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.     

TRPM1 Is Mutated in Patients with Autosomal-Recessive Complete Congenital Stationary Night Blindness

Night vision requires signaling from rod photoreceptors to adjacent bipolar cells in the retina. Mutations in the genes NYX and GRM6, expressed in ON bipolar cells, lead to a disruption of the ON bipolar cell response. This dysfunction is present in patients with complete X-linked and autosomal-recessive congenital stationary night blindness (CSNB) and can be assessed by standard full-field electroretinography (ERG), showing severely reduced rod b-wave amplitude and slightly altered cone responses. Although many cases of complete CSNB (cCSNB) are caused by mutations in NYX and GRM6, in ∼60% of the patients the gene defect remains unknown. Animal models of human diseases are a good source for candidate genes, and we noted that a cCSNB phenotype present in homozygous Appaloosa horses is associated with downregulation of TRPM1. TRPM1, belonging to the family of transient receptor potential channels, is expressed in ON bipolar cells and therefore qualifies as an excellent candidate. Indeed, mutation analysis of 38 patients with CSNB identified ten unrelated cCSNB patients with 14 different mutations in this gene. The mutation spectrum comprises missense, splice-site, deletion, and nonsense mutations. We propose that the cCSNB phenotype in these patients is due to the absence of functional TRPM1 in retinal ON bipolar cells.     

A Mutation in SLC24A1 Implicated in Autosomal-Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder that can be associated with impaired night vision. The last decade has witnessed huge progress in ophthalmic genetics, including the identification of three genes implicated in the pathogenicity of autosomal-recessive CSNB. However, not all patients studied could be associated with mutations in these genes and thus other genes certainly underlie this disorder. Here, we report a large multigeneration family with five affected individuals manifesting symptoms of night blindness. A genome-wide scan localized the disease interval to chromosome 15q, and recombination events in affected individuals refined the critical interval to a 10.41 cM (6.53 Mb) region that harbors SLC24A1, a member of the solute carrier protein superfamily. Sequencing of all the coding exons identified a 2 bp deletion in exon 2: c.1613_1614del, which is predicted to result in a frame shift that leads to premature termination of SLC24A1 (p.F538CfsX23) and segregates with the disorder under an autosomal-recessive model. Expression analysis using mouse ocular tissues shows that Slc24a1 is expressed in the retina around postnatal day 7. In situ and immunohistological studies localized both SLC24A1 and Slc24a1 to the inner segment, outer and inner nuclear layers, and ganglion cells of the retina, respectively. Our data expand the genetic basis of CSNB and highlight the indispensible function of SLC24A1 in retinal function and/or maintenance in humans.     

Recessive Mutations of the Gene TRPM1 Abrogate ON Bipolar Cell Function and Cause Complete Congenital Stationary Night Blindness in Humans

Complete congenital stationary night blindness (cCSNB) is associated with loss of function of rod and cone ON bipolar cells in the mammalian retina. In humans, mutations in NYX and GRM6 have been shown to cause the condition. Through the analysis of a consanguineous family and screening of nine additional pedigrees, we have identified three families with recessive mutations in the gene TRPM1 encoding transient receptor potential cation channel, subfamily M, member 1, also known as melastatin. A number of other variants of unknown significance were found. All patients had myopia, reduced central vision, nystagmus, and electroretinographic evidence of ON bipolar cell dysfunction. None had abnormalities of skin pigmentation, although other skin conditions were reported. RNA derived from human retina and skin was analyzed and alternate 5′ exons were determined. The most 5′ exon is likely to harbor an initiation codon, and the protein sequence is highly conserved across vertebrate species. These findings suggest an important role of this specific cation channel for the normal function of ON bipolar cells in the human retina.     

Characterization and Developmental Expression of Pax9, a Paired-Box-Containing Gene Related to Pax1

Pax9, a recently identified mouse paired-box-containing gene, is highly homologous to Pax1 and belongs to the same subfamily as Pax1, Hup48, PAX9, and pox meso. Two overlapping cDNA clones spanning the entire coding region of Pax9 were isolated and sequenced. A comparison of the Pax1 and -9 protein sequences reveals a high degree of similarity even outside the paired box, while the carboxy-terminus of the two proteins diverges completely. We demonstrate that Pax9 can bind to the e5 sequence from the Drosophila even skipped promoter, which is also recognized by Pax1. We analyzed the expression of Pax9 during embryo-genesis of wildtype, Undulated short-tail (Un^s), and Danforth's short tail (Sd) mice. In wildtype embryos Pax9 is expressed in the pharyngeal pouches and their derivatives, the developing vertebral column, the tail, the head, and the limbs. Expression of Pax9 is unaffected in Un^s embryos, in which the Pax1 gene is deleted, arguing that expression of Pax9 is not dependent on Pax1. The expression of Pax9 is lost in the caudal part of Sd homozygous embryos, suggesting that expression of Pax9 in the vertebral column independent on the notochord. These results indicate that both Pax9 and -1 may act in parallel during morphogenesis of the vertebral column.     

FXY2/MID2, a Gene Related to the X-Linked Opitz Syndrome Gene FXY/MID1, Maps to Xq22 and Encodes a FNIII Domain-Containing Protein That Associates with Microtubules

Opitz G/BBB syndrome (OS) is a genetically heterogeneous disorder with an X-linked locus and an autosomal locus linked to 22q11.2. OS affects multiple organ systems with often variable severity even between siblings. The clinical features, which include hypertelorism, cleft lip and palate, defects of cardiac septation, hypospadias, and anorectal anomalies, indicate an underlying disturbance of the developing ventral midline of the embryo. The gene responsible for X-linked OS, FXY/MID1, is located on the short arm of the human X chromosome within Xp22.3 and encodes a protein with both an RBCC (RING finger, B-box, coiled coil) and a B30.2 domain. The Fxy gene in mice is also located on the X chromosome but spans the pseudoautosomal boundary in this species. Here we describe a gene closely related to FXY/MID1, called FXY2, which also maps to the X chromosome within Xq22. The mouse Fxy2 gene is located on the distal part of the mouse X chromosome within a region syntenic to Xq22. Analysis of genes flanking both FXY/MID1 and FXY2 (as well as their counterparts in mouse) suggests that these regions may have arisen as a result of an intrachromosomal duplication on an ancestral X chromosome. We have also identified in both FXY2 and FXY/MID1 proteins a conserved fibronectin type III domain located between the RBCC and B30.2 domains that has implications for understanding protein function. The FXY/MID1 protein has previously been shown to colocalize with microtubules, and here we show that the FXY2 protein similarly associates with microtubules in a manner that is dependent on the carboxy-terminal B30.2 domain.     

Characterization of CaGSP1, the Candida albicans RAN/GSP1 homologue

Gsp1p is a small nuclear-located GTP binding protein from the yeast Saccharomyces cerevisiae. It is highly conserved among eucaryotic cells and is involved in numerous cellular processes, including nucleocytoplasmic trafficking of macromolecules. To learn more about the GSP1 structure/function, we have characterized its Candida albicans homologue. CaGsp1p is 214 amino acids long and displays 91% identity to the ScGsp1p. There is functional complementation in S. cerevisiae, and its mRNA is constitutively expressed in the diploid C. albicans grown under various physiological conditions. Disruption of both alleles was not possible, suggesting that it could be an essential gene, but heterozygous mutants exhibited genomic instability.     

Characterization of a staurosporine- and temperature-sensitive mutant, stt1, of Saccharomyces cerevisiae: STT1 is allelic to PKC1

Summary Staurosporine is an antibiotic that specifically inhibits protein kinase C. Fourteen staurosporine- and temperature-sensitive (stt) mutants of Saccharomyces cerevisiae were isolated and characterized. These mutants were divided into ten complementation groups, and characterized for their cross-sensitivity to K-252a, neomycin, or CaCl₂, The STT1 gene was cloned and sequenced. The nucleotide sequence of the STT1 gene revealed that STT1 is the same gene as PKC1. The STT1 gene conferred resistance to staurosporine on wild-type cells, when present on a high copy number plasmid. STT1/stt1::HIS3 diploid cells were more sensitive to staurosporine than STT1/STT1 diploid cells. Analysis of temperature-sensitive stt1 mutants showed that the STT1 gene product functioned in S or G2/M phase. These results suggest that a protein kinase (the STT1 gene product) is one of the essential targets of staurosporine in yeast cells.     

Comparative Genomic Sequence Analysis of the FXR Gene Family: FMR1, FXR1, and FXR2

Mutations in the X-linked gene FMR1 cause fragile X syndrome, the leading cause of inherited mental retardation. Two autosomal paralogs of FMR1 have been identified, and are known as FXR1 and FXR2. Here we describe and compare the genomic structures of the mouse and human genes FMR1, FXR1, and FXR2. All three genes are very well conserved from mouse to human, with identical exon sizes for all but two FXR2 exons. In addition, the three genes share a conserved gene structure, suggesting they are derived from a common ancestral gene. As a first step towards exploring this hypothesis, we reexamined the Drosophila melanogaster gene Fmr1, and found it to have several of the same intron/exon junctions as the mammalian FXRs. Finally, we noted several regions of mouse/human homology in the noncoding portions of FMR1 and FXR1. Knowledge of the genomic structure and sequence of the FXR family of genes will facilitate further studies into the function of these proteins.     

Isolation and Characterization of IS1181, an Insertion Sequence from Staphylococcus aureus

The repeated nucleotide sequence isolated from a methicillin-resistant Staphylococcus aureus isolate displays the characteristic features of an insertion sequence and was named IS1181. It has a size of 1512 bp and consists of a 1359-bp open reading frame that encodes a 439-amino-acid protein which is predicted to be highly basic and 23-bp terminal inverted complementary repeated sequences exhibiting six mismatches. The three copies of IS1181 isolated from distinct parts of the chromosome of S. aureus, BM3121, are flanked at their ends by 8-bp direct repeats, suggesting a duplication of the target sequence. IS1181 exhibits similarities with IS1165 from Leuconostoc mesenteroides and IS1001 from Bordetella parapertusis. IS1181 was detected in at least two to eight copies in 41 of the 52 S. aureus isolates tested, whereas none of the 26 coagulase-negative staphylococci, 24 streptococci, or 11 enterococci analyzed carried nucleotide sequences hybridizing with IS1181.     

Characterization of the Function of the NIN1 Gene Product of Saccharomyces cerevisiae

The nin1-1 mutant has been isolated as a temperature-sensitive mutant whose nucleus arrested at G2 phase and eventually disintegrated upon temperature upshift. In this study, a genetic event occurring in the nin1-1 mutant was found to be a frameshift mutation, resulting in a truncated protein smaller than the wild-type Nin1 protein. We found new phenotypes associated with the nin1-1 mutation: (i) rates of mitotic recombination and chromosome/plasmid loss in the nin1-1 strain were higher than those in the wild-type strain, and (ii) the mutant was more sensitive to uv irradiation than the wild-type strain. We found dotted structures in the cytoplasm of the wild-type cells by indirect immunofluorescence microscopy using the anti-Nin1 antibody. Similar results were obtained when we analyzed the localization of Nin1-β-galactosidase fusion protein formed in the cells expressing the NIN-lacZ fusion gene, which is active as NIN1, with anti-β-galactosidase antibody. The subcellular fractionation method revealed that Nin1 protein was not localized in a particular fraction of the cell lysate.     

The Structure and Expression of the Murine Wildtype p53-Induced Phosphatase 1 (Wip1) Gene

The human wildtype p53-induced phosphatase 1 (Wip1; GenBank symbol Ppm1d) gene encodes a type 2C protein phosphatase (PP2C) that is induced by ionizing radiation in a p53-dependent manner. We have cloned and sequenced the mouse Wip1 gene and its encoded mRNA. The mouse Wip1 gene is composed of six exons and spans over 36 kb of DNA. The mouse cDNA sequence predicts a 598-amino-acid protein with a molecular mass of roughly 66 kDa. Comparison of human and mouse Wip1 sequences revealed 83% overall identity at the amino acid level. The 5′-flanking region of exon 1 had promoter elements characteristic of a housekeeping gene. The Wip1 coding sequences share conserved functional regions with other PP2Cs from a diverse array of species. Expression of Wip1 mRNA was detected ubiquitously in adult and embryonic tissues, though expression in the testis was much higher than in other tissues. Wip1 has been mapped near the p53 gene on mouse chromosome 11.