Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26)

Abstract

Retinitis pigmentosa (RP), the main cause of adult blindness, is a genetically heterogeneous disorder characterized by progressive loss of photoreceptors through apoptosis. Up to now, 39 genes and loci have been implicated in nonsyndromic RP, yet the genetic bases of >50% of the cases, particularly of the recessive forms, remain unknown. Previous linkage analysis in a Spanish consanguineous family allowed us to define a novel autosomal recessive RP (arRP) locus, RP26, within an 11-cM interval (17.4 Mb) on 2q31.2-q32.3. In the present study, we further refine the RP26 locus down to 2.5 Mb, by microsatellite and single-nucleotide polymorphism (SNP) homozygosity mapping. After unsuccessful mutational analysis of the nine genes initially reported in this region, a detailed gene search based on expressed-sequence-tag data was undertaken. We finally identified a novel gene encoding a ceramide kinase (CERKL), which encompassed 13 exons. All of the patients from the RP26 family bear a homozygous mutation in exon 5, which generates a premature termination codon. The same mutation was also characterized in another, unrelated, Spanish pedigree with arRP. Human CERKL is expressed in the retina, among other adult and fetal tissues. A more detailed analysis by in situ hybridization on adult murine retina sections shows expression of Cerkl in the ganglion cell layer. Ceramide kinases convert the sphingolipid metabolite ceramide into ceramide-1-phosphate, both key mediators of cellular apoptosis and survival. Ceramide metabolism plays an essential role in the viability of neuronal cells, the membranes of which are particularly rich in sphingolipids. Therefore, CERKL deficiency could shift the relative levels of the signaling sphingolipid metabolites and increase sensitivity of photoreceptor and other retinal cells to apoptotic stimuli. This is the first genetic report suggesting a direct link between retinal neurodegeneration in RP and sphingolipid-mediated apoptosis.

Figure 1

The RP26 locus and the CERKL gene and protein. A, RP26 physical map of the 12.5-Mb cosegregation interval between markers D2S2261 and D2S273. B, Localization of CERKL within the candidate region (“CR”) defined by homozygosity mapping. Genes (rectangles) and the transcription direction (arrowheads) are indicated. C, CERKL exon/intron structure. The location of the R275X null mutation in exon 5 is shown.

Figure 2

Protein alignment of human (“h”) and murine (“m”) CERKL and CERK. Identical residues are highlighted in black, and conservative positions are in gray. The solid black line underlines the conserved COG1597 (LCB5) domain detected after comparison with the NCBI Conserved Domain Database. Murine CERKL sequence was assembled from GenBank sequences BY742285 and BC046474, human CERK sequence from AB079066, and murine CERK sequence from AB79067.

Figure 3

Unrooted phylogenetic tree of several members of the diacylglycerol, sphingosine, and ceramide kinases, including the human and murine CERKL sequences described in the present study (boxed). The tree was obtained with the neighbor-joining algorithm of the program ClustalX version 1.64b. Numbers show the values for the bootstrap analysis. SPHKs = sphingosine kinases; CERKs = ceramide kinases; and DGKs = diacylglycerol kinases. Abbreviations and GenBank accession numbers are as follows: human sphingosine kinase 1 (hSPHK1), AAF73423; murine sphingosine kinase 1 (mSPHK1), AAC61697; human sphingosine kinase 2 (hSPHK2), AAF74124; murine sphingosine kinase 2 (mSPHK2), AAF74125; Drosophila sphingosine kinase (dSPHK), AAF48045; Ciona intestinalis sphingosine kinase (ciSPHK), AK112588; yeast sphingosine kinase Lcb4 (Lcb4p), NP_014814; yeast sphingosine kinase Lcb5 (Lcb5p), NP_013361; Arabidopsis thaliana sphingosine kinase (AtSPHK), AY128394; human ceramide kinase (hCERK), AB079066; murine ceramide kinase (mCERK), AB079067; Ciona intestinalis ceramide kinase (ciCERK), AK112750; Drosophila ceramide kinase (dCERK), AAF52040; human ceramide kinase-like (hCERKL), AY357073; murine ceramide kinase-like (mCERKL), BY742285 and BC046474; Caenorhabditis elegans ceramide kinase (cCERK), AAC67466; human diacylglycerol kinase β subunit (hDGKb), Q9Y6T7; murine diacylglycerol kinase β subunit (mDGKb), XP_147651; human diacylglycerol kinase epsilon subunit (hDGKe), NP_003638; murine diacylglycerol kinase epsilon subunit (mDGKe), NP_062378); Caenorhabditis elegans diacylglycerol kinase 1 (cDGK1), NP_508190; Arabidopsis thaliana diacylglycerol kinase 1 (AtDGK1), Q39017; Drosophila diacylglycerol kinase 2 (dDGK2), Q09103; and Drosophila diacylglycerol kinase 1 (dDGK1), Q01583.

Figure 4

A, DNA sequence of exon 5 in an unaffected individual and patient VI.15 of the P2 family, showing the homozygous nonsense R257X mutation. B, Pedigrees of P2 and E1 families, showing the ARMS analysis of R257X mutation. A specific primer was designed to generate an AvaII restriction site in the amplified wild-type allele. All RP-affected individuals are homozygous for the mutant, nonrestricted allele. Individuals whose DNA samples were not available are indicated by a number sign (#).

Figure 5

CERKL and GAPD (control) RT-PCR expression analysis on several human tissues, including retina

Figure 6

A, Cerkl in situ hybridization on murine eye sections with the antisense riboprobe. B, Cerkl in situ hybridization on murine eye sections with the sense riboprobe (negative control). C, Rhodopsin in situ hybridization on murine eye sections (positive control). RPE = retinal pigment epithelium; PhR = photoreceptor cell layer; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer.