BACKGROUND
vitiligo is an acquired hypomelanotic disorder characterized by circumscribed depigmented macules in the skin resulting from the loss of melanocytes.
autoimmunity has been identified as the major etiological factor in vitiligo, although many other factors including infections, stress, neural abnormalities, aberrant melanocyte function, and genetic susceptibility have been implicated  <cit> .
the smyth line  chicken is the only animal model for autoimmune vitiligo that spontaneously displays all clinical and biological manifestations of the human disorder  <cit> .
like other autoimmune diseases, sl vitiligo  is multi-factorial in nature and involves the interplay of genetic, immune system, and environmental-factors.
slv susceptibility is manifested in part in an inherent melanocyte defect and loss of melanocytes is due to melanocyte-specific cell-mediated and humoral immune activities  <cit> .
recent genome wide association studies  in humans to understand the role of genetic components in a variety of autoimmune diseases including vitiligo have identified hundreds of loci harboring risk alleles  <cit> .
several gwas results identified vitiligo susceptible loci in human populations .
however, most susceptible loci identified by gwas results were found in regulatory regions of gene expression, therefore the identified associations were not sufficient to identify the causal gene or deduce alterations caused by risk variants, which generally do not induce profound changes to genes .
recently, the encyclopedia of dna elements  of mammalian species suggested that ~90% of disease associated genetic variations in human lie in noncoding regions, while only ~10% of variations in coding regions were causative mutations associated with human disease  <cit> .
nevertheless, the identification of potential coding mutations that alter protein functionalities is a prerequisite process to understand disease etiology.
moreover, the functional study of candidate genetic risk factors is almost impossible without appropriate model systems.
the sl chicken is an excellent model to conduct a functional verification study of candidate genes that underlie genetic susceptibility for vitiligo due to the tractable, definite phenotype, the high vitiligo incidence in the population , the feasibility of in vivo characterization and the relatively short generation time.
recently, microarray analysis examined global gene expression during slv development and provided comprehensive information at the transcriptome level that supported the multifactorial etiology of vitiligo  <cit> .
in this study, whole genome resequencing analysis using an illumina platform was performed to more deeply investigate the genetic aspects of slv expression in comparison with the parental brown line  of chickens from which the sl was originally derived.
bl chickens retain vitiligo susceptibility but with a very low  incidence rate of vitiligo development  <cit> , although none of the bl chickens used in this study had vitiligo.
millions of single nucleotide polymorphisms  were identified by genome resequencing and only potentially causal genes containing non-synonymous mutations that can induce amino acid changes in proteins were focused on in this study.
RESULTS
genome resequencing for bl and sl chickens
genome sequencing of pooled dna from  <dig> non-vitiliginous bl and sl chickens each with confirmed slv produced ~ <dig> and  <dig> million sequence reads of 200 bp, respectively .
of those, ~80% of the reads were used for sequence alignment, while 20% of sequence reads were not aligned.
therefore, genome coverage for bl and sl reached  <dig> x and  <dig> x, respectively, of the red jungle fowl chicken genome.
the total number of snps was  <dig>  and  <dig>  million  for the bl and sl genome, respectively.
the large number of snps per examined chicken line was based on data of at least  <dig> read coverage depths .
most snps were found on the larger chromosomes , including chr  <dig> through  <dig> and z  .
to identify genetic biomarkers that are responsible for the incidence of slv, unique snps that are found in sl only were selected by removing snps that overlapped with those found in the parental bl.
then, mutations with ≥75% snp rates were chosen as reliable marker snps.
since the objective of this study was to identify mutational snps uniquely found in sl compared to the parental bl, the filtering process used did not involve a typical snp calling and filtering method based on quality score   <cit> .
instead, snp filtering was conducted by removing overlapping snps found in both bl and sl, and applying fixed %snp rates  as described in methods.
as a result, a total of ~ <dig> million unique snps were identified throughout the sl chicken genome .
over  <dig>  snps were found in chr  <dig>   <dig>   <dig>  and z while chr  <dig> did not contain any unique snps for sl.
when snps were grouped by the feature types of chromosome regions, ~50% of snps were in the intergenic  regions and  <dig>  snps were found in cds sequences  .
most genes containing snps in regulatory regions, not in cds regions, identified by human gwas studies were also observed to contain unique snps in the current sl study .
around 60% of sl snps in protein coding cds regions were synonymous mutations that did not induce amino acid changes.
to identify potentially causal mutations that induce protein coding alterations, snp analysis focused on snps leading to changes in amino acid sequences.
using this approach, a total of  <dig> snps were identified that could induce non-synonymous-, frameshift-, nonsense-, and no-start-changes in the cds region , suggesting that the  <dig> snps are part of the genetic components in functional protein coding regions that may drive the high incidence rate of slv.
of the  <dig> candidate snps that are associated with amino acid changes, snps showing ≥ <dig> read depths  were chosen for the further analysis.
using this approach,  <dig> snps remained .
to reduce false positives due to possible errors in the assembly process, re-scanning of each snp position for the  <dig> potentially more reliable protein coding snps was conducted using the seqman-pro viewer program.
this process yielded  <dig> more reliable snps that were chosen as candidate marker snps for further analysis .table  <dig>
results of illumina sequencing and assembly
bl  1
sl2
<dig> means the parental line, no vitiligo.
<dig> sl, with vitiligo.
number of unique snps per chromosome found in vitiliginous sl chickens compared to non-vitiliginous bl chickens.
numbers are indicated for bars not clearly visible.
summary of snps in slv.
a) number of snps categorized by chromosomal region in sl chickens.
b) number of snps categorized by type of amino acid sequence changes.
the  <dig> reliable marker snps that induced amino acid changes showing
≥ <dig> read depths
contig id, chromosome  numbers, reference position , reference base , called  base, impact , snp%, feature name , dna change, amino acid  change, depths, and five columns for snp counts  are indicated.
snp validation using pcr and sanger sequencing
since pooled dna samples of  <dig> chickens for each line were used for genome sequencing, individual snps were subjected to the verification process with larger bird populations.
for this,  <dig> snps were randomly chosen from the  <dig> candidate marker snps showing ≥ <dig> read depths and were subjected to snp verification analysis using pcr and sanger sequencing to detect snp positions with larger numbers of birds; specifically  <dig> non-vitiliginous bl chickens and  <dig> sl chickens exhibiting vitiligo.
the results clearly showed differential frequencies of nucleotide bases in the  <dig> snp positions between bl and sl chickens .
thus, the  <dig> snps known to induce amino acid changes can become potential genetic biomarkers for vitiligo in sl chickens.table  <dig>
verification of  <dig> snps using pcr and sanger sequencing in larger numbers of non-vitiliginous parental bl  vs. vitiliginous sl  chickens
all_ct
all_del
ins_a_30%
ins_a_100%
c:a = 3:1
c:a = 1:4
all_g
g:t = 1:1
c:t = 10:1
c:t = 1:10
t:c = 4:1
t:c = 1:7
t:c = 10:1
all_c
all_t
t:c = 1:10
c:a = 4:1
c:a = 1:10
all_g
g:a = 1:10
all_c
all_g
all_g
g:c = 1:10
all_g
g:a = 1:4
all_a
a:t = 1:4
ratios between reference base and snp  in large chicken populations were bolded.
fs; frameshift mutation.
bioinformatic analyses of genes containing amino acid change snps
amino acid changes may have impacts on the functional interpretations for vitiligo induction in sl chickens.
the ingenuity pathway analysis  program generated bioinformatics data sets including functional groups  and gene networks for genes containing amino acid changes in sl chicken.
the  <dig> snps were found in  <dig> genes encompassing known- and unknown functions, chromosomal open reading frames, and hypothetical proteins .
functional roles
genes were categorized in  <dig> functional groups .
of these, six functional groups are of particular interest to autoimmune vitiligo development, including dermatological diseases/conditions, inflammatory response, inflammatory disease, immunological disease, immune cell trafficking, and infectious disease .
the functional group of genes for dermatological diseases/conditions contained the following genes: adamts <dig> ; aspm ; atp6v0a <dig> ; brca <dig> ; col12a <dig> ; grm <dig> ; lrp <dig> ; mki <dig> ; obscn ; plau ; rnf <dig> ; stab <dig> ; and xirp <dig> .
general and dermatological disease related functions for these genes are summarized in table  <dig> table  <dig>
vitiligo related functions of candidate genes
interestingly, a recent report by nikolaev et al.
<cit>  indicated that amino acid changes found in aspm, lrp <dig>  stab <dig>  and xirp <dig> proteins were associated with human melanoma by exome sequencing.
melanocytes in vitiligo also exhibit morphological and biological melanocyte defects/alterations compared to melanocytes from individuals with normal pigmentation  <cit> .
while these alterations may be different from those observed in melanoma, e.g.
slower growth, and higher sensitivity to oxidative stress of cultured melanocytes  <cit> , alterations in amino acid sequences found in homolog proteins but different residues may result in opposite phenotypes of dermatological diseases/conditions  <cit> .
in addition to these molecules, brca <dig>  grm <dig>  mki <dig>  and obscn associated with slv are also known to be associated with human melanoma.
the relationship between candidate genes and other dermatological diseases including melanoma is summarized in table  <dig>
gene networks
gene network analysis, which represents the intermolecular connections among interacting genes based on functional knowledge inputs, was performed on genes with amino acid changes in slv chickens using the ipa program.
the gene network analysis was carried out using the simplest setting of  <dig> focus molecules to facilitate and summarize the intermolecular connections .
a discussion of the  <dig> gene networks is provided below and gene information for focus molecules in each network is listed in additional file 4: table s <dig> table  <dig>
associated network functions of candidate genes
functions associated with  <dig> networks are listed.
score means the number of network eligible molecules out of differentially expressed genes.
gene network # <dig>  molecular interactions among important focus molecules are displayed.
gray symbols show the genes found in the list of snp while white symbols indicate neighboring genes that are functionally associated, but not included, in the gene list of snp.
symbols for each molecule are presented according to molecular functions and type of interactions.
gene network # <dig>  molecular interaction and symbols are the same as the description in figure  <dig>
gene network # <dig>  molecular interaction and symbols are the same as the description in figure  <dig>
gene network # <dig>  molecular interaction and symbols are the same as the description in figure  <dig>
gene network # <dig>  molecular interaction and symbols are the same as the description in figure  <dig>
gene network # <dig>  molecular interaction and symbols are the same as the description in figure  <dig>
gene network # <dig>  molecular interaction and symbols are the same as the description in figure  <dig>
candidate genes in network # <dig> are associated with signaling pathways of the mitogen activated protein kinase  and protein kinase c  connected to vegf  and pdgf  with plau in the center .
the top functions related to network # <dig> are cardiovascular disease, hematological disease, and cardiac infarction.
interestingly, molecules including lrp <dig>  plau, adamts <dig>  and grm <dig> that are part of network # <dig> were also identified as functional factors for melanoma as described above  <cit> .
additionally, mutations in the amino acid sequence of lrp <dig>  altered function of map2k <dig> and map2k <dig> induced by genetic mutations in melanoma patients  <cit> , and mutations in grm <dig> in mouse melanoma models  <cit>  were also reported.
the connections in network # <dig> therefore suggest genetic mutations that generated amino acid changes in lrp <dig>  plau, adamts <dig>  and grm <dig> may influence dermatological diseases, including vitiligo, through mapk and erk1/ <dig> signaling pathways.
the top functions of network # <dig> include cell cycle, dna replication, recombination and repair, and developmental disorder  and network # <dig> is involved in developmental disorder, endocrine system disorders, hereditary disorder .
most molecules in networks # <dig> and # <dig> directly bind to ubc .
ubiquitinylation, the covalent attachment of ubiquitin to proteins, regulates numerous cellular processes such as protein degradation and signal transduction.
recently, many ubiquitinylated proteins and their lysine ubiquitinylation site were identified using proteomic technologies in mammalian species .
indeed, in slv, the amino acid changes in lysine residues for cep <dig>  and api <dig>  were identified , suggesting that the various cellular functions including protein degradation by altered ubiquitinylation properties of proteins may play a significant role in the induction of vitiligo.
molecules in network # <dig> are involved in cell morphology, cellular function and maintenance, hair and skin development and function .
molecules in network # <dig> mainly interact with ifng , il <dig> , mapk <dig>  and calcium signaling pathways in addition to protein phosphatase  functions.
myo <dig> , kif18a , and casc <dig>  are known to directly bind to ppp1ca , suggesting that amino acid changes in those proteins may induce alterations  in protein phophorylation states in sl chickens, resulting in vitiligo development.
molecules interacting with ifng, il <dig>  mapk <dig> and calcium signaling pathways showed an indirect relationship, not a direct relationship with each other making it difficult to explain how amino acid changes in these molecules affect vitiligo induction in sl chicken.
molecules in network # <dig> also mainly bind to ubc as discussed in networks # <dig> and # <dig> and the functions include lipid metabolism, small molecule biochemistry, digestive system development and function .
network # <dig> contains molecules involved in hereditary disorder, ophthalmic disease, neurological disease .
in this network, zc2hc1a  and rufy <dig>  directly bind to app .
app is a precursor protein for beta-amyloide, which is the main constituent of amyloid plaques in the brains of alzheimer disease patients  <cit> .
rufy <dig> , which is a brain specific protein, regulates neuronal polarity by suppressing formation of surplus axons  <cit> .
though the binding of zc2hc1a and rufy <dig> to app was found during the progression of alzheimer disease  <cit> , the functional roles for this binding in the progression of this disease has not been characterized.
similarly, amino acid changes found in zc2hc1a and rufy <dig> are implicated in slv development possibly as a result of altered app binding properties.
ush2a  was included in network # <dig>  various mutations in ush2a have been identified in patients of usher syndrome type ii, which is characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa  <cit> .
vitiliginous sl chickens may also develop severe visual impairment and blindness due to autoimmune activity directed against choroidal melanocytes and subsequent damage to the retinal pigment epithelium.
<cit> .
taken together, the amino acid change in ush2a also may affect vitiligo progression and retinal depigmentation.
molecules in network # <dig> mainly function in cellular response to therapeutics, cellular assembly and organization, dna replication, recombination, and repair.
prkdc  and its interacting protein kinases are mainly involved in this network .
in addition to knock-out and inactive mutations, alteration of autophosphorylation capability by single amino acid change of prkdc has been known to influence rejoining of dna double stranded breaks in mammalian cells  <cit> , suggesting that vitiligo development may be affected by aberrant prkdc kinase activity due to an observed amino acid change.
vitiligo susceptible loci in human populations identified by several gwas  also showed several loci that induced amino acid changes in various proteins such as strn <dig> , dnah <dig> , kiaa <dig> , tyr , oca <dig> , ptpn <dig> , ifih <dig> , sla , cd <dig>  mc1r , ubash3a , c1qtnf <dig> , casp <dig> , and gzmb .
one similar mutation in ubash3a protein coding region from human  <cit>  was also found in the slv chicken model .
additionally, when the long list of  <dig> candidate amino acid altering snps  was considered, several genes, including ifih <dig> , cd <dig> antigen and dnah <dig> , matched those identified by human gwas studies , although the amino acid position and alterations did not match.
ubash3a is one of the two family members belonging to the t cell ubiquitin ligand  family and can negatively regulate t cell signaling  <cit> .
together with the ubc molecule discussed elsewhere in this paper, functions for ubash3a related to ubiquitinylation and t cell signaling pathway may be important for slv development.
ifih <dig> encodes an interferon-induced rna helicase involved in antiviral innate immune responses, associated with type  <dig> diabetes, graves’ disease, multiple sclerosis, psoriasis, and perhaps lupus .
cd <dig> encodes a cell surface glycoprotein with various functions, including a role in t cell development  <cit> , and is associated with lupus  <cit> .
dnah <dig> gene mutation is found in patients with primary ciliary dyskinesia   <cit> , a rare disease transmitted as an autosomal recessive trait and characterized by recurrent airway infections due to abnormal ciliary structure and function.
primary defects in the structure and function of sensory and motile cilia result in multiple ciliopathies  <cit> .
CONCLUSIONS
in this study, various potential genetic markers showing amino acid changes were identified in the slv model through genome re-sequencing.
when considering functionality based on the interpretation of factors involved, development of vitiligo appeared to be associated with the interactions among cytoskeletal factors , protein kinases , phosphatase , ubiquitinylation  and amyloid  production.
further functional validation study, such as allele specific expression of the candidate genes with candidate snps at the target tissues involved in slv development will be carried out using the sl chicken model for spontaneous autoimmune vitiligo.

 vitiligo is an acquired hypomelanotic disorder characterized by circumscribed depigmented macules in the skin resulting from the loss of melanocytes. autoimmunity has been identified as the major etiological factor in vitiligo, although many other factors including infections, stress, neural abnormalities, aberrant melanocyte function, and genetic susceptibility have been implicated. the smyth line chicken is the only animal model for autoimmune vitiligo that spontaneously displays all clinical and biological manifestations of the human disorder. like other autoimmune diseases, sl vitiligo is multi-factorial in nature and involves the interplay of genetic, immune system, and environmental-factors. slv susceptibility is manifested in part in an inherent melanocyte defect and loss of melanocytes is due to melanocyte-specific cell-mediated and humoral immune activities. recent genome wide association studies in humans to understand the role of genetic components in a variety of autoimmune diseases including vitiligo have identified hundreds of loci harboring risk alleles. several gwas results identified vitiligo susceptible loci in human populations. however, most susceptible loci identified by gwas results were found in regulatory regions of gene expression, therefore the identified associations were not sufficient to identify the causal gene or deduce alterations caused by risk variants, which generally do not induce profound changes to genes. recently, the encyclopedia of dna elements of mammalian species suggested that ~90% of disease associated genetic variations in human lie in noncoding regions, while only ~10% of variations in coding regions were causative mutations associated with human disease. nevertheless, the identification of potential coding mutations that alter protein functionalities is a prerequisite process to understand disease etiology. moreover, the functional study of candidate genetic risk factors is almost impossible without appropriate model systems. the sl chicken is an excellent model to conduct a functional verification study of candidate genes that underlie genetic susceptibility for vitiligo due to the tractable, definite phenotype, the high vitiligo incidence in the population, the feasibility of in vivo characterization and the relatively short generation time. recently, microarray analysis examined global gene expression during slv development and provided comprehensive information at the transcriptome level that supported the multifactorial etiology of vitiligo. in this study, whole genome resequencing analysis using an illumina platform was performed to more deeply investigate the genetic aspects of slv expression in comparison with the parental brown line of chickens from which the sl was originally derived. bl chickens retain vitiligo susceptibility but with a very low incidence rate of vitiligo development, although none of the bl chickens used in this study had vitiligo. millions of single nucleotide polymorphisms were identified by genome resequencing and only potentially causal genes containing non-synonymous mutations that can induce amino acid changes in proteins were focused on in this study.  genome resequencing for bl and sl chickens genome sequencing of pooled dna from non-vitiliginous bl and sl chickens each with confirmed slv produced ~ and million sequence reads of 200 bp, respectively. of those, ~80% of the reads were used for sequence alignment, while 20% of sequence reads were not aligned. therefore, genome coverage for bl and sl reached x and x, respectively, of the red jungle fowl chicken genome. the total number of snps was and million for the bl and sl genome, respectively. the large number of snps per examined chicken line was based on data of at least read coverage depths. most snps were found on the larger chromosomes, including chr through and z. to identify genetic biomarkers that are responsible for the incidence of slv, unique snps that are found in sl only were selected by removing snps that overlapped with those found in the parental bl. then, mutations with ≥75% snp rates were chosen as reliable marker snps. since the objective of this study was to identify mutational snps uniquely found in sl compared to the parental bl, the filtering process used did not involve a typical snp calling and filtering method based on quality score. instead, snp filtering was conducted by removing overlapping snps found in both bl and sl, and applying fixed %snp rates as described in methods. as a result, a total of ~ million unique snps were identified throughout the sl chicken genome. over snps were found in chr and z while chr did not contain any unique snps for sl. when snps were grouped by the feature types of chromosome regions, ~50% of snps were in the intergenic regions and snps were found in cds sequences. most genes containing snps in regulatory regions, not in cds regions, identified by human gwas studies were also observed to contain unique snps in the current sl study. around 60% of sl snps in protein coding cds regions were synonymous mutations that did not induce amino acid changes. to identify potentially causal mutations that induce protein coding alterations, snp analysis focused on snps leading to changes in amino acid sequences. using this approach, a total of snps were identified that could induce non-synonymous-, frameshift-, nonsense-, and no-start-changes in the cds region, suggesting that the snps are part of the genetic components in functional protein coding regions that may drive the high incidence rate of slv. of the candidate snps that are associated with amino acid changes, snps showing ≥ read depths were chosen for the further analysis. using this approach, snps remained. to reduce false positives due to possible errors in the assembly process, re-scanning of each snp position for the potentially more reliable protein coding snps was conducted using the seqman-pro viewer program. this process yielded more reliable snps that were chosen as candidate marker snps for further analysis.table results of illumina sequencing and assembly bl 1 sl2 means the parental line, no vitiligo. sl, with vitiligo. number of unique snps per chromosome found in vitiliginous sl chickens compared to non-vitiliginous bl chickens. numbers are indicated for bars not clearly visible. summary of snps in slv. a) number of snps categorized by chromosomal region in sl chickens. b) number of snps categorized by type of amino acid sequence changes. the reliable marker snps that induced amino acid changes showing ≥ read depths contig id, chromosome numbers, reference position, reference base, called base, impact, snp%, feature name, dna change, amino acid change, depths, and five columns for snp counts are indicated. snp validation using pcr and sanger sequencing since pooled dna samples of chickens for each line were used for genome sequencing, individual snps were subjected to the verification process with larger bird populations. for this, snps were randomly chosen from the candidate marker snps showing ≥ read depths and were subjected to snp verification analysis using pcr and sanger sequencing to detect snp positions with larger numbers of birds; specifically non-vitiliginous bl chickens and sl chickens exhibiting vitiligo. the results clearly showed differential frequencies of nucleotide bases in the snp positions between bl and sl chickens. thus, the snps known to induce amino acid changes can become potential genetic biomarkers for vitiligo in sl chickens.table verification of snps using pcr and sanger sequencing in larger numbers of non-vitiliginous parental bl vs. vitiliginous sl chickens all_ct all_del ins_a_30% ins_a_100% c:a = 3:1 c:a = 1:4 all_g g:t = 1:1 c:t = 10:1 c:t = 1:10 t:c = 4:1 t:c = 1:7 t:c = 10:1 all_c all_t t:c = 1:10 c:a = 4:1 c:a = 1:10 all_g g:a = 1:10 all_c all_g all_g g:c = 1:10 all_g g:a = 1:4 all_a a:t = 1:4 ratios between reference base and snp in large chicken populations were bolded. fs; frameshift mutation. bioinformatic analyses of genes containing amino acid change snps amino acid changes may have impacts on the functional interpretations for vitiligo induction in sl chickens. the ingenuity pathway analysis program generated bioinformatics data sets including functional groups and gene networks for genes containing amino acid changes in sl chicken. the snps were found in genes encompassing known- and unknown functions, chromosomal open reading frames, and hypothetical proteins. functional roles genes were categorized in functional groups. of these, six functional groups are of particular interest to autoimmune vitiligo development, including dermatological diseases/conditions, inflammatory response, inflammatory disease, immunological disease, immune cell trafficking, and infectious disease. the functional group of genes for dermatological diseases/conditions contained the following genes: adamts; aspm; atp6v0a; brca; col12a; grm; lrp; mki; obscn; plau; rnf; stab; and xirp. general and dermatological disease related functions for these genes are summarized in table  table vitiligo related functions of candidate genes interestingly, a recent report by nikolaev et al. indicated that amino acid changes found in aspm, lrp stab and xirp proteins were associated with human melanoma by exome sequencing. melanocytes in vitiligo also exhibit morphological and biological melanocyte defects/alterations compared to melanocytes from individuals with normal pigmentation. while these alterations may be different from those observed in melanoma, e.g. slower growth, and higher sensitivity to oxidative stress of cultured melanocytes, alterations in amino acid sequences found in homolog proteins but different residues may result in opposite phenotypes of dermatological diseases/conditions. in addition to these molecules, brca grm mki and obscn associated with slv are also known to be associated with human melanoma. the relationship between candidate genes and other dermatological diseases including melanoma is summarized in table  gene networks gene network analysis, which represents the intermolecular connections among interacting genes based on functional knowledge inputs, was performed on genes with amino acid changes in slv chickens using the ipa program. the gene network analysis was carried out using the simplest setting of focus molecules to facilitate and summarize the intermolecular connections. a discussion of the gene networks is provided below and gene information for focus molecules in each network is listed in additional file 4: table s table associated network functions of candidate genes functions associated with networks are listed. score means the number of network eligible molecules out of differentially expressed genes. gene network # molecular interactions among important focus molecules are displayed. gray symbols show the genes found in the list of snp while white symbols indicate neighboring genes that are functionally associated, but not included, in the gene list of snp. symbols for each molecule are presented according to molecular functions and type of interactions. gene network # molecular interaction and symbols are the same as the description in figure  gene network # molecular interaction and symbols are the same as the description in figure  gene network # molecular interaction and symbols are the same as the description in figure  gene network # molecular interaction and symbols are the same as the description in figure  gene network # molecular interaction and symbols are the same as the description in figure  gene network # molecular interaction and symbols are the same as the description in figure  candidate genes in network # are associated with signaling pathways of the mitogen activated protein kinase and protein kinase c connected to vegf and pdgf with plau in the center. the top functions related to network # are cardiovascular disease, hematological disease, and cardiac infarction. interestingly, molecules including lrp plau, adamts and grm that are part of network # were also identified as functional factors for melanoma as described above. additionally, mutations in the amino acid sequence of lrp altered function of map2k and map2k induced by genetic mutations in melanoma patients, and mutations in grm in mouse melanoma models were also reported. the connections in network # therefore suggest genetic mutations that generated amino acid changes in lrp plau, adamts and grm may influence dermatological diseases, including vitiligo, through mapk and erk1/ signaling pathways. the top functions of network # include cell cycle, dna replication, recombination and repair, and developmental disorder and network # is involved in developmental disorder, endocrine system disorders, hereditary disorder. most molecules in networks # and # directly bind to ubc. ubiquitinylation, the covalent attachment of ubiquitin to proteins, regulates numerous cellular processes such as protein degradation and signal transduction. recently, many ubiquitinylated proteins and their lysine ubiquitinylation site were identified using proteomic technologies in mammalian species. indeed, in slv, the amino acid changes in lysine residues for cep and api were identified, suggesting that the various cellular functions including protein degradation by altered ubiquitinylation properties of proteins may play a significant role in the induction of vitiligo. molecules in network # are involved in cell morphology, cellular function and maintenance, hair and skin development and function. molecules in network # mainly interact with ifng, il, mapk and calcium signaling pathways in addition to protein phosphatase functions. myo, kif18a, and casc are known to directly bind to ppp1ca, suggesting that amino acid changes in those proteins may induce alterations in protein phophorylation states in sl chickens, resulting in vitiligo development. molecules interacting with ifng, il mapk and calcium signaling pathways showed an indirect relationship, not a direct relationship with each other making it difficult to explain how amino acid changes in these molecules affect vitiligo induction in sl chicken. molecules in network # also mainly bind to ubc as discussed in networks # and # and the functions include lipid metabolism, small molecule biochemistry, digestive system development and function. network # contains molecules involved in hereditary disorder, ophthalmic disease, neurological disease. in this network, zc2hc1a and rufy directly bind to app. app is a precursor protein for beta-amyloide, which is the main constituent of amyloid plaques in the brains of alzheimer disease patients. rufy, which is a brain specific protein, regulates neuronal polarity by suppressing formation of surplus axons. though the binding of zc2hc1a and rufy to app was found during the progression of alzheimer disease, the functional roles for this binding in the progression of this disease has not been characterized. similarly, amino acid changes found in zc2hc1a and rufy are implicated in slv development possibly as a result of altered app binding properties. ush2a was included in network # various mutations in ush2a have been identified in patients of usher syndrome type ii, which is characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa. vitiliginous sl chickens may also develop severe visual impairment and blindness due to autoimmune activity directed against choroidal melanocytes and subsequent damage to the retinal pigment epithelium.. taken together, the amino acid change in ush2a also may affect vitiligo progression and retinal depigmentation. molecules in network # mainly function in cellular response to therapeutics, cellular assembly and organization, dna replication, recombination, and repair. prkdc and its interacting protein kinases are mainly involved in this network. in addition to knock-out and inactive mutations, alteration of autophosphorylation capability by single amino acid change of prkdc has been known to influence rejoining of dna double stranded breaks in mammalian cells, suggesting that vitiligo development may be affected by aberrant prkdc kinase activity due to an observed amino acid change. vitiligo susceptible loci in human populations identified by several gwas also showed several loci that induced amino acid changes in various proteins such as strn, dnah, kiaa, tyr, oca, ptpn, ifih, sla, cd mc1r, ubash3a, c1qtnf, casp, and gzmb. one similar mutation in ubash3a protein coding region from human was also found in the slv chicken model. additionally, when the long list of candidate amino acid altering snps was considered, several genes, including ifih, cd antigen and dnah, matched those identified by human gwas studies, although the amino acid position and alterations did not match. ubash3a is one of the two family members belonging to the t cell ubiquitin ligand family and can negatively regulate t cell signaling. together with the ubc molecule discussed elsewhere in this paper, functions for ubash3a related to ubiquitinylation and t cell signaling pathway may be important for slv development. ifih encodes an interferon-induced rna helicase involved in antiviral innate immune responses, associated with type diabetes, graves’ disease, multiple sclerosis, psoriasis, and perhaps lupus. cd encodes a cell surface glycoprotein with various functions, including a role in t cell development, and is associated with lupus. dnah gene mutation is found in patients with primary ciliary dyskinesia, a rare disease transmitted as an autosomal recessive trait and characterized by recurrent airway infections due to abnormal ciliary structure and function. primary defects in the structure and function of sensory and motile cilia result in multiple ciliopathies.  in this study, various potential genetic markers showing amino acid changes were identified in the slv model through genome re-sequencing. when considering functionality based on the interpretation of factors involved, development of vitiligo appeared to be associated with the interactions among cytoskeletal factors, protein kinases, phosphatase, ubiquitinylation and amyloid production. further functional validation study, such as allele specific expression of the candidate genes with candidate snps at the target tissues involved in slv development will be carried out using the sl chicken model for spontaneous autoimmune vitiligo. 
various potential genetic markers showing amino acid changes and potential roles in vitiligo development were identified in the slv chicken through genome resequencing. the results of the analysis of the snps clearly showed differential frequencies of nucleotide bases in the snp positions between bl and sl chickens. to understand the genetic components underlying the susceptibility to develop sl vitiligo, whole genome resequencing analysis was performed in slv chickens compared with non-vitiliginous parental brown line chickens, which maintain a very low incidence rate of vitiligo. eventually of the reliable marker snps, which can induce non-synonymous-, frameshift-, nonsense-, and no-start mutations in amino acid sequences in proteins, genes were chosen for further analysis. results of genome resequencing of pooled dna of each bl and sl chickens reached x and x coverage, respectively. bioinformatic analysis showed that the most reliable marker snps included genes involved in dermatological diseases/conditions such as adamts aspm, atp6v0a brca col12a grm lrp obscn, plau, rnf stab and xirp intermolecular gene network analysis revealed that candidate genes identified in slv play a role in networks centered on protein kinases, phosphatase, ubiquitinylation and amyloid production. of these, randomly chosen snps were examined for snp verification by pcr and sanger sequencing to detect snp positions in bl and sl chickens. the smyth line chicken is the only animal model for autoimmune vitiligo that spontaneously displays all clinical and biological manifestations of the human disorder. the genetic markers and bioinformatic interpretations of amino acid mutations found in slv chickens may provide insight into the genetic component responsible for the onset and the progression of autoimmune vitiligo and serve as valuable markers to develop diagnostic tools to detect vitiligo susceptibility. the total number of snps was and million in bl and sl genome, respectively. through a series of filtering processes, a total of ~ million unique snps were found in the sl alone.
 the smyth line chicken is the only animal model for autoimmune vitiligo that spontaneously displays all clinical and biological manifestations of the human disorder. to understand the genetic components underlying the susceptibility to develop sl vitiligo, whole genome resequencing analysis was performed in slv chickens compared with non-vitiliginous parental brown line chickens, which maintain a very low incidence rate of vitiligo.  illumina sequencing technology and reference based assembly on red jungle fowl genome sequences were used. results of genome resequencing of pooled dna of each bl and sl chickens reached x and x coverage, respectively. the total number of snps was and million in bl and sl genome, respectively. through a series of filtering processes, a total of ~ million unique snps were found in the sl alone. eventually of the reliable marker snps, which can induce non-synonymous-, frameshift-, nonsense-, and no-start mutations in amino acid sequences in proteins, genes were chosen for further analysis. of these, randomly chosen snps were examined for snp verification by pcr and sanger sequencing to detect snp positions in bl and sl chickens. the results of the analysis of the snps clearly showed differential frequencies of nucleotide bases in the snp positions between bl and sl chickens. bioinformatic analysis showed that the most reliable marker snps included genes involved in dermatological diseases/conditions such as adamts aspm, atp6v0a brca col12a grm lrp obscn, plau, rnf stab and xirp intermolecular gene network analysis revealed that candidate genes identified in slv play a role in networks centered on protein kinases, phosphatase, ubiquitinylation and amyloid production.  various potential genetic markers showing amino acid changes and potential roles in vitiligo development were identified in the slv chicken through genome resequencing. the genetic markers and bioinformatic interpretations of amino acid mutations found in slv chickens may provide insight into the genetic component responsible for the onset and the progression of autoimmune vitiligo and serve as valuable markers to develop diagnostic tools to detect vitiligo susceptibility. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2014 