Phenotypic Mutation 'drunk' (pdf version)
Alleledrunk
Mutation Type critical splice donor site (2 bp from exon)
Chromosome13
Coordinate59,512,323 bp (GRCm38)
Base Change A ⇒ T (forward strand)
Gene Agtpbp1
Gene Name ATP/GTP binding protein 1
Synonym(s) 2310001G17Rik, Nna1, 1700020N17Rik, 4930445M19Rik, 2900054O13Rik, 5730402G09Rik
Chromosomal Location 59,445,742-59,585,227 bp (-)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] NNA1 is a zinc carboxypeptidase that contains nuclear localization signals and an ATP/GTP-binding motif that was initially cloned from regenerating spinal cord neurons of the mouse.[supplied by OMIM, Jul 2002]
PHENOTYPE: Homozygotes show moderate ataxia due to degeneration of Purkinje cells of the cerebellum. Also, there is gradual degeneration of retina photoreceptor cells, olfactory bulb mitral cells and some thalamic neurons. Males have abnormal sperm and are sterile. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_023328 (isoform 1), NM_001048008 (isoform 2); MGI: 2159437

Mapped Yes 
Amino Acid Change
Institutional SourceBeutler Lab
Gene Model not available
SMART Domains Protein: ENSMUSP00000022040
Gene: ENSMUSG00000021557

DomainStartEndE-ValueType
low complexity region 362 391 N/A INTRINSIC
low complexity region 589 603 N/A INTRINSIC
Pfam:Peptidase_M14 851 1099 1.7e-13 PFAM
Predicted Effect probably benign
SMART Domains Protein: ENSMUSP00000105456
Gene: ENSMUSG00000021557

DomainStartEndE-ValueType
Pfam:V-ATPase_H_N 34 309 2.3e-7 PFAM
low complexity region 362 391 N/A INTRINSIC
low complexity region 589 603 N/A INTRINSIC
Predicted Effect probably benign
SMART Domains Protein: ENSMUSP00000126238
Gene: ENSMUSG00000021557

DomainStartEndE-ValueType
low complexity region 250 279 N/A INTRINSIC
low complexity region 477 491 N/A INTRINSIC
Predicted Effect probably benign
SMART Domains Protein: ENSMUSP00000130939
Gene: ENSMUSG00000021557

DomainStartEndE-ValueType
low complexity region 362 391 N/A INTRINSIC
low complexity region 589 603 N/A INTRINSIC
Pfam:Peptidase_M14 847 1123 1.2e-26 PFAM
Predicted Effect probably benign
Predicted Effect probably benign
Predicted Effect probably benign
Predicted Effect probably benign
Predicted Effect probably benign
Predicted Effect probably benign
SMART Domains Protein: ENSMUSP00000128589
Gene: ENSMUSG00000021557

DomainStartEndE-ValueType
Pfam:V-ATPase_H_N 34 309 2.4e-7 PFAM
low complexity region 362 391 N/A INTRINSIC
low complexity region 589 603 N/A INTRINSIC
low complexity region 787 795 N/A INTRINSIC
Predicted Effect probably benign
SMART Domains Protein: ENSMUSP00000132697
Gene: ENSMUSG00000021557

DomainStartEndE-ValueType
Pfam:V-ATPase_H_N 34 309 2.3e-7 PFAM
low complexity region 362 391 N/A INTRINSIC
low complexity region 589 603 N/A INTRINSIC
Predicted Effect probably benign
Meta Mutation Damage Score Not available question?
Is this an essential gene? Probably essential (E-score: 0.761) question?
Phenotypic Category
Phenotypequestion? Literature verified References
behavior/neurological
Candidate Explorer Status CE: no linkage results
Single pedigree
Linkage Analysis Data
Penetrance 100% 
Alleles Listed at MGI

All alleles(17) : Gene trapped(6) Transgenic(1) Spontaneous(6) Chemically induced(4)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00544:Agtpbp1 APN 13 59450172 missense probably damaging 1.00
IGL00808:Agtpbp1 APN 13 59462094 missense possibly damaging 0.84
IGL01298:Agtpbp1 APN 13 59504226 missense possibly damaging 0.77
IGL01628:Agtpbp1 APN 13 59508063 splice site probably benign
IGL01921:Agtpbp1 APN 13 59512483 missense possibly damaging 0.71
IGL02189:Agtpbp1 APN 13 59500461 missense probably benign 0.01
IGL02325:Agtpbp1 APN 13 59500489 missense probably benign 0.01
IGL02700:Agtpbp1 APN 13 59528419 missense probably damaging 1.00
IGL02821:Agtpbp1 APN 13 59482601 missense possibly damaging 0.69
IGL03130:Agtpbp1 APN 13 59474589 missense possibly damaging 0.73
IGL03167:Agtpbp1 APN 13 59532080 splice site probably benign
IGL03218:Agtpbp1 APN 13 59500207 missense possibly damaging 0.94
gru UTSW 13 59473746 missense probably damaging 1.00
rio UTSW 13 59525241 critical splice acceptor site probably benign
wobble UTSW 13 59474550 missense probably damaging 1.00
R0025:Agtpbp1 UTSW 13 59500200 missense probably benign 0.00
R0025:Agtpbp1 UTSW 13 59500200 missense probably benign 0.00
R0276:Agtpbp1 UTSW 13 59462031 missense possibly damaging 0.93
R0413:Agtpbp1 UTSW 13 59514152 missense probably benign 0.24
R0559:Agtpbp1 UTSW 13 59497000 missense probably benign 0.32
R0848:Agtpbp1 UTSW 13 59533939 intron probably benign
R0943:Agtpbp1 UTSW 13 59500602 missense probably benign
R1196:Agtpbp1 UTSW 13 59450318 unclassified probably benign
R1421:Agtpbp1 UTSW 13 59495575 missense possibly damaging 0.86
R1531:Agtpbp1 UTSW 13 59500634 synonymous probably null
R1833:Agtpbp1 UTSW 13 59465983 critical splice donor site probably null
R1864:Agtpbp1 UTSW 13 59450202 missense possibly damaging 0.92
R1994:Agtpbp1 UTSW 13 59531058 missense probably damaging 1.00
R1995:Agtpbp1 UTSW 13 59531058 missense probably damaging 1.00
R2001:Agtpbp1 UTSW 13 59475803 frame shift probably null
R2006:Agtpbp1 UTSW 13 59500321 missense probably benign 0.00
R2397:Agtpbp1 UTSW 13 59474569 missense probably benign 0.10
R2918:Agtpbp1 UTSW 13 59497015 missense possibly damaging 0.90
R3873:Agtpbp1 UTSW 13 59460596 missense possibly damaging 0.88
R3924:Agtpbp1 UTSW 13 59500407 missense probably benign 0.01
R4649:Agtpbp1 UTSW 13 59528399 missense possibly damaging 0.89
R4913:Agtpbp1 UTSW 13 59500072 missense probably damaging 1.00
R4933:Agtpbp1 UTSW 13 59500572 missense probably benign
R4969:Agtpbp1 UTSW 13 59500578 missense probably benign
R5066:Agtpbp1 UTSW 13 59474550 missense probably damaging 1.00
R5139:Agtpbp1 UTSW 13 59500213 missense probably damaging 0.99
R5194:Agtpbp1 UTSW 13 59500639 missense probably benign 0.19
R5269:Agtpbp1 UTSW 13 59473743 missense probably damaging 1.00
R5352:Agtpbp1 UTSW 13 59473746 missense probably damaging 1.00
R5558:Agtpbp1 UTSW 13 59482580 missense probably benign 0.05
R5687:Agtpbp1 UTSW 13 59500515 missense probably benign
R5824:Agtpbp1 UTSW 13 59466099 missense probably damaging 1.00
R5979:Agtpbp1 UTSW 13 59534046 nonsense probably null
R6109:Agtpbp1 UTSW 13 59473746 missense probably damaging 1.00
R6264:Agtpbp1 UTSW 13 59450300 missense possibly damaging 0.89
R6413:Agtpbp1 UTSW 13 59500020 missense possibly damaging 0.90
R6498:Agtpbp1 UTSW 13 59477040 missense possibly damaging 0.71
R6747:Agtpbp1 UTSW 13 59544353 intron probably null
R6950:Agtpbp1 UTSW 13 59450266 missense probably benign 0.32
R7030:Agtpbp1 UTSW 13 59504294 missense probably damaging 1.00
R7180:Agtpbp1 UTSW 13 59466038 missense probably benign 0.11
R7196:Agtpbp1 UTSW 13 59533180 missense possibly damaging 0.83
Mode of Inheritance Autosomal Recessive
Local Stock Embryos, gDNA
Repository

none

Last Updated 2018-05-22 9:29 AM by Anne Murray
Record Created unknown
Record Posted 2007-07-30
Phenotypic Description
The drunk phenotype was identified among ENU-induced G3 mutant mice; drunk mice display a severe ataxia of gait. Cerebellar Purkinje cells from homozygous drunk mice begin to degenerate beginning at approximately 3 weeks of age until virtually all cells are lost by 7 weeks of age. Degeneration of photoreceptor cells also begins at 6 weeks of age.
 
Homozygous drunk and wild type mice recover at the same rate after sciatic nerve crush operation (please see Background).
 
Male drunk mice are sterile due to a reduced number of abnormally shaped sperm cells. The drunk stock was maintained by breeding homozygous females with heterozygous males.

 

Nature of Mutation
The drunk mutation mapped to Chromosome 13, and corresponds to a T to A transversion in the donor splice site of intron 11 (GTAGGG -> GAAGGG) in the Agtpbp1 (hereafter Nna1) gene (position 44998 in Genbank genomic region NC_000079 for linear genomic DNA sequence of Agtpbp1). Sequencing of cDNA from drunk mice demonstrates that the mutation results in skipping of exon 11, thus destroying the reading frame after codon 302 (encoding glutamine) and creating a premature stop codon at codon 318.
 
     <--exon 10  <--exon 11 intron 11-->   exon 12-->
43219 ACTTCTCAA……CCGCCTGAAG GTAGGGGTG………………ACGACATTGATTTAG 49311
300   -T--S--Q-……-P--P--E-                …-T--T--L--I--*  318
       correct    deleted                    aberrant
 
The donor splice site of intron 11, which is destroyed by the drunk mutation, is indicated in blue lettering; the mutated nucleotide is indicated in red lettering.
Protein Prediction
Figure 1. Domain structure of NNA1. The drunk mutation corresponds to a T to A transversion in the donor splice site of intron 11 in the Agtpbp1 gene. A premature stop codon is predicted to truncate the protein after amino acid 318. Click on each mututation in red for more specific information.
Nna1 (nervous system nuclear protein induced by axotomy) is a 1218 amino acid-containing protein, translated from 25 exons with no alternative splicing (1;2). Nna1 is conserved in the bacterium Zymomonas mobilis, flies, worms, mice and humans (1). Nna1 has a number of predicted protein domains and motifs (1). The most prominent feature of Nna1 is the presence of a zinc carboxypeptidase-like sequence located within a highly conserved 300-amino acid region towards the C-terminus. This 300-amino acid region is 96% identical in mice and humans. The carboxypeptidase domain of Nna1 contains a zinc-binding motif, which is characterized by two conserved histidines and a catalytic glutamate residue, at positions H912, E915 and H1009 in Nna1. In this region, there are also consensus sequences for a tyrosine phosphorylation site, nuclear localization signal, and an ATP/GTP binding motif of the P-loop type. In the rest of the protein, there are several more predicted tyrosine phosphorylation sites, one nuclear localization signal, and consensus phosphorylation sites for protein kinase C, casein kinase II and cGMP/cAMP-dependent kinases. The N-terminal 450 amino acids of Nna1 are leucine-rich and possess weak homology to armadillo repeat proteins.
 
The drunk mutation causes the skipping of exon 11, creating a predicted premature stop codon after the insertion of fifteen aberrant amino acids. The mutation likely results in nonsense mediated decay of the aberrant transcript.
Expression/Localization
Nna1 mRNA is expressed throughout the brain, with prominent expression in cerebellar Purkinje and granule cells, mitral cells of the olfactory bulb, thalamic neurons, dorsal root ganglia, and hippocampal CA3 neurons (1;3). In the retina, Nna1 is expressed in photoreceptor cells (3). Nna1 is highly expressed in testes, in developing and mature sperm (3), and in heart, skeletal muscle and kidney (1). No Nna1 expression was found in ovary, liver, stomach, small intestine, lung, adrenal gland, spleen and thymus (1). During development, Nna1 is expression is found in the embryonic central and peripheral nervous systems (1).
 
Nna1 has two nuclear localization signals, and GFP-fusions with Nna1 localize to both the nucleus and the cytoplasm in primary cortical neurons (1). However, no zinc carboxypeptidases except AEBP1 have yet been found in the nucleus [discussed in (4)]. Definitive localization of the native Nna1 is unknown.
Background
Figure 2. Overview of cell types in the cerebellum.  The cerebellum functions in fine motor control.  Cellular components of the cerebellum include Purkinje cells, granule cells, mossy fibers, and deep cerebellar nuclei.  Mossy fibers enter the granule cell layer and synapse onto granule cells. Granule cells, which receive excitatory input from mossy fi bers, send axons into the molecular layer where they split to form parallel fi bers.  Purkinje cell bodies are oriented in a single layer of the cerebellum (Purkinje cell layer), with their characteristic, extensive dendritic arbors occupying the molecular layer within which they receive synaptic input from the parallel fibers.  Climbing fibers originating from the inferior olive split into multiple terminal branches that also innervate Purkinje cell dendrites in the molecular layer.  The deep cerebellar nuclei receive inhibitory input from Purkinje cells and excitatory input from mossy fibers and climbing fi bers. The deep cerebellar nuclear cells generate signals that can modify movements already begun.
Zinc carboxypeptidases in the nervous system generally serve regulatory roles, removing a C-terminal amino acid (usually arginine) from secreted proteins and peptides in order to activate or inhibit them. For example, carboxypeptidase E/H (CPE/H) proteolyses several neuropeptides including proenkephalin, proinsulin, proneurotensin, promelanin-concentrating hormone [discussed in (4)], and procholecystokinin [discussed in (5)]. A spontaneous mutation eliminating enzyme activity in CPE/H exists in the Cpe fat/fat mouse, which develops obesity (6). Other carboxypeptidases also function in the nervous system (4).
 
Nna1 was first identified in a screen for inducible genes in a sciatic nerve transection paradigm and it is expressed in spinal motor neurons undergoing axon regeneration. Its role in these events is currently unknown (1). Nna1 is also mutated in the spontaneously occurring pcd (Purkinje cell degeneration) mutants. There are currently eight known phenotypic alleles of pcd, out of which four have identified genetic lesions. In each of these four cases, protein levels are dramatically reduced by the genetic lesions (2;3). drunk is a new addition to the pcd allelic series.
 
The hallmark feature of pcd mice, as for drunk mice, is development of an ataxic gait between three and four weeks of age, which correlates with the onset of cerebellar Purkinje cell degeneration (7). Purkinje cells (Figure 2) proceed to deteriorate rapidly and die over the subsequent two week period. Distinct areas of the cerebellum display different rates of Purkinje cell degeneration, but all eventually die. Experiments with wild type-pcd chimeras demonstrated that this phenotype is cell autonomous (8). In addition to Purkinje cells, cerebellar granule cells (Figure 2) also display progressive death, with near normal numbers at three months declining to 5% by 20 months of age (7). Selected thalamic neurons also degenerate between postnatal days 50 and 60, and in addition, degeneration of retinal photoreceptors and olfactory bulb mitral cells progresses slowly over a year (3). Male pcd homozygous mice are sterile due to a reduction in the number of spermatozoa, which are sometimes abnormally shaped and immotile as well (3).
Putative Mechanism
The molecular mechanisms underlying the pcd phenotypes have been under investigation for decades, yet much is still unknown. Several groups published findings supporting a role for abnormally increased apoptosis in pcd mice. A 5-fold increase in the mRNA levels of c-fos, junB and krox-24, which are associated with neuronal apoptosis, has been detected specifically in cerebellar Purkinje cells of pcd mice at postnatal day 22, at the onset of cell death (9). These transcription factors may dictate cellular outcome by coordinating the expression of various anti- and pro-apoptotic proteins. In fact, mRNA levels of the anti-apoptotic protein Bcl-2 are reduced by 35% while those of the pro-apoptotic Bax remain unchanged in pcd mice at 22 days of age (9). Because Bcl-2 prevents apoptosis by binding and physically inhibiting Bax, a decrease in Bcl-2 may favor apoptosis by de-repressing Bax activity. Consistent with the hypothesis that pcd Purkinje cells undergo increased apoptosis, nuclear DNA fragmentation (10) and activated caspase-3 (11) have been observed in these cells.
 
However, genetic studies investigating the potential contribution of several cell death pathways demonstrated that Bax is not involved in Purkinje cell death of pcd mice, as pcd3jbax-/- mice still develop ataxia and lose Purkinje cells (4). The p53 pathway was also tested by generating homozygous ATM-null or Puma-null pcd mice. ATM serves to phosphorylate and activate p53 upon DNA damage, resulting in either DNA repair or apoptosis. Puma is a pro-apoptotic protein and a transcriptional target of p53. Neither combining ATM nor Puma mutations with the Nna1 mutation led to rescue of the pcd phenotype, strongly suggesting that the p53 pathway is not involved in pcd Purkinje cell death (4).
 
A recent report supports a role for ER stress in the pcd phenotype. The levels of endoplasmic reticulum (ER)-specific chaperone BiP, and the ER-stress related transcription factor CHOP are increased pcd mice at 23 and 26 days of age (11). Moreover, an unusual configuration of the ER with associated electron-dense particles was observed during the early characterization of pcd mice (12).
 
Finally, the putative carboxypeptidase substrate-binding site of Nna1 was demonstrated to be required for rescue of the pcd phenotype (13). Purkinje cell-specific transgenic mice expressing wild type Nna1 were generated and crossed with pcd3j homozygous mice. pcd3jNna1-transgenic mice showed no Purkinje cell degeneration or ataxia. Sequence comparisons of Nna1 with other carboxypeptidases and structural modeling predicted R962 as part of the catalytic site, while N970 and R971 constituted the substrate-binding site of Nna1. Significantly, Purkinje cell-specific pcd transgenic mice expressing a putative substrate-binding site mutant (Nna1N970A R971A) still displayed ataxia and Purkinje cell degeneration. These results indicate that N970 and R971 are essential for Nna1 to support Purkinje cell survival, and suggest that Nna1 is a true carboxypeptidase.
Primers Primers cannot be located by automatic search.
Genotyping
Drunk genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transition. The same primers are used for PCR amplification and for sequencing.
 
Primers
Drunk(F): 5’-TTCCAGTACCGCAGTGCTGAGCTGTG -3’
Drunk(R): 5’-CATGGCTCAGTGGGACTGTGGCAAG -3’
 
PCR program
1) 94°C             2:00
2) 94°C             0:30
3) 62°C             0:30
4) 72°C             1:00
5) repeat steps (2-4) 35X
6) 72°C             5:00
7) 4°C               ∞
 
The following sequence of 689 nucleotides (from Genbank genomic region NC_000079 for linear genomic sequence of Agtpbp1, sense strand) is amplified:
 
44764    ttccagt accgcagtgc tgagctgtgg tctaactctc atcatcttgt ttgaacagga
44821 gtgcttggcg gtcaggactc ttgatcctct tgtcaacaca tccagtctga taatgagaaa
44881 atgcttcccc aaaaaccgcc ttccgctccc caccattaaa agttctttcc acttccaatt
44941 gccaattatc cctgtgactg gacctgtggc ccagctctac agcttgccgc ctgaaggtag
45001 gggtgggcca tgggctgctg tgggagtccg tgactctgtt gtcgaaaatg tgtatctggt
45061 tgtttaatgt gcgcatgagg tgtgaaaata cacaaaaatt tgtcagcaat caatgtaaat
45121 attaaaagta taaggtggag acattatata agttgatgtc atacaagata gtgacattac
45181 attaagagga tgacatcaga tgctcagtat gtttgcgatt acctgtgtag ctttttaaaa
45241 gaattttaat tacacattgg tagtagcatc attattgtca ctatgtgggc atccacatat
45301 gttgtgtgtg ccatgtacat gcacatttgt gctcctgggt ctgtgagcac gtgagagtca
45361 gaggacagcc ttcagatgtt ggttcttccc tttaccaggt ggttgcaagg atcaaatgta
45421 tatcaggctt gccacagtcc cactgagcca tg
 
Primer binding sites are underlined; the mutated T is highlighted in red.
References
Science Writers Eva Marie Y. Moresco
Illustrators Diantha La Vine
AuthorsPia Viviani, Xin Du, Bruce Beutler
Edit History
2011-09-08 5:36 PM (current)
2011-01-07 9:04 AM
2010-11-02 8:46 AM
2010-11-02 8:45 AM
2010-11-02 8:44 AM
2010-01-18 12:00 AM