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Am J Hum Genet. 2009 Nov 13; 85(5): 730–736.
PMCID: PMC2775826

Mutations in TRPM1 Are a Common Cause of Complete Congenital Stationary Night Blindness


Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impaired night vision and variable decreased visual acuity. We report here that six out of eight female probands with autosomal-recessive complete CSNB (cCSNB) had mutations in TRPM1, a retinal transient receptor potential (TRP) cation channel gene. These data suggest that TRMP1 mutations are a major cause of autosomal-recessive CSNB in individuals of European ancestry. We localized TRPM1 in human retina to the ON bipolar cell dendrites in the outer plexifom layer. Our results suggest that in humans, TRPM1 is the channel gated by the mGluR6 (GRM6) signaling cascade, which results in the light-evoked response of ON bipolar cells. Finally, we showed that detailed electroretinography is an effective way to discriminate among patients with mutations in either TRPM1 or GRM6, another autosomal-recessive cCSNB disease gene. These results add to the growing importance of the diverse group of TRP channels in human disease and also provide new insights into retinal circuitry.

Main Text

Congenital stationary night blindness (CSNB), caused by defective signaling from photoreceptor to bipolar cells, is characterized by a reduced or absent b-wave and a normal a-wave in the electroretinogram (ERG). Two types of CSNB can be distinguished by use of the standard flash ERG: the “complete” form (cCSNB), also known as type 1 CSNB or CSNB1 (MIM 310500),1 is characterized by the complete absence of rod pathway function; and the “incomplete” form (icCSNB), also known as type 2 CSNB or CSNB2 (MIM 300071), is caused by impaired rod and cone pathway function. cCSNB is caused by postsynaptic defects in depolarizing or ON bipolar cell signaling, whereas the hyperpolarizing or OFF bipolar cell pathway is intact. CSNB segregates in X-linked and autosomal-recessive form. X-linked cCSNB is caused by mutations in NYX (MIM 300278), which encodes nyctalopin, a leucine-rich proteoglycan of unknown function.2–4 One form of autosomal-recessive CSNB is caused by mutations in GRM6 (MIM 604096), which encodes the metabotropic glutamate receptor 6 (mGluR6).5,6 Nyctalopin and mGluR6 are localized on the dendrites of ON bipolar cells7,8 and are required for signal transmission from photoreceptors to ON bipolar cells. Both are involved in modulating a nonspecific cation channel that has been identified through animal studies to be TrpM1.9,10 Modulation of TrpM1 leads to a change in the membrane potential of ON bipolar cells, thus making TrpM1 essential for ON bipolar cell function.

If TRPM1 (MIM 603576) indeed plays a role in ON bipolar cell signaling in humans, it should also be localized postsynaptically on the ON bipolar cell dendrites in the outer plexiform layer (OPL) of the retina (Figure 1A). To examine this, we reacted transverse sections of normal human retina with antibodies to TRPM1 and Ribeye (a presynaptic marker) or peanut agglutinin (PNA) (a marker for cone terminals) (Figure 1). Images of TRPM1 and PNA staining (Figure 1B) showed dense TRPM1 puncta closely aligned with PNA-positive cone photoreceptor terminals. Weaker TRPM1 staining in the inner nuclear layer was associated with bipolar cell somata. Staining for both TRPM1 and Ribeye (Figure 1C) showed closely associated, but nonoverlapping, labeling in large cone and small rod terminals. This localization of TRPM1 strongly resembles that of nyctalopin8 and indicated that it, too, is localized on rod ON bipolar cell dendrites.

Figure 1
TRPM1 Is Localized to the OPL in the Human Retina

Given this expression pattern and the role of TRPM1 in ON bipolar cell signaling in animals,9,10 we hypothesized that individuals with autosomal-recessive cCSNB lacking mutations in either GRM6 or NYX may have mutations in TRPM1. We subsequently screened eight female autosomal-recessive CSNB probands of European ancestry for mutations in TRPM1, after the other cCSNB genes had been excluded. We sequenced 26 exons (accession no. NM_002420_4) and adjacent splice sites and found TRPM1 mutations in six probands. All six probands had normal retinas on the basis of funduscopy (data not shown). Their clinical characteristics, including representative dark-adaptation curves and ISCEV (International Society for Clinical Electrophysiology of Vision) standard ERG, are presented in Table S1, Figure S1, and Figure S2 (available online). Five probands carried either homozygous or compound-heterozygous mutations, and in one proband we found only a single heterozygous mutation (Table S1). None of the described mutations were present in 210 control chromosomes.

Proband 1 was homozygous for a 36,445 bp deletion that includes exons 2–7 (Figure 2Ai). The deleted chromosome was present in both parents and was absent from the proband's unaffected sister. This deletion removes exons used in four additional isoforms recently described by Oancea et al.11 and should produce a null allele for all isoforms of TRPM1. Proband 2 (Figure 2Aii) was compound heterozygous with a single-bp deletion in exon 3 (c.83delA), resulting in a frameshift (p.Asn28Metf62) on one allele. The other allele carried a c.1600G>A transition in exon 14, which generates a p.Gly534Arg missense mutation. The proband's unaffected parents were carriers (mother for c.83delA; father for c.1600G>A), whereas the unaffected sister had no mutations. The parents were not aware of any possible consanguinity. Alamut analysis, which predicts mutation impact on function, classifies p.Gly534Arg as weak, although p.Gly534 is conserved in TRPM1 channels from human to frog (Figure 2B), suggesting functional importance. Proband 3 had a c.296T>C transversion in exon 4, which results in a p.Lys99Pro substitution on one allele, and a c.1832C>T transversion in exon 16, which results in p.Pro611His missense mutations (Figure 2Aiii) on the other allele. DNA samples from the parents were not available. However, the c.296T>C mutation was found in the unaffected sister of proband 3, and the c.1832C>T mutation was found in the unaffected brother. Alamut analysis predicts that both are likely to interfere with the function of the protein, and each contains highly conserved residues. Proband 4 carries a c.220C>T transition in exon 4, which results in a p.Arg74Cys substitution on one allele, and a c.1091T>G transversion in exon 9, which results in a p.Leu364Arg substitution (Figure 2Aiv) on the other allele. Only the c.220C>T mutation was found in proband 3's mother, and DNA was not available for her father. Alamut analysis predicts that both are highly conserved residues likely to interfere with the function of the protein, and each contains highly conserved residues (Figure 2B). We identified two mutations (c.3061+1G>A and c.3142G>A) in proband 5; the c.3142G>A mutation is located in the ion transport domain and, according to Alamut analysis, interferes with the function of the protein. The c.3061+1G>A mutation most likely affects the splicing pattern of the gene (deletion of exon 22 expected). Finally, we identified a single heterozygous splice-site mutation (c.2250+1G>A) in proband 6. The mutations that were predicted to alter splicing (probands 5 and 6) require further functional confirmation.

Figure 2
TRPM1 Mutations Are Present in Six Probands with Autosomal-Recessive cCSNB

Scotopic (rod), photopic (cone), mixed cone-rod, and 30 Hz photopic ERG responses were measured in control subjects (Figure S2A) and in cCSNB probands with GRM6, NYX, and TRPM1 mutations (Figures S2B–S2D), with the use of ISCEV standard conditions.12,13 In control subjects, the mixed cone-rod ERG has a negative-going a-wave, due to photoreceptor activation, preceding a positive-going b-wave, caused by ON bipolar cell depolarization (mixed; Figure S2A). Consistent with their diagnosis, all cCSNB probands showed mixed ERG responses of normal a-wave and no b-wave phenotype (mixed; Figure S2B–S2D). No scotopic ERG response could be identified. Their photopic, 30 Hz flicker ERG responses were normal, and oscillatory potential (OP) responses were normal to subnormal. So far, all of our probands had a phenotype similar to those of mouse models with mutations in Grm6, TrpM1,10,14 and Nyx,15 in which the absence of ON bipolar cell activity has been directly confirmed.8

In contrast to the standard flash ERG, a 15Hz flicker ERG paradigm can subdivide cCSNB patients with and without GRM6 mutations and provide insight into dysfunction in the various rod pathways6 (Figure 3). At low scotopic intensities, the response is dominated by the primary rod pathway (Figure 4Ai; rod → rod ON-BC → AII amacrine cell, whereas the response is dominated at high scotopic intensities by the secondary rod pathway (Figure 4Aii; rod →cone [via gap junctions between rod and cone pedicles] → BC).16,17 In control subjects (Figure 3A), the ERG amplitude decreases and then reaches a null at about −0.56 log scot td×s before increasing again at higher intensities. This occurs because of destructive interference, caused by out-of-phase signals from the primary and secondary rod pathways. None of the 15 Hz ERG responses of our TRPM1 probands showed a pattern similar to either the control subject (Figure 3A) or our autosomal-recessive cCSNB proband with GRM6 mutations (Figure 3B). Rather, the ERG responses of all TRPM1 probands (Figure 3D) were identical to those of cCSNB patients with NYX mutations (Figure 3C). Specifically, responses at low intensities were completely absent, whereas they were similar to normal at higher intensities, revealing the complete absence of the primary rod pathway and mildly reduced activity of the secondary rod pathway. As reported previously for autosomal-recessive cCSNB patients with GRM6 mutations, our GRM6 proband had responses at all intensities that were markedly dissimilar in phase as compared to normal responses.6 It has been suggested that this is due to the interaction between the secondary and tertiary rod pathways (Figure 4Aiii; rod → OFF-BCs).6

Figure 3
ERG Responses from Control Subjects and Probands with GRM6, NYX, and TRPM1 Mutations
Figure 4
Rod Signaling in the Mammalian Retina Travels via Three Routes

Distinct CSNB phenotypes also occur in retinal ganglion cell activity in Nyx and Grm6 mutant mice.18 Nyx mutant ganglion cells show spontaneous rhythmic bursting activity with a fundamental frequency of about 4 Hz; whereas the spontaneous activity of Grm6 mutant ganglion cells is the same as that of the wild-type.19 Furthermore, the spatial organization of all Nyx mutant ganglion cells is significantly altered, whereas Grm6 OFF ganglion cells have normal receptive field center/surround organization.18 Because the genes implicated in cCSNB are all located in the ON bipolar cell dendrites, our data imply that, in both humans and mice, the state of the ON bipolar cells differs between TRPM1 and NYX mutants and GRM6 mutants.

Figure 4B illustrates the currently identified proteins that function in ON bipolar cell signaling and cause a cCSNB phenotype in humans and/or mice. mGluR6 is the receptor sensing glutamate release by photoreceptors,20,21 GαO and Gβ5 are second messengers in this G protein signaling cascade.22,23 TRPM1 is most likely the cation channel that eventually modulates the light-evoked ON bipolar cell depolarization.9,10 Nyctalopin, whose function has not yet been conclusively established, may traffic and/or anchor TRPM1 to the membrane.24 Activation of mGluR6 leads to the closure of TRPM1 channels. Therefore, mutations in mGluR6 are likely to cause the TRPM1 channel to be open and conduct an inward current. In contrast, mutations in TRPM1 are likely to eliminate both the TRPM1 channel and the inward current. The simplest model predicts that ON bipolar cells in our probands with TRPM1 mutations will be hyperpolarized, whereas those with GRM6 mutations will be constitutively depolarized. This should differentially affect the sustained activity of AII amacrine cells, a vital component of information flow in the rod pathways. These changes could shift the balance between the remaining secondary and tertiary rod pathways and result in the differences that we observe in the 15 Hz ERG flicker response.

Overall, we investigated 11 autosomal-recessive cCSNB patients from nine families. Three patients from one family had mutations in GRM6. Six probands from six families had TRPM1 mutations. The remaining two patients may represent the opportunity to identify additional genes that regulate either TRPM1 channel activity or the mGluR6 signaling cascade. That said, our results indicate that TRPM1 mutations will account for more than 50% of autosomal-recessive cCSNB cases in our patient cohort of European ancestry. TRP channels are a diverse family of about 30 members that are now known to be expressed and participate in central and peripheral nervous system function. To date, mutations in these channels have rarely been associated with diseases of the nervous system. However, our results suggest that they may represent an unexplored target for these diseases.


This research was supported by grants from the Netherlands Organization for Scientific Research (NWO) (to M.K.), Algemene Nederlandse Vereniging ter Voorkoming van Blindheid (ANVVB) (to A.B.), ODAS (to M.G., M.B., and F.R.), and the U.S. National Institutes of Health (NIH) (R01EY12354 to R.G.G. and R01EY014701 to M.A.M.). We would like to thank all of the probands and their families for their collaboration in this project.

Supplemental Data

Document S1. Two Figures and Two Tables:

Web Resources

The URLs for data presented herein are as follows:

Note Added in Proof

The involvement of TrpM1 in ON bipolar cell function in mouse retina has been confirmed by Morgans et al.: TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells. PNAS. Published online October 27, 2009. doi: 10.1073/pnas.0908711106.


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