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Copyright © 2004, Genetics Society of America The Dominant white, Dun and Smoky Color Variants in Chicken Are Associated With Insertion/Deletion Polymorphisms in the PMEL17 Gene 1Present address: Section for Biology, Linköping University, SE-581 83 Linköping, Sweden. 2Corresponding author: Department of Medical Biochemistry and Microbiology, Uppsala University, BMC, Box 597, SE-751 24 Uppsala, Sweden. E-mail: leif.andersson/at/imbim.uu.se Communicating editor: C. Haley Received February 24, 2004; Accepted July 5, 2004.  This article has been cited by other articles in PMC. | |||||||||||||
Abstract Dominant white, Dun, and Smoky are alleles at the Dominant white locus, which is one of the major loci affecting plumage color in the domestic chicken. Both Dominant white and Dun inhibit the expression of black eumelanin. Smoky arose in a White Leghorn homozygous for Dominant white and partially restores pigmentation. PMEL17 encodes a melanocyte-specific protein and was identified as a positional candidate gene due to its role in the development of eumelanosomes. Linkage analysis of PMEL17 and Dominant white using a red jungle fowl/White Leghorn intercross revealed no recombination between these loci. Sequence analysis showed that the Dominant white allele was exclusively associated with a 9-bp insertion in exon 10, leading to an insertion of three amino acids in the PMEL17 transmembrane region. Similarly, a deletion of five amino acids in the transmembrane region occurs in the protein encoded by Dun. The Smoky allele shared the 9-bp insertion in exon 10 with Dominant white, as expected from its origin, but also had a deletion of 12 nucleotides in exon 6, eliminating four amino acids from the mature protein. These mutations are, together with the recessive silver mutation in the mouse, the only PMEL17 mutations with phenotypic effects that have been described so far in any species. | |||||||||||||
DOMINANT white is one of the major loci influencing plumage color in chicken (Smyth 1990). The Dominant white color was in fact one of the first traits to be investigated following Mendel's classical work (Bateson 1902) and the mutation was assigned the gene symbol I for its inhibiting effect on pigmentation (Hurst 1905). I is incompletely dominant to the wild-type allele, i. Dominant white is a breed characteristic of White Leghorns giving the birds a pure white plumage without any patterns or markings (Figure 1). Beak and shanks are yellow and the eye is brown since Dominant white affects only melanocytes of neural crest origin (Smyth 1990). A putative third allele at the Dominant white locus, denoted Smoky (I*S), has been identified. It arose in a White Leghorn line fixed for Dominant white and segregation data indicated that it is allelic to I (R. Okimoto, B. Payne and D. Salter, unpublished results). Smoky gives a grayish phenotype and is recessive to Dominant white but partially dominant to the wild-type allele (Figure 1). Dun (I*D) was identified as a spontaneous mutation in a Pit-gamecock bird (Ziehl and Hollander 1987). Segregation data indicated that Dun is allelic to Dominant white. Dun heterozygotes (I*D/i) show a Dun phenotype while homozygotes are whitish.
The I locus has been mapped to chicken linkage group E22C19W28 (Ruyter-Spira et al. 1996; Schmid et al. 2000). We have recently confirmed this assignment by linkage analysis using our intercross between the red jungle fowl and the White Leghorn chicken (Kerje et al. 2003a). Three genes, ERBB3, TUBAL1, and GLI, have been mapped to the E22C19W28 linkage group and their homologs are all located on chromosome 12 in human and chromosome 10 in mouse (Schmid et al. 2000). These chromosome regions in human and mouse harbor Silver encoding the melanocyte-specific PMEL17 protein also denoted GP100. PMEL17 is a type I integral membrane protein present in the melanosome and is a component of the fibrous striations upon which melanins are polymerized (Berson et al. 2003; Du et al. 2003). PMEL17 has a crucial role for the normal development of eumelanosomes. It has been shown that the proteolytic cleavage and processing of PMEL17 accompanies the restructuring of early eumelanosomes from amorphous rounded vesicles into elongated fibrillar structures (Kushimoto et al. 2001). A single-base insertion at the 3′-end of silver (encoding PMEL17) in mouse results in a premature stop codon and a truncation of the last 25 amino acids in the carboxyterminal cytoplasmic region of the protein (Martinez-Esparza et al. 1999; Solano et al. 2000). silver homozygotes show graying of the hair due to loss of follicular melanocytes (Quevedo et al. 1981). The truncated PMEL17 protein was found in different fractions of homogenized mouse cells due to misrouting of the mutated protein. Thus, PMEL17 was identified as a positional candidate gene for the Dominant white phenotype in chicken. (We have decided to denote this locus PMEL17 and not Silver, as Silver is already used as the locus designation for another classical plumage color locus in the chicken.) In this study we present strong evidence that Dominant white, Dun, and Smoky are all caused by mutations in PMEL17. Unique insertion/deletion polymorphisms (9–15 bp in length) were found in birds carrying these alleles. | |||||||||||||
MATERIALS AND METHODS Animals: We have generated a resource pedigree for gene mapping by crossing one red jungle fowl male and three White Leghorn females. Four F1 males and 37 F1 females were intercrossed and ~1000 F2 birds were hatched. The F2 generation showed a considerable variation in plumage color and digital photos of 814 F2 animals were taken and used for phenotypic classification (Kerje et al. 2003b). An F3 generation was generated by intercrossing 50 females with 50 males from the F2 population. A few F3 individuals heterozygous at PMEL17 were selected to produce 15 F4 embryos for tissue sampling. Samples for DNA and RNA isolation were collected 14 days after fertilization of the eggs. The PMEL17 genotypes of the embryos were determined by a DNA test.DNA samples used for sequencing are listed in Table 5. Breeds denoted by footnote b are from University of Arkansas and the Broiler line is a selection line derived from White Plymouth Rock (Dunnington and Siegel 1996).
Sequencing of genomic DNA and cDNA: Genomic DNA was isolated by standard methods, and primers to amplify the complete PMEL17 gene were designed on the basis of a chicken cDNA sequence (GenBank D88348). The gene was sequenced in five parts using the primer pairs P1fwd/P1rev, P2fwd/P2rev, P3fwd/P3rev or P3.1fwd/P3.1rev, P4fwd/P4rev, and P5fwd/P5rev (for primer sequences see supplementary Table S1 at http://www.genetics.org/supplemental/); the P3 and P3.1 primers were used to amplify genomic DNA and cDNA, respectively.The PCR for all primer pairs except P3 was performed in a total volume of 10 μl containing ~50 ng DNA, 1× PCR buffer (QIAGEN, Valencia, CA), 2.5 mm MgCl2, 200 μm dNTPs, 2 pmol of each primer, and 0.5 units HotStarTaq polymerase (QIAGEN). The thermocycling included 10 min at 95°, followed by 50 cycles with 30 sec at 95°, 30 sec at 65°, and 2 min at 72°, ending with 10 min at 72°. The PCR with P3 primers was performed as described above but with 1× PCR buffer II (Applied Biosystems, Foster City, CA) and 0.5 units AmpliTaqGold (Applied Biosystems). The thermocycling included 4 min at 95°, followed by 45 cycles with 30 sec at 95°, 30 sec at 58°, and 2 min at 72°, ending with 10 min at 72°. Most PMEL17 fragments were difficult to amplify by PCR due to a very high GC content (68.4% average GC content for the entire gene). These fragments were therefore cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) prior to sequencing with the T7 and M13R universal primers. For those amplicons for which direct sequencing could be applied, the PCR primers were used for sequncing. The MegaBACE sequencing kit (Amersham Biosciences, Uppsala, Sweden) was used for sequencing and fragments were electrophoresed using the MegaBACE 1000 capillary instrument (Amersham Biosciences) and analyzed with the Sequence Analysis software (Amersham Biosciences). Sequences were controlled, aligned, and compared using the Sequencher 3.1.1 program (Gene Codes, Ann Arbor, MI). PMEL17 genotyping and linkage mapping: PMEL17 segregation was scored using the polymorphic repeat in exon 7. PCR amplification using the Pmel_exon7fwd and Pmel_exon7rev primers (Table S1 at http://www.genetics.org/supplemental/) gave 490- and 574-bp fragments associated with the Dominant white and wild-type alleles, respectively. PCR was performed as described above in a total volume of 10 μl with 0.5 units HotStarTaq DNA polymerase (QIAGEN). Fragments were separated using a MegaBACE 1000 capillary instrument (Amersham Biosciences) and analyzed with the Genetic Profiler software (Amersham Biosciences). Microsatellite marker MCW317 was analyzed previously (Kerje et al. 2003a) and the same conditions were used for MCW188 in the present study.The MYG1 cDNA sequence was available in a chicken EST database (pgr1n.pk003.l20; http://www.chickest.udel.edu) and two single nucleotide polymorphisms (SNPs) were identified by partial sequencing of the gene. A fragment containing the SNPs was amplified by PCR with primers pyMYG1Bio and pyMYG1 (Table S1 at http://www.genetics.org/supplemental/) prior to pyrosequencing using the SNP reagent kit protocol (Pyrosequencing AB, Uppsala, Sweden). PCR was performed as described above with 0.5 units HotStarTaq DNA polymerase (QIAGEN) but with 1.75 mm MgCl2 and 5 pmol of each primer. The pyrosequencing primer, pyMYG1seq (Table S1 at http://www.genetics.org/supplemental/), was designed to anneal just before the first SNP. PMEL17, MCW188, and MYG1 were mapped in relation to the other markers previously genotyped in the pedigree (Kerje et al. 2003a). The CRIMAP program (Green et al. 1990) and the functions BUILD and FLIPS were used to test the order of the markers. CHROMPIC was used to reveal unlikely recombination events. The primer pair Pmel_exon10fwd and Pmel_exon10rev (Table S1 at http://www.genetics.org/supplemental/) was used for genotyping the insertion/deletion polymorphism in exon 10. The PCR was performed in a total volume of 10 μl containing the same amount of reagents as for the PCR using genomic DNA as described above. The thermocycling included 10 min at 95°, followed by 50 cycles with 30 sec at 95°, 30 sec at 60°, and 45 sec at 72°, ending with 5 min at 72°. Fragments were separated using a MegaBACE 1000 capillary instrument (Amersham Biosciences) and analyzed with the Genetic Profiler software (Amersham Biosciences). Expression analyses: Total RNA from 14-day-old whole embryos was isolated with the RNeasy mini kit according to the protocol for animal tissues (QIAGEN). The quality and concentration were checked with the Bioanalyzer (Agilent Technologies, Palo Alto, CA). Total RNA was treated with DNase according to the instructions for the DNA-free kit (Ambion, Austin, TX) prior to cDNA synthesis using the First-Strand cDNA Synthesis kit (Amersham Biosciences). cDNA and genomic DNA from PMEL17 heterozygotes were used for allele quantification with pyrosequencing. A 164-bp fragment containing a G/A SNP at nucleotide position 2184 in the gene was amplified using PCR with primers pyPmelBio and pyPmelrev (Table S1 at http://www.genetics.org/supplemental/). The PCR was performed in a total volume of 15 μl including ~20 ng cDNA, 1× PCR Buffer (QIAGEN), 2.5 mm MgCl2, 200 μm dNTPs, 3 pmol of each primer, and 0.75 units HotStarTaq polymerase (QIAGEN) and the following thermocycling was applied: 10 min at 95°, 45 cycles of 30 sec at 95°, 30 sec at 61°, and 30 sec at 72°, ending with 5 min at 72°. The sequencing primer, pyPmelseq (Table S1 at http://www.genetics.org/supplemental/) was designed to anneal just in front of the SNP. The difference in expression between the two alleles was determined by the peak heights in the pyrogram.In vitro expression of the PMEL17 transmembrane region: The so-called S-segment engineered into the lep gene in the pGEM1-based plasmid 67 (Hessa et al. 2003) was replaced with oligonucleotides encoding the predicted transmembrane domain of PMEL17 from wild type (WT; codons 707–749), Dominant white (WAP; codons 679–724), and Dun (codons 677–714) alleles. Each transmembrane segment of PMEL17 was generated by two sequential annealing reactions followed by a ligation reaction. First, six different annealing reactions composed of 13.6 μm of 5′ phosphorylated, complementing primer pairs were set up (for primer sequences see Table S1 at http://www.genetics.org/supplemental/). The annealing buffer was 20 mm Tris-HCl, pH 7.4, 2 mm MgCl2, and 50 mm NaCl. The reaction was incubated at 85° for 10 min and slowly cooled down to 30°. Second, equal amounts of annealed primers from the first annealing reaction were mixed together (for WT, annealing reactions 1, 2, and 3; for WAP, annealing reactions 1, 4, and 6; and for Dun, annealing reactions 1, 2, and 5) and incubated at 65° for 5 min and slowly cooled down to 30°. The ligation reaction was prepared by adding the annealed product, SpeI- and KpnI-digested plasmid 67, ligation buffer, and T4 ligase (Promega, Madison, WI). The reaction was incubated at room temperature overnight. After Escherichia coli transformation and plasmid isolation, each construct was confirmed by DNA sequencing.In vitro transcription and translation was performed as in Hessa et al. (2003), using 10 μl SP6 express TnT reaction mix (Promega), 0.5 μg of plasmid, 7.5 μCi 35S (Amersham Bioscience), and 1 μl (4 eq) of canine pancreatic microsomes (a gift from Masao Sakaguchi, Fukuoka, Japan), and incubating at 30° for 90 min. Translation products were analyzed by SDS-PAGE and the gel was imaged on a Fuji FLA-3000 phosphorimager (Fuji Instruments). | |||||||||||||
RESULTS Inheritance of plumage colors: The segregation at the Dominant white (I), Extended black (E), Barred (B), and Silver (S) loci among the ~800 F2 birds in our red jungle fowl × White Leghorn intercross has recently been reported (Kerje et al. 2003b). The segregation at the I locus did not deviate significantly from the expected 3:1 ratio, indicating that it is epistatic to the other color loci. A sex difference in plumage color due to the segregation at the sex-linked Silver and Barred loci was noted.Linkage analysis: The P3 primers were used to amplify a part of PMEL17 exon 7 from genomic DNA. Sequencing of PCR products revealed a difference in the number of a 72-bp repeat between the red jungle fowl and the White Leghorn. This polymorphism was used to map PMEL17 in relation to the Dominant white locus. The segregation at PMEL17 in the F2 generation did not deviate significantly from the expected 1:2:1 ratio (χ2 = 1.49, d.f. = 2, P = 0.47; see Table 2 for actual segregation data).
The linkage analysis included two loci, MCW317 and I, already typed in the pedigree, and three additional markers, MCW188, MYG1, and PMEL17; MYG1 was included to improve the comparative map for linkage group E22C19W28. All pairwise two-point LOD scores among these five loci were highly significant (Table 1). The two-point analysis did not indicate any recombination between Dominant white and PMEL17 among 773 informative offspring (Table 1). However, a closer examination of the association between PMEL17 genotypes and plumage color revealed three putative recombinants (Table 2). Three individuals classified as white, white with black spots, or cream (all assumed to carry the Dominant white allele) were scored as homozygous for the PMEL17 allele inherited from the red jungle fowl. However, CHROMPIC analysis revealed that these three animals (representing three different full-sib families) appeared as unlikely double recombinants. This implies that these three discrepancies from a complete association between PMEL17 and presumed I genotypes do not reflect true recombinants. The deviations may be due to a few genotype/phenotype mismatches in this large sample but it is also possible that a combination of genotypes at other loci affecting plumage color in rare cases may mimic the effect of the Dominant white allele. These three individuals were excluded from further analysis.
Multipoint analysis revealed the following map order and sex-averaged map distances (in Kosambi centimorgans) for this linkage group: MCW317 — 19.5 — MYG1 —12.8 — I/PMEL17 — 11.5 — MCW188 with no recombination between PMEL17 and I. This order was supported by a LOD score >6 compared with all other possible orders. Association between PMEL17 genotypes and plumage color: The very close linkage between PMEL17 and Dominant white allowed us to investigate the association between PMEL17/Dominant white genotypes and plumage color (Table 2). There was a marked sexual dimorphism for plumage color in this pedigree as reported in our previous study of the association between MC1R genotypes and variation in plumage color (Kerje et al. 2003b). Therefore, the data are presented separately for each sex. However, all individuals, irrespective of sex and scored as colored, were homozygous (J/J) for the jungle fowl allele at PMEL17 (Table 2). More than 90% of the females scored as PMEL17 W/W were purely white whereas only ~60% of the heterozygotes (J/W) were purely white. The Dominant white allele appeared less dominant in males since only ~30% of the animals scored as PMEL17 W/W and only ~20% of the J/W heterozygotes were purely white. This sex difference is at least partially explained by the different genotype distributions at the sex-linked Barred and Silver loci. Fifty percent of the female F2 birds were hemizygous for these dominant alleles inherited from the White Leghorn F0 females. In males, 50% of the F2 animals were heterozygous for Barred and Silver, but none were homozygous, and it is well known that males that are heterozygous at these loci are less white than homozygotes.Some phenotypes, such as white with black spots, were almost entirely composed of heterozygous animals. Most animals that scored as partially white expressed red colors. Sequence analysis of PMEL17 using genomic DNA: Sequencing of PMEL17 using genomic DNA from birds carrying the wild-type, Dominant white, Dun, or Smoky allele revealed a considerable amount of polymorphism (Table 3). The sequenced region started 32 bp before the start codon and included 111 bp of the 3′ UTR. Exon and intron borders were determined by comparing genomic and cDNA sequences obtained in this study with the chicken PMEL17 cDNA sequence D88348 and the EST sequences BU217288 and BU315027 (all from GenBank). The comparison revealed that the chicken gene has 11 exons like human Silver (PMEL17) but the gene is only 4.1 kb compared to 11.8 kb in human (http://www.ncbi.nlm.nih.gov/LocusLink/) due to smaller introns. We noted that the chicken D88348 GenBank sequence (Mochii et al. 1991) contains a likely sequencing error since it is missing the two last nucleotides in exon 8, which leads to a frameshift. We are convinced that the sequence reported in this article is correct since it was confirmed using both genomic DNA and cDNA, and it gives a better alignment with the corresponding mammalian homologs. The last 377 nucleotides of EST BU217288 do not show any similarity with any of the other chicken PMEL17 sequences and probably represent a cloning artifact (data not shown).
We identified a total of 56 SNPs and eight insertion/deletion polymorphisms across populations (Table 3). There was no sequence difference between the White Leghorn lines L13 from Sweden and ADOL from the United States. The White Leghorn and the red jungle fowl sequences differed from each other at six positions but only two of these were uniquely associated with the Dominant white allele, a synonymous substitution at position 1836 in exon 6 and a 9-bp insertion in exon 10. The Smoky allele was identical to Dominant white with the exception of a unique 12-bp deletion in exon 6. Dun was associated with a PMEL17 sequence that was clearly distinct from Dominant white and that possessed 13 unique SNP alleles and a unique 15-bp deletion in exon 10. The variable positions at the amino acid level are shown in Table 4. Seven missense mutations were observed in total but none was exclusively associated with the Dominant white or Smoky alleles. Three missense mutations were found only in the Dun allele. The repeat comprising 24 amino acids showed both sequence variation and variation in the number of repeated copies (Table 4). The polymorphic nature of this repeated region does not appear functionally important since there was no correlation between repeat-type composition and effect on plumage color. The 9-bp insertion, exclusively associated with Dominant white and Smoky, resulted in the insertion of the amino acid triplet WAP in the transmembrane region. Interestingly, the 15-bp deletion associated with Dun also alters the transmembrane region and leads to a deletion of five amino acids. The unique 12-bp deletion in exon 6, associated with Smoky, causes a deletion of four amino acids.
An alignment of the chicken sequence with the homologous sequences in humans and mouse shows that some parts of the PMEL17 protein are well conserved between birds and mammals whereas other parts are not, and these are hard to align (Figure 2). The proteolytic cleavage site dividing PMEL17 into an α- and β-chain (Berson et al. 2003) is well conserved between species. The Dominant white, Dun, and Smoky insertion/deletions all disrupt fairly well-conserved regions of PMEL17.
Sequence polymorphisms across breeds: The polymorphic 72-bp repeat in exon 7 and the insertion/deletion polymorphisms in exon 10 associated with Dominant white and Dun were screened across breeds (Table 5). A surprisingly high degree of variation in the number of repeats was found, ranging from one repeat in the Fayoumi to four in several breeds. It is also clear that Dominant white has the same composition of repeats, two A and one B repeat, as do Black Langshan and Rhode Island Red that carry the recessive i allele. This finding excludes the repeat region from being causative for the Dominant white phenotype at least on its own. In contrast, the insertion in exon 10 was found only in the three different breeds carrying Dominant white (one broiler and two White Leghorn lines) and in the Smoky allele assumed to be derived from Dominant white. The 15-bp deletion in exon 10 was found associated only with the Dun allele.Expression analysis of PMEL17: Total RNA was isolated from 2-week embryos from the F4 generation with the genotypes I/I, I/i, and i/i. Sequence analysis of the entire PMEL17 coding sequence from the two homozygotes (I/I and i/i) showed that both transcripts were expressed and the sequence differences detected using genomic DNA were confirmed. No other sequence difference or variant transcripts were detected.To investigate a possible difference in the level of PMEL17 expression associated with the I and i alleles, we took advantage of the synonymous SNP at nucleotide position 2184 in exon 6 that distinguishes the two alleles in our pedigree (Table 3). The relative expression of the two alleles in nine heterozygous individuals was quantified by pyrosequencing of RT-PCR products. The quantitative analysis of the peak heights indicated an 8% higher expression of the Dominant white allele whereas control reactions using genomic DNA from the same animals did not indicate a significant difference in amplification efficiency between alleles. However, it is very unlikely that this minor difference in expression level can cause the dramatic phenotypic difference observed between Dominant white and wild-type chicken (Figure 1). Membrane insertion of mutant PMEL17 transmembrane segments: To test whether the observed insertion/deletion polymorphisms in PMEL17 exon 10 of Dominant white and Dun disrupted the formation of the transmembrane helix, insertion into ER-derived rough microsomes was analyzed by in vitro transcription/translation (Figure 3). Briefly, the transmembrane segment of PMEL17 (denoted PMEL in Figure 3A) was inserted into the luminal P2 domain of the well-characterized integral membrane protein leader peptidase (Lep), where it is flanked by two Asn-X-Thr acceptor sites for N-linked glycosylation (G1, G2). The P2 domain is efficiently translocated into the lumen of the microsomes when wild-type Lep is translated in the presence of rough microsomes (Johansson et al. 1993). If the PMEL17-derived segment forms a transmembrane helix that is inserted into the microsomal membrane, only the G1 site is glycosylated by the lumenally disposed oligosaccharyl transferase (Figure 3A, top left), whereas both the G1 and the G2 sites are modified if the PMEL17-derived segment fails to form a transmembrane helix (Figure 3A, top right; cf. Sääf et al. 1998).
As shown in Figure 3B, the PMEL17-derived segments representing all three alleles (wild type, Dominant white, and Dun) were able to form transmembrane segments since the predominant form of the Lep-PMEL17 protein was glycosylated on only one site. For the Dun allele, a small fraction of the molecules was glycosylated on both sites, indicating that this variant is near the threshold where the formation of the transmembrane helix will be compromised. | |||||||||||||
DISCUSSION This study has shown that mutations in PMEL17 are causing the Dominant white, Dun, and Smoky plumage phenotypes in chicken. This conclusion is supported by a number of facts. All colored birds among the ~800 F2 progeny in our red jungle fowl/White Leghorn intercross were homozygous for the PMEL17 allele inherited from the red jungle fowl. All F2 birds expressing any black pigment carried at least one copy of the wild-type PMEL17 allele. Furthermore, an identical PMEL17 sequence was associated with Dominant white in two different White Leghorn lines and a broiler line. The lack of convincing recombinants among ~800 F2 birds and the complete association between the presence of Dominant white and certain PMEL17 sequence variants across breeds implies that the causative mutations must be in the very close vicinity of PMEL17. An inspection of the corresponding regions in human and mouse does not reveal any other candidate genes with an established function in the melanocyte. Furthermore, Dun arose independently of Dominant white but phenotypic similarities and segregation data indicate that the two mutations are allelic. The observation of a unique deletion in Dun occurring in the near vicinity of the WAP insertion associated with Dominant white is very intriguing. Finally, Smoky appeared in a White Leghorn line and is assumed to reflect a revertant allele at the Dominant white locus. The observation that this allele shares the PMEL17 sequence with Dominant white but in addition carries a unique 12-bp deletion in the PMEL17 exon 6 strongly supports our conclusion that PMEL17 causes these plumage color variants. The phenotypic expressions of Dominant white and Dun are also consistent with previous data on the role of PMEL17 in eumelanogenesis in the mouse. Our observation that all birds expressing any black pigment (eumelanin) carried at least one copy of the wild-type PMEL17 allele implies that Dominant white blocks the production of eumelanin but allows production of red pheomelanin. This result is in perfect agreement with the general knowledge that Dominant white as well as Dun primarily inhibits the production of black pigment (Ziehl and Hollander 1987; Smyth 1990). Interestingly, the silver mutation in the mouse also affects primarily the production of eumelanin (Silvers 1979). These phenotypic effects in the chicken and mouse mutants are consistent with the crucial role of PMEL17 (alias GP100) in the development of eumelanosomes but not of pheomelanosomes (Kobayashi et al. 1995). Our data strongly suggest that the observed insertion/deletion polymorphisms in exons 6 and 10 are causative for the Dominant white and Smoky phenotypes. The causative nature of these mutations is supported by the complete associations observed and by the fact that we have sequenced all exons and all introns. Furthermore, the expression analysis did not reveal any altered transcripts or any marked differences in expression levels in early embryos. Dun was found to be associated with three unique missense mutations at codons 35, 105, and 740 as well as with a unique deletion of five amino acids in the transmembrane region (Figure 2). The genetic data do not reveal which of these mutations is the causative one, but on the basis of the phenotypic similarity to Dominant white we postulate that the deletion in the transmembrane region is the most likely causative mutation. We used the TMHMM2.0 program (Krogh et al. 2001; http://www.cbs.dtu.dk/services/TMHMM/), developed for the prediction of transmembrane helices (TMH) in proteins, to evaluate the consequences of the insertion/deletion polymorphisms associated with Dominant white and Dun. The program predicted the location of a TMH at the expected position in wild-type chicken PMEL17 with a posterior probability of 0.8. The insertion of WAP in the protein encoded by Dominant white reduced the probability to 0.6, most likely because the insertion of a proline residue may disturb the formation of a TMH. Furthermore, the Dun-associated deletion reduced the probability for the formation of a TMH even more, to a probability value <0.4. However, our in vitro experiments showed that the sequences encoded by both Dominant white and Dun could form TMHs. However, these insertion/deletion polymorphisms in and near the transmembrane segment may well affect PMEL17 function in other ways. It has been shown that PMEL17 is an integral membrane protein that is already present in stage I premelanosomes and that the cleavage and processing of this protein into an Mα and Mβ subunit accompanies the restructuring of stage I melanosomes to stage II melanosomes with elongated fibrillar structures (Kushimoto et al. 2001; Berson et al. 2003). Even after PMEL17 has been cleaved to two subunits, the protein remains covalently tethered to its transmembrane domain (Berson et al. 2001). Thus, it is conceivable that the insertion/deletion polymorphisms associated with Dominant white and Dun lead to a failure in the proper integration of PMEL17 in the melanosomal membrane, which disrupts the normal formation of eumelanosomes. In fact, previous ultrastructural studies have shown that the eumelanosomes in melanocytes from I/i heterozygotes are scarce and disorganized (Brumbaugh 1971). Very few PMEL17 mutations with phenotypic effects have been described so far in comparison with the large number of functionally important mutations described for other major coat-color loci like Albino/TYR, Agouti, and Extension/MC1R. In fact, only one mutation (silver) has been described so far in the mouse (http://www.informatics.jax.org/) and no human PMEL17 mutation causing pigmentation disorders has yet been reported but it has been proposed as a likely candidate gene for some cases of human oculocutaneous albinism (http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=155550; December 2003). However, on the basis of the phenotypic effects in chicken and the established role for PMEL17 in the development of eumelanosomes but not of phaeomelanosomes, we postulate that this locus may be one of the loci causing red hair in humans. It is intriguing that Dominant white and Dun in chicken and silver in mouse are all insertion/deletions affecting the transmembrane or cytoplasmic region. No mutation with a phenotypic effect, except Smoky, has been found in the part of the protein forming the Mα subunit. Furthermore, no mutation causing a total loss of the PMEL17 protein has yet been found in any species despite the fact that such a mutation is expected to give an obvious pigmentation phenotype. This implies that such mutations may be lethal. PMEL17 may have a hitherto unknown crucial function outside the melanocytes. The absence of melanogenesis does not have a severe effect on survival as well documented by the viability of albino mutants in a variety of species. The suggestion that PMEL17 may have an important role in nonpigment cells gains some support by the fact that human EST sequences for PMEL17 have been obtained from a variety of tissues, although the majority of them are from melanoma cells (http://www.ncbi.nlm.nih.gov/UniGene). The rare occurrence of PMEL17 mutations suggests that the chicken mutations described here should be useful for structural/functional studies of this protein. An exciting topic for future studies will be to unravel how the four-amino-acid deletion associated with Smoky can partly rescue the defect caused by the WAP insertion in Dominant white. PMEL17 forms fibers after it has been cleaved by furin into an Mα and Mβ fragment. This process appears to be essential for the normal development of eumelanosomes. A reasonable interpretation therefore is that the insertion of the WAP amino acid triplet disrupts the process but it is partially restored by the Smoky deletion. This would resemble the trans-suppression of transthyretin misfolding in composite heterozygotes that protects from the development of amyloid disease in humans (Hammarstrom et al. 2001). The segregation analysis of PMEL17 alleles and plumage color in our F2 generation showed that Dominant white is required but not sufficient for the expression of the completely white phenotype. Homozygosity for Dominant white increased the incidence of white color but other loci contributed as well. At least three other major loci segregate in this cross: Extended black/MC1R, Silver, and Barred (Kerje et al. 2003b). We have previously shown that homozygosity for the Extended black (MC1R*E92K) allele increases the chance to develop a purely white phenotype, which may appear counterintuitive. However, it makes sense in light of the action of the Dominant white/PMEL17 mutation since the Extended black allele shifts melanin production toward eumelanogenesis, which in turn is severely inhibited by the effect of Dominant white. This is consistent with the fact that MC1R acts upstream of PMEL17 in the melanogenesis. Another major locus affecting plumage color in this intercross is the sex-linked Silver locus (not homologous to silver in the mouse!) where the dominant S allele inherited from the White Leghorn inhibits the expression of red pheomelanin. The causative gene for Silver has not yet been identified. | |||||||||||||
Acknowledgments We thank Ulla Gustavsson for excellent assistance with the sequencing. The study was funded by The Foundation for Strategic Environmental Research, the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, Wallenberg Consortium North, and the Swedish Foundation for Strategic Research. | |||||||||||||
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References
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