Title: A Missense Mutation in DHDDS, Encoding Dehydrodolichyl Diphosphate Synthase, Is Associated with Autosomal-Recessive Retinitis Pigmentosa in Ashkenazi Jews
Abstract: Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerations caused by mutations in at least 50 genes. Using homozygosity mapping in Ashkenazi Jewish (AJ) patients with autosomal-recessive RP (arRP), we identified a shared 1.7 Mb homozygous region on chromosome 1p36.11. Sequence analysis revealed a founder homozygous missense mutation, c.124A>G (p.Lys42Glu), in the dehydrodolichyl diphosphate synthase gene (DHDDS) in 20 AJ patients with RP of 15 unrelated families. The mutation was not identified in an additional set of 109 AJ patients with RP, in 20 AJ patients with other inherited retinal diseases, or in 70 patients with retinal degeneration of other ethnic origins. The mutation was found heterozygously in 1 out of 322 ethnically matched normal control individuals. RT-PCR analysis in 21 human tissues revealed ubiquitous expression of DHDDS. Immunohistochemical analysis of the human retina with anti-DHDDS antibodies revealed intense labeling of the cone and rod photoreceptor inner segments. Clinical manifestations of patients who are homozygous for the c.124A>G mutation were within the spectrum associated with arRP. Most patients had symptoms of night and peripheral vision loss, nondetectable electroretinographic responses, constriction of visual fields, and funduscopic hallmarks of retinal degeneration. DHDDS is a key enzyme in the pathway of dolichol, which plays an important role in N-glycosylation of many glycoproteins, including rhodopsin. Our results support a pivotal role of DHDDS in retinal function and may allow for new therapeutic interventions for RP. Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerations caused by mutations in at least 50 genes. Using homozygosity mapping in Ashkenazi Jewish (AJ) patients with autosomal-recessive RP (arRP), we identified a shared 1.7 Mb homozygous region on chromosome 1p36.11. Sequence analysis revealed a founder homozygous missense mutation, c.124A>G (p.Lys42Glu), in the dehydrodolichyl diphosphate synthase gene (DHDDS) in 20 AJ patients with RP of 15 unrelated families. The mutation was not identified in an additional set of 109 AJ patients with RP, in 20 AJ patients with other inherited retinal diseases, or in 70 patients with retinal degeneration of other ethnic origins. The mutation was found heterozygously in 1 out of 322 ethnically matched normal control individuals. RT-PCR analysis in 21 human tissues revealed ubiquitous expression of DHDDS. Immunohistochemical analysis of the human retina with anti-DHDDS antibodies revealed intense labeling of the cone and rod photoreceptor inner segments. Clinical manifestations of patients who are homozygous for the c.124A>G mutation were within the spectrum associated with arRP. Most patients had symptoms of night and peripheral vision loss, nondetectable electroretinographic responses, constriction of visual fields, and funduscopic hallmarks of retinal degeneration. DHDDS is a key enzyme in the pathway of dolichol, which plays an important role in N-glycosylation of many glycoproteins, including rhodopsin. Our results support a pivotal role of DHDDS in retinal function and may allow for new therapeutic interventions for RP. Retinitis pigmentosa (RP; MIM 268000) is the most common inherited retinal degeneration, with an estimated worldwide prevalence of 1:4000.1Rosenberg T. Epidemiology of hereditary ocular disorders.Dev. Ophthalmol. 2003; 37: 16-33Crossref PubMed Scopus (29) Google Scholar, 2Bundey S. Crews S.J. A study of retinitis pigmentosa in the City of Birmingham. II Clinical and genetic heterogeneity.J. Med. Genet. 1984; 21: 421-428Crossref PubMed Scopus (43) Google Scholar, 3Bunker C.H. Berson E.L. Bromley W.C. Hayes R.P. Roderick T.H. Prevalence of retinitis pigmentosa in Maine.Am. J. Ophthalmol. 1984; 97: 357-365Abstract Full Text PDF PubMed Scopus (328) Google Scholar The disease is highly heterogeneous and has several patterns of inheritance. At present, 35 genetic loci have been implicated in nonsyndromic autosomal-recessive RP (arRP), most of which account for a few percent of RP cases each. Although many of the early identified arRP genes were excellent candidates for the disease when mutated, mainly because of the function of the encoded protein (e.g., PDE6A4Huang S.H. Pittler S.J. Huang X. Oliveira L. Berson E.L. Dryja T.P. Autosomal recessive retinitis pigmentosa caused by mutations in the alpha subunit of rod cGMP phosphodiesterase.Nat. Genet. 1995; 11: 468-471Crossref PubMed Scopus (200) Google Scholar [MIM 180071] and PDE6B5McLaughlin M.E. Sandberg M.A. Berson E.L. 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Abu-Safieh L. Patel R.J. Papaioannou M.G. Inglehearn C.F. Keen T.J. Willis C. Moore A.T. et al.Mutations in HPRP3, a third member of pre-mRNA splicing factor genes, implicated in autosomal dominant retinitis pigmentosa.Hum. Mol. Genet. 2002; 11: 87-92Crossref PubMed Google Scholar) have been identified and provided new insight into processes that result in retinal degeneration. The reason for the retina-specific phenotype caused by mutations in these genes is still unclear. The Ashkenazi Jewish (AJ) population was established by Jews who originated in the Middle East and migrated to Europe, initially settling in Germany (the “Ashkenaz” region) at or before the 4th century. The AJ lived in closed communities in European countries and developed a unique culture and language (named Yiddish, which is based on a few different languages, including German, Hebrew, and Aramaic). After the Holocaust, the population size dropped from about 8.8 million individuals to only 2.8 million, and AJ immigrated out of Europe, mainly to the United States and the emerging state of Israel. AJ currently constitute the largest Jewish ethnic group in both countries. A large amount of effort was directed to study the genetic structure of the AJ population, in the context of other Jewish ethnic groups and Middle Eastern populations, at the Y chromosome,11Nebel A. Filon D. Brinkmann B. Majumder P.P. Faerman M. Oppenheim A. The Y chromosome pool of Jews as part of the genetic landscape of the Middle East.Am. J. Hum. Genet. 2001; 69: 1095-1112Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 12Behar D.M. Thomas M.G. Skorecki K. Hammer M.F. Bulygina E. Rosengarten D. Jones A.L. Held K. Moses V. Goldstein D. et al.Multiple origins of Ashkenazi Levites: Y chromosome evidence for both Near Eastern and European ancestries.Am. J. Hum. Genet. 2003; 73: 768-779Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar mitochondrial,13Behar D.M. Metspalu E. Kivisild T. Achilli A. Hadid Y. Tzur S. Pereira L. Amorim A. Quintana-Murci L. Majamaa K. et al.The matrilineal ancestry of Ashkenazi Jewry: Portrait of a recent founder event.Am. J. Hum. Genet. 2006; 78: 487-497Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar and genomic14Bray S.M. Mulle J.G. Dodd A.F. Pulver A.E. Wooding S. Warren S.T. Signatures of founder effects, admixture, and selection in the Ashkenazi Jewish population.Proc. Natl. Acad. Sci. USA. 2010; 107: 16222-16227Crossref PubMed Scopus (67) Google Scholar, 15Atzmon G. Hao L. Pe'er I. Velez C. Pearlman A. Palamara P.F. Morrow B. Friedman E. Oddoux C. Burns E. Ostrer H. Abraham's children in the genome era: Major Jewish diaspora populations comprise distinct genetic clusters with shared Middle Eastern Ancestry.Am. J. Hum. Genet. 2010; 86: 850-859Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar levels. Although consanguineous marriages are relatively uncommon among AJ (1.5% and rapidly declining),16Zlotogora J. Bach G. Munnich A. Molecular basis of mendelian disorders among Jews.Mol. Genet. Metab. 2000; 69: 169-180Crossref PubMed Scopus (30) Google Scholar, 17Cohen T. Vardi-Saliternik R. Friedlander Y. Consanguinity, intracommunity and intercommunity marriages in a population sample of Israeli Jews.Ann. Hum. Biol. 2004; 31: 38-48Crossref PubMed Scopus (45) Google Scholar most individuals who are affected by a rare AR disease in this ethnic group are homozygous for the disease-causing mutation, mainly because of a high rate of intracommunity marriages.17Cohen T. Vardi-Saliternik R. Friedlander Y. Consanguinity, intracommunity and intercommunity marriages in a population sample of Israeli Jews.Ann. Hum. Biol. 2004; 31: 38-48Crossref PubMed Scopus (45) Google Scholar Therefore, genetic analysis of hereditary diseases in the AJ population, via homozygosity mapping, can be highly efficient. In the present study, we used homozygosity mapping to identify the cause of disease in AJ families with arRP. The tenets of the Declaration of Helsinki were followed, and prior to donation of a blood sample, informed consent was obtained from all patients who participated in this study. DNA was extracted from the index patient, as well as from other affected and unaffected family members, with the FlexiGene DNA kit (QIAGEN). Whole-genome SNP analysis was initially performed on 11 patients with isolate or arRP who belong to eight different AJ families with either the Affymetrix 250K or 6.0 microarrays, and data analysis was performed with HomozygosityMapper. Patients from three of the families (MOL0400, MOL0565, and MOL0884; Table 1) had a shared homozygous region on chromosome 1p36.11 encompassing ∼2.32 Mb (Figure 1A ). The shared homozygous region contains 56 protein coding genes, none of which is expressed exclusively in the retina. While we were analyzing candidate genes for mutations in the shared homozygous region, Zuchner and collaborators reported the identification of a missense mutation in an AJ family with arRP in a gene encoding a key enzyme in the terpenoid backbone synthesis (S.Z. et al., abstract presented at The American Society of Human Genetics annual meeting 2010). One of the genes in the linked region on 1p36.11 was the dehydrodolichyl diphosphate synthase gene (DHDDS; MIM 608172), encoding the dehydrodolichyl diphosphate synthase (Figure 1B), which is predicted to take part in the above-mentioned biochemical pathway.18Endo S. Zhang Y.W. Takahashi S. Koyama T. Identification of human dehydrodolichyl diphosphate synthase gene.Biochim. Biophys. Acta. 2003; 1625: 291-295Crossref PubMed Scopus (48) Google Scholar Using the Primer3 software,19Rozen S. Skaletsky H.J. Primer3 on the WWW for general users and for biologist programmers.in: Krawetz S. Misener S. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ2000: 365-386Google Scholar we designed primers flanking all coding exons and exon-intron boundaries of DHDDS (accession number NM_024887.2) and performed sequence analysis in the three index patients. The analysis revealed a homozygous transition, c.124A>G (Figure 1C), expected to result in an amino acid (aa) substitution, p.Lys42Glu, in all three index cases. Screening the mutation in a set of 322 ethnically matched normal controls revealed one heterozygous individual, thus indicating a carrier frequency of 0.3% (95% confidence interval 0.07%–1.7%) in the AJ population. We subsequently screened this mutation in a set of 121 AJ patients with RP, 20 AJ patients with other inherited retinal diseases, and 70 non-AJ patients with retinal degeneration of other ethnic origins. The analysis revealed 12 additional index cases with RP that were homozygous for the c.124A>G mutation, all of AJ descent. In addition, we analyzed whole-genome SNP microarray data of patients from 124 consanguineous families with RP or Leber congenital amaurosis and identified 20 index cases homozygous to the DHDDS region. DHDDS sequencing analysis in these patients, as well as in an additional set of 20 index RP patients from nonconsanguineous families, did not reveal any potential pathogenic mutation. Statistical analyses with Fisher's exact test (based on allele frequency: the mutation was found in 1 out of 644 chromosomes in the control group versus 30 out of 246 chromosomes in patients) and chi-square (based on genotype frequency: the number of homozygous, heterozygous, and wild-type individuals was 0, 1, and 321, respectively, in controls versus 15, 0, and 108 in patients) showed a significant difference between patient and control groups (p < 0.001 for each comparison).Table 1Ashkenazi Jewish RP Families with the Homozygous p.Lys42Glu Mutation in DHDDSFamilyAffected SiblingsLevel of ConsanguinityaLevel of consanguinity is measured by the number of generations separating the spouse from the common ancestor (e.g., 2:2 denotes first cousins).Size of Homozygous Region (Mb)MOL03971NoneNPMOL040012:215.1MOL05652None3.8MOL07181None3.0MOL07351None3.0MOL07792None3.0MOL08843None3.7TB31/R541NoneNPTB61/R2261None3.4CHRD02622NoneNPCHRD06771None3.0CHRD33231None3.2CHRD34581None4.9CHRD40472None3.8CHRD51511None4.0NP denotes not performed.a Level of consanguinity is measured by the number of generations separating the spouse from the common ancestor (e.g., 2:2 denotes first cousins). Open table in a new tab NP denotes not performed. To better characterize the shared homozygous region, we performed whole-genome SNP analysis (with Affymetrix 6.0 arrays) on nine additional homozygous index cases (see Table 1 and Table S1 available online; whole-genome SNP array genotyping data are available by request). This allowed us to analyze the homozygous region in a total of 15 homozygous patients and 1 unaffected sibling representing 12 different AJ families. The analysis revealed an identical shared region of 1.68 Mb (26.4–28.08) on chromosome 1, harboring 41 protein-coding genes, including DHDDS. The shared haplotype contains 244 homozygous SNPs representing a founder RP-associated haplotype in the AJ population. No other shared homozygous regions (over 1 Mb) could be detected in this set of patients, nor in the original three families used for homozygosity mapping. The c.124A>G mutation affects a conserved amino acid residue (Lys42; Figure 1D) located in close proximity to a binding site of farnesyl diphosphate. The mutation substitutes the wild-type basic aa lysine with an acidic one, glutamic acid. The predicted effect of this substitution, with different tools designed to predict functional effects of missense changes, is not conclusive. Some tools did not predict a functional effect (e.g., PMUT: a neutral score of 0.4429; SAP: a neutral score of 0.53), whereas other tools did predict a functional effect (e.g., PolyPhen2: possibly damaging with a score of 0.206; SIFT: an intolerant score of 0.03; MutationTaster: a disease-causing prediction with a probability of 0.998). In summary, the p.Lys42Glu DHDDS mutation was identified in 15 (out of 123, 12%) AJ RP index patients in our cohort, and it cosegregated with the disease when additional family members were available for the study (Figure 2). Aiming to clinically characterize patients with the p.Lys42Glu DHDDS mutation, we performed full ophthalmologic examinations, electroretinography (ERG), kinetic visual fields, color vision testing, optical coherence tomography (OCT), and autofluoresence (AF) imaging, as previously described.20Beit-Ya'acov A. Mizrahi-Meissonnier L. Obolensky A. Landau C. Blumenfeld A. Rosenmann A. Banin E. Sharon D. Homozygosity for a novel ABCA4 founder splicing mutation is associated with progressive and severe Stargardt-like disease.Invest. Ophthalmol. Vis. Sci. 2007; 48: 4308-4314Crossref PubMed Scopus (31) Google Scholar, 21Jacobson S.G. Cideciyan A.V. Aleman T.S. Sumaroka A. Roman A.J. Gardner L.M. Prosser H.M. Mishra M. Bech-Hansen N.T. Herrera W. et al.Usher syndromes due to MYO7A, PCDH15, USH2A or GPR98 mutations share retinal disease mechanism.Hum. Mol. Genet. 2008; 17: 2405-2415Crossref PubMed Scopus (81) Google Scholar, 22Cideciyan A.V. Aleman T.S. Jacobson S.G. Khanna H. Sumaroka A. Aguirre G.K. Schwartz S.B. Windsor E.A. He S. Chang B. et al.Centrosomal-ciliary gene CEP290/NPHP6 mutations result in blindness with unexpected sparing of photoreceptors and visual brain: Implications for therapy of Leber congenital amaurosis.Hum. Mutat. 2007; 28: 1074-1083Crossref PubMed Scopus (108) Google Scholar Clinical evaluation of 18 patients showed a spectrum of findings (Table S2). The patients had visual acuity that ranged from light perception to 20/20 (LogMAR 0.0; Table S2). Funduscopic findings at various disease stages included waxy appearance of the optic nerve head, attenuation of retinal blood vessels, and bone spicule-like pigmentation (Figure S1). OCT imaging in early disease showed preserved central retinal photoreceptors but a decline in photoreceptor layer thickness with distance from the fovea (Figure 3E ), and occasionally the presence of cystoid macular edema (Figure S2). Kinetic visual fields revealed reduced peripheral function in the youngest patients studied and only small central islands of vision remaining later in life (Figures 3A and 3B and Figure S3). ERG responses were nondetectable in most patients (Table S2). The natural history of the disease in family MOL0884 can be gleaned from serial ocular data acquired from the second to the fourth decades of life in the three affected siblings (Figure 3). Symptoms occurred during the latter part of the second decade of life in all three siblings, and these related to decreasing night and side vision; loss of reading (central) vision was not an early complaint. Visual field extent was quantified via kinetic perimetry to a large target (Figures 3A and 3B); fields became progressively reduced with only a small central island remaining by ages 21–24. Best-corrected visual acuity had a more prolonged time course of loss but was 20/200 or worse in two siblings by age 30–31 (Figure 3C). Rod-mediated vision was measurable via dark-adapted chromatic sensitivities in all three patients at younger ages, but this progressively diminished until only cone-mediated function was detectable (Figure 3D). Photoreceptor topography in a wide expanse of central retina (Figure 3E) revealed that by the fourth decade of life, there was only a markedly thinned photoreceptor layer remaining in and around the fovea; in two of three siblings, photoreceptors were not detectable surrounding the foveal region. Patient MOL0884-4 (II:4 in Figure 2) also had a locus of detectable photoreceptors nasal to the optic nerve head (Figure 3E, P4), and this correlated with an area of limited rod sensitivity by dark-adapted psychophysical testing (Figure 3D). Near-infrared autofluoresence imaging showed islands of preserved retinal pigment epithelium corresponding in retinal location to the regions of preserved photoreceptors (Figure 3E, insets). Two of the patients were imaged with wide-angle view, and bone spicule-like pigment was visible (Figure S2). In addition, both parents and an asymptomatic sibling in family MOL0884 were examined clinically, with kinetic perimetry and standard ERGs. No abnormalities were detected, with the exception of a borderline rod ERG amplitude in the father's recording. DHDDS contains eight coding exons along a genomic region of ∼37 kb18Endo S. Zhang Y.W. Takahashi S. Koyama T. Identification of human dehydrodolichyl diphosphate synthase gene.Biochim. Biophys. Acta. 2003; 1625: 291-295Crossref PubMed Scopus (48) Google Scholar and is known to produce a few splice variants.23Rebl A. Anders E. Wimmers K. Goldammer T. Characterization of dehydrodolichyl diphosphate synthase gene in rainbow trout (Oncorhynchus mykiss).Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2009; 152: 260-265Crossref PubMed Scopus (13) Google Scholar No data, however, are available on its expression or function in the retina. To verify the retinal expression of DHDDS, we isolated RNA from the human retina with TRI-reagent (Sigma-Aldrich) and synthesized cDNA with the Verso cDNA kit (Thermo) in accordance with the manufacturer's protocol. PCR-specific primers (Table S3) were designed with Primer3, and RT-PCR analysis was performed with three primer sets that were designed to amplify different parts of the cDNA molecules (Figure 4A ). The analysis confirmed the expression of the full-length transcript in the retina, as well as three alternatively spliced variants, two of which are in-frame and are likely to encode a protein (Figure 4A): a NAGNAG sequence24Hiller M. Huse K. Szafranski K. Jahn N. Hampe J. Schreiber S. Backofen R. Platzer M. Widespread occurrence of alternative splicing at NAGNAG acceptors contributes to proteome plasticity.Nat. Genet. 2004; 36: 1255-1257Crossref PubMed Scopus (144) Google Scholar in the acceptor splice site of intron 8 and a transcript in which exon 6 is skipped. The third transcript had a frameshift addition of 23 nucleotides because of a cryptic acceptor splice site in intron 3, resulting in a premature stop codon (Figure 4A) within exon 4, and is therefore likely to be degraded by the nonsense-mediated mRNA decay or, alternatively, to produce a nonfunctional protein. Expressed sequence tags (ESTs) representing these variants were previously deposited in GenBank. To better characterize the distribution of DHDDS in human tissues, we performed RT-PCR analysis on cDNAs that were synthesized from RNA derived from 20 different human tissues (Clontech; category 636643, lot 8101369A), as well as the human retina (Figure 4B). Human PGM1 (MIM 171900) was used as a control. Ubiquitous expression of DHDDS was observed, with a band of higher intensity in the retinal sample as compared to other tissues (Figure 4B, right lane). The ubiquitous expression supports data obtained with RNA blot in a set of tissues that did not include ocular ones.18Endo S. Zhang Y.W. Takahashi S. Koyama T. Identification of human dehydrodolichyl diphosphate synthase gene.Biochim. Biophys. Acta. 2003; 1625: 291-295Crossref PubMed Scopus (48) Google Scholar To study the localization of the DHDDS enzyme in the human retina, we performed immunohistological studies with two commercially available anti-DHDDS antibodies (HPA026721 and HPA026727 [rabbit polyclonal, Sigma Life Science] at a final concentration of 1:15 and 1:175, respectively; secondary antibody Cy™2-conjugated donkey anti-rabbit IgG [1:200, Jackson ImmunoResearch Laboratories]). Prominent staining of the inner segments of photoreceptors was identified, which differed between rod and cone photoreceptors (Figure 5B , compared to the hematoxylin and eosin staining in Figure 5A and the negative control section in Figure 5C). Although the cone inner segments demonstrated a uniform signal, only the ellipsoid (distal region) and myoid (proximal region) of the rod photoreceptor inner segments were labeled, whereas the central part had much lower fluorescence (Figures 5D and 5E). The labeling of the cone inner segments is in accordance with a uniform distribution of cone inner segment organelles. In the rod inner segments, the myoid region contains the cellular protein synthesis machinery (including the Golgi apparatus and the endoplasmatic reticulum) in which N-glycosylation takes part, and the ellipsoid region contains mainly the mitochondria, which was shown to contain dolichol and dolichyl phosphate.25Ardail D. Lermé F. Louisot P. Dolichol kinase activity: A key factor in the control of N-glycosylation in inner mitochondrial membranes.Biochim. Biophys. Acta. 1990; 1024: 131-138Crossref PubMed Scopus (5) Google Scholar, 26Eggens I. Chojnacki T. Kenne L. Dallner G. Separation, quantitation and distribution of dolichol and dolichyl phosphate in rat and human tissues.Biochim. Biophys. Acta. 1983; 751: 355-368Crossref PubMed Scopus (89) Google Scholar The subcellular localization of DHDDS in rod photoreceptors is therefore in accordance with the expected distribution of the enzyme. DHDDS-positive staining was also evident in the basal aspect of retinal pigment epithelium cells (Figure 5E), and a weak staining was evident in other retinal layers (Figure 5B). The labeling pattern of DHDDS in the human retina is compatible with its function in dolichol metabolism (expected to occur in the photoreceptor inner segments), as well as the phenotype obtained (photoreceptor degeneration) as a result of the mutation identified in DHDDS. Dehydrodolichyl diphosphate (Dedol-PP) synthase is a highly conserved enzyme that can be found in all animal species, as well as in plants and bacteria. 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