Mutations in C10orf11, a Melanocyte-Differentiation Gene, Cause Autosomal-Recessive Albinism
2013; Elsevier BV; Volume: 92; Issue: 3 Linguagem: Inglês
10.1016/j.ajhg.2013.01.006
ISSN1537-6605
AutoresKaren Grønskov, Christopher M. Dooley, Elsebet Østergaard, Robert N. Kelsh, Lars Hestbjerg Hansen, Mitchell P. Levesque, Kaj Vilhelmsen, Kjeld Møllgård, Derek L. Stemple, Thomas Rosenberg,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoAutosomal-recessive albinism is a hypopigmentation disorder with a broad phenotypic range. A substantial fraction of individuals with albinism remain genetically unresolved, and it has been hypothesized that more genes are to be identified. By using homozygosity mapping of an inbred Faroese family, we identified a 3.5 Mb homozygous region (10q22.2–q22.3) on chromosome 10. The region contains five protein-coding genes, and sequencing of one of these, C10orf11, revealed a nonsense mutation that segregated with the disease and showed a recessive inheritance pattern. Investigation of additional albinism-affected individuals from the Faroe Islands revealed that five out of eight unrelated affected persons had the nonsense mutation in C10orf11. Screening of a cohort of autosomal-recessive-albinism-affected individuals residing in Denmark showed a homozygous 1 bp duplication in C10orf11 in an individual originating from Lithuania. Immunohistochemistry showed localization of C10orf11 in melanoblasts and melanocytes in human fetal tissue, but no localization was seen in retinal pigment epithelial cells. Knockdown of the zebrafish (Danio rerio) homolog with the use of morpholinos resulted in substantially decreased pigmentation and a reduction of the apparent number of pigmented melanocytes. The morphant phenotype was rescued by wild-type C10orf11, but not by mutant C10orf11. In conclusion, we have identified a melanocyte-differentiation gene, C10orf11, which when mutated causes autosomal-recessive albinism in humans. Autosomal-recessive albinism is a hypopigmentation disorder with a broad phenotypic range. A substantial fraction of individuals with albinism remain genetically unresolved, and it has been hypothesized that more genes are to be identified. By using homozygosity mapping of an inbred Faroese family, we identified a 3.5 Mb homozygous region (10q22.2–q22.3) on chromosome 10. The region contains five protein-coding genes, and sequencing of one of these, C10orf11, revealed a nonsense mutation that segregated with the disease and showed a recessive inheritance pattern. Investigation of additional albinism-affected individuals from the Faroe Islands revealed that five out of eight unrelated affected persons had the nonsense mutation in C10orf11. Screening of a cohort of autosomal-recessive-albinism-affected individuals residing in Denmark showed a homozygous 1 bp duplication in C10orf11 in an individual originating from Lithuania. Immunohistochemistry showed localization of C10orf11 in melanoblasts and melanocytes in human fetal tissue, but no localization was seen in retinal pigment epithelial cells. Knockdown of the zebrafish (Danio rerio) homolog with the use of morpholinos resulted in substantially decreased pigmentation and a reduction of the apparent number of pigmented melanocytes. The morphant phenotype was rescued by wild-type C10orf11, but not by mutant C10orf11. In conclusion, we have identified a melanocyte-differentiation gene, C10orf11, which when mutated causes autosomal-recessive albinism in humans. Autosomal-recessive albinism (oculocutaneous albinism [OCA (MIM 203100)]) is the most common condition among hypopigmentation disorders.1Tomita Y. Suzuki T. Genetics of pigmentary disorders.Am. J. Med. Genet. C. Semin. Med. Genet. 2004; 131C: 75-81Crossref PubMed Scopus (121) Google Scholar The disease is genetically heterogeneous and can be caused by mutations in at least four genes; however, a substantial fraction of individuals with albinism remain genetically unresolved.2Grønskov K. Ek J. Sand A. Scheller R. Bygum A. Brixen K. Brondum-Nielsen K. Rosenberg T. Birth prevalence and mutation spectrum in danish patients with autosomal recessive albinism.Invest. Ophthalmol. Vis. Sci. 2009; 50: 1058-1064Crossref PubMed Scopus (65) Google Scholar Melanin-producing cells originate from two lineages of progenitor cells: the outer neuroepithelium of the optic cup and the neural crest. Melanocyte development includes fate specification, migration, and differentiation in a temporally and spatially tightly controlled process.3Sommer L. Generation of melanocytes from neural crest cells.Pigment Cell Melanoma Res. 2011; 24: 411-421Crossref PubMed Scopus (95) Google Scholar, 4Murisier F. Beermann F. Genetics of pigment cells: Lessons from the tyrosinase gene family.Histol. Histopathol. 2006; 21: 567-578PubMed Google Scholar Melanogenesis involves a complex, but still only partially characterized, gene regulatory network.5Dupin E. Calloni G.W. Le Douarin N.M. The cephalic neural crest of amniote vertebrates is composed of a large majority of precursors endowed with neural, melanocytic, chondrogenic and osteogenic potentialities.Cell Cycle. 2010; 9: 238-249Crossref PubMed Scopus (65) Google Scholar, 6Greenhill E.R. Rocco A. Vibert L. Nikaido M. Kelsh R.N. An iterative genetic and dynamical modelling approach identifies novel features of the gene regulatory network underlying melanocyte development.PLoS Genet. 2011; 7: e1002265Crossref PubMed Scopus (47) Google Scholar, 7Hou L. Pavan W.J. Transcriptional and signaling regulation in neural crest stem cell-derived melanocyte development: Do all roads lead to Mitf?.Cell Res. 2008; 18: 1163-1176Crossref PubMed Scopus (148) Google Scholar, 8Lang D. Lu M.M. Huang L. Engleka K.A. Zhang M. Chu E.Y. Lipner S. Skoultchi A. Millar S.E. Epstein J.A. Pax3 functions at a nodal point in melanocyte stem cell differentiation.Nature. 2005; 433: 884-887Crossref PubMed Scopus (305) Google Scholar, 9Loftus S.K. Baxter L.L. Buac K. Watkins-Chow D.E. Larson D.M. Pavan W.J. Comparison of melanoblast expression patterns identifies distinct classes of genes.Pigment Cell Melanoma Res. 2009; 22: 611-622Crossref PubMed Scopus (14) Google Scholar, 10Schiaffino M.V. Signaling pathways in melanosome biogenesis and pathology.Int. J. Biochem. Cell Biol. 2010; 42: 1094-1104Crossref PubMed Scopus (142) Google Scholar, 11Tachibana M. MITF: A stream flowing for pigment cells.Pigment Cell Res. 2000; 13: 230-240Crossref PubMed Scopus (239) Google Scholar The four OCA types are distinguished on the basis of clinical and molecular findings. OCA type 1 (MIM 203100 and 606952) and OCA type 3 (MIM 203290) are directly involved in melanin synthesis through TYR (MIM 606933)-encoded tyrosinase and TYRP1 (MIM 115501)-encoded tyrosinase-related protein 1. OCA type 2 (MIM 203200) is caused by mutations in OCA2 (MIM 611409), encoding a melanosomal membrane protein, and OCA type 4 (MIM 606574) is due to mutations in SLC45A2 (MIM 606202), encoding a melanosomal membrane-associated transporter protein. Still, unknown genes are thought to be involved in this disorder. Fifteen individuals with autosomal-recessive albinism were examined at the outpatient eye clinic at the National Hospital in Tórshavn, Faroe Islands, and six of these were also examined at least once at the National Eye Clinic for the Visually Impaired, Denmark. The assessments consisted of a routine ophthalmological examination including motility, refraction, best Snellen visual acuity, slit lamp with transillumination, and ophthalmoscopy. Furthermore, Goldmann visual-field measurements, fundus photography, electroretinography, visual-evoked potentials (VEPs) with monocular and binocular flash and checkerboard stimulation, and bioccipital recording for the measurement of crossed asymmetry of the visual pathways were performed on eight affected persons. In one individual, ocular coherence tomography (OCT) of the macular region was performed. The study followed the guidelines of the Helsinki Declaration and was approved by the local ethics committee and by the Scientific Ethical Committee of the Faroe Islands, and it was contracted with the Biobank under the Faroese Ministry of Health. Affected individuals and healthy relatives were informed of the nature of the study, and written informed consent was obtained from all participants. Homozygosity mapping was performed with the Affymetrix SNP6.0 platform (Affymetrix, Santa Clara, CA, USA). The analysis was performed by AROS Applied Biotechnology A/S (Århus, Denmark). CNCHP files were generated with Genotyping Console (version 4.0) and analyzed by Chromosome Analysis Suite (version 1.2.2) software (Affymetrix) with default settings (minimum ten markers; loss of heterozygosity minimum physical size of 1 Mb). Five affected members from family OAR00201 were used for the initial analysis (Figure 1A). In addition, two unaffected family members were included for subtraction of nonrelevant homozygous regions. This revealed one homozygous region on chromosome 10 (chr10: 77,233,812–80,685,953; UCSC Genome Browser hg19). Three additional affected individuals (two sisters and one isolated case) were found to be homozygous in an overlapping region (Figure S1, available online). The region contains five protein-coding RefSeq genes: C10orf11 (MIM 614537), KCNMA1 (MIM 600150), DLG5 (MIM 604090), POLR3A (MIM 614258), and RPS24 (MIM 602412) (Figure S2). Furthermore, two genes (LOC100128292 and LOC100132987) encoding noncoding RNA molecules were located in the region. Mutations in KCNMA1 are a known cause of generalized epilepsy and paroxysmal dyskinesia (MIM 609446).12Du W. Bautista J.F. Yang H. Diez-Sampedro A. You S.A. Wang L. Kotagal P. Lüders H.O. Shi J. Cui J. et al.Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder.Nat. Genet. 2005; 37: 733-738Crossref PubMed Scopus (465) Google Scholar Mice with knockout of both Dlg5 alleles show renal cysts and hydrocephalus.13Nechiporuk T. Fernandez T.E. Vasioukhin V. Failure of epithelial tube maintenance causes hydrocephalus and renal cysts in Dlg5-/- mice.Dev. Cell. 2007; 13: 338-350Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar POLR3A encodes the largest and catalytic core component of RNA polymerase III, and mutations in POLR3A are known to cause leukodystrophy, hypomyelinating, 7, with or without oligodontia and/or hypogonadotropic hypogonadism (MIM 607694). Mutations in RPS24, encoding a ribosomal protein, are known to cause Diamond-blackfan anemia 3 (MIM 610629). Mutations in either of these genes seemed unlikely to cause albinism. We therefore started out by sequencing C10orf11 (primers are listed in Table S1). This revealed a homozygous nonsense mutation, c.580C>T (p.Arg194∗) (RefSeq accession number NM_032024.3), in all five affected individuals from family OAR00201. Analysis of a further eight unrelated affected individuals revealed five with the same mutation. This means that in nine families with autosomal-recessive albinism, probands from six of them were shown to have the nonsense mutation in C10orf11. Probands from the nine Faroese families had been previously analyzed in some or all of TYR, OCA2, SLC45A2, and TYRP1 (Table S2).The six families with the nonsense mutation were shown to share a common ancestor in both the paternal and maternal lines. Analysis of eight unaffected relatives from four families showed segregation in accordance with autosomal-recessive inheritance. Ninety-two control individuals from the Faroe Islands were analyzed by high-resolution melting analysis. In brief, a PCR product of 105 bp was amplified with primers 5′-GAAGTGTCGCTACGTTTACTATGG-3′ and 5′-TTCATGAAATGGGTGAAGGT-3′. Amplification products were analyzed in a 96-well LightScanner instrument (HR96, Biofire, Salt Lake City, UT, USA), and melting data were collected in the temperature range of 60°C –98°C. Data were analyzed with LightScanner software with Call-IT (version 2.0, Biofire). Three individuals were carriers of c.580C>T, corresponding to a carrier frequency of 3.3%. We next considered whether mutations in C10orf11 are a cause of autosomal-recessive albinism in other populations. Forty-eight genetically unexplained individuals who had autosomal-recessive albinism and who were residing in Denmark were screened for mutations in C10orf11. Of these, 42 had previously been screened for mutations in TYR, OCA2, TYRP1, and SLC45A2 (21 had no obvious mutations, 13 had one TYR mutation, 7 had one OCA2 mutation, and 1 had one TYR mutation and one OCA2 mutation),2Grønskov K. Ek J. Sand A. Scheller R. Bygum A. Brixen K. Brondum-Nielsen K. Rosenberg T. Birth prevalence and mutation spectrum in danish patients with autosomal recessive albinism.Invest. Ophthalmol. Vis. Sci. 2009; 50: 1058-1064Crossref PubMed Scopus (65) Google Scholar whereas the remaining six persons had no prior investigation. In one individual, we found a mutation in C10orf11. This person (individual 76121), originating from Lithuania, was apparently homozygous for a 1 bp duplication (c.66dupC [p.Ala23Argfs∗39]); however, a deletion on one allele cannot be ruled out and neither can a preferential amplification of one allele because of a sequence variation on the second allele (although primers were checked by the SNPCheck program for minimizing this risk). The possibility of a second mutation in linkage disequilibrium with the C10orf11 mutations contributing to the phenotype also cannot be ruled out. Most of the affected individuals had a light Northern complexion with a tendency to lighter pigmentation than that of their relatives. Eye symptoms were predominant: nystagmus and iris transillumination were present in all subjects. The phenotypic characteristics of nine affected individuals are shown in Table 1. Extremely sparse pigmentation of the peripheral ocular fundus was seen (Figure 2). Visual acuity varied between 6/9 and 3/60. VEP recordings showed crossed asymmetry of the cortical visual response in all tested individuals (n = 8). Photophobia was not a major problem. Hair color varied from pale blond to dark brown.Table 1Clinical Signs and Symptoms in Nine Individuals with Homozygous C10orf11 MutationsIndividual IDMutationSnellen Visual AcuityRefractionIris TransilluminationScalp Hair ColorMisroutingMiscellaneousPN0224c.[580C>T];[580C>T]R 6/60L 6/60R +6.50 −2.00 × 0°L +6.00 −1.00 × 10°extensiveblond with reddish tintNDesotropia 25° in R eyePN0220c.[580C>T];[580C>T]R 6/36L 6/36R +6.50 −2.00 × 160°L +6.50 −2.00 × 30°extensiveblond+esotropia 15° in L eyePN0278c.[580C>T];[580C>T]R 6/18L 6/18R +1.50 −3.00 × 0°L +1.50 −3.00 × 0°extensivedark brown+dark brown eyebrows and lashes, slow tanningPN0207c.[580C>T];[580C>T]R 6/30L 6/24R +3.00 −3.50 × 15°L +3.00 −3.00 × 165°extensiveblond+nonePN0302c.[580C>T];[580C>T]R 3/60L 3/60R +0.50 −2.00 × 10°L +0.50 −2.00 × 10°only peripheralblond+cone-rod dystrophy,aPN0302 and PN0303 are sisters and, in addition to the C10orf11 mutation, had a deleterious homozygous PCDH21 mutation causing cone-rod dystrophy.14 blond eyebrows and lashesPN0303c.[580C>T];[580C>T]R 6/30L 6/30L +5.00 −1.50 × 130°L +7.00 −1.50 × 130°extensiveblond+cone-rod dystrophyaPN0302 and PN0303 are sisters and, in addition to the C10orf11 mutation, had a deleterious homozygous PCDH21 mutation causing cone-rod dystrophy.14PN0295c.[580C>T];[(580C>T)]R 3/60L 3/60R +7.50 −3.50 × 20°L +7.50 −3.00 × 15°extensivewhite+exotropia in R eyePN0449c.[580C>T];[(580C>T)]R 6/36L 6/36R +5.00 −1.00 × 170°L +4.50 −1.50 × 20°extensivedark blond+none76121c.[66dupC];[(66dupC)]R 6/30L 6/30R +2.25 −3.00 × 175°L +2.50 −3.00 × 5°extensivedark blond+corneal surgery (Moscow, 1982)Individuals whose ID number begins with “PN” belong to the population of the Faroe Islands. Individuals PN0224 (III2) and PN0220 (II8) belong to the index pedigree. Individuals PN0278, PN0207, PN0295, and PN0449 stated to have no affected relatives. Individual 76121 was born in Lithuania by Lithuanian parents. None of the individuals showed signs of Wardenburg syndrome, i.e., white forelock, premature graying of the hair, dystopia canthorum, hypopigmented or hyperpigmented areas of skin, craniofacial dysmorphism, or sensineural deafness. The following abbreviations are used: R, right; L, left; and ND, no data.a PN0302 and PN0303 are sisters and, in addition to the C10orf11 mutation, had a deleterious homozygous PCDH21 mutation causing cone-rod dystrophy.14Ostergaard E. Batbayli M. Duno M. Vilhelmsen K. Rosenberg T. Mutations in PCDH21 cause autosomal recessive cone-rod dystrophy.J. Med. Genet. 2010; 47: 665-669Crossref PubMed Scopus (51) Google Scholar Open table in a new tab Individuals whose ID number begins with “PN” belong to the population of the Faroe Islands. Individuals PN0224 (III2) and PN0220 (II8) belong to the index pedigree. Individuals PN0278, PN0207, PN0295, and PN0449 stated to have no affected relatives. Individual 76121 was born in Lithuania by Lithuanian parents. None of the individuals showed signs of Wardenburg syndrome, i.e., white forelock, premature graying of the hair, dystopia canthorum, hypopigmented or hyperpigmented areas of skin, craniofacial dysmorphism, or sensineural deafness. The following abbreviations are used: R, right; L, left; and ND, no data. C10orf11 encodes a 198 amino acid protein containing three leucine-rich repeats (LRRs) and one LRR C-terminal (LRRCT) domain (Figure 1B). LRRs containing proteins cover a broad spectrum of functions and include cell adhesion and signaling, extracellular-matrix assembly, platelet aggregation, neuronal development, RNA processing, and immune response.15Bella J. Hindle K.L. McEwan P.A. Lovell S.C. The leucine-rich repeat structure.Cell. Mol. Life Sci. 2008; 65: 2307-2333Crossref PubMed Scopus (342) Google Scholar The localization of C10orf11 in human tissue specimens was investigated in samples of human embryonic and fetal eye and skin tissues. These were obtained either from the archives of the Department of Cellular and Molecular Medicine at the University of Copenhagen or from legal abortions performed at the Department of Obstetrics and Gynecology, Frederiksberg Hospital. Oral and written information was given, and informed consent was obtained from all contributing women and was approved by the Regional Committee on Biomedical Research Ethics of Copenhagen and Frederiksberg (KF (01) 258206). Samples from the adult human eye were also included. Late in the human embryonic period, neural crest cells migrate into the mesenchyme of the developing dermis, where they differentiate into C10orf11-positive melanocyte precursors (melanoblasts) (Figures 3A and 3B ). Later, the majority migrate to the dermoepidermal junction, penetrate the basal membrane, and differentiate into strongly stained melanocytes in the basal layer of epidermis and in hair follicles (Figure 3C). All control sections were negative (Figure S3). Parallel sections that included retinal pigment epithelium (RPE) in the developing human eye showed no reactivity to C10orf11 (Figure S4). This localization pattern is consistent with a gene that is important for and acts cell autonomously in melanocyte differentiation and function. To advance our understanding of the function of C10orf11, we employed the zebrafish (Danio rerio) as a model organism. Zebrafish embryos were obtained via natural spawning essentially as described.16Westerfield M. The Zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio).Fourth Edition. University of Oregon Press, Eugene2000Google Scholar Replicates of morpholino experiments were carried out in WIK, TLF, and SAT zebrafish backgrounds. A single zebrafish homolog, c10orf11, is known and shows 69% similarity at both the nucleotide and amino acid levels (Figure 4A). mRNA expression was investigated by in situ hybridization, which was essentially carried out as previously stated (Zebrafish Model Organism Database). Oligonucleotides (c10orf11 and c10orf11_T7) (Table S3) were designed for generating a c10orf11 PCR product. Antisense probes were synthesized from the PCR products with the use of digoxigenin-labeled nucleotide triphosphates and T7 polymerase. Sense probes were produced in a similar manner and were used for negative controls. Expression of c10orf11 was seen in migrating neural crest cells (Figures 4B–4D). This suggests that c10orf11 might have a conserved role in pigment cell development in zebrafish. To test whether the expressing cells included melanoblasts, we examined c10orf11 expression in mitfa mutant embryos. Mitfa is required for specification, maintenance, and differentiation of the melanocyte lineage.17Lister J.A. Close J. Raible D.W. Duplicate mitf genes in zebrafish: Complementary expression and conservation of melanogenic potential.Dev. Biol. 2001; 237: 333-344Crossref PubMed Scopus (142) Google Scholar In contrast to wild-type siblings, mitfaw2 mutants18Lister J.A. Robertson C.P. Lepage T. Johnson S.L. Raible D.W. nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate.Development. 1999; 126: 3757-3767PubMed Google Scholar showed highly reduced c10orf11 expression and no detectable expression in migrating neural crest cells, clearly consistent with expression in melanoblasts. Residual expression in cells that we interpret as premigratory neural crest cells (Figures 4E and 4F, arrowheads) indicates that expression might be initially induced by a mitfa-independent mechanism. These data encouraged us to explore a role for c10orf11 in melanocyte development by knockdown with morpholino antisense oligonucleotides (MOs) (Figures 4G and 4H). Three morpholinos were designed to specifically knockdown zebrafish c10orf11 (ENSDART00000145499): ATG-MO19Ekker S.C. Morphants: A new systematic vertebrate functional genomics approach.Yeast. 2000; 17: 302-306Crossref PubMed Google Scholar to block any transcript including potential maternal transcript, Ex-skip MO to block splicing, and Int_Ret_MO20Draper B.W. Morcos P.A. Kimmel C.B. Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: A quantifiable method for gene knockdown.Genesis. 2001; 30: 154-156Crossref PubMed Scopus (305) Google Scholar to block zygotic transcripts. We also incorporated the use of the Tp53 MO and a standard control morpholino when stated. See Table S3 for sequences of morpholinos. The Tp53 MO was used to decrease apoptosis triggered by nonspecific morpholino-induced Tp53 activation.21Langheinrich U. Hennen E. Stott G. Vacun G. Zebrafish as a model organism for the identification and characterization of drugs and genes affecting p53 signaling.Curr. Biol. 2002; 12: 2023-2028Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar All morpholinos were produced by GeneTools (Philomath, OR, USA). To control the efficacy of the splice-blocking Ex-skip MO, we performed RT-PCR on cDNA (Transcriptor High Fidelity cDNA Synthesis Kit, Roche, Basel, Switzerland) generated from 24 hpf (hours postfertilization) morphant embryos by using oligonucleotides ex2F and ex2R (Table S3). It was possible to confirm a splice skipping of exon 2 via RT-PCR band shift (Figure 4I). We focused on the early pigment cell phenotype because morphant larvae were observed to go into developmental arrest at 48–72 hpf. Melanization of the RPE was first seen at 24 hpf in wild-type animals and was followed by the melanization of the neural-crest-derived melanocytes soon after; by 32 hpf, melanocytes migrated away from the dorsal neural tube and were dispersed throughout most of the head, anterior trunk, and yolk sac (Figure 4G). In the morphants, there was a strong reduction in the apparent number of pigmented melanocytes, and the remaining cells showed substantially decreased pigmentation (Figure 4H). Epidermal blistering along ventral and dorsal fin folds was also prominent among most morphants at this stage (Figure 4H). Similar phenotypic results were obtained with ATG-MO, as well as when ATG-MO and Ex-skip MO were mixed at half concentrations (data not shown). To test whether melanoblasts are present but not pigmented, we performed in situ hybridization with the melanoblast-specific marker dopachrome tautomerase (dct [MIM 191275]) at 26 hpf by using a probe generated with the primers dct and dct_T7 (Table S3 and Figures 4J and 4K). This showed a partial reduction in both dct expression levels and the apparent melanoblast number in the c10orf11 morphants, indicating that c10orf11 is important for multiple aspects of melanocyte development, including differentiation. However, MO knockdown of c10orf11 in zebrafish did not completely abolish pigmented melanocytes. This is consistent with the phenotype observed in the human probands, who showed some pigmentation in hair and skin. Furthermore, some residual endogenous expression of c10orf11 was observed in the embryos (Figure 4I). To complement our knockdown studies, we then cloned the full-length zebrafish c10orf11 cDNA into the pGEMT cloning vector (Promega, Madison, WI, USA) by using primers 5′-CGTCAGTGGGAGTCTTCGT-3′ and 5′-CTCGCGTGTCTATCCACTGA-3′ on zebrafish cDNA from 24 hpf embryos. The c10orf11 cDNA was then subcloned by restriction digestion with EcoRI and then by T4-DNA ligation into the PCS2+ expression vector. Clones with the correct orientation and no coding mutations were identified by sequencing. We then created two truncated versions, p.Lys54∗ and p.Arg230∗ (Figure S5), mimicking the alterations identified in humans. We generated capped c10orf11 mRNA (SP6 message-machine, Life Technologies, Paisley, UK) of all three forms and then carried out a series of injections into zebrafish embryos at the 1-cell stage. All three c10orf11 mRNAs were injected at titrated concentrations for the establishment of an effective concentration (data not shown). When injected at two different amounts (100 pg and 200 pg per embryo), wild-type c10orf11 mRNA failed to give obvious ectopic phenotypes, whereas embryos injected with p.Arg230∗ were dwarfed and lacked a notochord in about half the cases (Figure 4M). The c10orf11 Ex-skip MO was coinjected with wild-type c10orf11, p.Lys54∗, or p.Arg230∗ for testing the ability to rescue the morphant phenotype (Figure 4L). When the same c10orf11 mRNAs were coinjected with Ex-skip MO, the wild-type mRNA, unlike truncated versions p.Lys54∗ and p.Arg230∗, was able to significantly rescue the morphant phenotype (p < 0.010) (Figure 4N). Currently, the likely biological function of C10orf11 remains poorly understood. Studies by Wada and collaborators show that an homolog of human C10orf11 is involved in beta-catenin (MIM 116806) signaling in early Ciona intestinalis embryos and suggest that C10orf11 functions upstream of or parallel to beta-catenin.22Wada S. Hamada M. Kobayashi K. Satoh N. Novel genes involved in canonical Wnt/beta-catenin signaling pathway in early Ciona intestinalis embryos.Dev. Growth Differ. 2008; 50: 215-227Crossref PubMed Scopus (17) Google Scholar The Wnt/beta-catenin pathway plays an important role in fundamental biological processes involving cellular adhesion, tissue morphogenesis, and oncogenesis.23Mulholland D.J. Dedhar S. Coetzee G.A. Nelson C.C. Interaction of nuclear receptors with the Wnt/beta-catenin/Tcf signaling axis: Wnt you like to know?.Endocr. Rev. 2005; 26: 898-915Crossref PubMed Scopus (346) Google Scholar Beta-catenin has been shown to be required for melanocyte specification and has the transcription-factor-encoding Mitfa or MITF (MIM 156845) as a downstream target,24Widlund H.R. Horstmann M.A. Price E.R. Cui J. Lessnick S.L. Wu M. He X. Fisher D.E. Beta-catenin-induced melanoma growth requires the downstream target Microphthalmia-associated transcription factor.J. Cell Biol. 2002; 158: 1079-1087Crossref PubMed Scopus (229) Google Scholar, 25Dorsky R.I. Raible D.W. Moon R.T. Direct regulation of nacre, a zebrafish MITF homolog required for pigment cell formation, by the Wnt pathway.Genes Dev. 2000; 14: 158-162PubMed Google Scholar, 26Hari L. Brault V. Kléber M. Lee H.Y. Ille F. Leimeroth R. Paratore C. Suter U. Kemler R. Sommer L. Lineage-specific requirements of beta-catenin in neural crest development.J. Cell Biol. 2002; 159: 867-880Crossref PubMed Scopus (270) Google Scholar perhaps suggesting that C10orf11 might function upstream of MITF. However, our preliminary zebrafish results showing decreased expression of c10orf11 in mitfa mutants indicate that c10orf11 expression lies downstream of mitfa. MITF plays a critical role in the development of specific cell types, including neural-crest-derived melanocytes and optic-cup-derived RPE cells, and furthermore functions as a master regulator of melanocyte development, including the regulation of all core enzymes necessary for melanin biogenesis, such as tyrosinase, tyrosinase-related protein 1, and dopachrome tautomerase.27Wan P. Hu Y. He L. Regulation of melanocyte pivotal transcription factor MITF by some other transcription factors.Mol. Cell. Biochem. 2011; 354: 241-246Crossref PubMed Scopus (75) Google Scholar Mutations in MITF cause Waardenburg syndrome (type 2A [MIM 193510]) and show an autosomal-dominant inheritance. Waardenburg syndromes are auditory-pigmentary disorders characterized by congenital sensorineural hearing loss and pigmentary disturbances of iris, hair, and skin. We would expect that mutations in a gene functioning upstream of MITF would cause a more severe phenotype than OCA. More experiments are needed for determining the position of C10orf11 in the melanogenesis gene regulatory network. Because the precise relationship between MITF and C10orf11 in melanocyte development and differentiation is still an area of ongoing study, the reason that the affected individuals have an albino phenotype is not completely clear at this time. In conclusion, we have identified mutations in a melanocyte-differentiation gene, C10orf11, in individuals with autosomal-recessive albinism. Mutational analysis of C10orf11 in larger cohorts of individuals with albinism is needed for estimating the contribution of this gene to these diseases. The study was supported by a grant from The Danish Association of the Blind. We thank Søren Petersen, Alma Dedic, Pia Skovgaard, and Jette Bune Rasmussen (Kennedy Center, Copenhagen University Hospital, Rigshospitalet) for technical assistance. We would also like to thank Hans-Martian Maischein of the Nuesslein-Volhard lab for sending us mitfaw2 embryos. The expert technical assistance of Hanne Hadberg and Pernille S. Froh (Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen) is gratefully acknowledged. Download .pdf (.96 MB) Help with pdf files Document S1. Figures S1–S5 and Tables S1–S3 The URLs for data presented herein are as follows:ExonPrimer, http://ihg.gsf.de/ihg/ExonPrimer.htmlHuman Genome Variation Society, http://www.hgvs.org/mutnomen/Online Mendelian Inheritance in Man (OMIM), http://www.omim.orgRefSeq, http://www.ncbi.nlm.nih.gov/RefSeqSNPCheck, https://ngrl.manchester.ac.uk/SNPCheckV3/snpcheck.htmUCSC Genome Browser, http://genome.ucsc.edu/Zebrafish Model Organism Database, http://zfin.org
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