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A Missense Mutation within the Helix Initiation Motif of the Keratin K71 Gene Underlies Autosomal Dominant Woolly Hair/Hypotrichosis

      Woolly hair (WH) is an abnormal variant of tightly curled hair, which is frequently associated with hypotrichosis. Non-syndromic forms of WH can show either autosomal-dominant WH (ADWH) or autosomal-recessive WH (ARWH) inheritance patterns. ARWH has recently been shown to be caused by mutations in either the lysophosphatidic acid receptor 6 (LPAR6) or lipase H (LIPH) gene. More recently, a mutation in the keratin K74 (KRT74) gene has been reported to underlie ADWH. Importantly, all of these genes are abundantly expressed in the inner root sheath (IRS) of human hair follicles. Besides these findings, the molecular mechanisms underlying hereditary WH have not been fully disclosed. In this study, we identified a Japanese family with ADWH and associated hypotrichosis. After exclusion of known causative genes, we discovered the heterozygous mutation c.422T>G (p.Phe141Cys) within the helix initiation motif of the IRS-specific keratin K71 (KRT71) gene in affected family members. We demonstrated that the mutant K71 protein led to disruption of keratin intermediate filament formation in cultured cells. To our knowledge, it is previously unreported that the KRT71 mutation is associated with a hereditary hair disorder in humans. Our findings further underscore the crucial role of the IRS-specific keratins in hair follicle development and hair growth in humans.

      Abbreviations

      ADWH
      autosomal-dominant woolly hair
      ARWH
      autosomal-recessive woolly hair
      HF
      hair follicle
      HIM
      helix initiation motif
      HTM
      helix termination motif
      IIF
      indirect immunofluorescence
      IRS
      inner root sheath
      KIF
      keratin intermediate filament
      KRT71
      keratin K71
      LIPH
      lipase H
      LPA
      2-acyl-lysophosphatidic acid
      LPAR
      lysophosphatidic acid receptor 6
      PA-PLA1α
      phosphatidic acid-selective phospholipase A1α
      SEM
      scanning electron microscopy
      WB
      western blot
      WH
      woolly hair
      Wt
      wild-type

      Introduction

      The mammalian hair follicle (HF) has a highly complex structure with several distinct cell layers. The growth of the hair shaft is supported and molded by the inner root sheath (IRS), the companion layer, and the outer root sheath. The IRS is composed of three layers: the IRS cuticle, the Huxley layer, and the Henle layer. During the anagen (growth) phase, matrix cells in the bulb portion, which are derived from the stem cell niche in the bulge, actively proliferate and differentiate into these distinct cell layers, except for the outer root sheath (
      • Shimomura Y.
      • Christiano A.M.
      Biology and genetics of hair.
      ). Recent advances in molecular genetics have led to the identification of numerous genes expressed in the HF. Furthermore, mutations in some of these genes have been shown to underlie hereditary hair disorders in humans, which are largely classified into syndromic and non-syndromic forms.
      In syndromic forms, hair symptoms appear as part of a broader syndrome that can exhibit other skin symptoms and/or various systemic manifestations, whereas affected individuals with the non-syndromic forms only show hair phenotype. In the past decade, several causative genes for non-syndromic forms of hypotrichosis have been identified, including corneodesmosin (CDSN) (hypotrichosis simplex of the scalp 1; OMIM 146520) (
      • Levy-Nissenbaum E.
      • Betz R.C.
      • Frydman M.
      • et al.
      Hypotrichosis simplex of the scalp is associated with nonsense mutations in CDSN encoding corneodesmosin.
      ), desmoglein 4 (DSG4) (localized autosomal-recessive hypotrichosis (LAH) 1; OMIM 607903) (
      • Kljuic A.
      • Bazzi H.
      • Sundberg J.P.
      • et al.
      Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris.
      ), desmocollin 3 (DSC3) (hypotrichosis and recurrent skin vesicles; OMIM 613102) (
      • Ayub M.
      • Basit S.
      • Jelani M.
      • et al.
      A homozygous nonsense mutation in the human desmocollin-3 (DSC3) gene underlies hereditary hypotrichosis and recurrent skin vesicles.
      ), and an inhibitory upstream open reading frame in the 5′-untranslated region of the hairless (U2HR) (Marie Unna hereditary hypotrichosis; OMIM 146550) (
      • Wen Y.
      • Liu Y.
      • Xu Y.
      • et al.
      Loss-of-function mutations of an inhibitory upstream ORF in the human hairless transcript cause Marie Unna hereditary hypotrichosis.
      ). Furthermore, generalized hypotrichosis simplex (OMIM 605389) has recently been reported to be caused by mutations in either adenomatosis polyposis coli downregulated 1 (APCDD1) (
      • Shimomura Y.
      • Agalliu D.
      • Vonica A.
      • et al.
      APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex.
      ) or ribosomal protein L21 (RPL21) genes (
      • Zhou C.
      • Zang D.
      • Jin Y.
      • et al.
      Mutation in ribosomal protein L21 underlies hereditary hypotrichosis simplex.
      ).
      In addition to these genes, a total of three genes have been reported to be associated with non-syndromic forms of hereditary woolly hair (WH). WH is defined as an abnormal variant of tightly curled hair and is considered to be a hair growth deficiency (
      • Chien A.J.
      • Valentine M.C.
      • Sybert V.P.
      Hereditary woolly hair and keratosis pilaris.
      ). There are both syndromic and non-syndromic forms of WH. The non-syndromic forms of WH can show either an autosomal-dominant WH (ADWH) or autosomal-recessive WH (ARWH) inheritance pattern (
      • Salamon T.
      Über eine familie mit recessiver Kraushaarigkeit, hypotrichose und anderen anomalien.
      ;
      • Hutchinson P.E.
      • Cairns R.J.
      • Wells R.S.
      • et al.
      Woolly hair. Clinical and general aspects.
      ). It is known that WH is frequently associated with hypotrichosis, and it has been shown that mutations in lysophosphatidic acid receptor 6 (LPAR6) gene, also known as P2RY5, underlie ARWH with or without hypotrichosis (ARWH1/LAH3; OMIM 278150) (
      • Pasternack S.M.
      • von Kügelgen I.
      • Aboud K.A.
      • et al.
      G protein–coupled receptor P2Y5 and its ligand LPA are involved in maintenance of human hair growth.
      ;
      • Shimomura Y.
      • Wajid M.
      • Ishii Y.
      • et al.
      Disruption of P2RY5, an orphan G protein–coupled receptor, underlies autosomal recessive woolly hair.
      ). Furthermore, it has been reported that recessively inherited mutations in lipase H (LIPH) gene can also show similar WH/hypotrichosis phenotypes (ARWH2/LAH2; OMIM 604379) (
      • Kazantseva A.
      • Goltsov A.
      • Zinchenko R.
      • et al.
      Human hair growth deficiency is linked to a genetic defect in the phospholipase gene LIPH.
      ;
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Mutations in the lipase H (LIPH) gene underlie autosomal recessive woolly hair/hypotrichosis.
      ).
      The LIPH gene encodes a phosphatidic acid-selective phospholipase A1α (PA-PLA1α), which produces 2-acyl-lysophosphatidic acid (LPA) from phosphatidic acid (
      • Sonoda H.
      • Aoki J.
      • Hiramatsu T.
      • et al.
      A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid.
      ). The LPAR6 gene encodes a G protein–coupled receptor LPA6, also known as P2Y5, which has recently been shown to be a receptor of LPA (
      • Pasternack S.M.
      • von Kügelgen I.
      • Aboud K.A.
      • et al.
      G protein–coupled receptor P2Y5 and its ligand LPA are involved in maintenance of human hair growth.
      ;
      • Yanagida K.
      • Masago K.
      • Nakanishi H.
      • et al.
      Identification and characterization of a novel lysophosphatidic acid receptor, p2y5/LPA6.
      ). As both genes are abundantly expressed in the human HFs, particularly in the IRS, PA-PLA1α/LPA/LPA6 signaling is believed to play a crucial role in HF development and hair growth in humans (
      • Shimomura Y.
      • Wajid M.
      • Ishii Y.
      • et al.
      Disruption of P2RY5, an orphan G protein–coupled receptor, underlies autosomal recessive woolly hair.
      ,
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Mutations in the lipase H (LIPH) gene underlie autosomal recessive woolly hair/hypotrichosis.
      ). More recently, heterozygous mutations in the keratin K74 (KRT74) gene, also known as K6irs4, have been reported to underlie ADWH (OMIM 194300) (
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Autosomal dominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture.
      ) and associated hypotrichosis (hypotrichosis simplex of the scalp 2; OMIM 613981) (
      • Wasif N.
      • ul-Hassan Naqvi S.K.
      • Basit S.
      • et al.
      Novel mutations in the keratin-74 (KRT74) gene underlie autosomal dominant woolly hair/hypotrichosis in Pakistani families.
      ). The KRT74 gene encodes type II epithelial keratin K74, which is predominantly expressed in the Huxley layer of the IRS (
      • Langbein L.
      • Rogers M.A.
      • Praetzel S.
      • et al.
      K6irs1, K6irs2, K6irs3, and K6irs4 represent the inner-root-sheath-specific type II epithelial keratins of the human hair follicle.
      ). In addition to the KRT74 gene, three other type II epithelial keratin genes, keratin K71 (KRT71; K6irs1), keratin K72 (KRT72; K6irs2), and keratin K73 (KRT73; K6irs3), have been mapped on human chromosome 12q13, and their precise expression patterns in the IRS have been characterized in detail (
      • Langbein L.
      • Rogers M.A.
      • Praetzel S.
      • et al.
      K6irs1, K6irs2, K6irs3, and K6irs4 represent the inner-root-sheath-specific type II epithelial keratins of the human hair follicle.
      ).
      Keratins are a major structural component of the HF and contribute to form keratin intermediate filaments (KIFs) through heterodimerization between type I (acidic) and type II (basic to neutral) keratins (
      • Coulombe P.A.
      • Omary M.B.
      ‘Hard’ and ‘soft’ principles defining the structure, function and regulation of keratin intermediate filaments.
      ;
      • Moll R.
      • Divo M.
      • Langbein L.
      The human keratins: biology and pathology.
      ). All keratin proteins share a common structural organization composed of three domains: the N-terminal head domain; the central α-helical rod domain; and the C-terminal tail domain. The N terminus and C terminus of the central α-helical rod domain are known as the helix initiation motif (HIM) and helix termination motif (HTM), respectively, and these are highly conserved and critical for heterodimerization (
      • Coulombe P.A.
      • Omary M.B.
      ‘Hard’ and ‘soft’ principles defining the structure, function and regulation of keratin intermediate filaments.
      ). Indeed, most pathogenic mutations for keratin diseases have been identified within either the HIM or the HTM of various keratins (Human Intermediate Filament Database: http://www.interfil.org). For example, the KRT74 mutation identified in a Pakistani family with ADWH was a non-conservative amino-acid substitution (p.Asn148Lys) within the HIM of the K74 protein (
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Autosomal dominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture.
      ).
      Other than these recent findings, the molecular basis of hereditary hair disorders has not yet been fully elucidated. In this study, we identified a Japanese family with ADWH/hypotrichosis and found a heterozygous missense mutation in the KRT71 gene.

      Results

      Identification of Japanese family with ADWH/hypotrichosis

      A 5-year-old Japanese girl visited our hospital because of her hair symptoms (III-1; Figure 1a). She exhibited tightly curled scalp hairs since birth (Figure 1b and c). Her scalp hairs were short and stopped growing at a few inches, while neither hair shaft fragility nor follicular papule was evident. She also showed reduced density of scalp hairs, eyebrows, and eyelashes (Figure 1b and c). She showed normal facial features, teeth, nails, and sweating. In addition, she did not have palmoplantar keratoderma, heart disease, or mental retardation. There was no consanguinity between the parents. Notably, both her father (II-1) and her paternal grandfather (I-1) were also affected, suggesting an AD inheritance (Figure 1a). Both affected individuals I-1 and II-1 originally showed WH and hypotrichosis, similarly to the affected girl (III-1), but their hair symptoms gradually improved with aging, resulting in only WH with almost normal hair density (data not shown). Under scanning electron microscopy (SEM), the hair shaft of the affected girl (III-1) did not show disease-specific anomalies such as monilethrix (
      • Ito M.
      • Hashimoto K.
      • Katsuumi K.
      • et al.
      Pathogenesis of monilethrix: computer stereography and electron microscopy.
      ;
      • Shimomura Y.
      • Sakamoto F.
      • Kariya N.
      • et al.
      Mutations in the desmoglein 4 gene are associated with monilethrix-like congenital hypotrichosis.
      ). Nonetheless, her hair shafts frequently showed longitudinal grooves (Figure 1d), as compared with seven age-matched Japanese control individuals (Figure 1e; data not shown), suggesting abnormal hair growth. Collectively, we diagnosed the family as having a non-syndromic form of ADWH/hypotrichosis.
      Figure thumbnail gr1
      Figure 1A Japanese family with autosomal-dominant wooly hair (ADWH)/hypotrichosis. (a) Family pedigree. (b, c) Clinical features of affected individual III-1. Note that her scalp hairs are short, sparse, and tightly curled. Her eyebrows are also sparse. (d) SEM observation of a hair shaft from affected individual III-1. A longitudinal groove is indicated by the white arrow. (e) SEM observation of a hair shaft of an age-matched healthy Japanese individual. SEM, scanning electron microscopy. (d, e) Bars=14.2μm.

      Identification of heterozygous mutation in KRT71 gene

      To confirm the molecular basis of the disease, we performed direct sequencing analyses of the candidate genes. Using the genomic DNA from family members, we first sequenced the LPAR6, LIPH, and KRT74 genes, in which mutations are known to underlie non-syndromic forms of WH (
      • Shimomura Y.
      • Wajid M.
      • Ishii Y.
      • et al.
      Disruption of P2RY5, an orphan G protein–coupled receptor, underlies autosomal recessive woolly hair.
      ,
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Mutations in the lipase H (LIPH) gene underlie autosomal recessive woolly hair/hypotrichosis.
      ,
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Autosomal dominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture.
      ). None of these genes, however, had any sequence variants (data not shown). We also analyzed the CDSN, U2HR, APCDD1, and RPL21 genes, which are causative genes for AD forms of hereditary hypotrichosis (
      • Levy-Nissenbaum E.
      • Betz R.C.
      • Frydman M.
      • et al.
      Hypotrichosis simplex of the scalp is associated with nonsense mutations in CDSN encoding corneodesmosin.
      ;
      • Wen Y.
      • Liu Y.
      • Xu Y.
      • et al.
      Loss-of-function mutations of an inhibitory upstream ORF in the human hairless transcript cause Marie Unna hereditary hypotrichosis.
      ;
      • Shimomura Y.
      • Agalliu D.
      • Vonica A.
      • et al.
      APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex.
      ;
      • Zhou C.
      • Zang D.
      • Jin Y.
      • et al.
      Mutation in ribosomal protein L21 underlies hereditary hypotrichosis simplex.
      ). Nonetheless, we found no mutations in any of these genes (data not shown). We then decided to sequence three IRS-specific type II keratin genes, KRT71, KRT72, and KRT73, which are located near the KRT74 gene on human chromosome 12q13 (Figure 2a). Although the affected girl (III-1) did not have mutations in either the KRT72 or the KRT73 genes (data not shown), she carried a heterozygous nucleotide change c.422T>G in exon 1 of the KRT71 gene, which was predicted to result in substitution of phenylalanine with cysteine at codon 141 of K71 protein, and thus designated as p.Phe141Cys (Figure 2b). Screening assays with the restriction enzyme HhaI demonstrated that her affected father also had the mutation heterozygously, whereas her unaffected mother and 200 healthy control Japanese individuals (400 chromosomes) did not carry it (Figure 2c; data not shown). Phe residue at position 141 is located within the HIM of K71 protein (Figure 3a), and is completely conserved among all human type II keratin members (Figure 3b).
      Figure thumbnail gr2
      Figure 2Identification of heterozygous missense mutation in the keratin K71 (KRT71) gene. (a) Physical map of the four inner root sheath (IRS)-specific type II keratin genes KRT71–KRT74 on human chromosome 12q13. The position and direction of the genes are indicated by arrows. The KRT71 gene is colored in red. The expression patterns of these genes in the IRS are stated below the genes: IRS-cu, IRS-cuticle; Hu, Huxley layer; He, Henle layer. (b) Identification of a heterozygous mutation c.422T>G (p.Phe141Cys) in exon 1 of the KRT71 gene of the affected individual III-1. (c) Screening assays for the KRT71 mutation with the restriction enzyme HhaI. PCR products from the mutant allele, 682bp in size, were digested into 516 and 166bp fragments. Affected individuals (II-1 and III-1) are colored in red. C, control individuals; MWM, molecular weight markers.
      Figure thumbnail gr3
      Figure 3Mutation p.Phe141Cys occurred within the highly conserved helix initiation motif (HIM) of K71 protein. (a) Schematic representation of K71 protein. The location of mutation p.Phe141Cys in human K71 is shown above the scheme and is indicated in red. The sites of all K71 mutations identified in the other species are shown below the scheme. Of these, mutations in the Rco3 mice and the Re cats are recessively inherited, whereas all the others are dominant mutations. The HIM and HTM are colored in yellow and blue, respectively. Ca, Caracul; Cal4, Caracul-like 4; Rco, reduced coat; Re, Rex. Rgsc689 is a chemically induced mutation (
      • Kikkawa Y.
      • Oyama A.
      • Ishii R.
      • et al.
      A small deletion hotspot in the type II keratin gene mK6irs1/Krt2–6g on mouse chromosome 15, a candidate for causing the wavy hair of the caracul (Ca) mutation.
      ). (b) Multiple amino-acid sequence alignment of the HIM of human type II keratins. K71 (K6irs1) is indicated in red. The Phe residue at amino-acid position 12 in the HIM is highlighted in green.

      Mutant K71 protein disrupts KIF formation in cultured cells

      To investigate whether the mutation p.Phe141Cys in K71 protein affects its function, we transfected the expression vectors for either wild-type (Wt) or p.Phe141Cys mutant (Mut) K71 protein into HEK293T (human embryonic kidney), HaCaT (human epidermal keratinocyte), and PtK2 (kangaroo rat kidney) cells. We first performed western blots (WBs) with anti-K71 antibody, which showed no differences in expression levels between the Wt- and the Mut-K71 proteins in the three cell lines (Supplementary Figure S1 online). We then performed indirect immunofluorescence (IIF) studies in HaCaT and PtK2 cells. In HaCaT cells, the Wt-K71 protein efficiently underwent KIF formation with endogenous K14 protein (41 of 50 K71-positive cells observed) (Figure 4a–c). In contrast, the Mut-K71 protein showed an aberrant expression pattern around the nuclei, leading to collapse of the KIF network in 43 of 50 K71-positive cells observed (Figure 4d–f). Similar results were also obtained in PtK2 cells (Supplementary Figure S2 online), but as compared with the expression patterns in HaCaT cells, the Mut-K71 protein showed a tendency to form aggregates with endogenous K18 protein around the nuclei, leading to disruption of the KIF network in PtK2 cells (45 of 50 K71-positive cells observed) (Supplementary Figure S2 online). These data strongly suggest that the non-conservative amino-acid change p.Phe141Cys within the HIM of K71 protein resulted in mislocalization and severely affected heterodimer formation with type I keratins.
      Figure thumbnail gr4
      Figure 4Mutant K71 protein disrupts endogenous keratin intermediate filament (KIF) formation in HaCaT cells, and K71 colocalizes with PA-PLA1α in the inner root sheath (IRS) of human hair follicle (HF). (ac) Ectopically expressed wild-type (Wt)-K71 protein forms a KIF network via heterodimerization with endogenous K14 protein. (df) The p.Phe141Cys mutant (Mut)-K71 protein causes a collapse of the endogenous KIF network around the nucleus. (gi) Double IIF studies with anti-K71 and anti-PA-PLA1α antibodies showed that both K71 and PA-PLA1α proteins were co-expressed in the IRS of human HF. (h) Expression of PA-PLA1α was also weakly detected in the hair shaft. (c, f, i) Counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) is shown in blue. Bars=(a) 20μm and (g) 100μm.

      Expression of K71 and PA-PLA1α finely overlaps in IRS of human HFs

      Finally, we performed double IIF on normal human scalp skin sections with anti-K71 and anti-PA-PLA1α (LIPH) antibodies, which specifically recognized both ectopically expressed proteins and endogenous proteins on WBs (Supplementary Figures S1 and S3 online). The results showed that PA-PLA1α protein was abundantly expressed in all three layers of the IRS of human HFs, which finely overlapped with K71 (Figure 4g–i). Expression of PA-PLA1α was also weakly detected in the hair shaft (Figure 4h), which is consistent with the expression patterns of the LIPH-mRNA reported previously (
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Mutations in the lipase H (LIPH) gene underlie autosomal recessive woolly hair/hypotrichosis.
      ).

      Discussion

      In this study, we identified a Japanese family with ADWH/hypotrichosis. After we had excluded known causative genes for non-syndromic forms of hereditary WH and AD forms of hypotrichosis through direct sequencing analyses, we decided to search for mutations in the IRS-specific type II keratin genes, KRT71, KRT72, and KRT73 (
      • Langbein L.
      • Rogers M.A.
      • Praetzel S.
      • et al.
      A novel epithelial keratin, hK6irs1, is expressed differentially in all layers of the inner root sheath, including specialized Huxley cells (Flügelzellen) of the human hair follicle.
      ). Several lines of evidence enabled us to hypothesize that a mutation might exist in one of these genes. First, mutations in the KRT71 gene have been shown to underlie wavy/curly coat phenotypes in mice (
      • Kikkawa Y.
      • Oyama A.
      • Ishii R.
      • et al.
      A small deletion hotspot in the type II keratin gene mK6irs1/Krt2–6g on mouse chromosome 15, a candidate for causing the wavy hair of the caracul (Ca) mutation.
      ;
      • Peters T.
      • Sedlmeier R.
      • Büssow H.
      • et al.
      Alopecia in a novel mouse model RCO3 is caused by mK6irs1 deficiency.
      ;
      • Runkel F.
      • Klaften M.
      • Koch K.
      • et al.
      Morphologic and molecular characterization of two novel Krt71 (Krt2–6g) mutations: Krt71rco12 and Krt71rco13.
      ;
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Autosomal dominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture.
      ), rats (
      • Kuramoto T.
      • Hirano R.
      • Kuwamura M.
      • et al.
      Identification of the rat Rex mutation as a 7-bp deletion at splicing acceptor site of the Krt71 gene.
      ), and cats (
      • Gandolfi B.
      • Outerbridge C.A.
      • Beresford L.G.
      • et al.
      The naked truth: Sphynx and Devon Rex cat breed mutations in KRT71.
      ) (Figure 3a). Secondly, it has been reported that non-synonymous coding SNP in the KRT71 gene is strongly associated with curly coat phenotype in dogs (
      • Cadieu E.
      • Neff M.W.
      • Quignon P.
      • et al.
      Coat variation in the domestic dog is governed by variants in three genes.
      ) (Figure 3a). Thirdly, mutations in the IRS-specific KRT74 gene, a neighborhood gene of KRT71 (Figure 2a), have recently been reported to underlie ADWH/hypotrichosis in humans (
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Autosomal dominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture.
      ;
      • Wasif N.
      • ul-Hassan Naqvi S.K.
      • Basit S.
      • et al.
      Novel mutations in the keratin-74 (KRT74) gene underlie autosomal dominant woolly hair/hypotrichosis in Pakistani families.
      ). Finally, both LPAR6 and LIPH, the causative genes for ARWH/hypotrichosis, are abundantly expressed in the IRS of human HFs (
      • Shimomura Y.
      • Wajid M.
      • Ishii Y.
      • et al.
      Disruption of P2RY5, an orphan G protein–coupled receptor, underlies autosomal recessive woolly hair.
      ,
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Mutations in the lipase H (LIPH) gene underlie autosomal recessive woolly hair/hypotrichosis.
      ).
      Strikingly, we discovered heterozygous missense mutation c.422T>G (p.Phe141Cys) in the KRT71 gene in affected individuals of the family (Figure 2b and c). The mutation was not detected in 200 Japanese control individuals (Figure 2c; data not shown). It is worth noting that the mutation is located within the HIM of K71 protein (Figure 3a). Phe141 (amino-acid position 12 in the HIM) is fully conserved among all human type II keratins (Figure 3b). Furthermore, substitutions at amino-acid position 12 in the HIM of other type II keratins underlie several AD genodermatoses in humans (
      • Stephens K.
      • Ehrlich P.
      • Weaver M.
      • et al.
      Primers for exon-specific amplification of the KRT5 gene: identification of novel and recurrent mutations in epidermolysis bullosa simplex patients.
      ;
      • Smith F.J.
      • McKenna K.E.
      • Irvine A.D.
      • et al.
      A mutation detection strategy for the human keratin 6A gene and novel missense mutations in two cases of pachyonychia congenita type 1.
      ;
      • Terrinoni A.
      • Smith F.J.
      • Didona B.
      • et al.
      Novel and recurrent mutations in the genes encoding keratins K6a, K16 and K17 in 13 cases of pachyonychia congenita.
      ;
      • Virtanen M.
      • Gedde-Dahl Jr., T.
      • Mörk N.J.
      • et al.
      Phenotypic/genotypic correlations in patients with epidermolytic hyperkeratosis and the effects of retinoid therapy on keratin expression.
      ;
      • Liao H.
      • Sayers J.M.
      • Wilson N.J.
      • et al.
      A spectrum of mutations in keratins K6a, K16 and K17 causing pachyonychia congenita.
      ) (Supplementary Table S1 online). Of these, substitutions from Phe to Cys at this amino-acid position have been found in K1 and K6A proteins as pathogenic mutations for epidermolytic hyperkeratosis and pachyonychia congenita type 1, respectively (
      • Virtanen M.
      • Gedde-Dahl Jr., T.
      • Mörk N.J.
      • et al.
      Phenotypic/genotypic correlations in patients with epidermolytic hyperkeratosis and the effects of retinoid therapy on keratin expression.
      ;
      • Liao H.
      • Sayers J.M.
      • Wilson N.J.
      • et al.
      A spectrum of mutations in keratins K6a, K16 and K17 causing pachyonychia congenita.
      ) (Supplementary Table S1 online). In addition, all dominant mutations in the KRT71 gene responsible for wavy coat phenotype in mice have been identified in either the HIM or HTM of K71 protein (
      • Kikkawa Y.
      • Oyama A.
      • Ishii R.
      • et al.
      A small deletion hotspot in the type II keratin gene mK6irs1/Krt2–6g on mouse chromosome 15, a candidate for causing the wavy hair of the caracul (Ca) mutation.
      ;
      • Runkel F.
      • Klaften M.
      • Koch K.
      • et al.
      Morphologic and molecular characterization of two novel Krt71 (Krt2–6g) mutations: Krt71rco12 and Krt71rco13.
      ;
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Autosomal dominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture.
      ) (Figure 3a).
      Our expression studies in cultured cells clearly demonstrated that the p.Phe141Cys mutant K71 resulted in disruption of the KIF network, most likely in a dominant-negative manner (Figure 4a–f and Supplementary Figure S2 online). These data strongly suggest that c.422T>G (p.Phe141Cys) in the KRT71 gene is not a non-consequential polymorphism, but is a pathogenic mutation for ADWH/hypotrichosis in our family. Previous studies in Ca and Rco mice showed that the mutant K71 proteins disrupted KIF formation in the IRS of mouse pelage HFs (
      • Kikkawa Y.
      • Oyama A.
      • Ishii R.
      • et al.
      A small deletion hotspot in the type II keratin gene mK6irs1/Krt2–6g on mouse chromosome 15, a candidate for causing the wavy hair of the caracul (Ca) mutation.
      ;
      • Peters T.
      • Sedlmeier R.
      • Büssow H.
      • et al.
      Alopecia in a novel mouse model RCO3 is caused by mK6irs1 deficiency.
      ;
      • Runkel F.
      • Klaften M.
      • Koch K.
      • et al.
      Morphologic and molecular characterization of two novel Krt71 (Krt2–6g) mutations: Krt71rco12 and Krt71rco13.
      ). It would be much more convincing if we could observe a similar phenomenon in patient HFs; however, we were unable to obtain permission for skin biopsy from the patients. We hope that other dermatologists worldwide will identify additional families with ADWH/hypotrichosis carrying mutations in the KRT71 gene, which will provide precious information to further characterize the phenotypes and pathological features resulting from KRT71 mutations. In addition, some families with ADWH/hypotrichosis may carry mutations in a type I keratin partner of K71, as mutations in two IRS-specific type I keratin genes, keratin K25 (KRT25) and keratin K27 (KRT27), have been reported to cause wavy coat phenotype in mice (
      • Tanaka S.
      • Miura I.
      • Yoshiki A.
      • et al.
      Mutations in the helix termination motif of mouse type I IRS keratin genes impair the assembly of keratin intermediate filament.
      ).
      The affected girl with the KRT71 mutation showed symptoms in both scalp hairs and facial hairs (Figure 1b and c), whereas all KRT74 mutations reported to date showed an obvious phenotype in scalp hairs alone (
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Autosomal dominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture.
      ;
      • Wasif N.
      • ul-Hassan Naqvi S.K.
      • Basit S.
      • et al.
      Novel mutations in the keratin-74 (KRT74) gene underlie autosomal dominant woolly hair/hypotrichosis in Pakistani families.
      ), suggesting that KRT71 mutations can exhibit a more severe phenotype than KRT74 mutations. This may be because K71 is expressed in all three layers of the IRS, whereas K74 expression is restricted to the Huxley layer (
      • Langbein L.
      • Rogers M.A.
      • Praetzel S.
      • et al.
      K6irs1, K6irs2, K6irs3, and K6irs4 represent the inner-root-sheath-specific type II epithelial keratins of the human hair follicle.
      ). It is noted that in mice K71 is expressed only in the Henle and the Huxley layers of the IRS (
      • Aoki N.
      • Sawada S.
      • Rogers M.A.
      • et al.
      A novel type II cytokeratin, mK6irs, is expressed in the Huxley and Henle layers of the mouse inner root sheath.
      ), and mouse hairs are apparently shorter and thinner than those of humans, which may explain why similar K71 mutations cause a milder phenotype (wavy or curly hair) in mice instead of a more prominent phenotype (WH) in humans.
      We (MI and YS) and others have recently reported that most Japanese patients with ARWH/hypotrichosis carry common founder mutations in the LIPH gene (
      • Shimomura Y.
      • Ito M.
      • Christiano A.M.
      Mutations in the LIPH gene in three Japanese families with autosomal recessive woolly hair/hypotrichosis.
      ;
      • Shinkuma S.
      • Akiyama M.
      • Inoue A.
      • et al.
      Prevalent LIPH founder mutations lead to loss of P2Y5 activation ability of PA-PLA1alpha in autosomal recessive hypotrichosis.
      ). The clinical features of some affected Japanese individuals with the LIPH mutations are indistinguishable from those of the affected girl with the KRT71 mutation (
      • Shimomura Y.
      • Ito M.
      • Christiano A.M.
      Mutations in the LIPH gene in three Japanese families with autosomal recessive woolly hair/hypotrichosis.
      , unpublished data). Although expression of LIPH-mRNA was previously determined in the hair shaft and the IRS of human HFs by in situ hybridization (
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Mutations in the lipase H (LIPH) gene underlie autosomal recessive woolly hair/hypotrichosis.
      ), its expression at the protein level had never been investigated in human HFs. We (AI and JA) have recently produced an antibody that specifically recognizes endogenous human PA-PLA1α (Supplementary Figure S3 online). Double IIF with anti-K71 and anti-PA-PLA1α antibodies clearly revealed that the expression of both proteins finely overlapped in the IRS of human HFs (Figure 4g–i). Importantly, LPA6, which is another causative molecule of ARWH/hypotrichosis, is also predominantly expressed in the IRS of human HFs (
      • Shimomura Y.
      • Wajid M.
      • Ishii Y.
      • et al.
      Disruption of P2RY5, an orphan G protein–coupled receptor, underlies autosomal recessive woolly hair.
      ). Most recently, we (AI and JA) reported that PA-PLA1α-knockout mice showed a wavy coat phenotype, similarly to humans with LIPH mutations (
      • Inoue A.
      • Arima N.
      • Ishiguro J.
      • et al.
      LPA-producing enzyme PA-PLA1 α regulates hair follicle development by modulating EGFR signalling.
      ). It is worth noting that K71 expression was significantly reduced in the skin of the knockout mice (
      • Inoue A.
      • Arima N.
      • Ishiguro J.
      • et al.
      LPA-producing enzyme PA-PLA1 α regulates hair follicle development by modulating EGFR signalling.
      ). Moreover, a series of studies have revealed that the PA-PLA1α/LPA/LPA6 axis regulates differentiation and maturation of mouse HFs via a signaling pathway composed of tumor necrosis factor-α converting enzyme, transforming growth factor-α, and epidermal growth factor receptor (
      • Inoue A.
      • Arima N.
      • Ishiguro J.
      • et al.
      LPA-producing enzyme PA-PLA1 α regulates hair follicle development by modulating EGFR signalling.
      ). These data are consistent with previous reports that mutant mice defective in tumor necrosis factor-α converting enzyme, transforming growth factor-α, and epidermal growth factor receptor also exhibited wavy coat phenotypes (
      • Luetteke N.C.
      • Qiu T.H.
      • Peiffer R.L.
      • et al.
      TGF alpha deficiency results in hair follicle and eye abnormalities in targeted and waved-1 mice.
      ,
      • Luetteke N.C.
      • Phillips H.K.
      • Qiu T.H.
      • et al.
      The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.
      ;
      • Mann G.B.
      • Fowler K.J.
      • Gabriel A.
      • et al.
      Mice with a null mutation of the TGF alpha gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation.
      ;
      • Peschon J.J.
      • Slack J.L.
      • Reddy P.
      • et al.
      An essential role for ectodomain shedding in mammalian development.
      ). Taken together, it can be postulated that PA-PLA1α/LPA/LPA6 signaling is involved in regulating the KRT71 expression through the activation of epidermal growth factor receptor. Further studies need to be performed to disclose the actual relationships between these WH-associated genes in humans.
      To our knowledge, it is previously unreported that a mutation in the human KRT71 gene underlies a hereditary hair disorder. Our findings will provide new insight into the field of genotrichoses (
      • Betz R.C.
      • Cabral R.M.
      • Christiano A.M.
      • et al.
      Unveiling the roots of monogenic genodermatoses: genotrichoses as a paradigm.
      ), and will further underscore the crucial roles of the IRS in HF development and hair growth in humans.

      Materials and Methods

      Source of DNA

      After obtaining written informed consent, we collected peripheral blood samples from the family members, as well as from 200 unrelated healthy control individuals of Japanese origin in tubes with EDTA. The study protocol was approved by the Ethics Committee of Niigata University School of Medicine and adhered to the Declaration of Helsinki Principles. Isolation of the genomic DNA from the samples was performed in accordance with standard techniques.

      Mutation analysis

      Using the genomic DNA of the family members as templates, all the coding exons and exon–intron boundaries of LPAR6, LIPH, APCDD1, KRT71, KRT72, KRT73, KRT74, CDSN, and RPL21 genes were amplified by PCR using primers reported previously (
      • Levy-Nissenbaum E.
      • Betz R.C.
      • Frydman M.
      • et al.
      Hypotrichosis simplex of the scalp is associated with nonsense mutations in CDSN encoding corneodesmosin.
      ;
      • Shimomura Y.
      • Wajid M.
      • Ishii Y.
      • et al.
      Disruption of P2RY5, an orphan G protein–coupled receptor, underlies autosomal recessive woolly hair.
      ,
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Mutations in the lipase H (LIPH) gene underlie autosomal recessive woolly hair/hypotrichosis.
      ,
      • Shimomura Y.
      • Agalliu D.
      • Vonica A.
      • et al.
      APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex.
      ,
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Autosomal dominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture.
      ). Primers for the RPL21 gene were shown in Supplementary Table S2 online. We also performed PCR amplification of the U2HR gene, considering the possibility of Marie Unna hereditary hypotrichosis (
      • Wen Y.
      • Liu Y.
      • Xu Y.
      • et al.
      Loss-of-function mutations of an inhibitory upstream ORF in the human hairless transcript cause Marie Unna hereditary hypotrichosis.
      ). The amplified PCR products were directly sequenced in an ABI Prism 3,100 Automated Sequencer (PE Applied Biosystems, Foster City, CA), using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems).

      Screening assays for KRT71 mutation

      Mutation c.422T>G in the KRT71 gene resulted in generating a restriction enzyme site for HhaI, which was used for screening assays. The 682-bp sequences including exon 1 of the KRT71 gene were PCR amplified using previously described primers (
      • Shimomura Y.
      • Wajid M.
      • Petukhova L.
      • et al.
      Autosomal dominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture.
      ). PCR products were subsequently digested using HhaI restriction enzyme at 37°C for 3hour, and run on 1.5% agarose gels.

      Generation of expression vectors

      Using first-strand cDNA from plucked hairs of a healthy Japanese individual as a template, full-length coding sequences of the human KRT71 and LIPH genes were amplified by PCR using gene-specific primers (Supplementary Table S3 online). The PCR product for the KRT71 gene was cloned into EcoRI and KpnI sites of the mammalian expression vector pCXN2.1 (
      • Niwa H.
      • Yamamura K.
      • Miyazaki J.
      Efficient selection for high-expression transfectants with a novel eukaryotic vector.
      ;
      • Noguchi K.
      • Ishii S.
      • Shimizu T.
      Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family.
      ). The generated vector was designated pCXN2.1-K71-Wt. The mutation c.422T>G (p.Phe141Cys) was subsequently introduced into the pCXN2.1-K71-Wt vector using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and mismatch primers shown in Supplementary Table S3 online. The PCR product for the LIPH gene was cloned into the EcoRI and XhoI sites of pCXN2.1.

      Transient transfection, WBs, and IIF studies

      HaCaT (human epidermal keratinocyte), PtK2 (kangaroo rat kidney), and HEK293T (human embryonic kidney) cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), 100IUml−1 penicillin, and 100μgml−1 streptomycin. Cells were plated in 35-mm dishes or 8-well chamber slides (Nalge Nunc International, Rochester, NY) on the day before transfection. In line with the manufacturer's instructions, expression vectors were transfected with Lipofectamine 2000 (Invitrogen), and were further cultured for 24hours. Total cell lysates of the cultured cells and scalp skin from a healthy Japanese individual was prepared as described previously (
      • Shimomura Y.
      • Agalliu D.
      • Vonica A.
      • et al.
      APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex.
      ). Samples were mixed with Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) containing 5% β-mercaptoethanol, boiled at 95°C for 10minutes, and separated by 10% SDS-PAGE. WBs, as well as IIF in cultured cells, were performed as described previously (
      • Shimomura Y.
      • Agalliu D.
      • Vonica A.
      • et al.
      APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex.
      ). For IIF on 4% paraformaldehyde-fixed paraffin sections of normal human scalp skin, deparaffinized sections were autoclaved at 121°C for 10minutes in citrate buffer (pH 6.0), which were subsequently treated with 0.1% trypsin at 37°C for 3minutes. Subsequently, sections were blocked with 10% normal goat serum at room temperature for 30minutes before the application of primary antibodies. The primary antibodies used were rabbit polyclonal anti-K71 (diluted 1:5,000 in WB and 1:2,000 in IIF, respectively) (
      • Aoki N.
      • Sawada S.
      • Rogers M.A.
      • et al.
      A novel type II cytokeratin, mK6irs, is expressed in the Huxley and Henle layers of the mouse inner root sheath.
      ), mouse monoclonal anti-K14 (clone LL002; diluted 1:2,000; GenWay Biotech, San Diego, CA), mouse monoclonal anti-K18 (clone 2F8; diluted 1:50; Sigma-Aldrich, St Louis, MO), rabbit polyclonal anti-β-actin (diluted 1:3,000; Sigma-Aldrich), and rat monoclonal anti-human PA-PLA1α (clone 1A2; diluted 1:100 in WB and 1:10 in IIF, respectively) (produced by A.I. and J.A.). In IIF studies in cultured cells, filament formation in a total of 50 K71-positive cells was evaluated for each transfection.

      ACKNOWLEDGMENTS

      We are grateful to Drs Satoshi Ishii (Tokyo University, Japan) and Junichi Miyazaki (Osaka University, Japan) for supplying the pCXN2.1 vector. This work was supported by the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT), a Grant-in-Aid for Scientific Research on Innovative Areas (MEXT KAKENHI 11001497), and a grant from the Kanae Foundation for the Promotion of Medical Science to YS.

      SUPPLEMENTARY MATERIAL

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

      REFERENCES

        • Aoki N.
        • Sawada S.
        • Rogers M.A.
        • et al.
        A novel type II cytokeratin, mK6irs, is expressed in the Huxley and Henle layers of the mouse inner root sheath.
        J Invest Dermatol. 2001; 116: 359-365
        • Ayub M.
        • Basit S.
        • Jelani M.
        • et al.
        A homozygous nonsense mutation in the human desmocollin-3 (DSC3) gene underlies hereditary hypotrichosis and recurrent skin vesicles.
        Am J Hum Genet. 2009; 85: 515-520
        • Betz R.C.
        • Cabral R.M.
        • Christiano A.M.
        • et al.
        Unveiling the roots of monogenic genodermatoses: genotrichoses as a paradigm.
        J Invest Dermatol. 2012; 132: 906-914
        • Cadieu E.
        • Neff M.W.
        • Quignon P.
        • et al.
        Coat variation in the domestic dog is governed by variants in three genes.
        Science. 2009; 326: 150-153
        • Chien A.J.
        • Valentine M.C.
        • Sybert V.P.
        Hereditary woolly hair and keratosis pilaris.
        J Am Acad Dermatol. 2006; 54: 35-39
        • Coulombe P.A.
        • Omary M.B.
        ‘Hard’ and ‘soft’ principles defining the structure, function and regulation of keratin intermediate filaments.
        Curr Opin Cell Biol. 2002; 14: 110-122
        • Gandolfi B.
        • Outerbridge C.A.
        • Beresford L.G.
        • et al.
        The naked truth: Sphynx and Devon Rex cat breed mutations in KRT71.
        Mamm Genome. 2010; 21: 509-515
        • Hutchinson P.E.
        • Cairns R.J.
        • Wells R.S.
        • et al.
        Woolly hair. Clinical and general aspects.
        Trans St Johns Hosp Dermatol Soc. 1974; 60: 160-177
        • Inoue A.
        • Arima N.
        • Ishiguro J.
        • et al.
        LPA-producing enzyme PA-PLA1 α regulates hair follicle development by modulating EGFR signalling.
        EMBO J. 2011; 30: 4248-4260
        • Ito M.
        • Hashimoto K.
        • Katsuumi K.
        • et al.
        Pathogenesis of monilethrix: computer stereography and electron microscopy.
        J Invest Dermatol. 1990; 95: 186-194
        • Kazantseva A.
        • Goltsov A.
        • Zinchenko R.
        • et al.
        Human hair growth deficiency is linked to a genetic defect in the phospholipase gene LIPH.
        Science. 2006; 314: 982-985
        • Kikkawa Y.
        • Oyama A.
        • Ishii R.
        • et al.
        A small deletion hotspot in the type II keratin gene mK6irs1/Krt2–6g on mouse chromosome 15, a candidate for causing the wavy hair of the caracul (Ca) mutation.
        Genetics. 2003; 165: 721-733
        • Kljuic A.
        • Bazzi H.
        • Sundberg J.P.
        • et al.
        Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris.
        Cell. 2003; 113: 249-260
        • Kuramoto T.
        • Hirano R.
        • Kuwamura M.
        • et al.
        Identification of the rat Rex mutation as a 7-bp deletion at splicing acceptor site of the Krt71 gene.
        J Vet Med Sci. 2010; 72: 909-912
        • Langbein L.
        • Rogers M.A.
        • Praetzel S.
        • et al.
        A novel epithelial keratin, hK6irs1, is expressed differentially in all layers of the inner root sheath, including specialized Huxley cells (Flügelzellen) of the human hair follicle.
        J Invest Dermatol. 2002; 118: 789-799
        • Langbein L.
        • Rogers M.A.
        • Praetzel S.
        • et al.
        K6irs1, K6irs2, K6irs3, and K6irs4 represent the inner-root-sheath-specific type II epithelial keratins of the human hair follicle.
        J Invest Dermatol. 2003; 120: 512-522
        • Levy-Nissenbaum E.
        • Betz R.C.
        • Frydman M.
        • et al.
        Hypotrichosis simplex of the scalp is associated with nonsense mutations in CDSN encoding corneodesmosin.
        Nat Genet. 2003; 34: 151-153
        • Liao H.
        • Sayers J.M.
        • Wilson N.J.
        • et al.
        A spectrum of mutations in keratins K6a, K16 and K17 causing pachyonychia congenita.
        J Dermatol Sci. 2007; 48: 199-205
        • Luetteke N.C.
        • Phillips H.K.
        • Qiu T.H.
        • et al.
        The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.
        Genes Dev. 1994; 8: 399-413
        • Luetteke N.C.
        • Qiu T.H.
        • Peiffer R.L.
        • et al.
        TGF alpha deficiency results in hair follicle and eye abnormalities in targeted and waved-1 mice.
        Cell. 1993; 73: 263-278
        • Mann G.B.
        • Fowler K.J.
        • Gabriel A.
        • et al.
        Mice with a null mutation of the TGF alpha gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation.
        Cell. 1993; 73: 249-261
        • Moll R.
        • Divo M.
        • Langbein L.
        The human keratins: biology and pathology.
        Histochem Cell Biol. 2008; 129: 705-733
        • Niwa H.
        • Yamamura K.
        • Miyazaki J.
        Efficient selection for high-expression transfectants with a novel eukaryotic vector.
        Gene. 1991; 108: 193-199
        • Noguchi K.
        • Ishii S.
        • Shimizu T.
        Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family.
        J Biol Chem. 2003; 278: 25600-25606
        • Pasternack S.M.
        • von Kügelgen I.
        • Aboud K.A.
        • et al.
        G protein–coupled receptor P2Y5 and its ligand LPA are involved in maintenance of human hair growth.
        Nat Genet. 2008; 40: 329-334
        • Peschon J.J.
        • Slack J.L.
        • Reddy P.
        • et al.
        An essential role for ectodomain shedding in mammalian development.
        Science. 1998; 282: 1281-1284
        • Peters T.
        • Sedlmeier R.
        • Büssow H.
        • et al.
        Alopecia in a novel mouse model RCO3 is caused by mK6irs1 deficiency.
        J Invest Dermatol. 2003; 121: 674-680
        • Runkel F.
        • Klaften M.
        • Koch K.
        • et al.
        Morphologic and molecular characterization of two novel Krt71 (Krt2–6g) mutations: Krt71rco12 and Krt71rco13.
        Mamm Genome. 2006; 17: 1172-1182
        • Salamon T.
        Über eine familie mit recessiver Kraushaarigkeit, hypotrichose und anderen anomalien.
        Hautarzt. 1963; 14: 540-544
        • Shimomura Y.
        • Agalliu D.
        • Vonica A.
        • et al.
        APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex.
        Nature. 2010; 464: 1043-1047
        • Shimomura Y.
        • Christiano A.M.
        Biology and genetics of hair.
        Ann Rev Genomics Hum Genet. 2010; 11: 109-132
        • Shimomura Y.
        • Ito M.
        • Christiano A.M.
        Mutations in the LIPH gene in three Japanese families with autosomal recessive woolly hair/hypotrichosis.
        J Dermatol Sci. 2009; 56: 205-207
        • Shimomura Y.
        • Sakamoto F.
        • Kariya N.
        • et al.
        Mutations in the desmoglein 4 gene are associated with monilethrix-like congenital hypotrichosis.
        J Invest Dermatol. 2006; 126: 1281-1285
        • Shimomura Y.
        • Wajid M.
        • Ishii Y.
        • et al.
        Disruption of P2RY5, an orphan G protein–coupled receptor, underlies autosomal recessive woolly hair.
        Nat Genet. 2008; 40: 335-339
        • Shimomura Y.
        • Wajid M.
        • Petukhova L.
        • et al.
        Mutations in the lipase H (LIPH) gene underlie autosomal recessive woolly hair/hypotrichosis.
        J Invest Dermatol. 2009; 129: 622-628
        • Shimomura Y.
        • Wajid M.
        • Petukhova L.
        • et al.
        Autosomal dominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture.
        Am J Hum Genet. 2010; 86: 632-638
        • Shinkuma S.
        • Akiyama M.
        • Inoue A.
        • et al.
        Prevalent LIPH founder mutations lead to loss of P2Y5 activation ability of PA-PLA1alpha in autosomal recessive hypotrichosis.
        Hum Mutat. 2010; 31: 602-610
        • Smith F.J.
        • McKenna K.E.
        • Irvine A.D.
        • et al.
        A mutation detection strategy for the human keratin 6A gene and novel missense mutations in two cases of pachyonychia congenita type 1.
        Exp Dermatol. 1999; 8: 109-114
        • Sonoda H.
        • Aoki J.
        • Hiramatsu T.
        • et al.
        A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid.
        J Biol Chem. 2002; 277: 34254-34263
        • Stephens K.
        • Ehrlich P.
        • Weaver M.
        • et al.
        Primers for exon-specific amplification of the KRT5 gene: identification of novel and recurrent mutations in epidermolysis bullosa simplex patients.
        J Invest Dermatol. 1997; 108: 349-353
        • Tanaka S.
        • Miura I.
        • Yoshiki A.
        • et al.
        Mutations in the helix termination motif of mouse type I IRS keratin genes impair the assembly of keratin intermediate filament.
        Genomics. 2007; 90: 703-711
        • Terrinoni A.
        • Smith F.J.
        • Didona B.
        • et al.
        Novel and recurrent mutations in the genes encoding keratins K6a, K16 and K17 in 13 cases of pachyonychia congenita.
        J Invest Dermatol. 2001; 117: 1391-1396
        • Virtanen M.
        • Gedde-Dahl Jr., T.
        • Mörk N.J.
        • et al.
        Phenotypic/genotypic correlations in patients with epidermolytic hyperkeratosis and the effects of retinoid therapy on keratin expression.
        Acta Dermatol Venereol. 2001; 81: 163-170
        • Wasif N.
        • ul-Hassan Naqvi S.K.
        • Basit S.
        • et al.
        Novel mutations in the keratin-74 (KRT74) gene underlie autosomal dominant woolly hair/hypotrichosis in Pakistani families.
        Hum Genet. 2011; 129: 419-424
        • Wen Y.
        • Liu Y.
        • Xu Y.
        • et al.
        Loss-of-function mutations of an inhibitory upstream ORF in the human hairless transcript cause Marie Unna hereditary hypotrichosis.
        Nat Genet. 2009; 41: 228-233
        • Yanagida K.
        • Masago K.
        • Nakanishi H.
        • et al.
        Identification and characterization of a novel lysophosphatidic acid receptor, p2y5/LPA6.
        J Biol Chem. 2009; 284: 17731-17741
        • Zhou C.
        • Zang D.
        • Jin Y.
        • et al.
        Mutation in ribosomal protein L21 underlies hereditary hypotrichosis simplex.
        Hum Mutat. 2011; 32: 710-714