Advertisement

Mutations in ABCB6 Cause Dyschromatosis Universalis Hereditaria

  • Author Footnotes
    8 These authors contributed equally to this work.
    Caie Zhang
    Footnotes
    8 These authors contributed equally to this work.
    Affiliations
    Core Laboratory, Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

    Department of Dermatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Author Footnotes
    8 These authors contributed equally to this work.
    Duanzhuo Li
    Footnotes
    8 These authors contributed equally to this work.
    Affiliations
    Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Author Footnotes
    8 These authors contributed equally to this work.
    Jianguo Zhang
    Footnotes
    8 These authors contributed equally to this work.
    Affiliations
    BGI-Shenzhen, Shenzhen, Guangdong, China

    T-Life Research Center, Fudan University, Shanghai, China
    Search for articles by this author
  • Xingping Chen
    Affiliations
    Department of Dermatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Mi Huang
    Affiliations
    Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Stephen Archacki
    Affiliations
    Center for Cardiovascular Genetics, Department of Molecular Cardiology, and Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA
    Search for articles by this author
  • Yuke Tian
    Affiliations
    Core Laboratory, Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Weiping Ren
    Affiliations
    Department of Dermatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Aihua Mei
    Affiliations
    Department of Dermatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Qingyan Zhang
    Affiliations
    BGI-Shenzhen, Shenzhen, Guangdong, China
    Search for articles by this author
  • Mingyan Fang
    Affiliations
    BGI-Shenzhen, Shenzhen, Guangdong, China
    Search for articles by this author
  • Zheng Su
    Affiliations
    BGI-Shenzhen, Shenzhen, Guangdong, China
    Search for articles by this author
  • Ye Yin
    Affiliations
    BGI-Shenzhen, Shenzhen, Guangdong, China
    Search for articles by this author
  • Dongxian Liu
    Affiliations
    Department of Dermatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Yingling Chen
    Affiliations
    Department of Dermatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Xiukun Cui
    Affiliations
    Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Chang Li
    Affiliations
    Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Huanming Yang
    Affiliations
    BGI-Shenzhen, Shenzhen, Guangdong, China
    Search for articles by this author
  • Qing Wang
    Affiliations
    Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Jun Wang
    Affiliations
    BGI-Shenzhen, Shenzhen, Guangdong, China

    Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Search for articles by this author
  • Mugen Liu
    Correspondence
    Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China
    Affiliations
    Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Yunhua Deng
    Correspondence
    Department of Dermatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China
    Affiliations
    Department of Dermatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
    Search for articles by this author
  • Author Footnotes
    8 These authors contributed equally to this work.
      Dyschromatosis universalis hereditaria (DUH) is a pigmentary genodermatosis characterized by a mixture of hyperpigmented and hypopigmented macules distributed randomly over the body. No causative genes have been reported to date. In this study, we investigated a large five-generation Chinese family with DUH. After excluding the two known DUH loci, we performed genome-wide linkage analysis and identified a DUH locus on chromosome 2q33.3-q36.1 with a maximum LOD score of 3.49 with marker D2S2382. Exome sequencing identified a c.1067T>C (p.Leu356Pro) mutation in exon 3 of ABCB6 (ATP-binding cassette subfamily B, member 6) in the DUH family. Two additional missense mutations, c.508A>G (p.Ser170Gly) in exon 1 and c.1736G>A (p.Gly579Glu) in exon 12 of ABCB6, were found in two out of six patients by mutational screening using sporadic DUH patients. Immunohistologic examination in biopsy specimens showed that ABCB6 is expressed in the epidermis and had a diffuse cytoplasmic distribution. Examination of subcellular localization of wild-type ABCB6 in a B16 mouse melanoma cell line revealed that it is localized to the endosome-like compartment and dendrite tips, whereas disease-causing mutations of ABCB6 resulted in its retention in the Golgi apparatus. Our studies identified ABCB6 as the first pathogenic gene associated with DUH. These findings suggest that ABCB6 may be a physiological factor for skin pigmentation.

      Abbreviations

      ABCB6
      ATP-binding cassette subfamily B, member 6
      DSH
      dyschromatosis symmetrica hereditaria
      DUH
      dyschromatosis universalis hereditaria

      INTRODUCTION

      Dyschromatosis universalis hereditaria [DUH; Online Mendelian Inheritance in Man (OMIM) 127500] is a rare autosomal dominant genodermatosis initially described by
      • Ichikawa T.
      • Hiraga Y.
      A previously undescrided anomaly of pigmentation dyschromatosis universalis hereditaria.
      . It is characterized by asymptomatic hyperpigmented and hypopigmented macules that occur in a generalized distribution over the trunk, limbs, and sometimes the face (
      • Hawsawi K.
      • Aboud K.
      • Ramesh V.
      • et al.
      Dyschromatosis universalis hereditaria: report of a case and review of the literature.
      ;
      • Sethuraman G.
      • Srinivas C.R.
      • Souza M.
      • et al.
      Dyschromatosis universalis hereditaria.
      ). These lesions of irregular size and shape always appear in infancy or early childhood (
      • Urabe K.
      • Hori Y.
      Dyschromatosis.
      ;
      • Hawsawi K.
      • Aboud K.
      • Ramesh V.
      • et al.
      Dyschromatosis universalis hereditaria: report of a case and review of the literature.
      ;
      • Sethuraman G.
      • Srinivas C.R.
      • Souza M.
      • et al.
      Dyschromatosis universalis hereditaria.
      ). The trunk and extremities are the dominant sites containing the changes in the skin. Facial lesions can be seen in ∼50% of affected individuals, but involvement of palms and soles is unusual (
      • Hawsawi K.
      • Aboud K.
      • Ramesh V.
      • et al.
      Dyschromatosis universalis hereditaria: report of a case and review of the literature.
      ). Abnormalities of hair and nails have also been reported (
      • Sethuraman G.
      • Srinivas C.R.
      • Souza M.
      • et al.
      Dyschromatosis universalis hereditaria.
      ). DUH may be associated with abnormalities of dermal connective tissue, nerve tissue, or other systemic complications (
      • Hawsawi K.
      • Aboud K.
      • Ramesh V.
      • et al.
      Dyschromatosis universalis hereditaria: report of a case and review of the literature.
      ;
      • Bukhari I.A.
      • El-Harith E.A.
      • Stuhrmann M.
      • et al.
      Dyschromatosis universalis hereditaria as an autosomal recessive disease in five members of one family.
      ).
      Several human genetic diseases, such as dyschromatosis symmetrica hereditaria (DSH; OMIM 127400) and xeroderma pigmentosum (OMIM 278700), show some overlap with DUH. The characteristic clinical features of DSH also include a mixture of hyperpigmented and hypopigmented macules of various sizes, but these lesions are always limited to the dorsal aspects of the extremities. To date, DSH and DUH have not been found together in the same pedigree. The double-stranded RNA-specific adenosine deaminase gene (ADAR1 or DSRAD) has been identified as the causative gene of DSH (
      • Miyamura Y.
      • Suzuki T.
      • Kono M.
      • et al.
      Mutations of the RNA-specific adenosine deaminase gene (DSRAD) are involved in dyschromatosis symmetrica hereditaria.
      ), yet none of the ADAR1 mutations have been identified among DUH individuals (
      • Suzuki N.
      • Suzuki T.
      • Inagaki K.
      • et al.
      Mutation analysis of the ADAR1 gene in dyschromatosis symmetrica hereditaria and genetic differentiation from both dyschromatosis universalis hereditaria and acropigmentatio reticularis.
      ). These results may indicate that DSH is completely different from DUH, although it has been suggested to be a phenotypical variation. Xeroderma pigmentosum resembles DUH dyschromatosis, but DUH does not show the atrophy, xerosis, or skin cancers usually observed in xeroderma pigmentosum patients.
      DUH cases have been reported from different regions in the world. Most published DUH pedigrees show it to have an autosomal dominant pattern of inheritance (
      • Xing Q.H.
      • Wang M.T.
      • Chen X.D.
      • et al.
      A gene locus responsible for dyschromatosis symmetrica hereditaria (DSH) maps to chromosome 6q24.2-q25.2.
      ;
      • Nuber U.A.
      • Tinschert S.
      • Mundlos S.
      • et al.
      Dyschromatosis universalis hereditaria: familial case and ultrastructural skin investigation.
      ), although autosomal recessive inheritance and a few sporadic cases have been documented (
      • Stuhrmann M.
      • Hennies H.C.
      • Bukhari I.A.
      • et al.
      Dyschromatosis universalis hereditaria: evidence for autosomal recessive inheritance and identification of a new locus on chromosome 12q21-q23.
      ). Two chromosome segments have been reported to be associated with DUH: 6q24.2-q25.2 in two Chinese families (
      • Miyamura Y.
      • Suzuki T.
      • Kono M.
      • et al.
      Mutations of the RNA-specific adenosine deaminase gene (DSRAD) are involved in dyschromatosis symmetrica hereditaria.
      ;
      • Xing Q.H.
      • Wang M.T.
      • Chen X.D.
      • et al.
      A gene locus responsible for dyschromatosis symmetrica hereditaria (DSH) maps to chromosome 6q24.2-q25.2.
      ;
      • Suzuki N.
      • Suzuki T.
      • Inagaki K.
      • et al.
      Mutation analysis of the ADAR1 gene in dyschromatosis symmetrica hereditaria and genetic differentiation from both dyschromatosis universalis hereditaria and acropigmentatio reticularis.
      ) and 12q21-q23 in an Arab family (
      • Stuhrmann M.
      • Hennies H.C.
      • Bukhari I.A.
      • et al.
      Dyschromatosis universalis hereditaria: evidence for autosomal recessive inheritance and identification of a new locus on chromosome 12q21-q23.
      ). No causative genes have been identified thus far. In this study, we identified the ATP-binding cassette transporter ABCB6 (ATP-binding cassette subfamily B, member 6) as a pathogenic gene associated with DUH. Three ABCB6 mutations were found in a large Chinese DUH family, as well as in two sporadic cases of DUH. Our investigation showed that ABCB6 is expressed in human epidermis, and our mutations have a clinical correlation to this skin genodermatosis.

      RESULTS

      Clinical features

      We characterized a five-generation Chinese family with DUH, in which the disease was transmitted in an autosomal dominant manner (Figure 1a). The proband (V1) was a 9-year-old boy who had normal skin at birth. Hyperpigmented and hypopigmented macules appeared initially on his trunk at the age of 2 years, and then gradually extended to involve his face, neck, and limbs. He did not report any pruritus or pain. Examination of the skin showed motley hyperpigmented and hypopigmented macules that nearly involved his whole body. The lesions occurred in a symmetrical pattern and were most obvious on the face, neck, trunk, and the dorsa of his hands and feet (Figure 1b–e). His palms and soles, oral mucosa, hair, nails, and teeth were normal, as were the rest of his physical exam and routine blood work. Histopathological examination of a skin biopsy from the pigmented macules showed a pigmented basal layer of the epidermis, pigmentary incontinence in the papillary dermis, and some melanophages and lymphocytes in the upper dermis (Supplementary Figure S1 online). None of the affected members in this family were found to have skin cancer or ocular defects, following an ophthalmologic examination.
      Figure thumbnail gr1
      Figure 1Pedigree and clinical features. (a) Pedigree and haplotype analysis. The disease haplotype is indicated by a black bar. Black symbols represent affected individuals, and open symbols represent unaffected individuals. Circles and squares indicate female and male individuals, respectively. The arrow indicates the proband in the family. (be) Clinical characteristics of the proband with dyschromatosis universalis hereditaria (DUH). The proband, a 9-year-old boy, was involved with motley hyperpigmented and hypopigmented macules on his (b) face and neck, (d) trunk, and the (e) dorsa of hands and (c) feet in a symmetrical pattern.

      Linkage mapping of DUH to chromosome 2q33.3-q36.1

      Genomic DNA was isolated from blood samples of patients and unaffected family members. Linkage and haplotype analyses were carried out to test the linkage of the family with two known DUH loci. All individuals were genotyped using four microsatellite markers spanning the locus on 12q21-q23 (D12S326, D12S1708, D12S351, and D12S346), and three markers spanning the locus on 6q24.2-q25.2 (D6S1654, D6S441, and D6S1577). However, negative LOD scores were obtained for these loci (data not shown).
      After excluding the pathogenic gene in the DUH family from two known candidate loci, a genome-wide linkage scan using microsatellite markers was performed as described previously (
      • Dai X.
      • Gao Y.
      • Xu Z.
      • et al.
      Identification of a novel genetic locus on chromosome 8p21.1-q11.23 for idiopathic basal ganglia calcification.
      ;
      • Wang C.
      • Li Y.
      • Shi L.
      • et al.
      Mutations in SLC20A2 link familial idiopathic basal ganglia calcification with phosphate homeostasis.
      ). Given the inheritance pattern observed in our family, LOD scores were calculated under the assumption of autosomal dominant inheritance with a penetrance of 100%. Linkage and haplotype analyses found that the disease-causative gene in the DUH family was localized in the 2q33.3-q36.1 region, between D2S325 and D2S126 (Figure 1a). Multipoint analysis gave a maximum LOD score of 3.49 at D2S2382.

      Exome sequencing identifies a candidate gene

      We subjected the exomes of two affected (IV13 and V1) and one unaffected (IV6) individual to exome sequencing. Approximately 4.23 billion bases per individual were sequenced, and 3.75 billion bases were acceptable. The mean coverage of each sample was × 59.2 (Supplementary Table S1 online). Nonsynonymous/splice acceptor and donor site/insertion or deletion (NS/SS/Indel) variants were identified and quality filtered. A variant for the disease-related pathogenic mutation was defined as one that exclusively existed in the two patients but appeared neither in the control of the family nor in the databases, including dbSNP129, the 1000 Genome Project, HapMap, and YH databases (Table 1). Twenty-nine variants remained and were located on multiple chromosomes. Among them, only two variants in two genes (c.1067T>C in ABCB6 and c.2584C>T in ZNF142) were located in the 2q33.3-q36.1 region. Sanger sequencing and cosegregation analysis showed that the c.1067T>C mutation in exon 5 of ABCB6 occurred in all affected members, but not in any of the unaffected individuals of the family (Supplementary Table S2 online). The other variant, c.2584C>T mutation in exon 8 of ZNF142, was excluded by cosegregation analysis, and was found to be a single-nucleotide polymorphism (SNP) in the newly released SNP build 137 from the University of California Santa Cruz (UCSC) Genome Browser website. The results suggest that ABCB6 may be the disease-causing mutation in DUH.
      Table 1Overview of all variants identified by exome sequencing in two affected (IV13 and V1) patients and one unaffected (IV6) individual
      DUH-IV6DUH-IV13DUH-V1
      Total SNPs and indels44,478+3,12341,904+2,90843,660+2,850
      Functional SNPs and indels7,567+4317,482+4607,430+434
      Filtered_dbSNP932+147977+168972+163
      Filtered_dbSNP_1000genomes699+73620+92582+87
      Filtered_dbSNP_1000genomes_Hapmap8695+73541+92511+87
      Filtered_dbSNP_1000genomes_Hapmap8_YH663+72533+91503+86
      Filtered_dbSNP_1000genomes_Hapmap8_YH_DUH-IV60443+59240+44
      Shared by all cases29
      Within the linkage region2 (ABCB6 and ZNF142)
      Abbreviations: ABCB6, ATP-binding cassette subfamily B, member 6; dbSNP, single-nucleotide polymorphism database; DUH, dyschromatosis universalis hereditaria; indels, insertions or deletions; SNP, single-nucleotide polymorphism.
      Each cell indicates the number of candidate variants identified against different databases.

      Mutation of the ABCB6 gene in DUH patients

      The c.1067T>C mutation in ABCB6 leads to a proline to leucine substitution at codon 356 (p.Leu356Pro) in the ABCB6 protein. Further direct DNA sequence analysis with a panel of 500 unaffected control individuals matched for the geographical location did not detect this mutation. To confirm that ABCB6 may be the DUH disease–causing gene, we screened all of the exons of ABCB6 in six sporadic DUH patients by direct DNA sequence. Two additional missense mutations, c.508A>G (p.Ser170Gly) in exon 1 and c.1736G>A (p.Gly579Glu) in exon 12 (Figure 2a) of ABCB6, were identified in two of these six cases. Neither of the ABCB6 mutations was found in 400 control individuals or the public SNP databases on the 1000 Genomes Project and UCSC Genome Browser websites. All three of the mutations occurred in evolutionarily conserved amino acids of ABCB6 (Figure 2b). These data suggest that ABCB6 is a causative gene for DUH.
      Figure thumbnail gr2
      Figure 2ABCB6 mutations cause dyschromatosis universalis hereditaria (DUH). (a) The mutations in ABCB6 and their Sanger sequencing tracing, including c.1067T>C (p.Leu356Pro) (reverse complement), c.508A>G (p.Ser170Gly), and c.1736G>A (p.Gly579Glu). (b) The mutations in ABCB6 occurred in the evolutionarily conserved regions. A partial sequence of ABCB6 was compared with other species orthologs. Arrows indicate the location of the three mutations identified in DUH patients. (c) Immunohistologic staining of human skin shows that ABCB6 is localized in the basal layers of the epidermis and has a diffuse cytoplasmic distribution. The right panel indicates a higher magnification of an insert in the left panel. Original magnifications × 100 and × 400. Nuclei are counterstained with hematoxylin. (d) Western blot analysis shows the expression of ABCB6 in HaCaT and A375 cells. ABCB6, ATP-binding cassette subfamily B, member 6; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

      Immunohistologic staining and western blotting show ABCB6 expression in human epidermis cell

      ABCB6 is reported to be widely expressed in many tissues, with its highest expression in the heart and skeletal muscles (
      • Mitsuhashi N.
      • Miki T.
      • Senbongi H.
      • et al.
      MTABC3, a novel mitochondrial ATP-binding cassette protein involved in iron homeostasis.
      ). To examine the expression level of ABCB6 in skin, we performed immunohistologic staining with a polyclonal antibody against ABCB6 on normal human skin harvested from the abdominal area. Immunohistochemical staining of human normal skin showed that ABCB6 localized to the epidermis and had a diffuse cytoplasmic distribution (Figure 2c).
      To confirm the ABCB6 expression in melanocytes and keratinocytes, HaCaT (human keratinocyte line) and A375 cells (a human malignant melanoma cell line) were lysed in a Nonidet P-40 lysis buffer. A measure of 10μg total protein was separated via SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were probed with anti-ABCB6 or glyceraldehyde-3-phosphate dehydrogenase antibodies. Western blot results showed that ABCB6 was expressed in HaCaT and A375 cells (Figure 2d).

      Retention of mutant ABCB6 in the Golgi apparatus

      To investigate whether the mutations of ABCB6 are associated with DUH, we examined the subcellular localization of wild-type and mutant ABCB6 proteins in B16 cell lines by immunofluorescent staining. The wild-type ABCB6 and three mutant forms of ABCB6 (p.Ser170Gly, p.Leu356Pro, and p.Gly579Glu) were tagged with enhanced green fluorescent protein (EGFP) fusion proteins and then transfected into mouse B16 cells, respectively. The localization of the various fusion proteins was determined by confocal microscopy. As expected, wild-type ABCB6 localized to the endosome-like compartment, and its accumulation in the dendrites could be seen clearly (
      • Kiss K.
      • Brozik A.
      • Kucsma N.
      • et al.
      Shifting the paradigm: the putative mitochondrial protein ABCB6 resides in the lysosomes of cells and in the plasma membrane of erythrocytes.
      ). At the same time, mutant ABCB6 accumulated primarily in the perinuclear area instead of the dendrite tips (Figure 3).
      Figure thumbnail gr3
      Figure 3Subcellular localization of wild-type and mutated ABCB6 in B16-F1 cells. Cotransfected DsRed-monomer-tag Golgi marker galactosyl-transferase (GalT) with GFP-tag mutant-type or wild-type ABCB6 into B16-F1 cell, respectively. Golgi body is indicated by red color. Nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI; blue color). Mutant-type or wild-type (WT) ABCB6 are indicated by enhanced green fluorescent protein (EGFP; green color). Arrows indicate the dendrites. Scale bar=10μm. ABCB6, ATP-binding cassette subfamily B, member 6.
      To examine the exact subcellular localization of mutant ABCB6 in the perinuclear area, we cotransfected DsRed-monomer-tag Golgi marker galactosyl-transferase (GalT) with GFP-tag mutant-type or wild-type ABCB6 into B16-F1 cells. Confocal microscopy showed that most of the mutated ABCB6 accumulated in the Golgi body instead of the dendrites, whereas most of the wild-type ABCB6 proteins did not colocalize to the Golgi body (Figure 3).

      DISCUSSION

      DUH is a heterogeneous disease. Previous studies reported two loci responsible for DUH: 6q24.2-q25.2 and 12q21-q237 (
      • Xing Q.H.
      • Wang M.T.
      • Chen X.D.
      • et al.
      A gene locus responsible for dyschromatosis symmetrica hereditaria (DSH) maps to chromosome 6q24.2-q25.2.
      ;
      • Stuhrmann M.
      • Hennies H.C.
      • Bukhari I.A.
      • et al.
      Dyschromatosis universalis hereditaria: evidence for autosomal recessive inheritance and identification of a new locus on chromosome 12q21-q23.
      ). However, no pathogenic DUH gene has been found to date. Here we performed an integrated approach combining linkage analysis, whole-exome sequencing, and mutational analyses, and found three mutations in ABCB6 in patients with DUH. Our study identified a disease-causing gene that is responsible for DUH.
      ABCB6 is an ATP-binding cassette transporter and is reported to regulate porphyrin synthesis (
      • Krishnamurthy P.C.
      • Du G.
      • Fukuda Y.
      • et al.
      Identification of a mammalian mitochondrial porphyrin transporter.
      ). It localizes to the outer mitochondrial membrane and is a mammalian mitochondrial porphyrin transporter. However, recent studies revealed that ABCB6 is glycosylated in multiple cell types and is also found in the classical secretory pathway, which includes the endoplasmic reticulum, Golgi apparatus, plasma membrane, and exosomes (
      • Watabe H.
      • Valencia J.C.
      • Le Pape E.
      • et al.
      Involvement of dynein and spectrin with early melanosome transport and melanosomal protein trafficking.
      ;
      • Kiss K.
      • Brozik A.
      • Kucsma N.
      • et al.
      Shifting the paradigm: the putative mitochondrial protein ABCB6 resides in the lysosomes of cells and in the plasma membrane of erythrocytes.
      ;
      • Wang L.
      • He F.
      • Bu J.
      ABCB6 mutations cause ocular coloboma.
      ).
      DUH is considered to be attributable to a deficiency of melanin synthesis and/or melanosome sorting (
      • Kim N.S.
      • Im S.
      • Kim S.C.
      Dyschromatosis universalis hereditaria: an electron microscopic examination.
      ;
      • Nuber U.A.
      • Tinschert S.
      • Mundlos S.
      • et al.
      Dyschromatosis universalis hereditaria: familial case and ultrastructural skin investigation.
      ;
      • Wang G.
      • Li C.Y.
      • Gao T.W.
      • et al.
      Dyschromatosis universalis hereditaria: two cases in a Chinese family.
      ). Skin pigment melanin is synthesized by melanocytes that are located in the basal layer of the epidermis. In melanocytes, melanin synthesis takes place within melanosomes that are derived from the endoplasmic reticulum. Melanosomes bud off the Golgi complex, move to the dendrites, and are extruded into surrounding keratinocytes (
      • Sturm R.A.
      • Teasdale R.D.
      • Box N.F.
      Human pigmentation genes: identification, structure and consequences of polymorphic variation.
      ).
      ABCB6 is reported to be present in exosomes released from reticulocytes (
      • Kiss K.
      • Brozik A.
      • Kucsma N.
      • et al.
      Shifting the paradigm: the putative mitochondrial protein ABCB6 resides in the lysosomes of cells and in the plasma membrane of erythrocytes.
      ). Exosomes are vesicles contained in multivesicular bodies that are secreted upon fusion of multivesicular bodies with the cell surface plasma membrane (
      • Simons M.
      • Raposo G.
      Exosomes–vesicular carriers for intercellular communication.
      ). Exosome secretion has been found to relate to the melanosome transport machinery. Many melanogenic proteins are sorted in exosomes, including Pmel17/gp100, TYRP1 (tyrosinase-related protein 1), and MART-1 (melanoma antigen recognized by T cells) (
      • Andre F.
      • Schartz N.E.
      • Movassagh M.
      • et al.
      Malignant effusions and immunogenic tumour-derived exosomes.
      ;
      • Theos A.C.
      • Truschel S.T.
      • Tenza D.
      • et al.
      A lumenal domain-dependent pathway for sorting to intralumenal vesicles of multivesicular endosomes involved in organelle.
      ). A recent study shows that Rab27 functions in the exosome secretion pathway as well (
      • Ostrowski M.
      • Carmo N.B.
      • Krumeichet S.
      Rab27a and Rab27b control different steps of the exosome secretion pathway.
      ). These observations are consistent with our examination of subcellular localization of ABCB6 in the B16 cell line. Our finding that wild-type ABCB6 protein is clearly expressed in the dendrites of B16 cells implies that it may have a key role in the melanosome transport to surrounding keratinocytes. A mutant ABCB6 protein, which fails to localize to the dendrites, could disrupt melanosome transport and cause DUH. Previous studies have indicated that ABCB6 is a half-transporter (
      • Krishnamurthy P.C.
      • Du G.
      • Fukuda Y.
      • et al.
      Identification of a mammalian mitochondrial porphyrin transporter.
      ), and thus mutant ABCB6 may bind to the normal type and interfere with melanogenesis, which may explain the dominant negative effect.
      ABCB6-null mutations have been reported in humans (
      • Helias V.
      • Saison C.
      • Ballif B.A.
      • et al.
      ABCB6 is dispensable for erythropoiesis and specifies the new blood group system Langereis.
      ), and ABCB6 knockout mice have been generated (
      • Ulrich D.L.
      • Lynch J.
      • Wang Y.
      • et al.
      ATP-dependent mitochondrial porphyrin importer ABCB6 protects against phenylhydrazine toxicity.
      ). No skin pigmentation disorders have been described to date. Recently, two mutations in ABCB6 associated with ocular coloboma have been reported (
      • Wang L.
      • He F.
      • Bu J.
      ABCB6 mutations cause ocular coloboma.
      ). No ocular defects were found in our DUH family or in two sporadic patients. Further studies might reveal the relationship between the genotype of ABCB6 deficiencies and the phenotypic heterogeneity.
      In conclusion, we identified the ATP-binding cassette transporter ABCB6 as the first protein involved in DUH. With whole-exome and Sanger sequencing in a DUH family and two sporadic members with DUH, respectively, we identified three mutations in the gene that encodes the ATP-binding cassette transporter (ABCB6). We showed that ABCB6 is expressed in the epidermis. In the mouse melanoma B16 cell line, ABCB6 protein was distributed in an endosome-like pattern and was rich in the dendrites. DUH disease–causing mutations in ABCB6 caused its retention in the Golgi body. Our findings suggest that ABCB6 may be a previously unreported physiological factor for skin pigmentation. Further studies on the function of ABCB6 will not only uncover the pathogenic mechanism for DUH but will also help us understand melanogenesis and skin pigmentation.

      MATERIALS AND METHODS

      Informed consent

      Our study was conducted according to the Declaration of Helsinki Principles. Written informed consent from all subjects was obtained. The study protocol was approved by the ethics committee of Tongji Hospital, and College of Life Science and Technology, Huazhong University of Science and Technology.

      Linkage analysis

      The microsatellite markers from the Linkage Mapping Set MD-10 (Applied Biosystems, Foster City, CA) were genotyped using an ABI 3100 Genetic Analyzer. Genotypes were analyzed by the GeneMapper 2.5 Software program (Applied Biosystems). LOD score was calculated with the GeneHunter 2.1 software (http://www.mybiosoftware.com).

      Exome sequencing

      We obtained 15μg DNA samples from each of the three members of the DUH family (one control and two cases) and sheared the DNA by sonication. The sheared genomic DNA of each individual was then hybridized with an Agilent 38M sequence capture array (Agilent Technologies, Santa Clara, CA) to enrich the exonic DNA in each library.
      Exon-enriched DNA was sequenced by the Illumina Hiseq 2000 platform according to the manufacturer’s instructions (Illumina, San Diego, CA). Raw image files were processed by the Illumina pipeline for base calling and generating the reads set.
      The sequencing reads were aligned to the NCBI human reference genome (NCBI36.3) using SOAPaligner. We collected reads that were aligned to the designed target regions for SNP identification and subsequent analysis. The consensus sequence and quality of each allele was calculated by SOAPsnp (http://soap.genomics.org.cn/soapsnp.html; BGI. Shenzhen, Guangdong, China). The low-quality variations were filtered out using the following criteria: (1) quality score ≥20 (Q20); (2) average copy number at the allele site ≤2; (3) distance of two adjacent SNPs ≥5bp; and (4) sequencing depth ≥4 and ≤500.

      Sanger sequencing

      Primers used in ABCB6 variants validation and mutation analysis are listed in Supplementary Table S2 online. PCR products from genomic DNA were sequenced using an ABI3730XL DNA Analyzer (International Equipment Trading, Vernon Hills, IL). Sanger sequencing was used to confirm the presence and identity of variants in the candidate gene identified via exome sequencing and to screen the candidate gene in additional sporadic cases with DUH.

      Immunohistochemistry

      To examine the expression of ABCB6 in skin, normal abdomen skin samples were stained with a polyclonal antibody against ABCB6. Formalin-fixed, paraffin-embedded wax blocks (3mm) were cut with a microtome (RM2016, Leica, Brunswick, Germany) and applied to amino alkylsilane–coated slides. The sections were dried overnight at 37°C. The slides were dewaxed and dehydrated. Heat-mediated antigen retrieval was carried out, and endogenous peroxidase was blocked with 0.3% hydrogen peroxide for 10min. Staining was carried out with the REALTM EnVision+/HRP RABBIT (Dako Denmark A/S, Shanghai, China). Light microscopy was carried out with the ordinary optics microscope (XSP-C204, COIC, Chongqing, China).

      Western blot

      Human HaCaT and human A375 Cells were lysed in a Nonidet P-40 lysis buffer (0.15M NaCl, 1% NP-40, and 0.05M Tris-HCl, pH 8.0) treated with a mixture of protease inhibitors (0.25mM phenylmethylsulfonyl fluoride, 10mg/ml aprotinin and leupeptin, and 1mM dithiothreitol). A measure of 10μg of total protein was separated via 12% SDS-PAGE and transferred to a nitrocellulose membrane (Whatman, London, UK). The membranes were probed with anti-ABCB6 or glyceraldehyde-3-phosphate dehydrogenase antibodies (Ptglab, Wuhan, China).

      Construction of expression vectors

      Wild-type human ABCB6 complementary DNA (Ptglab) was cloned into the eukaryotic expression vector pEGFP-N1 (Clontech, Mountain View, CA). Next, we performed site-directed mutagenesis to generate the three ABCB6 mutations associated with DUH (p.Ser170Gly, p.Leu356Pro, and p.Gly579Glu). All constructs were verified by Sanger sequencing.

      Confocal microscopy

      B16-F1 cells were transfected with pEGFP-ABCB6 wild type or mutant type. After 48hours, cells were fixed in 4% paraformaldehyde for 30minutes, and then washed twice with phosphate-buffered saline. Samples were viewed with a confocal microscope (Leica TCS SP2 AOBS MP microscope system). Images were assembled using Adobe Photoshop CS3 (Adobe Systems, San Jose, CA).

      Internet resources

      UCSC Genome Browser website (http://genome.ucsc.edu/)

      ACKNOWLEDGMENTS

      We thank the patients and their family members for their enthusiastic participation. This study was supported by grants from the National Natural Science Foundation of China (31171228, 81071357, 31071106, and 81270983) and Scientific Research Foundation for Returned Scholars, Ministry of Education of China (2009-1001).

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

      REFERENCES

        • Andre F.
        • Schartz N.E.
        • Movassagh M.
        • et al.
        Malignant effusions and immunogenic tumour-derived exosomes.
        Lancet. 2002; 360: 295-305
        • Bukhari I.A.
        • El-Harith E.A.
        • Stuhrmann M.
        • et al.
        Dyschromatosis universalis hereditaria as an autosomal recessive disease in five members of one family.
        J Eur Acad Dermatol Venereol. 2006; 20: 628-629
        • Dai X.
        • Gao Y.
        • Xu Z.
        • et al.
        Identification of a novel genetic locus on chromosome 8p21.1-q11.23 for idiopathic basal ganglia calcification.
        Am J Med Genet B Neuropsychiatr Genet. 2010; 5: 1305-1310
        • Hawsawi K.
        • Aboud K.
        • Ramesh V.
        • et al.
        Dyschromatosis universalis hereditaria: report of a case and review of the literature.
        Pediatr Dermatol. 2002; 19: 523-526
        • Helias V.
        • Saison C.
        • Ballif B.A.
        • et al.
        ABCB6 is dispensable for erythropoiesis and specifies the new blood group system Langereis.
        Nat Genet. 2012; 44: 170-173
        • Ichikawa T.
        • Hiraga Y.
        A previously undescrided anomaly of pigmentation dyschromatosis universalis hereditaria.
        Jap J Dermatol. 1933; 34: 360-364
        • Kim N.S.
        • Im S.
        • Kim S.C.
        Dyschromatosis universalis hereditaria: an electron microscopic examination.
        J Dermatol. 1997; 24: 161-164
        • Kiss K.
        • Brozik A.
        • Kucsma N.
        • et al.
        Shifting the paradigm: the putative mitochondrial protein ABCB6 resides in the lysosomes of cells and in the plasma membrane of erythrocytes.
        PLoS One. 2012; 7: e37378
        • Krishnamurthy P.C.
        • Du G.
        • Fukuda Y.
        • et al.
        Identification of a mammalian mitochondrial porphyrin transporter.
        Nature. 2006; 443: 586-589
        • Mitsuhashi N.
        • Miki T.
        • Senbongi H.
        • et al.
        MTABC3, a novel mitochondrial ATP-binding cassette protein involved in iron homeostasis.
        J Biol Chem. 2000; 275: 17536-17540
        • Miyamura Y.
        • Suzuki T.
        • Kono M.
        • et al.
        Mutations of the RNA-specific adenosine deaminase gene (DSRAD) are involved in dyschromatosis symmetrica hereditaria.
        Am J Hum Genet. 2003; 73: 693-699
        • Nuber U.A.
        • Tinschert S.
        • Mundlos S.
        • et al.
        Dyschromatosis universalis hereditaria: familial case and ultrastructural skin investigation.
        Am J Med Genet A. 2004; 125: 261-266
        • Ostrowski M.
        • Carmo N.B.
        • Krumeichet S.
        Rab27a and Rab27b control different steps of the exosome secretion pathway.
        Nat Cell Biol. 2010; 12 (1–13): 19-30
        • Sethuraman G.
        • Srinivas C.R.
        • Souza M.
        • et al.
        Dyschromatosis universalis hereditaria.
        Clin Exp Dermatol. 2002; 27: 477-479
        • Simons M.
        • Raposo G.
        Exosomes–vesicular carriers for intercellular communication.
        Curr Opin Cell Biol. 2009; 21: 575-581
        • Stuhrmann M.
        • Hennies H.C.
        • Bukhari I.A.
        • et al.
        Dyschromatosis universalis hereditaria: evidence for autosomal recessive inheritance and identification of a new locus on chromosome 12q21-q23.
        Clin Genet. 2008; 73: 566-572
        • Sturm R.A.
        • Teasdale R.D.
        • Box N.F.
        Human pigmentation genes: identification, structure and consequences of polymorphic variation.
        Gene. 2001; 277: 49-62
        • Suzuki N.
        • Suzuki T.
        • Inagaki K.
        • et al.
        Mutation analysis of the ADAR1 gene in dyschromatosis symmetrica hereditaria and genetic differentiation from both dyschromatosis universalis hereditaria and acropigmentatio reticularis.
        J Invest Dermatol. 2005; 124: 1186-1192
        • Theos A.C.
        • Truschel S.T.
        • Tenza D.
        • et al.
        A lumenal domain-dependent pathway for sorting to intralumenal vesicles of multivesicular endosomes involved in organelle.
        Dev Cell. 2006; 10: 343-354
        • Ulrich D.L.
        • Lynch J.
        • Wang Y.
        • et al.
        ATP-dependent mitochondrial porphyrin importer ABCB6 protects against phenylhydrazine toxicity.
        J Biol Chem. 2012; 287: 12679-12690
        • Urabe K.
        • Hori Y.
        Dyschromatosis.
        Semin Cutan Med Surg. 1997; 16: 81-85
        • Wang C.
        • Li Y.
        • Shi L.
        • et al.
        Mutations in SLC20A2 link familial idiopathic basal ganglia calcification with phosphate homeostasis.
        Nat Genet. 2012; 12: 254-256
        • Wang G.
        • Li C.Y.
        • Gao T.W.
        • et al.
        Dyschromatosis universalis hereditaria: two cases in a Chinese family.
        Clin Exp Dermatol. 2005; 30: 494-496
        • Wang L.
        • He F.
        • Bu J.
        ABCB6 mutations cause ocular coloboma.
        Am J Hum Genet. 2012; 90: 40-48
        • Watabe H.
        • Valencia J.C.
        • Le Pape E.
        • et al.
        Involvement of dynein and spectrin with early melanosome transport and melanosomal protein trafficking.
        J Invest Dermatol. 2008; 128: 162-174
        • Xing Q.H.
        • Wang M.T.
        • Chen X.D.
        • et al.
        A gene locus responsible for dyschromatosis symmetrica hereditaria (DSH) maps to chromosome 6q24.2-q25.2.
        Am J Hum Genet. 2003; 73: 377-382