Phenotypic Mutation 'mire' (pdf version)
Allelemire
Mutation Type critical splice donor site (2 bp from exon)
Chromosome10
Coordinate79,564,947 bp (GRCm39)
Base Change T ⇒ A (forward strand)
Gene Hcn2
Gene Name hyperpolarization-activated, cyclic nucleotide-gated K+ 2
Synonym(s) HAC1, trls
Chromosomal Location 79,552,468-79,571,942 bp (+) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The protein encoded by this gene is a hyperpolarization-activated cation channel involved in the generation of native pacemaker activity in the heart and in the brain. The encoded protein is activated by cAMP and can produce a fast, large current. Defects in this gene were noted as a possible cause of some forms of epilepsy. [provided by RefSeq, Jan 2017]
PHENOTYPE: Mice homozygous for mutant alleles exhibit decreased body weight, behavioral/neurological abnormalities, and tremors or absence seizures. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_008226; MGI:1298210

MappedYes 
Amino Acid Change
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000020581 ] [ENSMUSP00000097113 ]   † probably from a misspliced transcript
AlphaFold O88703
PDB Structure HCN2J 443-645 in the presence of cGMP [X-RAY DIFFRACTION]
HCN2I 443-640 in the presence of cAMP, selenomethionine derivative [X-RAY DIFFRACTION]
HCN2J 443-645 in the presence of cAMP, selenomethionine derivative [X-RAY DIFFRACTION]
Structure and rearrangements in the carboxy-terminal region of SpIH channels [X-RAY DIFFRACTION]
HCN2-I 443-460 E502K in the presence of cAMP [X-RAY DIFFRACTION]
X-ray structure of cysteine-free fragment of mHCN2 C-terminal region from amino acids 443-630 including C508N, C584S, and C601S mutations [X-RAY DIFFRACTION]
HCN2I 443-640 apo-state [X-RAY DIFFRACTION]
Trip8b-1a#206-567 interacting with the carboxy-terminal seven residues of HCN2 [X-RAY DIFFRACTION]
SMART Domains Protein: ENSMUSP00000020581
Gene: ENSMUSG00000020331

DomainStartEndE-ValueType
low complexity region 4 47 N/A INTRINSIC
low complexity region 106 128 N/A INTRINSIC
Pfam:Ion_trans_N 140 183 5e-23 PFAM
Pfam:Ion_trans 184 447 3.3e-24 PFAM
low complexity region 448 459 N/A INTRINSIC
Blast:cNMP 460 492 9e-13 BLAST
cNMP 517 630 4.79e-22 SMART
low complexity region 727 765 N/A INTRINSIC
low complexity region 778 800 N/A INTRINSIC
low complexity region 804 838 N/A INTRINSIC
Predicted Effect probably null
SMART Domains Protein: ENSMUSP00000097113
Gene: ENSMUSG00000020331

DomainStartEndE-ValueType
low complexity region 4 47 N/A INTRINSIC
low complexity region 106 128 N/A INTRINSIC
Pfam:Ion_trans_N 139 215 2.6e-47 PFAM
Pfam:Ion_trans 219 435 1.5e-20 PFAM
low complexity region 448 459 N/A INTRINSIC
Blast:cNMP 460 492 9e-13 BLAST
cNMP 517 630 4.79e-22 SMART
low complexity region 727 765 N/A INTRINSIC
low complexity region 778 800 N/A INTRINSIC
low complexity region 804 838 N/A INTRINSIC
Predicted Effect probably null
Meta Mutation Damage Score 0.9495 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Unknown
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(12) : Chemically induced (ENU)(3) Chemically induced (other)(1) Spontaneous(3) Targeted(5)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00945:Hcn2 APN 10 79569637 nonsense probably null
IGL01339:Hcn2 APN 10 79564902 missense probably damaging 1.00
IGL02183:Hcn2 APN 10 79560647 critical splice donor site probably null
asombrarse UTSW 10 79560445 missense probably damaging 1.00
curveball UTSW 10 79560620 missense probably damaging 1.00
curveball2 UTSW 10 79569607 nonsense probably null
R0269:Hcn2 UTSW 10 79570075 unclassified probably benign
R0671:Hcn2 UTSW 10 79570066 splice site probably null
R1879:Hcn2 UTSW 10 79562023 missense probably benign 0.03
R1913:Hcn2 UTSW 10 79566777 missense probably benign 0.14
R4051:Hcn2 UTSW 10 79569521 splice site probably null
R4052:Hcn2 UTSW 10 79569521 splice site probably null
R4328:Hcn2 UTSW 10 79560445 missense probably damaging 1.00
R4507:Hcn2 UTSW 10 79560620 missense probably damaging 1.00
R4518:Hcn2 UTSW 10 79560536 missense probably benign 0.17
R4578:Hcn2 UTSW 10 79560282 splice site probably null
R5334:Hcn2 UTSW 10 79562125 missense probably damaging 0.99
R5788:Hcn2 UTSW 10 79552945 missense possibly damaging 0.48
R6131:Hcn2 UTSW 10 79569742 missense probably damaging 1.00
R6457:Hcn2 UTSW 10 79569607 nonsense probably null
R6547:Hcn2 UTSW 10 79552986 missense probably benign 0.29
R6851:Hcn2 UTSW 10 79564947 critical splice donor site probably null
R7276:Hcn2 UTSW 10 79564934 missense possibly damaging 0.95
R7706:Hcn2 UTSW 10 79570017 missense possibly damaging 0.78
R7893:Hcn2 UTSW 10 79560245 missense probably damaging 1.00
R8208:Hcn2 UTSW 10 79566778 missense possibly damaging 0.94
R8677:Hcn2 UTSW 10 79560619 missense probably benign 0.28
R9333:Hcn2 UTSW 10 79561991 missense possibly damaging 0.56
R9527:Hcn2 UTSW 10 79570706 missense probably benign 0.05
R9594:Hcn2 UTSW 10 79560559 missense probably damaging 1.00
R9602:Hcn2 UTSW 10 79562128 missense probably benign 0.05
R9604:Hcn2 UTSW 10 79564787 missense probably damaging 1.00
X0024:Hcn2 UTSW 10 79569954 missense probably damaging 1.00
Mode of Inheritance Unknown
Local Stock
Repository
Last Updated 2019-09-04 9:25 PM by Anne Murray
Record Created 2019-05-12 6:56 PM by Bruce Beutler
Record Posted 2019-05-16
Phenotypic Description

Figure 1. Mire mice exhibited reduced body weights compared to wild-type littermates. Scaled body weights 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 mire phenotype was identified among G3 mice of the pedigree R6851, 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 42 mutations (X-axis) identified in the G1 male of pedigree R6851. Weight 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 42 mutations. The body weight phenotype was linked to mutations in two genes on chromosome 10: Hcn2 and Osbpl8. The mutation in Hcn2 was presumed causative as the phenotype in the mire mice mimicked that of other Hcn2 mutant mouse models [see curveball and (1;2)]. The Hcn2 mutation is a T to A transversion at base pair 79,729,113 (v38) on chromosome 10, or base pair 12,480 in the GenBank genomic region NC_000076 within the splice donor site of intron 4 (2-base pairs from exon 4 out of 8 total exons). Linkage was found with a recessive model of inheritance, wherein one variant homozygote departed phenotypically from 10 homozygous reference mice and 14 heterozygous mice with a P value of 9.676 x 10-6 (Figure 2).

The effect of the mutation at the cDNA and protein levels has not been examined, but the mutation is predicted to result in the use of a cryptic splice site in intron 4. The resulting transcript would have a 90-base pair insertion of intron 4, which would cause an in-frame insertion of 30 aberrant amino acids after amino acid 452 of the protein (which is normally 863 amino acids long).

C57BL/6J:
          <--exon 3      <--exon 4 intron 4-->          exon 5-->
1161 ……AACAACATGGTG ……TACCAGGAGAAG gtcagaggagggaggctg…… TACAAGCAAGTA……
376  ……-N--N--M--V- ……-Y--Q--E--K-                      -Y--K--Q--V-……

 

The donor splice site of intron 4, which is destroyed by the mire mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red.

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 3. Domain organization and topology of mouse HCN2. HCN2 has six transmembrane domains and cytoplasmic N- and C-termini. The ion-conducting pore region is between transmembrane domains 5 and 6. The HCN2 C-terminus has two domains: a C-linker domain and the cyclic nucleotide-binding domain (CNBD). The mire mutation is within the splice donor site of intron 4. This image is interactive. Other mutations found in HCN2 are noted in red. Click on each muation for more information.

Hcn2 encodes hyperpolarization-activated cyclic nucleotide-gated (HCN) channel 2 (HCN2). The HCN channels (i.e., HCN1, HCN2, HCN3, and HCN4) are members of the voltage-gated potassium ion channel superfamily present mainly in neurons and heart cells (3). The HCN channels are complexes of four HCN subunits arranged around the central pore; the HCN proteins can form homomeric or heteromeric channels with other members of the HCN family (4-6).

HCN2 has six transmembrane domains and cytoplasmic N- and C-termini (Figure 3). Transmembrane domain 4 is a positively-charged voltage sensor. The ion-conducting pore region is between transmembrane domains 5 and 6. The HCN2 C-terminus has two domains: a C-linker domain composed of six α-helices separated by short loops and the CNBD. The C-linker domain connects the CNBD to the transmembrane domains.  The CNBD mediates modulation by cyclic nucleotides (e.g., cyclic adenosine monophosphate [cAMP]) (7).

The mire mutation is predicted to cause an in-frame insertion of 30 aberrant amino acids after amino acid 452 of the protein (which is normally 863 amino acids long). The insertion would occur after TM6 and before the C-linker.

Please see the record curveball for more information about Hcn2.

Putative Mechanism

HCN2 is a regulator of nociceptor excitability. In nociceptive neurons, HCN2 modulates the generation of action potentials in response to inflammation. For example, upon prostaglandin E2 (PGE2) binding to a G protein-coupled receptor coupled to Gs, adenylate cyclase is activated, which elevates cAMP. The elevation of cAMP shifts the activation curve of HCN2 in the positive direction, which causes a HCN2-dependent tonic inward current (termed Ih) to be activated at the resting potential. The Ih current is essential for cardiac and neuronal pacemaker activity, dendritic integration of synaptic transmission, and the setting of resting potentials (8).

Loss-of-function mutations in HCN2 have been linked to idiopathic generalized epilepsies in patients (9;10). In addition, gain-of-function mutations are linked to polygenic epilepsy (11). Mutations in HCN2 have been correlated with increased incidence of febrile seizures (12). The mutant HCN2 channels exhibited faster kinetics with higher temperatures and subsequent increased rate of availability of the current.

Hcn2-deficient (Hcn2-/-) mice were hypoactive and smaller than their wild-type littermates (1;2;13). The Hcn2-/- mice exhibited absence epilepsy, ataxia, and sinus arrhythmia (13). Hcn2-/- mice also showed impaired balance and coordination (1;2).

The reduced size phenotype observed in the mire mice indicates a loss of HCN2-associated function.

Primers PCR Primer
mire_pcr_F: CAATTGCAGAACCACTCGTG
mire_pcr_R: GAACACTCAGAACTGGTCCC

Sequencing Primer
mire_seq_F: GCTCTTCAAGGCCATGAGC
mire_seq_R: ACTGGTCTTGTTGGTTCTGAAC
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 437 nucleotides is amplified (chromosome 10, + strand):


1   caattgcaga accactcgtg gagcgagctc tactcgttcg cgctcttcaa ggccatgagc
61  cacatgctgt gcatcggcta cgggcggcag gcgcccgaga gcatgacaga catctggctg
121 accatgctca gcatgatcgt aggcgccacc tgctatgcca tgttcattgg gcacgccact
181 gcgctcatcc agtccctgga ttcgtcacgg cgccaatacc aggagaaggt cagaggaggg
241 aggctgtgtc ttcggtctga gtggggaggt ggccctgaat ctggggtggg gaagcagccc
301 ctggttctaa gggcttctgt aagagctgag gagagagtca aagggaggac ccaggtaggg
361 gtagatgagg ggaggggagg agcccaccgg gttcagaacc aacaagacca gtccctgggg
421 accagttctg agtgttc


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

References
Science Writers Anne Murray
Illustrators Diantha La Vine
AuthorsZhao Zhang and Bruce Beutler