMUSP00000084320
Gene: ENSMUSG00000004872
AA Change: V263A

DomainStartEndE-ValueType
PAX 34 159 1.99e-91 SMART
low complexity region 164 185 N/A INTRINSIC
HOX 219 281 6.6e-27 SMART
Pfam:Pax7 346 391 5.3e-27 PFAM
Predicted Effect probably damaging

PolyPhen 2 Score 0.999 (Sensitivity: 0.14; Specificity: 0.99)
(Using ENSMUST00000087086)
Meta Mutation Damage Score 0.9310 question?
Is this an essential gene? Probably essential (E-score: 0.822) question?
Phenotypic Category Autosomal Dominant
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(41) : Chemically and radiation induced (1) Chemically induced (ENU)(5) Chemically induced (other) (1) Radiation induced(5) Spontaneous (6) Targeted(23)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01642:Pax3 APN 1 78173300 critical splice donor site probably null
IGL02249:Pax3 APN 1 78171962 missense probably damaging 0.98
IGL02271:Pax3 APN 1 78171969 missense probably damaging 1.00
IGL02376:Pax3 APN 1 78108929 missense probably damaging 1.00
IGL02530:Pax3 APN 1 78098424 missense possibly damaging 0.87
IGL02950:Pax3 APN 1 78079997 missense probably benign 0.06
Widget UTSW 1 78099227 critical splice donor site probably null
R0049:Pax3 UTSW 1 78080141 missense probably damaging 1.00
R0049:Pax3 UTSW 1 78080141 missense probably damaging 1.00
R0523:Pax3 UTSW 1 78172078 missense possibly damaging 0.83
R1575:Pax3 UTSW 1 78080121 missense probably benign 0.00
R1831:Pax3 UTSW 1 78108977 missense probably damaging 1.00
R1934:Pax3 UTSW 1 78080117 missense possibly damaging 0.90
R2420:Pax3 UTSW 1 78173501 splice site probably null
R2473:Pax3 UTSW 1 78099227 critical splice donor site probably null
R4430:Pax3 UTSW 1 78171961 missense probably damaging 1.00
R4693:Pax3 UTSW 1 78173383 missense probably benign 0.00
R4818:Pax3 UTSW 1 78108869 missense probably damaging 1.00
R4860:Pax3 UTSW 1 78169093 missense possibly damaging 0.78
R4860:Pax3 UTSW 1 78169093 missense possibly damaging 0.78
R5302:Pax3 UTSW 1 78098249 missense possibly damaging 0.88
R5475:Pax3 UTSW 1 78080055 missense probably benign 0.06
R5855:Pax3 UTSW 1 78098288 missense probably damaging 0.99
R6102:Pax3 UTSW 1 78108984 missense probably damaging 1.00
R6190:Pax3 UTSW 1 78169186 missense possibly damaging 0.63
R6856:Pax3 UTSW 1 78109056 missense probably damaging 1.00
R7065:Pax3 UTSW 1 78170648 splice site probably null
R7547:Pax3 UTSW 1 78099231 nonsense probably null
R8059:Pax3 UTSW 1 78080003 missense probably benign 0.04
R8224:Pax3 UTSW 1 78098327 missense probably damaging 1.00
R8312:Pax3 UTSW 1 78172006 missense probably damaging 1.00
R8324:Pax3 UTSW 1 78170426 missense probably damaging 1.00
R9319:Pax3 UTSW 1 78080079 missense probably benign
R9759:Pax3 UTSW 1 78170415 missense probably damaging 1.00
Z1176:Pax3 UTSW 1 78099227 critical splice donor site probably null
Mode of Inheritance Autosomal Dominant
Local Stock
Repository
Last Updated 2019-09-04 9:43 PM by Anne Murray
Record Created 2016-03-04 12:11 PM by Carlos Reyna
Record Posted 2016-06-03
Phenotypic Description
Figure 1. The Nidoqueen mice exhibit variable white belly spotting.

The Nidoqueen phenotype was identified among N-ethyl-N-nitrosourea (ENU)-induced mice of the pedigree R4818, some of which exhibited variable white spotting of the fur (Figure 1).

Nature of Mutation

Whole exome HiSeq sequencing of the G1 grandsire identified 62 mutations. Among these, only one affected a gene with known effects on pigmentation, Pax3. The mutation in Pax3 was presumed to be causative because the Nidoqueen pigmentation phenotype mimics other known alleles of Pax3 (see MGI for a list of Pax3 alleles as well as the entry for Widget). The Pax3 mutation is a T to C transition at base pair 78,132,232 (v38) on chromosome 1, or base pair 65,316 in the GenBank genomic region NC_000067 for the Pax3 gene.

The mutation corresponds to residue 1,175 in the mRNA sequence NM_008781 within exon 5 of 8 total exons and residue 1,175 in the mRNA sequence NM_001159520 within exon 5 of 9 total exons.

 

65300 CTTACCGAGGCCCGAGTGCAGGTCTGGTTTAGC

258   -L--T--E--A--R--V--Q--V--W--F--S-

Genomic numbering corresponds to NC_000067. The mutated nucleotide is indicated in red.  The mutation results in a valine (V) to alanine (A) substitution at position 263 (V263A) in both variants of the PAX3 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.956).

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 2. The domain structure of mouse PAX3. The Nidoqueen mutation is indicated. Abbreviations: PD, paired domain; OCT, octapeptide motif, HD, homeodomain; TA, transactivation domain.

Paired box (PAX) 3 [PAX3; alternatively, melanocyte specific factor (MSF)] is a member of the PAX family of transcription factors (1). The paired domain (PD; amino acids 34-159) and homeodomain (HD; amino acids 219-281) of PAX3 are both DNA-binding domains that can act independently or together to bind target genes [Figure 2; (2;3)]. PAX3 also has an octapeptide motif (HSIDGILS; amino acids 186-193) and a C-terminal transcription activation (TA) domain (amino acids 282-484) (4).

The Nidoqueen mutation results in a valine to alanine substitution at amino acid 263, which is located in the HD. The PAX3 HD folds into a prototypical HD fold of three α-helices (α1, α2, and α3), connected by short loops, and an extended N-terminal arm that is disordered in the absence of DNA (5). Upon interaction with DNA, the arm inserts into the major groove to interact with bases in both strands of the DNA as well as with the sugar-phosphate backbone.

For more information about Pax3, please see the record for Widget.

Putative Mechanism

The PAX protein family regulates pattern formation, morphogenesis, cellular differentiation, and organogenesis by activating (or repressing) genes that encode secreted proteins, cell surface receptors, cell cycle regulators, and transcription factors. PAX3 is essential for normal development of the peripheral nervous system, the trigeminal (cranial nerve V), superior (IX), and jugular (X) ganglia as well as the frontal, ophthalmic, and spinal accessory nerves (6;7). Mutations in PAX3 are linked to craniofacial-deafness-hand syndrome [OMIM: #122880; (8;9)] and alveolar rhabdomyosarcoma 2 [OMIM: #268220; (10)] as well as Waardenburg syndrome (WS) type 1 [WS1; OMIM: #193500; (11)] and WS type 3 (WS3; alternatively, Klein-Waardenburg syndrome; OMIM: #148820) (12;13)].

During melanocyte development in the embryo, PAX3 and SOX10 (mutated in Dalmatian) are both required for the expression of Mitf, which is required for melanoblast survival after migration from the dorsal neural tube to the migration staging area (14-16). PAX3, SOX10, MITF, and LEF1 bind directly to the Dct promoter, which encodes an enzyme necessary for melanin pigment production in melanocytes (17-19). PAX3 and MITF share the same binding site within the Dct promoter and compete for occupancy (17).

A spontaneous white belly spot phenotype was observed in a mouse, designated as Splotch, and attributed to a spontaneous splice acceptor site mutation in Pax3 (20). Homozygous Splotch mice are embryonic lethal by embryonic day (E) 14 due to neural tube closure defects in the lumbo-sacral area (i.e., spina bifida), absence (or reduction in size) of the dorsal root ganglia, exencephaly, abnormal formation of the cardiac outflow tract, and absent limb musculature (21). Heterozygous Splotch mice are viable and exhibit variable white belly spotting. Several other Splotch alleles, including two ENU-induced alleles, have also been identified; each of these mutants display variable white belly spotting in the heterozygote (22;23). Similar to other Pax3 mutants, heterozygous Nidoqueen mice exhibit variable white belly spotting. Pigment defects in Pax3 mutants are attributed to reduced melanoblast numbers, not in defects through the migratory pathway (24).

Primers PCR Primer
Nidoqueen_pcr_F: TCTAAGCACTGTTCACTGTTGC
Nidoqueen_pcr_R: ACCTCAGTCAGATGAAGGCTC

Sequencing Primer
Nidoqueen_seq_F: AGCACTGTTCACTGTTGCTAATATGG
Nidoqueen_seq_R: CAGATGAAGGCTCCGATATTGACTC
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 412 nucleotides is amplified (chromosome 1, - strand):


1   acctcagtca gatgaaggct ccgatattga ctctgaacct gatttaccgc tgaagaggaa
61  gcagcgcagg agcagaacca ccttcacggc agagcagctg gaggaactgg agcgggcttt
121 cgagagaacc cactacccag acatttacac cagggaggag ctggcccaga gggcgaagct
181 taccgaggcc cgagtgcagg tactgatgcc cggatgtcgg ggacttcctc ccatccaggg
241 aggaatctcc ggggtctcct catgggaacc gttttcgttg tttgtttgat cttactaagt
301 agcccgggct ggtacggatt caaaggcctc ctaaacgctg accttacaac atacagccaa
361 gaagtgtttg aagccaagaa aaccatatta gcaacagtga acagtgctta ga


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

References
Science Writers Anne Murray
Illustrators Peter Jurek
AuthorsCarlos Reyna, Jamie Russell, and Bruce Beutler