|Molecular Vision 2000;
Received 4 October 1999 | Accepted 27 January 2000 | Published 22 February 2000
Evaluation of human diacylglycerol kinasei, DGKI, a homolog of Drosophila rdgA, in inherited retinopathy mapping to 7q
Sara J. Bowne,1
Lori S. Sullivan,1 Li
Ding,2 Elie Traer,2 Stephen M. Prescott,2 David G.
Birch,3 Avril Kennan,4 Peter Humphries,4
Stephen P. Daiger1
1Human Genetics Center, School of Public Health, and Department of Ophthalmology and Visual Science, The University of Texas-Houston Health Science Center, Houston, TX; 2Huntsman Cancer Center Institute, The University of Utah, Salt Lake City, UT; 3Retina Foundation of the Southwest, Dallas, TX; 4Genetics Department, Trinity College, Dublin, Ireland
Correspondence to: Stephen P. Daiger, PhD, P.O. Box 20334, Houston, TX, 77225-0334; Phone: (713) 500-9829; Fax: (713) 500-0900; email: email@example.com
Purpose: To determine the genomic organization of diacylglycerol kinasei and to test whether defects in this gene are present in individuals affected with autosomal dominant retinitis pigmentosa (adRP). Diacylglycerol kinasei has been mapped to the RP10 locus on 7q and shows 49% sequence similarity to the Drosophila DGK2 rdgA gene. Since mutations in the DGK2 rdgA gene cause photoreceptor degeneration in Drosophila, it is possible that mutations in diacylglycerol kinasei could be responsible for human retinal degeneration.
Methods: DNA sequence from genomic clones containing diacylglycerol kinasei was compared with the cDNA sequence to identify intron/exon boundaries. Single-strand conformational analysis and PCR product sequencing were used to screen members of one family previously mapped to the RP10 locus and 47 small unmapped families with autosomal dominant retinitis pigmentosa.
Results: Diacylglycerol kinasei is divided into 35 exons with the initiation codon being present in exon 2. Mutational analysis found a missense change (Lys153Phe) in three adRP families; however, it did not segregate with disease in one of the families. Silent substitutions were seen in codons 865 and 875. Intronic variation was detected in the amplifications of exons 3,5,18, 28, and 32; these do not affect splice site consensus sequences. Typing of a polymorphic variant detected in intron 31 in members of the RP10 family gave a LOD score of -4.2 at 0% recombination.
Conclusions: No evidence of disease-associated mutations was found in any of the samples tested. Based on the linkage data and mutation screening, diacylglycerol kinasei is excluded as a candidate for the RP10 form of adRP and cannot be a frequent cause of other forms of adRP. Mutations in diacylglycerol kinasei may yet be the cause of recessive forms of retinal degeneration in humans, either in homozygous or compound heterozygous forms. The data provided here will permit testing of this hypothesis.
Retinitis pigmentosa (RP) is a group of clinically and genetically heterogeneous hereditary disorders that affect the photoreceptors and retinal pigment epithelium. To date, thirteen autosomal recessive (arRP), eleven autosomal dominant (adRP) and six X-linked (XlRP) loci have been mapped. Of the eleven adRP loci, only five disease-associated genes have been identified; rhodopsin, peripherin/RDS, CRX, NRL, and RP1 (RetNet, 1 October 1999). One of the unidentified adRP loci, RP10, has been mapped to 7q31-q35 using linkage data from three independent families [1-4].
Recently a novel retinal-expressed diacylglycerol kinase (DGK), diacylglycerol kinasei (DGKI), has been identified and mapped by fluorescence in situ hybridization to 7q32.2-q33 . DGK controls diacylglycerol levels by phosphorylating diacylglycerol into phosphatidic acid. Through this interaction, DGK plays an important role in complex lipid biosynthesis and signal transduction [5-7]. Diacylglycerol kinasei is a member of the DGK type IV subfamily, which is characterized by the presence of four ankyrin repeats located at the C-terminus and a MARCKs phosphorylation site . DGKI has 49% sequence similarity with another type IV subfamily member, the Drosophila DGK2 rdgA gene .
The Drosophila DGK2 rdgA gene was first identified by Masai et al. in 1993  as the mutant gene responsible for the retinal degeneration in the homozygous rdgA fruit fly. Studies have shown that the photoreceptor degeneration in the rdgA mutant is due to the disruption of the subrhabdomeric cisternae which are perforated extensions of the endoplasmic reticulum. Subrhabdomeric cisternae play an important role in the maintenance of fly photoreceptors by transporting phospholipids to the photoreceptor membrane. Recent studies have indicated that the rdgA protein is associated with subrhabdomeric cisternae, and it is the reduced levels of this diacylglycerol kinase in the rdgA mutant that cause a deficiency of phosphatidic acid and subsequent photoreceptor degeneration [9,10].
DGKI has the highest sequence similarity of any known human gene to the Drosophila rdgA gene, and is the most likely rdgA ortholog. Due to the high sequence similarity of human DGKI to the Drosophila DGK2 rdgA gene, and proximity of the diacylglycerol kinasei locus to the RP10 disease region, it is possible that mutations in the DGKI gene could be responsible for human retinal degeneration. To test this hypothesis we determined the genomic organization of DGKI for subsequent mutation testing of affected individuals. Samples from one RP10 family, UTAD045, and 47 small adRP families were tested for mutations.
Genomic clones were isolated by Genome Systems (St. Louis, MS) using fragments of the DGKI cDNA and genomic primers. Positive genomic clones were analyzed by restriction mapping and Southern blot analysis. Southern blots of the positive genomic clones were probed with cDNA and positive bands were subcloned into conventional vectors and sequenced by automated dideoxy sequencing. Alternatively, some intron/exon boundaries were identified by direct sequencing of P1, BAC and PAC genomic clones. Comparison of the genomic sequence with the cDNA sequence identified intron/exon boundaries. Intron/exon boundaries for exons 3 through 35 were identified using the above method. The 3' boundary of exon 2 was determined using Vectorette (Genosys, Woodlands, TX) modification of a CEPH YAC in the region and PCR cycle sequencing with the AmplicycleTM Sequencing Kit (Perkin Elmer, Branchburg, NJ). All boundaries were consistent with conserved splice site sequences. PCR amplification in genomic DNA was used to determine the location of the initiation codon. All unpublished sequences from this study have been deposited in GenBank (accession numbers AF219907-AF219939). Portions of the DGKI genomic sequence have been recently deposited in GenBank as part of the large-scale sequencing of 7q for the Human Genome Project. BLAST analysis of these sequences confirms the location of several intron/exon junctions.
Patients and Families
Members of the large American RP10 family, UTAD045, were identified and blood collected as previously described [2,3]. Members of 47 small adRP families were ascertained at either (1) the Anderson Vision Research Center, Retina Foundation of the Southwest, Dallas or (2) the Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles. Informed consent was obtained from all subjects. For each family member tested, a clinical examination revealed the presence of RP. Autosomal dominant inheritance was established through pedigree evaluation.
DNA was isolated from peripheral blood using standard extraction procedures. Individual exons were amplified by PCR with AmpliTaq Gold polymerase (Perkin Elmer) using the primers in Table 1 and standard cycling parameters. PCR products were radiolabeled by incorporating 1mCi 32P-dCTP (Amersham Pharmacia Biotech, Piscataway, NJ). The amplified products of exons 3, 21, and 29 were digested with Sma I, Sau3 I, and Pal I, respectively, in order to reduce fragment sizes. PCR products were denatured and separated on 0.6% MDE gels (FMC Bioproducts, Rockland, ME) at room temperature and 4 °C. Gels were dried and subjected to autoradiography after electrophoresis.
The underlying DNA change for each SSCP variant was determined by PCR product sequencing. Genomic DNA from patients was amplified by PCR using the conditions described above. PCR product sequencing was performed using either the SequenaseTM PCR Product Sequencing Kit (Amersham) or the AmplicycleTM Sequencing Kit (Perkin Elmer). PCR product was treated with Exonuclease I and Shrimp Alkaline Phosphatase (United States Biochemical, Cleveland, OH) prior to sequencing with the AmplicycleTM Sequencing Kit. Samples were run on either 6% Acryl-a-MixTM (Promega) or 6% LongRangerTM (FMC Bioproducts) denaturing gels.
To insure that sequence variants present in the RP10 family were not missed due to the limitations of SSCP analysis, automated dideoxy sequencing of PCR product was performed using DNA from two affected and one unaffected member of UTAD045. Sequencing was terminated once linkage analysis of the polymorphism in intron 31 (see below) excluded diacylglycerol kinasei as the cause of disease in UTAD045. All coding sequences except exons 2 and 27 were tested prior to the termination of sequencing.
SSCP and sequencing analysis revealed the presence of a sequence polymorphism in intron 31 at 3130-39 (G->A). This polymorphism was typed in members of UTAD045 using EcoR I digestion of PCR product. After digestion, PCR products were visualized on 2% agarose gels (Promega).
In this study we determined that DGKI is encoded in 35 exons with the initiation codon beginning in exon 2. Comparison of genomic sequence from clones containing DGKI with the cDNA sequence was used to determine the intron/exon junctions for exons 3 through 35. The 3' intron/exon junction of exon 2 was identified using alternative methods. Intron/exon junctions for exons 2 through 35 are described in Table 2. The exact location of exon 1 and the 5' exon 2 intron/exon junction could not be determined due to the presence of several repetitive sequences in the 5' region of the DGKI cDNA. No information regarding this region was present in the databases. The genomic structure and flanking intronic sequence of DGKI was used to design primers for mutational analysis. PCR primers for exon 2 were designed based on the cDNA sequence and contain all but the first 17 bp of the coding sequence.
Samples from the American RP10 family, UTAD045, and 47 small adRP families without mutations in known adRP genes were tested for mutations in DGKI. Mutational analysis found one missense substitution, two silent substitutions, and five intronic variants. None of these variants appear to be disease-causing, and linkage testing of one of the polymorphic intronic variants in UTAD045 excludes mutations in DGKI as the cause of RP10.
Members of three small adRP families had a Lys153Phe (CTT->TTT) missense substitution in exon 3. Further testing demonstrated that this substitution did not segregate with disease in one of the families tested. Two silent substitutions, Thr865Thr (ACA->ACG) and Glu875Glu (GAA->GAG) were also identified in the samples tested. The Glu875Glu substitution is polymorphic in the African-American population (data not shown).
Intronic variation was detected in amplifications for exons 3, 5, 18, 28, and 32. None of these variants affect the splice site consensus sequence. The G->A transition at 3130-39 in intron 31 occurred at polymorphic levels in the population. This two-allele marker was typed in members of UTAD045. Recombinant individuals were detected in UTAD045 and linkage analysis of this marker with disease gave a LOD score of -4.2 at 0% recombination. Table 3 summarizes the sequence variants detected in this study.
No apparent disease-causing mutations were identified in DGKI, in adRP families. It is unlikely that the missense sequence change (Lys153Phe) seen in several of the families is disease causing since it does not segregate with disease in at least one of the families tested. None of the intronic variants detected in this study affect consensus splice sequences so they are also unlikely causes of disease. Identification of the sequence polymorphism in intron 31 allowed linkage testing between the disease locus in the UTAD045 family and DGKI. A LOD score of -4.2 at 0% recombination excludes DGKI as the possible cause of disease in this family, although portions of the gene 3' of intron 31 are not formally excluded.
DGKI is excluded as the cause of RP10 by mutation screening and linkage analysis. Further, it is unlikely that mutations in DGKI are a common cause of other forms of autosomal dominant RP. It is possible though, that mutations in DGKI may yet be the cause of retinal degeneration in humans, either in homozygous or compound heterozygous forms. In order for disease to manifest in the rdgA Drosophila, both alleles of the diacylglycerol kinase have to be defective, therefore it is possible that both alleles of DGKI must also be mutated in humans to cause retinal degeneration . Data presented here will make it possible to determine if mutations in DGKI are the cause of disease in patients with autosomal recessive RP or other forms of retinopathy.
We thank members of the UTAD045 family for their continued participation in this study. This work was supported by grants from the Foundation Fighting Blindness and the George Gund Foundation, the William Stamps Farish Fund, the M.D. Anderson Foundation, and grant EY07142 from the National Eye Institute-National Institutes of Health.
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