Phenotypic Mutation 'demure' (pdf version)
Alleledemure
Mutation Type missense
Chromosome2
Coordinate163,706,040 bp (GRCm39)
Base Change A ⇒ T (forward strand)
Gene Rims4
Gene Name regulating synaptic membrane exocytosis 4
Synonym(s) Rim4
Chromosomal Location 163,701,671-163,760,603 bp (-) (GRCm39)
MGI Phenotype PHENOTYPE: Mice homozygous for an ENU-induec allele exhibit reduced body weight. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_1832023; MGI:2674366

MappedYes 
Amino Acid Change Valine changed to Glutamic Acid
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000045637]
AlphaFold P60191
SMART Domains Protein: ENSMUSP00000045637
Gene: ENSMUSG00000035226
AA Change: V198E

DomainStartEndE-ValueType
C2 129 232 1.42e-11 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 0.994 (Sensitivity: 0.69; Specificity: 0.97)
(Using ENSMUST00000044734)
Meta Mutation Damage Score 0.8896 question?
Is this an essential gene? Probably nonessential (E-score: 0.121) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance 6/10 
Alleles Listed at MGI

All Mutations and Alleles(4) : Gene trapped(2) Targeted(2)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL01627:Rims4 APN 2 163706022 missense probably damaging 1.00
IGL01980:Rims4 APN 2 163707702 splice site probably benign
diminutive UTSW 2 163706785 critical splice donor site probably null
R0115:Rims4 UTSW 2 163706040 missense probably damaging 0.99
R0152:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R0153:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R0173:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R0238:Rims4 UTSW 2 163705945 missense probably benign 0.03
R0238:Rims4 UTSW 2 163705945 missense probably benign 0.03
R0481:Rims4 UTSW 2 163706040 missense probably damaging 0.99
R0702:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R0735:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R0973:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R0973:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R0974:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R1013:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R1014:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R1017:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R1104:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R1209:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R1401:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R1554:Rims4 UTSW 2 163721042 missense probably damaging 1.00
R1618:Rims4 UTSW 2 163705849 missense possibly damaging 0.65
R2104:Rims4 UTSW 2 163706785 critical splice donor site probably null
R2171:Rims4 UTSW 2 163706046 splice site probably null
R3611:Rims4 UTSW 2 163721126 missense possibly damaging 0.50
R3735:Rims4 UTSW 2 163705905 missense possibly damaging 0.88
R3836:Rims4 UTSW 2 163760573 missense possibly damaging 0.86
R4685:Rims4 UTSW 2 163706914 nonsense probably null
R4849:Rims4 UTSW 2 163707463 missense probably benign 0.11
R4873:Rims4 UTSW 2 163707443 missense probably null 0.00
R4875:Rims4 UTSW 2 163707443 missense probably null 0.00
R5337:Rims4 UTSW 2 163707763 missense probably benign 0.00
R5415:Rims4 UTSW 2 163760596 missense probably benign 0.26
R5646:Rims4 UTSW 2 163705937 nonsense probably null
R6487:Rims4 UTSW 2 163706817 missense possibly damaging 0.93
R7213:Rims4 UTSW 2 163705981 missense probably benign 0.00
R7814:Rims4 UTSW 2 163760548 missense probably benign 0.05
R7849:Rims4 UTSW 2 163705974 missense probably damaging 1.00
Mode of Inheritance Autosomal Recessive
Local Stock Sperm, gDNA
Repository
Last Updated 2019-09-04 9:45 PM by Anne Murray
Record Created 2015-06-19 5:03 PM by Jeff SoRelle
Record Posted 2016-11-08
Phenotypic Description

Figure 1. Demure mice exhibit reduced body weights. Scaled body weight data are shown. Abbreviations: REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The demure phenotype was identified among G3 mice of the pedigree R0115, some of which showed reduced body weights compared to wild-type littermates (Figure 1).

Nature of Mutation

Figure 2. Linkage mapping of the reduced body weights using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 86 mutations (X-axis) identified in the G1 male of pedigree R0115. Weight 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 86 mutations. The body weight phenotype was linked to a mutation in Rims4: a T to A transversion at base pair 163,864,120 (v38) on chromosome 2, or base pair 54,871 in the GenBank genomic region NC_000068 encoding Rims4. Linkage was found with a recessive model of inheritance, wherein 10 variant homozygous mice departed phenotypically from 22 homozygous reference mice and 31 heterozygous mice (P = 3.317 x 10-6; Figure 2).

The mutation corresponds to residue 593 in the NM_1832023 mRNA sequence in exon 6 of 6 total exons. 

 

577 GGCAAAGTCCTGCAGGTAATCGTGTGGGGAAAC

193 -G--K--V--L--Q--V--I--V--W--G--N-

The mutated nucleotide is indicated in red.  The mutation results in a valine (V) to glutamic acid (E) substitution at position 198 (V198E) in the RIMS4 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.994).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 3. Domain organization of RIMS4. RIMS4 has a C2 domain. The demure mutation results in a valine (V) to glutamic acid (E) substitution at position 198 (V198E). This image is interactive; click to view other mutations in Rims4.

Rims4 encodes RIMS4 (protein regulating synaptic membrane exocytosis 4; alternatively, RIM4γ), a member of the RIM (Rab-3 interacting molecules) family of synaptic proteins that regulate normal neurotransmitter release. There are four isoforms of the RIM proteins: RIM1 to RIM4. The RIM1 and RIM2 isoforms have longer α and β variants and a short γ variant that differ in their protein domain organization. RIM3 and RIM4 only have γ variants. RIMS4 is highly homologous to RIM2γ and RIM3γ.

RIMS4 has a C2 domain at amino acids 129-232 (Figure 3). The C2 domain putatively binds liprins, which are adaptor proteins that regulate the active zone size (1;2). The C2 domain also binds to synaptotagmin 1, which regulates neurotransmitter release (3;4). The RIM proteins bind Rab3 via their N-terminal domain, which is absent in RIMS4. Also, a central domain in RIM binds Cav2 (N and P/Q) channels to recruit the channels to the active zones.

The demure mutation results in a valine (V) to glutamic acid (E) substitution at position 198 (V198E); residue 198 is within the C2 domain.

Expression/Localization

RIMS4 is primarily synthesized in the brain (5). Little to no RIMS4 expression was observed in the heart, spleen, lung, liver, skeletal muscle, kidney, or testis from the rat. RIM proteins are typically localized to active zones; the localization of RIMS4 is unknown.

Background
Figure 4. RIM proteins function in synaptic vesicle exocytosis. Synaptic vesicles translocate to the active zone, tether to the presynaptic membrane, and are primed. In response to calcium influx, the vesicles undergo exocytosis and release neurotransmitters into the synaptic cleft, where the neurotransmitters bind to their receptors in the postsynaptic membrane. Exocytosis involves the complex interactions among several evolutionarily conserved proteins. RIM (Rab-3 interacting molecules), RIM binding protein (RIM-BP), mammalian uncoordinated–13 (Munc-13), and α-liprins form a single large protein complex that constitutes the core of the active zone.

Synaptic exocytosis involves synaptic vesicle docking and fusion with the presynaptic membrane (Figure 4). Synaptic vesicle exocytosis occurs in the active zone, which contains clustered Cav2-type voltage-gated Ca2+ channels. In response to calcium influx, the synaptic vesicles undergo exocytosis and release neurotransmitters into the synaptic cleft, whereby the neurotransmitters can bind to their receptors in the postsynaptic membrane.

The RIM proteins are adaptor proteins that were identified as effectors of Rab3, which is a synaptic vesicle protein that binds GTP and regulates neurotransmitter release (6;7). The RIM proteins putatively regulate neurotransmitter release by interacting with synaptic proteins (e.g., Rab3). The RIM, RIM-BP, Munc13, and liprin proteins form a single large protein complex that forms the core of the active zone. RIM proteins bind Rab3, Rab27, Munc13, and Cav2 channels. The liprins function as a link between the RIM/RIM-BP/Munc13 complex and adhesion molecules of the receptor phosphotyrosine phosphatase family. Synaptic vesicle fusion with the presynaptic membranes is dependent on the interactions between SNARE proteins on the synaptic vesicle and syntaxin-1 and SNAP-25.

The function of RIMS4 is unknown.

Putative Mechanism

The cause of the low body weight in the demure mice is unknown.

Primers PCR Primer
demure_pcr_F: TCCAATGTCCAATGTGCCCCTG
demure_pcr_R: TGTCTCAGCCCACTGTCATAGAGC

Sequencing Primer
demure_seq_F: TTAAGATCGCTCGCCACAGG
demure_seq_R: CCACTGTCATAGAGCAGGAC
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 647 nucleotides is amplified (chromosome 2, - strand):


1   tgtctcagcc cactgtcata gagcaggaca gaattaagag atgagataca ggacagccaa
61  tgaggagcct gcagccccag attcacgggc cagctccgag aggatgctga ccaggagatg
121 ggaaagggct acagagaaag attggtcgga acaagaatag cagctaacag tgtgctgtta
181 tctctacttt ataaaaatgt ttcatgtggg gttccaaggt cccctgcggg ggtagcaggg
241 aaaggggtca tgtggcagat gtggggacat caccagccga gcagccctgc ccagctcagt
301 tgcaagacgt aaccagccgt ggttttcgtc ctgcaggtaa tcgtgtgggg aaactacggg
361 cggatggagc ggaagcagtt catgggcgtg gctcgagtcc tgctggagga actggacttg
421 accaccctgg ccgtgggctg gtacaagctc ttccccacct cctccatggt ggacccagcc
481 acaggtccgc tgcttcggca ggcatcccag ctgtccctcg agagcaccgt ggggccctgt
541 ggcgagcgat cttaatgccg gggaggggga ggggctccgc gagatggcct ggagaccacc
601 cagccctgac ctgggacccc aggcccaggg gcacattgga cattgga


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
AuthorsJeff SoRelle and Bruce Beutler