Molecular Vision 2010; 16:425-437 <http://www.molvis.org/molvis/v16/a48>
Received 28 December 2009 | Accepted 5 March 2010 | Published 12 March 2010

© 2010 Molecular Vision

TRPM1 mutations are associated with the complete form ofcongenital stationary night blindness
Makoto Nakamura,1 Rikako Sanuki,2,3 Tetsuhiro R. Yasuma,1 Akishi Onishi,2,3 Koji M. Nishiguchi,1Chieko Koike,2,4 Mikiko Kadowaki,2 Mineo Kondo,1 Yozo Miyake,1,5 Takahisa Furukawa2,3
1Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Department of Developmental

Biology, Osaka Bioscience Institute, Osaka, Japan; 3JST, CREST, Suita, Osaka, Japan; 4JST, PRESTO, Kawaguchi, Saitama, Japan;
5Department of Orthoptics and Vision Science, Aichi Shukutoku University, Nagoya, Japan

Purpose: To identify human transient receptor potential cation channel, subfamily M, member 1 (TRPM1) gene mutations
in patients with congenital stationary night blindness (CSNB).
Methods: We analyzed four different Japanese patients with complete CSNB in whom previous molecular examination
revealed no mutation in either nyctalopin (NYX) or glutamate receptor, metabotropic 6 (GRM6). The ophthalmologic
examination included best-corrected visual acuity, refraction, biomicroscopy, ophthalmoscopy, fundus photography,
Goldmann kinetic perimetry, color vision tests, and electroretinography (ERG). Exons 2 through 27 and the exon-intron
junction regions of human TRPM1 were sequenced.
Results: Five different mutations in human TRPM1 were identified. Mutations were present in three unrelated patientswith complete CSNB. All three patients were compound heterozygotes. Fundus examination revealed no abnormalities
other than myopic changes, and the single bright-flash, mixed rod-cone ERG showed a “negative-type” configuration witha reduced normal a-wave and a significantly reduced b-wave amplitude. Our biochemical and cell biologic analyses suggestthat the two identified IVS mutations lead to abnormal TRPM1 protein production, and imply that the two identified
missense mutations lead to the mislocalization of the TRPM1 protein in bipolar cells (BCs).
Conclusions: Human TRPM1 mutations are associated with the complete form of CSNB in Japanese patients, suggestingthat TRPM1 plays an essential role in mediating the photoresponse in ON BCs in humans as well as in mice.

The complete form of congenital stationary night
blindness (CSNB) is a subtype of Schubert-Bornschein CSNB
in which the fundus is essentially normal except for myopicchanges [1-5]. From early childhood, patients with complete
CSNB lack rod function and experience night blindness.
Nystagmus and amblyopia sometimes accompany the othersymptoms, and the clinical course is stationary. Bestcorrected visual acuity is mildly reduced, and high tomoderate myopia is usually found. Electroretinogram (ERG)examinations reveal absent depolarization of neuron by light(ON)-responses (b-wave), and previous extensive physiologic
studies indicated that the pathology in complete CSNB lies inthe dysfunction of the depolarizing ON bipolar cell (BC).
There are two hereditary patterns for complete CSNB: Xlinked recessive and autosomal recessive [4].
To date, two genes, the leucine-rich proteoglycan
nyctalopin
gene(NYX)—encoding
theglycosylphosphatidylinositol (GPI)-anchored extracellular
protein nyctalopin [6,7]—and the GRM6 gene—encoding themetabotropic glutamate receptor mGluR6—have been
Correspondence to: Takahisa Furukawa, Department ofDevelopmental Biology, Osaka Bioscience Institute, JST, CREST,
Suita, Osaka, Japan; Phone: +81668724853; FAX: +81668723933;
email: furukawa@obi.or.jp

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identified as the mutated gene in X-linked recessive andautosomal recessive complete CSNB [8-10], respectively.
Both nyctalopin and mGluR6 proteins are distributed on thepostsynaptic ON BCs and are required for the depolarizationof the cell. The NYX gene appears to be the major, and possibly
only, causative gene for X-linked recessive complete CSNB
since NYX gene mutations were identified in the majority of
X-linked recessive families with complete CSNB. In contrast,
GRM6 gene mutations have been found in only some of theautosomal recessive families with complete CSNB, indicating
the existence of other unknown genes for autosomal recessive
complete CSNB.
We previously identified a mouse transient receptor
potential cation channel, subfamily M, member 1 (Trpm1)
homolog of human TRPM1 [11]. Trpm1 is alternatively
spliced, resulting in the production of a long form protein(trpm1-L) and a short N-terminal form devoid oftransmembrane segments (trpm1-S). Although mouse trpm1S was previously identified as melastatin [12], mouse trpm1L has not been identified. Without distinction between trpm1L or trpm1-S, trpm1 has been reported to be detected in humanprimary melanocytes [13], poorly metastatic melanoma celllines [14,15], mouse retinal pigment epithelium (RPE) [16],and subsets of ON and hyperpolarization of neuron by light(OFF) BCs [17,18]. We found that trpm1-L localization isdevelopmentally restricted to the dendritic tips of ON BCs inMolecular Vision 2010; 16:425-437 <http://www.molvis.org/molvis/v16/a48>© 2010 Molecular Visionco-localization with mGluR6 [11,19]. Trpm1 null mutant
mice completely lose the photoresponse of ON BCs. Trpm1L channel activity is negatively regulated by activated Go in
the mGluR6 cascade, and we showed that Trpm1-L is acomponent of the ON BC transduction channel. Interestingly,
the trpm1-deficient mice showed similar electrophysiologicalresponses to the responses of those with complete CSNB—
significantly reduced b-waves—which led us to examine
whether the gene is mutated in human patients with complete
CSNB.
Very recently three groups reported the association of
TRPM1 mutations with CSNB [20-22]. Independently fromthese studies, in our present study, we identified five different
novel mutations in the human TRPM1 gene: IVS2–3C>G,
IVS8+3_6delAAGT, R624C (c.1870C>T), S882X (c.
2645C>A), and F1075S (c.3224T>C). In addition, ourbiochemical and cell biologic analyses suggested that the two
intron mutations were likely to result in abnormal protein
production by abnormal splicing, and the two missense
mutations lead to the mislocalization of the TRPM1 protein
in BCs. Fundus examination revealed no abnormalities otherthan myopic changes, and the single bright-flash, mixed rodcone ERG showed a “negative-type” configuration with areduced normal a-wave and a significantly reduced b-wave
amplitude.

TABLE 1. THE PRIMER SETS USED IN THIS STUDY.
Exon
2
2
3–1
3–1
4–2
4–2
4
4
5
5
6
6
7
7
8
8
9
9
10
10
11
11
12
12
13
13
14
14
15
15
16
16
17
17
18
18
19
19
20
20
21
21
22
22
23
23
24
24
25
25
26
26
27–1
27–1
27–2
27–2
27–3
27–3
27–4
27–4
27–5
27–5
27–6
27–6

Direction
Forward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReversePrimer sequence (5′-3′)
GCTTGGGAATGTATTGGTCC
TTCTGGACTCCTCTTTCTGC
TTGCTAATGAAGGGGAAAGGTGAAAGCTGTGATCCTAGTG
TACCCAACAGATTCCTATGG
TTGTGGATTCAGAGCTTTGG
TGCTTGGACATGAGCAATAGTATTAGGAAGCTCTTTGGGGGATCTGTAGGGAATGTAGGG
CAACTGCATCTGAATCAACG
TATTGTGCTTGCTTCCCTTG
GCAATCAATCTCCTTCTCAGTACTCTTCCCCATACTTCAG
CGTTTGGAATGTCTAAGCAG
CCACAGCAAAGTCTCAAATCAATAACCCTGTCATGCACACCAGTCTCTGTGGTCATTTTG
TTCATGCTGAGACTGCTCAAG
AAGCCAGCAATGCTTAGTTCTGTCACAAGGGAGAACATAC
ATGTGTGCACATACAGAGGTGAAGCAAGGACAAGAACCATACCCTTTCCTCTGTGTTTCCGCACAATATGCACCAGTGACTACTGACCTCAAGTGATCTG
TTCATTATCTCCTGTGCCTG
CTGTTACATGAACTCCCAAG
TTGTTGAAAAGCAAGCGAGG
CCATTCGTATGTCCACTATCGAGTAGTATCGTATATTCGC
AGTTCTGGAACTCTTTGTTG
AGAGACAAGTACTCAGGATG
GGTGGATATGCCTGTCTAAGAAAGTGCTCAGTTCAGCCTG
GGCTCCTAGGTTATTGAATAGTGGTACAAGAAATCTCAACTTCTAATGGTGCCTGTGTGCCAATGTTAGCCAGAGATCTC
GTGTCAACCTGAAAGAAAACTTTCAGTAAGGAGCCATATC
ATGACCTGGAATGTTCCATC
GCCTGTTCTTATGTCCTAACTATAATGAGCCAGGGTGAAC
TTTGGAACTGATCATCTGCC
CTTTTCTCTCAACCATCATC
ACAGTGAATTTGTGGTTCTCAGCTGGGGCTACAGAGTTTATATCTTGGGAGCGTTCTGAGGTAACTTTGACTGCTCTGGG
CGAGCAAGTAGTTGAGTGAG
GGGCTGTGAAATTCTCATTG
AACTCTGGCATCCCCCATAG
CATGTTAGGGGATTGTGAAGATACGTTGCCTCACATTCAGCAGAATGGTGAATGCTCTTGGAAAGGTGAAGACTGTTCTGAAAAAAACCTGTTCCTTCCG
TTTGTCGTTTCCACTGTTAGGAAGAAACTATTTCCCCAAGGAATATCTGTGCTATGAGAGTAGAGTACAGTTCAATCACGATGGATTTCACATTCCTAGG
TCTGTGAAGCCAGATCAAACGTTTAGATGGCCAAGATGACMETHODS
Subjects: Four separate patients with complete CSNB (#204,#373, #437, and #484) in whom previous molecularexamination revealed no mutation in either NYX or GRM6
among 11 separate patients were analyzed. They were all
male, and their ages were 26, 9,19, and 27 years, respectively.
They complained of night blindness from early childhood, andotherwise had no health problems. All individuals examined
had been under observation at the Department ofOphthalmology of Nagoya University, Nagoya, Japan. The
research protocol was designed in compliance with the
Declaration of Helsinki and approved by the institutional
review board. Written informed consent was obtained fromthe subjects after an explanation of the purpose of this study
was provided. Briefly, the protocol entails molecular analysis
of causative genes from blood samples of patients withcomplete CSNB and in vitro analysis including expression
assay and cytological assay using the samples without any
disadvantages for the patients.
DNA analysis of patients with complete CSNB: Genomic
DNA was extracted from the peripheral blood leukocytes.
Exons 2 through 27 of TRPM1 were amplified by polymerase
chain reaction (PCR) using the DNA Thermal Cycler 9700
(Applied Biosystems, Foster City, CA). Primers were
designed by and purchased from Invitrogen (Carlsbad, CA).
The primer sets used in this study are shown in Table 1. For
all exons, 100 ng of genomic DNA was amplified in a 50 µl
reaction with 0.5 µM of each primer, 0.2 mM of each dNTP,
and AmpliTaq Gold polymerase (Applied Biosystems). The
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PCR conditions were as follows: 5 min at 94 °C; 35 cycles at
94 °C for 30 s, followed by 30 s at 54 °C, and 45 s at 72 °C;
and a final extension step at 72 °C for 7 min. The PCR products
were directly sequenced using an ABI PRISM 3100 Genetic
Analyzer (Applied Biosystems) and BigDye Terminator v3.1
Cycle Sequencing Kit (Applied Biosystems). To search forpolymorphisms, exons 3, 8, 16, 21, and 24 of TRPM1 from
200 alleles (50 men and 50 women) from unrelated, normal
Japanese individuals were directly sequenced.

solution (5 μg/μl) was injected into the subretinal space of P0mouse pups, and square electric pulses (80 V; five 50 ms
pulses with 950 ms intervals) were applied with electrodes.
Wild-type (WT) and mutant forms of TRPM1 (R624C andF1075S) containing 3× Flag at the C-terminus were expressedfrom the mouse mGluR6 promoter (mGluR6-TRPM1–
3×Flag). To generate this construct, the chicken β-actin gene
(CAG) promoter region in CAG-TRPM1 was replaced with
the 9.5 kb fragment of mGluR6 5′ upstream genomic sequence[24]. mGluR6-TRPM1–3×Flag (4 μg/μl) and CAG-GFP(1 μg/μl) were co-electroporated and harvested at P14.
Immunohistochemistry to observe the subcellular localizationof TRPM1–3×Flag expressed in the electroporated ON BCs
was performed on sections as previously described [23].
Mouse anti-Flag antibody (1:500), rabbit anti-PKCα antibody(1:20,000; Sigma), and rat anti-GFP antibody (1:1,000;
Nacalai Tesque) were used. The signal intensity of Flag
immunostaining at the dendritic tips and somas of ON BCs
was measured using AxioVision software (Carl Zeiss,
Oberkochen, Germany). The ON BCs that showed more
intensity in the dendritic tips than in the somas were counted.
One hundred electroporated ON BCs were counted from three
retinas independently prepared for each construct.
Clinical evaluations in patients associated with TRPM1
mutations: The ophthalmologic examination included bestcorrected visual acuity, refraction, biomicroscopy,
ophthalmoscopy, fundus photography, Goldmann kinetic
perimetry, color vision testing, and electroretinography [25].
Standardized full-field ERGs were elicited by Ganzfeld
stimuli after pupillary dilation with 0.5% tropicamide and0.5% phenylephrine hydrochloride and 20 min of darkadaptation. The rod (scotopic) ERGs were elicited by a blue
stimulus with a luminance of 5.2×10−3 cd/sec•m2. The mixed
rod-cone, single flash (bright white) ERGs were elicited by awhite stimulus of 44.2 cd/sec•m2. The photopic (cones) singleflash ERGs and 30 Hz flicker ERGs were elicited by white
stimuli at a luminance of 4 cd/sec•m2 and 0.9 cd/sec•m2,
respectively, on a white background of 68 cd/m2. The single
flash bright white ERGs were recorded again after 17 h of dark
adaptation.
ERG recordings in the trpm1 null mutant (trpm1–/–) mice: We
generated trpm1–/– mice as described previously [11]. Our
methods for recording the ERGs from the mice have been
described in detail [26]. In brief, the mice were dark-adapted
overnight, and then anesthetized with an intramuscular
injection of 70 mg/kg ketamine and 14 mg/kg xylazine. ERGs
were recorded with a gold-wire loop electrode placed on theanesthetized cornea. The mice were placed in a Ganzfeld bowl
and stimulated with stroboscopic stimuli of 1.0 log cd-s/m2(photopic units) maximum intensity. Four levels of stimulus
intensity ranging from −5.0 to 1.0 log cd-s/m2 were used forthe dark-adapted ERG recordings, and four levels of stimulus
intensity ranging from −0.5 to 1.0 log cd-s/m2 were used forthe light-adapted ERGs. The light-adapted ERGs were

TRPM1 splicing minigene constructs: DNA fragments of
TRPM1 exon 2 with its downstream splice donor region(nucleotide position in GenBank # NC_000015: 24738–
25055; exon 2+SD) and exon 8 with its downstream splicedonor region (39020–39392; exon 8+SD) were PCRamplified using human genomic DNA as a PCR template;
SacI and KpnI sites were appended to the 5′ and 3′ ends,
respectively. A partial fragment of β-globin (HBB) intron 2(614–1166 in GenBank # NC_000011) was PCR-amplified;
KpnI and SalI sites were appended to the 5′ and 3′ ends,
respectively. DNA fragments of TRPM1 exon 3 with its
upstream splice acceptor region (31325–31691; SA+exon 3)
and exon 9 with its splice acceptor region (39976–40280; SA+exon 9) were PCR-amplified using human genomic DNA asa PCR template; SalI and BamHI sites were appended to the
5′ and 3′ ends, respectively. The exon 2+SD, HBB intron, andSA+exon 3 fragments (exon 2+3 construct), or exon 8+SD,
HBB intron, and SA+exon 9 (exon 8+9 construct) were ligatedand subcloned into the SacI and BamHI sites of pBluescript
KS+ (Stratagene), containing three repeats of the Flag-tag
between the BamHI and NotI sites. The generated exon 2+3
construct and exon 8+9 construct were digested with SacI and
NotI and subcloned into SacI and BamHI digested pEGFP-C3
and pEGFP-C2 (Clontech, Palo Alto, CA), respectively, eachwith a synthesized BglII-NotI linker.
Transfection and western blots analysis: HEK293T cells were
cultured in D-MEM containing 10% fetal bovine serum (FBS;
Nissui, Tokyo, Japan). These cells were grown under 5%
carbon dioxide at 37 °C. Transfections were performed using
the calcium phosphate method. Transfected cells wereincubated at 37 °C for 48 h, and harvested for further analysis.
Cell extract proteins were separated by SDS–PAGE, andtransferred to a polyvinylidene difluoride membrane (ATTO,
Tokyo Japan). The membrane was incubated with a mouseanti-Flag antibody (1:1,000; Sigma, St Louis, MO), a rabbit
anti-β−gal antibody (1:10,000; Chemicon, Temecula, CA), ora mouse anti-β−actin antibody (1:2,000; Sigma), andincubated with a horseradish peroxidase-conjugated goat antimouse or -rabbit IgG (1:10,000; Zymed Laboratories, San
Francisco, CA). The bands were visually developed using
Chemi-Lumi One L (Nacalai Tesque, Kyoto, Japan).
In vivo electroporation and section immunohistochemistry:
In vivo electroporation was performed at postnatal day 0 (P0)
as described previously [23]. Approximately 0.3 μl of DNA
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They were interpreted as likely pathogenic because thearginine at position 624 and the phenylalanine at 1075 are wellconserved among the TRPM subfamily, suggesting theimportance of these amino acid residues for TRPM function(Figure 1E). The two mutations were not found in an analysisof 100 Japanese normal controls. None of the five mutations
mentioned above have been reported in a SNP database.

recorded on a rod-suppressing white background of 1.3 log
cd-s/m2 (photopic units). The amplitude of the a-wave wasmeasured from the baseline to a set time of 8 msec after the
initial negative wave for the dark-adapted ERG, and 11 msec
for the light-adapted ERG. The b-wave amplitude wasmeasured from the negative trough of the a-wave to the
maximal positive peak.
Animal care: All procedures conformed to the ARVO
Statement for the Use of Animals in Ophthalmic and Vision
Research, and these procedures were approved by theInstitutional Safety Committee on Recombinant DNA
Experiments and the Animal Research Committee of Osaka
Bioscience Institute. The mice were housed in a temperaturecontrolled room with a 12h:12h light-dark cycle. Fresh waterand rodent diet were available at all times.

Analysis of protein expression from TRPM1 mutant
minigenes: Our previous expression analysis of mouse
TRPM1 showed that the long form (TRPM1-L), whichfunctions as a cation channel, is predominantly expressed in
the retina [11], suggesting that human patient samples for
directly analyzing splicing are not readily available. To
overcome this limitation, minigenes for expression in tissue
culture cells were designed; these minigenes included the
upstream exons (exon 2 or 8) fused with EGFP, downstreamexons (exon 3 or 9) fused with 3×Flag-tags, and a HBB intron,
which is often used in splicing assays (an exon 2+3 minigene
and an exon 8+9 minigene; Figure 2A). Since the signals
required for constitutive splicing are usually within ~100
nucleotides of splice junctions [11], we took ~200 nucleotide
regions for both splicing donors and acceptors to ensure thesplicing assay. We also generated mutated minigenes
containing the IVS2–3C>G mutation in the exon 2+3
minigene or the IVS8+3_6delAAGT mutation in the exon 8+9
minigene. Minigene splicing was analyzed in HEK293T cells
since they have been used extensively to study constitutive
RNA splicing. HEK293T cells were transfected withminigene DNA and a lacZ expression internal control vector,
and western blotting was performed on whole cell extracts
isolated 48 h later. We then analyzed protein products of the
EGFP-exon 2+3 fusion and the EGFP-exon 8+9 fusion
proteins by western blot analysis of whole cell extracts
transfected with the WT and mutated minigenes. We detected
an expected 45 kDa or 39 kDa band with the anti-Flag
antibody in HEK293T cell extracts transfected with the WT
exon 2+3 minigene or the exon 8+9 minigene (Figure 2B,C).
In contrast, we did not detect any bands in either of the cell
extracts transfected with the mutated minigenes. We detected
the β-gal bands of the transfection internal control, and the βactin bands of the cell extract control in both the WT andmutant minigene transfected cells. These results showed thatboth the IVS2–3C>G and IVS8+3_6delAAGT mutations
abrogate normal splicing and lead to abnormal protein
production, suggesting that these trpm1 alleles are loss-offunction alleles.

RESULTSDNA analysis of the complete CSNB patients: Five different
mutations in TRPM1 were identified: IVS2–3C>G,
IVS8+3_6delAAGT, R624C (c.1870C>T), S882X (c.
2645C>A), and F1075S (c.3224T>C). Mutations were
present in three unrelated patients with complete CSNB(#373, #437, and #484). All three patients were compound
heterozygotes. Their pedigrees and the DNA sequencechromatogram for the patients harboring these mutations arepresented in Figure 1A,B.
The two intron changes, IVS2–3C>G and
IVS8+3_6delAAGT (Figure 1C), were interpreted to bepathogenic because no RNA splicing activities were detected
when each of the two changes was present (see splicing assay
section). They were also predicted to affect RNA splicing, asassessed by an online splice-site prediction tool. The donorsite prediction score from the software for the exon 8 donorsite in TRPM1 was 0.99 for the WT sequence, whereas it wasunder 0.01 for the corresponding mutated sequence with the
IVS8+3_6delAAGT sequence change. The acceptor site
prediction score for the exon 3 acceptor site in TRPM1 was
0.98 for the WT sequence, whereas it was 0.44 for the mutatedsequence with the IVS2–3C>G sequence change. The twomutations were not found in an analysis of 100 Japanesenormal controls.
The S882X mutation was considered to be a pathogenicnull mutation since mRNA containing the predicted early stopcodon would most likely be subject to nonsense mediateddecay without being translated into a protein [27]. Even if thetranslated protein was produced, it would lack the C-terminus
region of TRPM1, including transmembrane domains 4–6,
which is very likely to lead to a loss of function as a cation
channel (Figure 1D). This change was not found among the
100 healthy control individuals we examined. The two
missense changes in the TRPM1 gene, R624C and F1075S,
were located in the N-terminal region and in the C-terminalend of the transmembrane domain, respectively (Figure 1D).

In vivo analysis of intracellular localization of TRPM1
protein carrying missense mutations: The TRPM1 missense
mutations R624C and F1075S are located in the N-terminal
and the C-terminal end of the six-transmembrane domains(6TM), respectively (Figure 1D). Mutations in these regions
often cause misfolding/mislocalization of the protein, and/or
a defect in channel activity [28-32]. To determine the428Molecular Vision 2010; 16:425-437 <http://www.molvis.org/molvis/v16/a48>© 2010 Molecular Vision

Figure 1. Compound heterozygous TRPM1 mutations identified in patients #373, #437, and #484. A: Sequence chromatograms showing themutations: IVS2–3C>G in patient #373 (a), IVS8+3_6delAAGT in patient #484 (b), Arg624Cys (c. 1870C>T) in patients #437 and #484 (c),Ser882Ter (c. 2645C>A) in patient #437 (d), and Phe1075Ser (c. 3224T>C) in patient #373(e). B: Complete CSNB pedigrees of Japanesepatients #373, #437, and #484. These three patients are compound heterozygotes of TRPM1 mutations. C: Exon structures of human
TRPM1. The first methionine (Start) and a stop codon (Stop) of the TRPM1 open reading frame are indicated. All mutations found in thisstudy are shown. D: Putative topology of the human TRPM1. All mutations found in this study are illustrated. The six transmembrane domains
are indicated as TM1-TM6. E: Alignment of R624 and F1075 in TRPM proteins. Sequence alignment of TRPM1 from human, bovine, mouse,chick, and TRPM2, TRPM4, and TRPM7 from human. Amino acid residues R624 and F1075 are boxed. The asterisks indicate completely
conserved residues.

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Figure 2. Analysis of protein expression
from TRPM1 mutant minigenes. A:
Schematic of TRPM1 minigenes. The
minigene IVS2–3C>G contains an
EGFP gene, exon 2, the exon 2 splicedonor region, a portion of the β-globin(HBB) intron 2, the exon 3 splice
acceptor region, and exon 3, and uses the
CMV promoter and SV40 polyA sitefrom the pEGFP-C3 vector. The
minigene IVS8+3_6delAAGT contains
an EGFP gene, exon 8, the exon 8 splicedonor region, a portion of the HBB
intron 2, the exon 9 splice acceptor
region, and exon 9, and uses the CMV
promoter and SV40 polyA site from thepEGFP-C2 vector. Mutations of IVS2–
3C>G and IVS8+3_6delAAGT are
shown. B-C: western blots of HEK293
cell extracts transfected with minigenes
IVS2–3C>G(B)
or
IVS8+3_6delAAGT(C).
WT
represents protein from a wild-type
minigene transfection, and asterisks
represent spliced EGFP detected by ananti-Flag antibody. β-Gal and β-actin
were used for transfection control andloading control, respectively.

functional relevance of R624C and F1075S TRPM1 residues,
we mutagenized the TRPM1 cDNA to introduce R624C andF1075S mutations. We first prepared expression vectors
encoding R624C or F1075S mutant proteins attached with a
Flag-tag at the C-terminus, and expressed them both, as wellas WT vectors, in HEK293T cells. We did not observe asignificant difference in the protein level between the mutants

and WT by western blotting, suggesting that the protein
stability of R624C and F1075S was not affected (data notshown). We then prepared expression vectors of R624C- andF1075S-TRPM1 cDNAs fused with the mouse mGluR6 9.5 kb
promoter that drives ON BC-specific expression (Figure 3A).
We electroporated each of them as well as the WT vector into
the P0 mouse retinas. We examined the localization of the430

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expressed TRPM1 mutant proteins in ON BCs at P14 whenthe endogenous mouse TRPM1 is preferentially localized tothe dendritic tips [11,19]. The electroporated human TRPM1
was visualized by an anti-Flag tag antibody. TRPM1 WT was
localized at the soma (arrow) and dendritic tips (arrowheads)of the ON BCs (Figure 3B,C). On the other hand, theimmunostaining signals of both the R624C and the F1075S
TRPM1 proteins were significantly weaker than those of theWT protein (Figure 3D-G). We quantified the number ofelectroporated ON BCs in which the WT TRPM1 protein was
localized more in the dendritic tips than somas. About 70% of
ON BCs that electroporated with WT TRPM1 showed abrighter TRPM1 signal at the dendritic tips, and the signals on
the dendritic tips were dramatically reduced in both mutant
proteins (Figure 3H). These results may imply that R624C andF1075S mutations lead to mislocalization of TRPM1 protein
and are responsible for the protein trafficking of TRPM1channel proteins. However, it should be noted that there is also
a possibility that the transfected TRPM1 proteins are
misfolded due to the addition of the 3xFlag tag, leading to theundetectability of Flag-tagged mutant proteins specifically inthe dendrites. Future analysis is required to obtain aconclusive result on this point.
Clinical characteristics of the complete CSNB patientsassociated with TRPM1 mutations: The clinicalcharacteristics of the three complete CSNB patients with
TRPM1 mutations are summarized in Table 2. All the patients
showed the typical clinical features of complete CSNB. They
complained of night blindness from their early childhood, andthe best-corrected visual acuities were normal in two patients(#373 and #437) and mildly reduced in one (#484). The
refractive errors were highly or moderately myopic, andastigmatism was present in all of the patients (Table 2). Mildnystagmus and esotropia was observed in the patient with
reduced visual acuity (#484). Slit lamp examination revealed
no abnormality, and all fundus examinations revealed noabnormalities in the posterior pole other than myopic changes,
including the tilted discs and temporal pallor of the optic discs(Figure 4). Goldmann kinetic visual fields and color vision
tests with the Ishihara plates, the AO H-R-R
pseudoisochromatic plates, and Farnsworth D-15 panel test
were conducted in two patients (#437 and #484) with normal
results. No family history was obtained from the patients(Figure 1B).
The full-field scotopic (rod) ERGs elicited by a blue light
were non-recordable after 20 min of dark-adaptation (Figure
5B). The photopic ERG showed an a-wave with apparently
normal implicit time and a b-wave with delayed implicit time
(Figure 5C). The amplitudes of the 30 Hz flicker ERG were
within normal range (Figure 5D). The single bright-flash,mixed rod-cone ERG elicited by a white stimulus had a“negative-type” configuration with a reduced normal a-wave
and with a significantly reduced b-wave amplitude (Figure
5A). The oscillatory potentials were reduced.

ERG analysis of the trpm1 null mutant (trpm1–/–) mice: To
address a possible role of TRPM1 in ON BC function, we
previously generated trpm1 null mutant (trpm1–/–) mice bytargeted gene disruption [11]. Next, to compare thephysiologic retinal function of trpm1–/– between humans andmice, we recorded mouse ERGs in 8-week-old mice (Figure6). The dark-adapted (rod) ERGs elicited by different stimulus
intensities from WT and trpm1–/– mice are shown in Figure
6A. The amplitudes of the a-wave, originating from thephotoreceptors, were approximately equal for both types ofmice (Figure 6B). In contrast, the dark-adapted ERG b-wave,
originating mainly from the ON BCs, in trpm1–/– mice was notpresent at the lower intensities, and only a very small positive
inflection was seen at the highest stimulus intensity of 1.0 log
cd-s/m2 (Figure 6C). The ERGs elicited by different stimulus
intensities from light-adapted WT and trpm1–/– mice areshown in Figure 6D. The b-wave was severely depressed orabsent leaving only the a-wave in the trpm1–/– mouse (Figure
6E,F). These ERG results are similar to those obtained byother groups [19,33], and suggested that the function of bothrod and cone ON BCs, and the signal transmission from rod
and cone photoreceptors to BCs were severely impaired in
trpm1–/– mice. In contrast, the presence of a normal a-wave
indicated that the rod and cone photoreceptors werefunctioning normally in this mutant mouse.
DISCUSSION
An essential step in intricate visual processing is thesegregation of visual signals into ON and OFF pathways byretinal BCs [34]. The release of glutamate fromphotoreceptors modulates the photoresponse of ON BCs[35] via metabotropic glutamate receptor 6 (mGluR6) and the
G-protein (Go) that regulates a cation channel [36-38]. We
recently reported that we identified a mouse trpm1 long form
(trpm1-L) and found that TRPM1-L localization isdevelopmentally restricted to the dendritic tips of ON BCs in
co-localization with mGluR6 [11]. Trpm1 null mutant micecompletely lose the photoresponse of ON BCs. TRPM1-L
channel activity is negatively regulated by activated Go in themGluR6 cascade. These results suggest that TRPM1-L is acomponent of the ON BC transduction channel. Theseobservations led us to examine whether the gene is mutatedin human patients with complete CSNB in the current study.
To identify human TRPM1 gene mutations in CSNB
patients, we analyzed four separate Japanese patients withcomplete CSNB in whom previous molecular examination
revealed no mutation in either the NYX or GRM6 genes. In thecurrent study, we identified five different mutations in
TRPM1: IVS2–3C>G, IVS8+3_6delAAGT, R624C (c.
1870C>T), S882X (c.2645C>A), and F1075S (c.3224T>C).
Mutations were present in three unrelated patients withcomplete CSNB (#373, #437, and #484). Using a minigene
expression assay, we showed that two intron mutations,431

Molecular Vision 2010; 16:425-437 <http://www.molvis.org/molvis/v16/a48>© 2010 Molecular Vision

Figure 3. In vivo electroporation of WT,
R624C, and F1075S TRPM1 expression
vectors fused with the mGluR6
promoter. A: DNA constructs used forelectroporation. CAG-GFP was coelectroporated to identify electroporated
regions. WT and mutant forms of
TRPM1 fused with 3×Flag wereexpressed under the mouse mGluR6 9.5
kb
promoter.
B-G:
Sectionimmunohistochemistry of the P14
retinas electroporated in vivo at P0 with
CAG-GFP and mGluR6-TRPM1–
3×Flag.
The
sections
were
immunostained with PKCα (blue), GFP(green), and Flag (red) antibodies (B,D, and F). Immunostaining with Flag
was visualized separately (C, E and G).
Arrows represent the soma andarrowheads represent the dendritic tipsof ON BC. Scale bar: 10 μm. H.
Composition of electroporated ON BC(GFP+ PKCα+), where bright Flagsignal was observed in the dendrites.
The error bars represent standard
deviation (SD). Asterisks show that thedifferences are statistically significant(n=3) (Student’s t-test, p<0.05).

432

Molecular Vision 2010; 16:425-437 <http://www.molvis.org/molvis/v16/a48>© 2010 Molecular Vision

TABLE 2. CLINICAL CHARACTERISTICS IN COMPLETE CSNB WITH TRPM1 MUTATIONS.
Mutation

Corrected
visual acuity

Refraction

ColorvisionFundusappearance

Visualfield

Nightblindness

Nystagmus

Strabismus

1.0 (OD)−8.25 −2.75 X 40 (OD)

ND

MyopicND+−

Orthophoria

1.0 (OS)−8.25 −0.50 X 180 (OS)−4.50 −2.25 X 120 (OD)
−4.50 −2.75 X 50 (OS)NormalMyopicNormal+−Orthophoria−13.00 −3.00 X 90 (OD)

NormalMyopicNormal++Esotropia

patient #373 (Male, 9 years old)
IVS3–3C>G,F1075S

patient #437 (Male, 19 years old)
R624C, S882X

1.0 (OD)
1.0 (OS)patient #484 (Male, 27 years old)
IVS9+3_6delA
0.5 (OD)
AGT, R624C

Figure 4. Fundus photographs of patients with mutations of TRPM1. A-C: Fundus images of patient #373 (A), patient #437 (B), and patient#484 (C). All fundus examinations revealed no abnormalities in the posterior pole other than myopic changes, including the tilted discs andtemporal pallor of the optic discs. The patient’s number is indicated in each photograph.

IVS2–3C> and IVS8+3_6delAAGT, can cause splicing
abnormalities leading to defects in protein production. Since
these two mutations are located in the N-terminus region of
TRPM1, these mutations are likely to produce a loss-offunction allele of TRPM1. The nonsense mutation S882X (c.
2645C>A) is located between TM2 and TM3. Thus, thetruncated TRPM1 protein is likely to be non-functional as achannel. Regarding the two missense mutations, R624C (c.
1870C>T) and F1075S (c.3224T>C), we observed failure of
the transportation of the missense mutant channels to thedendritic tips. However, ~20% of ON BCs could transport the
mutant forms of TRPM1, suggesting that these ON BCs arestill active. Considering that human patients carrying thesemutations still have cone ERG responses, this fraction might
be cone ON BCs converging inputs from cone photoreceptors.
Since these two amino acid residues are evolutionarily
conserved among the TRPM subfamily, these amino acidresidues are indispensable for the physiologic functions of
TRPM channels. For example, R671Q mutation in the
Drosophila TRPM1 homolog (trp14) resulted in a significant
reduction of ERG response [39]. This mutation is locatedbetween the 6TM and the TRP domain [40], which is close to
the F1075 residue of human TRPM1. Although this region is
not in the functional domains, it still could be responsible forthe physiologic function of the TRPM channel.
The scotopic ERG b-waves recorded using Ganzfeld
stimuli were nonrecordable, and the photopic ERG showednormal a-wave amplitude, but the amplitude of the b-wave isreduced or absent in the three affected human individuals.
There seems to be no apparent genotype-phenotype
correlation in our patients with TRPM1 mutations. Full-field
ERG results showed no detectable post-receptoral ONpathway function for the three patients. The ERG amplitudesof patient #484 were smaller than those of the other twopatients, but the reductions were considered to be due to highmyopia (−12 to −13 D) [41] or other unknown reasons. The
function of the inner retina was further analyzed using L-M
cone ERGs elicited by rectangular, 100–125 msec duration
light stimuli under light-adapted conditions in one patient, andas a result, the amplitude of the b-wave at light onset (ONresponse) was significantly reduced, but the d-wave amplitude(OFF-response) at light offset was unaffected with normal
433

Molecular Vision 2010; 16:425-437 <http://www.molvis.org/molvis/v16/a48>© 2010 Molecular Vision

Figure 5. Full-field ERGs of patientswith mutations of the TRPM1 gene. AD: Full-field ERGs recorded in a normal
subject and three affected individuals.
The single bright-flash, mixed rod-coneERGs showed a “negative-type”
configuration with a reduced normal awave and a significantly reduced bwave amplitude (A). The scotopic ERGs
showed no response after 20 min ofdark-adaptation (B). The photopic
ERGs showed an apparent a-wave withnormal implicit time and a b-wave withdelayed implicit time (C). The
amplitudes of the 30 Hz flicker ERGs
were within normal range (D). The
oscillatory potentials were reduced.
Arrowheads: stimulus onset. Eachpatient’s number is noted at left.

Figure 6. ERG of WT and trpm1–/– mice.
A-F: ERGs recorded from 8-week-old
mice. Dark-adapted (A) and lightadapted (D) ERGs were elicited by four
different stimulus intensities in both WT
and trpm1–/– mice (n=5). Amplitudes ofdark-adapted (B) and light-adapted (E)ERG a-waves as a function of thestimulus
intensity
areshown.
Amplitudes of dark-adapted (C) andlight-adapted (F) ERG b-waves as afunction of the stimulus intensity are
shown. The bars represent the standard
error of the mean (SEM). Asterisks
show that the differences are statisticallysignificant(Mann–Whitneytest,
p<0.05). The trpm1–/– mouse had anormal a-wave, but a severely depressedb-wave for both dark- and light-adapted
ERGs.

strongly indicated that TRPM1 is essential for the function of
ON BCs in the visual pathway.
The clinical features of patients with complete CSNB are
similar even when the causative gene is different. Most
patients complain of night blindness while showing normal
fundi accompanied by highly myopic refractive errors [4,5].
The best-corrected visual acuity is mildly reduced, orsometimes normal. We could not clarify any difference in
ERG responses that were recorded according to ISCEV

OPs on the OFF-responses (data not shown). Theseelectrophysiological results indicated that the pathology incomplete CSNB with TRPM1 mutations lay in the dysfunctionof the depolarizing ON BCs because it is generally consideredthat the positive ON-response (b-wave) reflects the
depolarizing ON BCs. The impairment in the ON-response
was also observed in the trpm1–/– mice in which b-wave
amplitude for both scotopic and photopic conditions werecompletely defective. These results in human and mice434

Molecular Vision 2010; 16:425-437 <http://www.molvis.org/molvis/v16/a48>© 2010 Molecular Vision

5.

protocol among those with different gene defects. To date, theonly phenotypic difference among complete CSNB patientswith different gene mutations has been detected using a
scotopic 15 Hz-flicker ERG with increasing intensities [42].
It was reported that patients with TRPM1 mutations showed
15 Hz-flicker ERG responses similar to those with NYX
mutations, but different from those with GRM6 mutations or
normal control subjects, suggesting some difference in the rod
pathway [21].
Very recent studies reported that TRPM1 gene mutations
were the major cause of AR complete CSNB in patients with
Caucasian ancestors [20-22]. We previously identified NYX
mutations in five families and GRM6 mutations in two
families among 11 Japanese families with complete CSNB
[43,44], and in the current study, we found TRPM1 mutationsin three of the remaining four families. From these results we
confirmed that the gene was responsible for patients withcomplete CSNB. It is likely that one of the three genes that is
localized at the dendritic terminals of ON BCs andcontributing the cell activity, TRPM1, GRM6, or NYX, would
responsible for most patients with complete CSNB.
In conclusion, we identified five different mutations in
the TRPM1 gene in three unrelated Japanese patients withcomplete CSNB. TRPM1 is essential for the depolarizing ON
BC function in humans as well as in mice.

6.

7.

8.

9.

ACKNOWLEDGMENTS
We thank S. Nakanishi for providing us the MG6-Z vector.
We also thank M. Joukan, A. Tani, T. Tsujii, A. Ishimaru, Y.
Saioka, K. Sone and S. Kennedy for technical assistance. This
work was supported by CREST from Japan Science andTechnology Agency, and a Grant for Molecular Brain
Science, a Grant-in-Aid for Scientific Research (B) from
Ministry of Education, Culture, Sports, Science andTechnology, and The Takeda Science Foundation, The
Uehara Memorial Foundation, Novartis Foundation, Mochida
Memorial Foundation for Medical and Pharmaceutical
Research, The Naito Foundation.

10.

11.

12.

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