Journal of Heredity Advance Access originally published online on June 15, 2005
Journal of Heredity 2005 96(7):774-776; doi:10.1093/jhered/esi079
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Chromosomal Assignment of the Canine Melanophilin Gene (MLPH): A Candidate Gene for Coat Color Dilution in Pinschers
From the Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Bünteweg 17p, 30559 Hannover, Germany (Philipp and Leeb); UMR6061, CNRS, Université de Rennes1, 2 av. Pr. Léon Bernard, 35043 Rennes Cedex, France (Quignon and André); and College of Veterinary Medicine, 4700 Hillsborough Street, North Carolina State University, Raleigh, NC 27606 (Scott and Breen)
Address correspondence to Tosso Leeb at the address above, or e-mail: tosso.leeb{at}tiho-hannover.de.
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Abstract |
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Pinschers affected by coat color dilution show a specific pigmentation phenotype. The dilute pigmentation phenotype leads to a silver-blue appearance of the eumelanin-containing fur and a pale sandy color of pheomelanin-containing fur. In Pinscher breeding, dilute black-and-tan dogs are called "blue," and dilute red or brown animals are termed "fawn" or "Isabella fawn." Coat color dilution in Pinschers is sometimes accompanied by hair loss and a recurrent infection of the hair follicles. In human and mice, several well-characterized genes are responsible for similar pigment variations. To investigate the genetic cause of the coat color dilution in Pinschers, we isolated BAC clones containing the canine ortholog of the known murine color dilution gene Mlph. RH mapping of the canine MLPH gene was performed using an STS marker derived from BAC sequences. Additionally, one MLPH BAC clone was used as probe for FISH mapping, and the canine MLPH gene was assigned to CFA25q24.
Coat color dilution in black-and-tan Pinschers (Doberman Pinschers, German Pinschers, Miniature Pinschers) leads to the so-called blue pigmentation phenotype, characterized by a silver-blue shade of the black fur areas. Similarly, coat color dilution is responsible for the Isabella fawn phenotype on brown-and-red or red Pinschers. Color dilution in Pinschers is inherited as a Mendelian autosomal recessive trait. Although there are no severe impairments known, this pigmentation variation is of clinical relevance because Pinschers with coat color dilution show an increased prevalence of color dilution alopecia (CDA), also called blue Doberman syndrome. CDA is characterized by a progressive loss of hair, which is sometimes accompanied by recurrent bacterial infections of the hair follicles (folliculitis). The exposed skin of CDA affected dogs is often dry and scaly as well as sensitive to sunburn or extreme cold.
In human and mice, genes are already known that lead to similar pigment variations. In the mouse three mutants with coat color dilution called dilute, ashen, and leaden, respectively, are well characterized. Three different genes are mutated in these strains: In dilute mice the myosin Va gene (Myo5a) is mutated (Mercer et al. 1991), whereas in ashen or leaden mice the Rab27a gene or the melanophilin gene (Mlph) is mutated, respectively (Matesic et al. 2001; Wilson et al. 2000). The proteins encoded by these three genes are part of the melanosome transport complex. The pigment synthesis itself is functioning normal in these three mouse mutants, but the transport of pigment granules is impaired. This leads to an accumulation of melanosomes around the melanocytes' nuclei as well as large clumps of pigment in the hair shafts. In human, mutations in one of the three genes MYO5A, RAB27A, or MLPH, respectively, lead to the rare autosomal recessively inherited Griscelli syndrome (Menasche et al. 2000, 2003; Pastural et al. 1997). We have previously mapped MYO5A and RAB27A in dogs (Philipp et al. 2003a,b) and report here the chromosomal assignment of MLPH.
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Materials and Methods |
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BAC Library Screening
The canine BAC Library RPCI-81 (Li et al. 1999) was screened with a 382-bp human cDNA probe corresponding to the 5'-end of the MLPH gene. The probe was generated by polymerase chain reaction (PCR) from the human cDNA clone IRAKp961H0816, which was provided by the Resource Center/Primary Database of the German Human Genome Project (www.rzpd.de). Three positive BAC clones, termed RP81-102L9, RP81-203J24, and RP81-380K13 were isolated. To confirm the identity of the isolated BAC clones, DNA was isolated, digested with EcoRI, separated on an agarose gel, and transferred to a nylon membrane. Hybridization of this membrane with the MLPH cDNA probe confirmed that the clones indeed contained parts of the MLPH gene. BAC end sequences of the clones were determined on a Licor 4200 automated sequencer (Licor Biosciences, Bad Homburg, Germany). The BAC end sequences were used for comparative mapping to the human genome by BLAST searching and RP81-203J24 was selected for further analysis. Random plasmid shotgun subclones of RP81-203J24 were prepared using the TOPO Shotgun cloning Kit (Invitrogen, Karlsruhe, Germany). The shotgun plasmid subclones were sequenced with M13 universal and reverse primers on a MegaBace automated sequencer (Amersham Biosciences, Freiburg, Germany). The BAC insert size was determined by pulsed field gel electrophoresis (PFGE). Genomic sequences from the BAC clone were deposited under accession number AJ920047 in the EMBL nucleotide database.
Chromosomal Localization of BAC Clone RP81-203J24
Fluorescence in situ hybridization (FISH) was carried out with 25 ng fluorochrome-labeled BAC DNA from the chosen clones. Labeling was performed by nick translation to incorporate Spectrum Green-dUTP or Spectrum Orange-dUTP as described previously (Breen et al. 2001). The signal resulting from RP81-203J24 was observed at the distal end on CFA25, and this location was confirmed by subsequent colocalization of RP81-203J24 with a known single-locus probe (SLP) for CFA 25 (RP81-375L21) form a panel of chromosome-specific SLPs reported previously (Thomas et al. 2003). For radiation hybrid (RH) mapping primers MLPH_F (5'-TGAGCGTTGTGTCCGTAGTC-3') and MLPH_R (5'-CTCACTCCTTGTGGGCACTT-3') were designed from the partial sequence information of a BAC subclone. These primers generated a 192-bp product from the MLPH intron 10 on dog genomic DNA. The 118 DNAs of the RHDF 5000-2 panel (Vignaux et al. 1999) were subjected to PCR amplification with the MLPH_F and MLPH_R as described (Priat et al. 1998). The typing data obtained in duplicate were incorporated in the radiation hybrid map (Guyon et al. 2003) using the Multi Map package (Matise et al. 1994).
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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A library screen with a human MLPH cDNA probe resulted in the isolation of three canine BAC clones, from which RP81-203J24 was selected for further analysis. The insert size of this clone was estimated at 190 kb from PFGE. The SP6 BAC end sequence of RP81-203J24 had no significant match to the human genome. The T7 BAC end sequence had a BLAST match to HSA2 at 238.011 Mb in positive orientation (NCBI Map Viewer, build 35.1, E-value 9e-12). This BLAST hit was thus located
167 kb proximal of the MLPH gene in the human genome. Given the insert size of RP81-203J24 and the observation that genomic distances in the dog are often somewhat smaller than in human, this BLAST hit was entirely consistent with the hypothesis that RP81-203J24 contained at least parts of the canine MLPH gene. Furthermore, this BLAST hit indicated that RP81-203J24 also contained the entire COL6A3 gene that spans from 238.014 to 238.105 Mb on HSA2 (NCBI Map Viewer, build 35.1). To confirm these comparative mapping results, 96 randomly chosen shotgun plasmid sublones from the BAC clone were also sequenced and subjected to BLAST analyses. The BLAST results confirmed that BAC clone RP81-203J24 contained at least parts of the COL6A3 and MLPH gene. The most distal BLAST hit of the plasmid subclone sequences to the human genome was at 238.230 Mb on HSA2, which corresponds to intron 10 of the human MLPH gene. Thus we concluded that RP81-203J24 contained the entire canine COL6A3 gene and the first 10 out of 16 MLPH exons assuming that these genes have a conserved genomic organization between human and dog. The comparative analyses also indicated that the 190-kb dog clone shared homology with at least 219 kb of human DNA sequence, which is consistent with the slightly smaller genome size of the dog compared to human. From one of the plasmid subclone sequences that were located in intron 10 of the canine MLPH gene (based on the comparative mapping data) an intragenic MLPH PCR primer pair was designed and used for RH mapping. Two point analysis placed MLPH next to marker RDC1 on CFA25 with a lod score of 16.9. The next closest markers are BAC_381-H21 and BAC_375-L21 with lod scores of 14.4 and 13.8, respectively. The BAC clone RP81-203J24 was also fluorescently labeled and used for FISH mapping on canine metaphase chromosomes. Dual color FISH of the MLPH BAC clone and a known CFA25q24 probe allowed the precise assignment of MLPH to the distal portion of CFA25q24 (Figure 1). The chromosomal localization of MLPH is in good agreement with the known human–dog comparative map as the distal end of CFA25 shows conserved synteny with HSA2q36–q37.
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During our work the canine genome sequence became available. BLAST analyses indicates that RP81-203J24 spans from 51.011 to 51.208 Mb on CFA25 (NCBI Map Viewer, build 1.1). Two genes are currently annotated in this interval of the dog genome, COL6A3 and a gene termed "similar to Slp homolog lacking C2 domains-a (LOC486176)." The human ortholog of "Slp homolog lacking C2 domains-a (Slac2-a)" is in fact identical to the human MLPH gene, and the official gene symbol is now MLPH. Thus our data provide experimental evidence that LOC486176 is really a gene that should be called MLPH to avoid confusion with nonstandardized gene names.
The chromosomal assignment of MLPH as a candidate gene for coat color dilution will allow linkage analysis in suitable pedigrees with known and new markers and could thus facilitate the molecular elucidation of canine coat color mutations.
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Acknowledgments |
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This paper was delivered at the 2nd International Conference on the "Advances in Canine and Feline Genomics: Comparative Genome Anatomy and Genetic Disease," Universiteit Utrecht, Utrecht, The Netherlands, October 14–16, 2004.
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Footnotes |
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Corresponding Editor: Bernard van Oost
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Breen M, Jouquand S, Renier C, Mellersh CS, Hitte C, Holmes NG, Cheron A, Suter N, Vignaux F, Bristow AE, and others, 2001. Chromosome-specific single-locus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/linkage map of the domestic dog genome to all chromosomes. Genome Res 11:1784–1795.
Guyon R, Lorentzen TD, Hitte C, Kim L, Cadieu E, Parker HG, Quignon P, Lowe JK, Renier C, Gelfenbeyn B, and others, 2003. A 1-Mb resolution radiation hybrid map of the canine genome. Proc Natl Acad Sci USA 100:5296–5301.
Li R, Mignot E, Faraco J, Kadotani H, Cantanese J, Zhao B, Lin X, Hinton L, Ostrander EA, Patterson DF, and de Jong PJ, 1999. Construction and characterization of an eightfold redundant dog genomic bacterial artificial chromosome library. Genomics 58:9–17.[CrossRef][Web of Science][Medline]
Matesic LE, Yip R, Reuss AE, Swing DA, O'Sullivan TN, Fletcher CF, Copeland NG, and Jenkins NA, 2001. Mutations in Mlph, encoding a member of the Rab effector family, cause the melanosome transport defects observed in leaden mice. Proc Natl Acad Sci USA 98:10238–10243.
Matise TC, Perlin M, and Chakravarti A, 1994. Automated construction of genetic linkage maps using an expert system (MultiMap): a human genome linkage map. Nat Genet 6:384–90.[CrossRef][Web of Science][Medline]
Menasche G, Pastural E, Feldmann J, Certain S, Ersoy F, Dupuis S, Wulffraat N, Bianchi D, Fischer A, Le Deist F, and de Saint Basile G, 2000. Mutations in Rab 27A cause Griscelli syndrome associated with hemaphagocytic syndrome. Nat Genet 25:173–176.[CrossRef][Web of Science][Medline]
Menasche G, Ho CH, Sanal O, Feldmann J, Tezcan I, Ersoy F, Houdusse A, Fischer A, and de Saint Basile G, 2003. Griscelli syndrome restricted to hypopigmentation results from melanophilin defect (GS3) or a Myo5A F-exon deletion (GS1). J Clin Invest 112:450–456.[CrossRef][Web of Science][Medline]
Mercer JA, Seperack PK, Strobel MC, Copeland NG, and Jenkins NA, 1991. Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature 349:709–713.[CrossRef][Medline]
Pastural E, Barrat FJ, Dufourq-Lagelouse R, Certain S, Sanal O, Jabado N, Seger R, Griscelli C, Fischer A, and de Saint Basile G, 1997. Griscelli disease maps to chromosome 15q21 and is associated with mutations in the myosin -VA gene. Nat Genet 16:289–292.[CrossRef][Web of Science][Medline]
Priat C, Hitte C, Vignaux F, Renier C, Jiang Z, Jouquand S, Cheron A, André C, and Galibert F, 1998. A whole-genome radiation hybrid map of the dog genome. Genomics 54:361–378.[CrossRef][Web of Science][Medline]
Philipp U, Quignon P, Scott A, Rak S, André C, Breen M, and Leeb T, 2003a. Assignment of the canine myosin Va gene (MYO5A) to chromosome 30q14 by fluorescence in situ hybridization and radiation hybrid mapping. Cytogenet Genome Res 101:92C.[Medline]
Philipp U, Scott A, Quignon P, André C, Breen M, and Leeb T, 2003b. Assignment of the RAB27A gene to canine chromosome 30q15.1 by fluorescence in situ hybridization and radiation hybrid mapping. Cytogenet Genome Res 101:92E.[Medline]
Thomas R, Smith KC, Ostrander EA, Galibert F, and Breen M, 2003. Chromosome aberrations in canine multicentric lymphomas detected with comparative genomic hybridisation and a panel of single locus probes. Brit J Cancer 89:1530–1537.[CrossRef][Web of Science][Medline]
Vignaux F, Hitte C, Priat C, Chuat JC, André C, and Galibert F, 1999. Construction and optimization of a dog whole-genome radiation hybrid panel. Mamm Genome 10:888–894.[CrossRef][Web of Science][Medline]
Wilson SM, Yip R, Swing DA, O'Sullivan TN, Zhang Y, Novak EK, Swank RT, Russell LB, Copeland NG, and Jenkins NA, 2000. A mutation in Rab27A causes the vesicle transport defects observed in ashen mice. Proc Natl Acad Sci USA 97:7933–7938.
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