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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. jdermsci.2014.05.008. References [1] Castori M, Sinibaldi L, Mingarelli R, Lachman R, Rimoin D, Dallapiccola B. Pachydermoperiostosis: an update. Clin Genet 2005;68:477–86. [2] Touraine A, Solente G, Golé L. Un syndrome ostéodermopathique: la pachydermie plicaturée avec pachypé riostose des extrémités. Presse Med 1935;43:1820–4. [3] Uppal S, Diggle C, Carr I, Fishwick C, Ahmed M, Ibrahim G, et al. Mutations in 15-hydroxyprostaglandin dehydrogenase cause primary hypertrophic osteoarthropathy. Nat Genet 2008;40:789–93. [4] Sasaki T, Niizeki H, Shimizu A, Shiohama A, Hirakiyama A, Okuyama T, et al. Identification of mutations in the prostaglandin transporter gene SLCO2A1 and its phenotype-genotype correlation in Japanese patients with pachydermoperiostosis. J Dermatol Sci 2012;68:36–44. [5] Zhang Z, Xia W, He J, Zhang Z, Ke Y, Yue H, et al. Exome sequencing identifies SLCO2A1 mutations as a cause of primary hypertrophic osteoarthropathy. Am J Hum Genet 2012;90:125–32. [6] Diggle CP, Parry DA, Logan CV, Laissue P, Rivera C, Restrepo CM, et al. Prostaglandin transporter mutations cause pachydermoperiostosis with myelofibrosis. Hum Mutat 2012;33:1175–81. [7] Zhang Z, He JW, Fu WZ, Zhang CQ, Zhang ZL. Mutations in the SLCO2A1 gene and primary hypertrophic osteoarthropathy: a clinical and biochemical characterization. J Clin Endocrinol Metab 2013;98:E923–33. [8] Niizeki H, Shiohama A, Sasaki T, Seki A, Kabashima K, Otsuka A, et al. The novel SLCO2A1 heterozygous missense mutation p. E427 K and nonsense mutation p.R603* in a female patient with pachydermoperiostosis with an atypical phenotype. Br J Dermatol 2014;170:1187–9. [9] Seifert W, Kühnisch J, Tüysüz B, Specker C, Brouwers A, Horn D. Mutations in the prostaglandin transporter encoding gene SLCO2A1 cause primary hypertrophic osteoarthropathy and isolated digital clubbing. Hum Mutat 2012;33:660–4. [10] Matsuda Y, Maruta T, Ide C, Watanabe A, Komori K, Sakai Y, et al. A case of early stage of pachydermoperiostosis preceded by acromegaly. Jap J Clin Exp Med (Rinsho to kenkyu) 2008;85:1475–8 [in Japanese].
H. Niizeki* Department of Dermatology, National Center for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan A. Shiohamaa,b Department of Dermatology, Keio University School of Medicine, Tokyo, Japan, bLaboratory of Gene Medicine, Keio University School of Medicine, Tokyo, Japan
a
T. Sasaki Center for Integrated Medical Research, Keio University School of Medicine, Tokyo, Japan A. Seki Department of Orthopedics, National Center for Child Health and Development, Tokyo, Japan K. Kabashima, A. Otsuka Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan
Letter to the Editor The natural trait of the curvature of human hair is correlated with bending of the hair follicle and hair bulb by a structural disparity in the root sheath
The shape of human hair is one of the most distinguishable characteristics among various populations and commonly
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K. Kosaki Department of Orthopedic Surgery, University of Tokyo, Tokyo, Japan A. Ogo Department of Metabolism and Endocrinology, Clinical Research Institute, National Hospital Organization Kyushu Medical Center, Fukuoka, Japan T. Yamada Department of Dermatology, Himeji Red Cross Hospital, Hyogo, Japan M. Miyasaka Department of Radiology, National Center for Child Health and Development, Tokyo, Japan K. Matsuoka Department of Pathology, National Center for Child Health and Development, Tokyo, Japan A. Hirakiyama, T. Okuyama Department of Laboratory Medicine, National Center for Child Health and Development, Tokyo, Japan M. Matsuda Department of Dermatology, National Center for Child Health and Development, Tokyo, Japan K. Nakabayashi Department of Reproductive Biology, National Research Institute for Child Health and Development, Tokyo, Japan K. Tanese Department of Dermatology, Keio University School of Medicine, Tokyo, Japan A. Ishiko Department of Dermatology, School of Medicine, Toho University, Ota-ku, Tokyo, Japan M. Amagai Department of Dermatology, Keio University School of Medicine, Tokyo, Japan J. Kudoh Laboratory of Gene Medicine, Keio University School of Medicine, Tokyo, Japan * Corresponding author. Tel.: +81 3 3416 0181; fax: +81 3 5494 7909. E-mail address: [email protected] (H. Niizeki). Received 9 December 2013 Received in revised form 12 May 2014 Accepted 13 May 2014 http://dx.doi.org/10.1016/j.jdermsci.2014.05.008
classified according to geographic regions and ethnic differences. Two hypotheses have been proposed as key determinants of human hair shape based on morphological features. One proposes that hair shape is determined solely by the shape of the hair follicle (HF) [1], straight hair being generated from straight cylindrical HFs whereas highly curled hair is produced by curved HFs. The second hypothesis proposes that hair shape is programmed by the hair bulb (HB) based on results from organ culture, suggesting that an asymmetric differentiation and proliferation of each follicular layer beginning in the HB and mechanical stress on the concave side of the HB are closely related to the formation of hair shape [2].
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Here we show the morphological characteristics of human HFs from various natural hair shapes using a collection of isolated HFs (Supplementary Table S1). The curvature parameter measured 3dimensionally using individual hair shafts differed significantly well-representing their global hair shape in each group (Supplementary Fig. S1). Characterization of the morphology of HFs in scalp biopsies showed no obvious differences in the morphology of
isolated HFs between straight and wavy hair, whereas curly hairs were dramatically curved and bent at the HB, showing a retrocurved structure as reported previously (Fig. 1A) [2]. The curvatures of all HFs and HBs (defined in Supplementary Fig. S2) were plotted and classified according to hair shape for quantitative investigation. The curvatures of HFs and HBs in curly hairs plotted in a distinct higher value area, while those of straight and wavy
[(Fig._1)TD$IG]
Fig. 1. Morphological characterization of HFs depending on hair shape. (A) Typical examples of global hair shape and corresponding HFs from scalp biopsies. Scale bars = 1 mm. (B) The curvatures of HFs and HBs were measured 2-dimensionally and are plotted based on the shape of the hair shaft. (C) The curvature of total HFs and HBs, defined as 0.53 mm from the tip, are compared among the hair shapes. Asterisks (*) indicate a significant difference (p < 0.001) compared to the other two groups by one-way ANOVA (Tukey–Kramer test). Values of curvature are means SD. (D) Representative HFs were stained with hematoxylin and eosin to illustrate their typical morphologies. At low magnification, HFs from curly hairs have areas with asymmetrical thickness along the bent portions of their structures (arrowheads). The arrow indicates the location of complicated membrane-like structures on the concave side of the HBs from curly hairs. Scale bars = 200 mm.
[(Fig._2)TD$IG]
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Fig. 2. Structural alterations of basement membrane and inner root sheath in curly hair detected by immunohistochemistry. (A) Folded basement membrane and localization of proliferative cells are located in the ORS of curly HFs. Staining of keratin-17 (K17), laminin alpha-5 (LAMA5), a-smooth muscle actin (a-SMA) and plakoglobin (PG) in straight and curly HFs are shown. Arrowheads identify LAMA5 that ectopically exists in the ORS on the concave side of HFs from curly hair. The open arrowhead indicates positive staining of a-SMA on the dermal side of the basement membrane in curly hair. Ki67-positive proliferative cells were detected on both sides of the epithelial component divided by the folded BM (dotted line) in HBs of curly hairs. Nuclei are identified in all images using DAPI staining (blue). Scale bars = 200 mm (B) The thickness of K17 staining in the HBs was compared in both the concave and the convex sides between straight and curly HFs. White bars indicate the thickness of straight hair, and gray bars are those of curly hair. (*) p < 0.05 (Student’s t-test) (C) Uneven distribution and asymmetric differentiation of IRS keratins are shown in curly hair. IRS keratins stained with type-I K25 and type-II K71, K74 reveal the inherent structure of the IRS in curly hair. Magnified areas show K25 staining of straight and curly hairs. Arrowheads indicate the region where cells in the Henle layer of the IRS terminally differentiate. Nuclei are identified in all images using DAPI staining (blue). Scale bars = 200 mm, 50 mm (in magnified images).
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hairs were almost identical in the lower value area (Fig. 1B). Supporting those data, the average values of those parameters showed that the curvatures of HFs and HBs in curly hairs were significantly higher than those of straight and wavy hairs (Fig. 1C). In contrast, no difference in those parameters was detected between straight and wavy hairs. Supplementry material related to this article found, in the online version, at http://dx.doi.org/10.1016/j. jdermsci.2014.06.003. The expression of structural proteins in the HBs was examined histologically. Hematoxylin-eosin staining revealed no significant difference between straight and wavy hairs (not shown) correlating with the structure of isolated HFs. However, in curly HFs, the concave sides of the HBs and the upper part of retrocurved HFs showed a thicker outer root sheath (ORS), companion layer (CL) and dermal sheath along the HF (Fig. 1D). In addition, enlargement of the ORS and CL was detected on the concave side of the HB following a multiple-layered ORS with differential expression of K17, in particular on the side where a folded extracellular membrane structure was seen in the ORS (Fig. 1D, arrow, Fig. 2A). The thickness of K17 staining in the HBs on the concave side of curly hair was thicker than that of straight hair and on the convex side (Fig. 2B). This folded membrane-like structure in the ORS was identified since the basement membrane (BM) located in the enlarged ORS at the concave side of curved HBs stained positively for laminin alpha5 and a-smooth muscle actin (Fig. 2A). On enlargement of the ORS following the ectopic presence of the folded BM, proliferative cells in the HB differed in curly hair. Significant numbers of Ki-67 positive proliferative cells were detected on both sides of the folded BM in the enlarged ORS, whereas fewer Ki-67 positive cells were interspersed along the single-layered ORS in straight hair (Fig. 2A). The inflection point where the retrocurved HFs of curly hairs change direction corresponded to the area where the Henle layer of the inner root sheath (IRS) undergoes terminal differentiation. A precise examination of the expression of IRS keratins showed differences in their distribution patterns among the hair shapes. First, in straight HFs, cells negative for K25, indicating terminal differentiation of Henle cells, occurred at the same height on both sides. In contrast, in curly hairs, terminally differentiated Henle cells appeared at a higher position on the convex side (Fig. 2C, open arrowheads). Second, in straight HFs, K25 localized homogeneously whereas a porous uneven distribution pattern of K25 was observed in curly HFs, especially at the Huxley layer (Fig. 2C). The average intensity of K25 staining in the IRS normalized with Trichohyalin at the height where Henle cells undergo terminal differentiation statistically differed in curly HFs (Supplementary Fig. S3). A similar uneven distribution of type II keratin K71 was detected in the Huxley layer of curly hair and the distribution pattern of K74 was moderately affected (Fig. 2C). Supplementry material related to this article found, in the online version, at http://dx.doi.org/10.1016/j. jdermsci.2014.06.003. Throughout this study, we found that curly hairs in subjects of African-descent depend on the shape and structure of the HF, whereas wavy hair with weaker curliness in other ethnic populations is independent of the morphological shape of the HF. In addition, we propose two novel characteristics highly correlating with curly HFs. First, the folded BM and dermal sheath were located only on the concave side of the ORS of curly hair. Some BM components are known to affect the proliferation of keratinocytes and anchor signaling molecules [3], which may cause the localization of proliferative cells at the concave side of curly HBs. The second characteristic is the
asymmetric differentiation of the Henle layer and the uneven distribution of keratins in the Huxley layer of the IRS. Among the defective mutations in mouse IRS keratins, variable patterns and aggregations of IRS keratins were observed, which result in twisted HFs [4–7]. Furthermore, linkage analysis of dog hair phenotypes has shown a linkage between curly hair and Krt71 gene locus [8]. In humans, mutations in IRS keratin genes (KRT74 and KRT71) have been reported in autosomal-dominant woolly hair [9,10]. Indeed, there have been indicated that several SNPs have differences in allele frequency between African-descent and other populations in IRS keratins [10]. We also found those polymorphisms in KRT71 and KRT25 genes among curly hair subjects in this study. The effect of those variations of IRS keratins is need to be examined to clarify their role in the manner of intermediate filaments as well as the determination of hair shape as natural traits.
Funding All funding for this research was supported by the Kao Corporation.
Conflicts of interest The authors have no conflicts of interest.
Acknowledgments We thank William L. Pickens for his continuous technical support. We appreciate Dr. Lutz Langbein and Dr. Juergen Schweizer (German Cancer Research Center, Heidelberg, Germany) for their critical review of the manuscript.
References [1] Lindelöf B, Forslind B, Hedblad MA, Kaveus U. Human hair form. Morphology revealed by light and scanning electron microscopy and computer aided threedimensional reconstruction. Arch Dermatol 1988;124:1359–63. [2] Thibaut S, Gaillard O, Bouhanna P, Cannell DW, Bernard BA. Human hair shape is programmed from the bulb. Br J Dermatol 2005;152:632–8. [3] Pouliot N, Saunders NA, Kaur P. Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration. Exp Dermatol 2002;11:387–97. [4] Kikkawa Y, Oyama A, Ishii R, Miura I, Amano T, Ishii Y, 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;165:721–33. [5] Peters T, Sedlmeier R, Büssow H, Runkel F, Lüers GH, Korthaus D, et al. Alopecia in a novel mouse model RCO3 is caused by mK6irs1 deficiency. J Invest Dermatol 2003;121:674–80. [6] Peters T, Sedlmeier R, Büssow H, Runkel F, Lüers GH, Korthaus D, et al. Morphologic and molecular characterization of two novel Krt71 (Krt2-6 g) mutations: Krt71rco12 and Krt71rco13. Mamm Genome 2006;17:1172–82. [7] Tanaka S, Miura I, Yoshiki A, Kato Y, Yokoyama H, Shinogi 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;90:703–11. [8] Cadieu E, Neff MW, Quignon P, Walsh K, Chase K, Parker HG, et al. Coat variation in the domestic dog is governed by variants in three genes. Science 2009;326:150–3. [9] Shimomura Y, Wajid M, Petukhova L, Kurban M, Christiano AM. Autosomaldominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture. Am J Hum Genet 2010;86:632–8. [10] Fujimoto A, Farooq M, Fujikawa H, Inoue A, Ohyama M, Ehama R, et al. A missense mutation within the helix initiation motif of the keratin K71 gene underlies autosomal dominant woolly hair/hypotrichosis. J Invest Dermatol 2012;132:2342–9.
Hiroshi Yoshidaa,*, Hiroyuki Taguchia Akira Hachiyaa
Letters to the Editor / Journal of Dermatological Science 75 (2014) 190–207
Takashi Kitaharaa Raymond E. Boissyb Marty O. Visscherc a Biological Science Laboratories, Kao Corporation, 2606 Akabane, Ichikai-Machi, Haga-Gun, Tochigi 321-3497, Japan, bDepartment of Dermatology, University of Cincinnati, Cincinnati, OH, USA, cThe Skin Sciences Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
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* Corresponding author. Tel.: +81 285 68 7491; fax: +81 285 68 7360. E-mail address: [email protected] (H. Yoshida). Received 9 December 2013 Received in revised form 12 May 2014 Accepted 13 May 2014 http://dx.doi.org/10.1016/j.jdermsci.2014.06.003
Letter to the Editor Dorfman-Chanarin syndrome without mental retardation caused by a homozygous ABHD5 splice site mutation that skips exon 6
Dorfman-Chanarin syndrome (DCS) is a rare autosomal recessive form of congenital ichthyosis, characterized by the presence of intracellular lipid droplets in multiple organs [1,2] (OMIM: 275630). Extra-cutaneous manifestations variably include fatty liver, myopathy, cataracts, and a variety of neurologic symptoms, such as mental retardation [3]. DCS patients often have mutations in ABHD5, which encodes abhydrolase domaincontaining 5 (ABHD5), an activator of adipose triglyceride lipase, leading to accumulation of triglycerides [4,5]. Herein, we report a case of DCS without mental retardation, caused by a homozygous ABDH5 splicing site mutation, which resulted in the skipping of the entire exon 6. Moreover, we suggest a genotype–phenotype correlation of this type of ABDH5 mutation and DCS without mental retardation. A 36-year-old Tunisian man with fine, gray to brown scales on his body (that were apparent since birth), came to our clinic (Fig. 1A). His parents were non-consanguineous. He had three siblings; one sister also had congenital ichthyosis, but no mental retardation. Our patient showed pruritus and hypohidrosis, but did not experience mental retardation, growth retardation, muscle weakness, microtia, hearing involvement, or cataracts. A laboratory test revealed the following parameters: WBC, 3400/ mL;PLT, 76,000/ mL;AST, 118 IU/L;ALT, 86 IU/L;LDH, 353 IU/L;g-GTP, 157 IU/ L;T-Bil, 1.7 mg/dL;D-Bil, 0.7 mg/dL; and CK, 663 IU/L. Computer tomography revealed low density in the whole liver and splenomegaly. The results of a liver biopsy previously performed at a different hospital revealed lipid deposits in the hepatocytes. Light microscopy of a skin lesion sample taken from the patient's trunk showed marked hyperkeratosis, with only a small number of parakeratotic cells (Fig. 1B). There were large cytoplasmic vacuoles containing amorphous material in the basal layer cells of the epidermis (Fig. 1C). A peripheral blood smear showed leucocytes containing prominent lipid vacuoles (Jordan's anomaly) (Fig. 1D), which is the pathognomonic finding in DCS. From these findings, the patient was diagnosed as DCS. Following ethical approval, informed consent was obtained in compliance with the Declaration of Helsinki guidelines. The entire coding regions of ABHD5, including the exon/intron boundaries, were sequenced using genomic DNA samples from the patient [3].
Abbreviations: ABHD5, abhydrolase domain-containing 5; ALT, alanine2oxoglutarate aminotransferase; AST, aspartate2-oxoglutarate aminotransferase; CK, creatine kinase; D-Bil, direct bilirubin; DCS, Dorfman-Chanarin syndrome; g-GTP, g-glutamyl transpeptidase; LDH, lactate dehydrogenase; PLT, platelets; T-Bil, total bilirubin; WBC, white blood cells.
The patient had a homozygous ABHD5 mutation c.773 1 G > A (Fig. 2A). c.773 1 G > A was previously reported as a pathogenic ABHD5 splice site mutation found in two Tunisian families [4]. However, the ABHD5 mRNA products derived from the mutant allele were not analyzed in detail. We therefore examined the consequences of this ABHD5 mutation. Reverse transcription polymerase chain reaction (RT-PCR) analysis using total RNA samples from the patient's plucked scalp hair showed two aberrantly-spliced mRNA products, but did not show any normally spliced mRNA products (Fig. 2B) (Supplementary Table S1) [6,7]. Sequencing of the aberrant cDNA fragments revealed that the major product skipped the entire exon 6 and the minor product skipped the 50 –95 bp of exon 6 (Fig. 2B). Thus, the splice-site mutation resulted predominantly in the skipping of the entire exon 6, leading to premature translation termination. This caused a truncation of ABHD5 into p.Ser258ArgfsX21. The minor product from the aberrant splicing, in which the 50 –95 bp of exon 6 was skipped, resulted into the formation of p.Gly259PhefsX65, although the amount of this minor product appeared to be very small (Fig. 2B). Interestingly, as shown in Fig. 2B, RT-PCR of the scalp hair of a normal subject also produced the alternatively spliced mRNA product, in which the entire exon 6 was skipped. This finding was consistent with a previous report [8]. Supplementry material related to this article found, in the online version, at 10.1016/j.jdermsci.2014.05.009. In order to confirm that the alternatively spliced mRNA product (in which the entire exon 6 was skipped) exists in normal fetal and adult brains as well as in normal adult livers, RT-PCR was conducted using total RNA derived from human tissue that was purchased from Takara Bio Inc. (Otsu, Japan). We found that the alternatively spliced ABHD5 mRNA product, in which the entire exon 6 was skipped, existed at higher levels in the normal fetal brain than in the normal adult brain, liver, or scalp hair (Fig. 2C). ABHD5 has been reported to colocalize with perilipin [9]. The truncated ABHD5 p.Ser258ArgfsX21 possibly dissociates from perilipin because the truncated ABHD5 lacks p.260Glu, which is a critical residue for binding to perilipin [9]. The clinical manifestations of one of the Tunisian patients with the c.773 1 G > A homozygous mutation, were previously described in detail, and they were found to be identical to those of the patient currently under investigation (Supplementary Table S2) [4]. The patient had bilateral cataracts and mild deafness, in addition to hepatomegaly, liver steatosis, and ichthyosis. However, this patient did not exhibit any signs of mental retardation. The laboratory parameters of the previously reported patient were similar to those of the patient currently under investigation [4]. Another DCS patient with a homozygous c.959 + 6 A > T mutation, leading predominantly to the skipping of the entire exon 6 of ABHD5, had ichthyosis and hepatomegaly, but also showed no signs of mental retardation (Supplementary Table S2) [8]. There is a notion that genotype– phenotype correlations are not possible in DCS [10]. However, we suggest that the ABHD5 splicing site mutation, which results into the skipping of the entire exon 6, is associated with a DCS phenotype that is not associated with mental retardation. Based on
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. jdermsci.2014.05.008. References [1] Castori M, Sinibaldi L, Mingarelli R, Lachman R, Rimoin D, Dallapiccola B. Pachydermoperiostosis: an update. Clin Genet 2005;68:477–86. [2] Touraine A, Solente G, Golé L. Un syndrome ostéodermopathique: la pachydermie plicaturée avec pachypé riostose des extrémités. Presse Med 1935;43:1820–4. [3] Uppal S, Diggle C, Carr I, Fishwick C, Ahmed M, Ibrahim G, et al. Mutations in 15-hydroxyprostaglandin dehydrogenase cause primary hypertrophic osteoarthropathy. Nat Genet 2008;40:789–93. [4] Sasaki T, Niizeki H, Shimizu A, Shiohama A, Hirakiyama A, Okuyama T, et al. Identification of mutations in the prostaglandin transporter gene SLCO2A1 and its phenotype-genotype correlation in Japanese patients with pachydermoperiostosis. J Dermatol Sci 2012;68:36–44. [5] Zhang Z, Xia W, He J, Zhang Z, Ke Y, Yue H, et al. Exome sequencing identifies SLCO2A1 mutations as a cause of primary hypertrophic osteoarthropathy. Am J Hum Genet 2012;90:125–32. [6] Diggle CP, Parry DA, Logan CV, Laissue P, Rivera C, Restrepo CM, et al. Prostaglandin transporter mutations cause pachydermoperiostosis with myelofibrosis. Hum Mutat 2012;33:1175–81. [7] Zhang Z, He JW, Fu WZ, Zhang CQ, Zhang ZL. Mutations in the SLCO2A1 gene and primary hypertrophic osteoarthropathy: a clinical and biochemical characterization. J Clin Endocrinol Metab 2013;98:E923–33. [8] Niizeki H, Shiohama A, Sasaki T, Seki A, Kabashima K, Otsuka A, et al. The novel SLCO2A1 heterozygous missense mutation p. E427 K and nonsense mutation p.R603* in a female patient with pachydermoperiostosis with an atypical phenotype. Br J Dermatol 2014;170:1187–9. [9] Seifert W, Kühnisch J, Tüysüz B, Specker C, Brouwers A, Horn D. Mutations in the prostaglandin transporter encoding gene SLCO2A1 cause primary hypertrophic osteoarthropathy and isolated digital clubbing. Hum Mutat 2012;33:660–4. [10] Matsuda Y, Maruta T, Ide C, Watanabe A, Komori K, Sakai Y, et al. A case of early stage of pachydermoperiostosis preceded by acromegaly. Jap J Clin Exp Med (Rinsho to kenkyu) 2008;85:1475–8 [in Japanese].
H. Niizeki* Department of Dermatology, National Center for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan A. Shiohamaa,b Department of Dermatology, Keio University School of Medicine, Tokyo, Japan, bLaboratory of Gene Medicine, Keio University School of Medicine, Tokyo, Japan
a
T. Sasaki Center for Integrated Medical Research, Keio University School of Medicine, Tokyo, Japan A. Seki Department of Orthopedics, National Center for Child Health and Development, Tokyo, Japan K. Kabashima, A. Otsuka Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan
Letter to the Editor The natural trait of the curvature of human hair is correlated with bending of the hair follicle and hair bulb by a structural disparity in the root sheath
The shape of human hair is one of the most distinguishable characteristics among various populations and commonly
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K. Kosaki Department of Orthopedic Surgery, University of Tokyo, Tokyo, Japan A. Ogo Department of Metabolism and Endocrinology, Clinical Research Institute, National Hospital Organization Kyushu Medical Center, Fukuoka, Japan T. Yamada Department of Dermatology, Himeji Red Cross Hospital, Hyogo, Japan M. Miyasaka Department of Radiology, National Center for Child Health and Development, Tokyo, Japan K. Matsuoka Department of Pathology, National Center for Child Health and Development, Tokyo, Japan A. Hirakiyama, T. Okuyama Department of Laboratory Medicine, National Center for Child Health and Development, Tokyo, Japan M. Matsuda Department of Dermatology, National Center for Child Health and Development, Tokyo, Japan K. Nakabayashi Department of Reproductive Biology, National Research Institute for Child Health and Development, Tokyo, Japan K. Tanese Department of Dermatology, Keio University School of Medicine, Tokyo, Japan A. Ishiko Department of Dermatology, School of Medicine, Toho University, Ota-ku, Tokyo, Japan M. Amagai Department of Dermatology, Keio University School of Medicine, Tokyo, Japan J. Kudoh Laboratory of Gene Medicine, Keio University School of Medicine, Tokyo, Japan * Corresponding author. Tel.: +81 3 3416 0181; fax: +81 3 5494 7909. E-mail address: [email protected] (H. Niizeki). Received 9 December 2013 Received in revised form 12 May 2014 Accepted 13 May 2014 http://dx.doi.org/10.1016/j.jdermsci.2014.05.008
classified according to geographic regions and ethnic differences. Two hypotheses have been proposed as key determinants of human hair shape based on morphological features. One proposes that hair shape is determined solely by the shape of the hair follicle (HF) [1], straight hair being generated from straight cylindrical HFs whereas highly curled hair is produced by curved HFs. The second hypothesis proposes that hair shape is programmed by the hair bulb (HB) based on results from organ culture, suggesting that an asymmetric differentiation and proliferation of each follicular layer beginning in the HB and mechanical stress on the concave side of the HB are closely related to the formation of hair shape [2].
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Here we show the morphological characteristics of human HFs from various natural hair shapes using a collection of isolated HFs (Supplementary Table S1). The curvature parameter measured 3dimensionally using individual hair shafts differed significantly well-representing their global hair shape in each group (Supplementary Fig. S1). Characterization of the morphology of HFs in scalp biopsies showed no obvious differences in the morphology of
isolated HFs between straight and wavy hair, whereas curly hairs were dramatically curved and bent at the HB, showing a retrocurved structure as reported previously (Fig. 1A) [2]. The curvatures of all HFs and HBs (defined in Supplementary Fig. S2) were plotted and classified according to hair shape for quantitative investigation. The curvatures of HFs and HBs in curly hairs plotted in a distinct higher value area, while those of straight and wavy
[(Fig._1)TD$IG]
Fig. 1. Morphological characterization of HFs depending on hair shape. (A) Typical examples of global hair shape and corresponding HFs from scalp biopsies. Scale bars = 1 mm. (B) The curvatures of HFs and HBs were measured 2-dimensionally and are plotted based on the shape of the hair shaft. (C) The curvature of total HFs and HBs, defined as 0.53 mm from the tip, are compared among the hair shapes. Asterisks (*) indicate a significant difference (p < 0.001) compared to the other two groups by one-way ANOVA (Tukey–Kramer test). Values of curvature are means SD. (D) Representative HFs were stained with hematoxylin and eosin to illustrate their typical morphologies. At low magnification, HFs from curly hairs have areas with asymmetrical thickness along the bent portions of their structures (arrowheads). The arrow indicates the location of complicated membrane-like structures on the concave side of the HBs from curly hairs. Scale bars = 200 mm.
[(Fig._2)TD$IG]
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Fig. 2. Structural alterations of basement membrane and inner root sheath in curly hair detected by immunohistochemistry. (A) Folded basement membrane and localization of proliferative cells are located in the ORS of curly HFs. Staining of keratin-17 (K17), laminin alpha-5 (LAMA5), a-smooth muscle actin (a-SMA) and plakoglobin (PG) in straight and curly HFs are shown. Arrowheads identify LAMA5 that ectopically exists in the ORS on the concave side of HFs from curly hair. The open arrowhead indicates positive staining of a-SMA on the dermal side of the basement membrane in curly hair. Ki67-positive proliferative cells were detected on both sides of the epithelial component divided by the folded BM (dotted line) in HBs of curly hairs. Nuclei are identified in all images using DAPI staining (blue). Scale bars = 200 mm (B) The thickness of K17 staining in the HBs was compared in both the concave and the convex sides between straight and curly HFs. White bars indicate the thickness of straight hair, and gray bars are those of curly hair. (*) p < 0.05 (Student’s t-test) (C) Uneven distribution and asymmetric differentiation of IRS keratins are shown in curly hair. IRS keratins stained with type-I K25 and type-II K71, K74 reveal the inherent structure of the IRS in curly hair. Magnified areas show K25 staining of straight and curly hairs. Arrowheads indicate the region where cells in the Henle layer of the IRS terminally differentiate. Nuclei are identified in all images using DAPI staining (blue). Scale bars = 200 mm, 50 mm (in magnified images).
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hairs were almost identical in the lower value area (Fig. 1B). Supporting those data, the average values of those parameters showed that the curvatures of HFs and HBs in curly hairs were significantly higher than those of straight and wavy hairs (Fig. 1C). In contrast, no difference in those parameters was detected between straight and wavy hairs. Supplementry material related to this article found, in the online version, at http://dx.doi.org/10.1016/j. jdermsci.2014.06.003. The expression of structural proteins in the HBs was examined histologically. Hematoxylin-eosin staining revealed no significant difference between straight and wavy hairs (not shown) correlating with the structure of isolated HFs. However, in curly HFs, the concave sides of the HBs and the upper part of retrocurved HFs showed a thicker outer root sheath (ORS), companion layer (CL) and dermal sheath along the HF (Fig. 1D). In addition, enlargement of the ORS and CL was detected on the concave side of the HB following a multiple-layered ORS with differential expression of K17, in particular on the side where a folded extracellular membrane structure was seen in the ORS (Fig. 1D, arrow, Fig. 2A). The thickness of K17 staining in the HBs on the concave side of curly hair was thicker than that of straight hair and on the convex side (Fig. 2B). This folded membrane-like structure in the ORS was identified since the basement membrane (BM) located in the enlarged ORS at the concave side of curved HBs stained positively for laminin alpha5 and a-smooth muscle actin (Fig. 2A). On enlargement of the ORS following the ectopic presence of the folded BM, proliferative cells in the HB differed in curly hair. Significant numbers of Ki-67 positive proliferative cells were detected on both sides of the folded BM in the enlarged ORS, whereas fewer Ki-67 positive cells were interspersed along the single-layered ORS in straight hair (Fig. 2A). The inflection point where the retrocurved HFs of curly hairs change direction corresponded to the area where the Henle layer of the inner root sheath (IRS) undergoes terminal differentiation. A precise examination of the expression of IRS keratins showed differences in their distribution patterns among the hair shapes. First, in straight HFs, cells negative for K25, indicating terminal differentiation of Henle cells, occurred at the same height on both sides. In contrast, in curly hairs, terminally differentiated Henle cells appeared at a higher position on the convex side (Fig. 2C, open arrowheads). Second, in straight HFs, K25 localized homogeneously whereas a porous uneven distribution pattern of K25 was observed in curly HFs, especially at the Huxley layer (Fig. 2C). The average intensity of K25 staining in the IRS normalized with Trichohyalin at the height where Henle cells undergo terminal differentiation statistically differed in curly HFs (Supplementary Fig. S3). A similar uneven distribution of type II keratin K71 was detected in the Huxley layer of curly hair and the distribution pattern of K74 was moderately affected (Fig. 2C). Supplementry material related to this article found, in the online version, at http://dx.doi.org/10.1016/j. jdermsci.2014.06.003. Throughout this study, we found that curly hairs in subjects of African-descent depend on the shape and structure of the HF, whereas wavy hair with weaker curliness in other ethnic populations is independent of the morphological shape of the HF. In addition, we propose two novel characteristics highly correlating with curly HFs. First, the folded BM and dermal sheath were located only on the concave side of the ORS of curly hair. Some BM components are known to affect the proliferation of keratinocytes and anchor signaling molecules [3], which may cause the localization of proliferative cells at the concave side of curly HBs. The second characteristic is the
asymmetric differentiation of the Henle layer and the uneven distribution of keratins in the Huxley layer of the IRS. Among the defective mutations in mouse IRS keratins, variable patterns and aggregations of IRS keratins were observed, which result in twisted HFs [4–7]. Furthermore, linkage analysis of dog hair phenotypes has shown a linkage between curly hair and Krt71 gene locus [8]. In humans, mutations in IRS keratin genes (KRT74 and KRT71) have been reported in autosomal-dominant woolly hair [9,10]. Indeed, there have been indicated that several SNPs have differences in allele frequency between African-descent and other populations in IRS keratins [10]. We also found those polymorphisms in KRT71 and KRT25 genes among curly hair subjects in this study. The effect of those variations of IRS keratins is need to be examined to clarify their role in the manner of intermediate filaments as well as the determination of hair shape as natural traits.
Funding All funding for this research was supported by the Kao Corporation.
Conflicts of interest The authors have no conflicts of interest.
Acknowledgments We thank William L. Pickens for his continuous technical support. We appreciate Dr. Lutz Langbein and Dr. Juergen Schweizer (German Cancer Research Center, Heidelberg, Germany) for their critical review of the manuscript.
References [1] Lindelöf B, Forslind B, Hedblad MA, Kaveus U. Human hair form. Morphology revealed by light and scanning electron microscopy and computer aided threedimensional reconstruction. Arch Dermatol 1988;124:1359–63. [2] Thibaut S, Gaillard O, Bouhanna P, Cannell DW, Bernard BA. Human hair shape is programmed from the bulb. Br J Dermatol 2005;152:632–8. [3] Pouliot N, Saunders NA, Kaur P. Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration. Exp Dermatol 2002;11:387–97. [4] Kikkawa Y, Oyama A, Ishii R, Miura I, Amano T, Ishii Y, 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;165:721–33. [5] Peters T, Sedlmeier R, Büssow H, Runkel F, Lüers GH, Korthaus D, et al. Alopecia in a novel mouse model RCO3 is caused by mK6irs1 deficiency. J Invest Dermatol 2003;121:674–80. [6] Peters T, Sedlmeier R, Büssow H, Runkel F, Lüers GH, Korthaus D, et al. Morphologic and molecular characterization of two novel Krt71 (Krt2-6 g) mutations: Krt71rco12 and Krt71rco13. Mamm Genome 2006;17:1172–82. [7] Tanaka S, Miura I, Yoshiki A, Kato Y, Yokoyama H, Shinogi 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;90:703–11. [8] Cadieu E, Neff MW, Quignon P, Walsh K, Chase K, Parker HG, et al. Coat variation in the domestic dog is governed by variants in three genes. Science 2009;326:150–3. [9] Shimomura Y, Wajid M, Petukhova L, Kurban M, Christiano AM. Autosomaldominant woolly hair resulting from disruption of keratin 74 (KRT74), a potential determinant of human hair texture. Am J Hum Genet 2010;86:632–8. [10] Fujimoto A, Farooq M, Fujikawa H, Inoue A, Ohyama M, Ehama R, et al. A missense mutation within the helix initiation motif of the keratin K71 gene underlies autosomal dominant woolly hair/hypotrichosis. J Invest Dermatol 2012;132:2342–9.
Hiroshi Yoshidaa,*, Hiroyuki Taguchia Akira Hachiyaa
Letters to the Editor / Journal of Dermatological Science 75 (2014) 190–207
Takashi Kitaharaa Raymond E. Boissyb Marty O. Visscherc a Biological Science Laboratories, Kao Corporation, 2606 Akabane, Ichikai-Machi, Haga-Gun, Tochigi 321-3497, Japan, bDepartment of Dermatology, University of Cincinnati, Cincinnati, OH, USA, cThe Skin Sciences Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
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* Corresponding author. Tel.: +81 285 68 7491; fax: +81 285 68 7360. E-mail address: [email protected] (H. Yoshida). Received 9 December 2013 Received in revised form 12 May 2014 Accepted 13 May 2014 http://dx.doi.org/10.1016/j.jdermsci.2014.06.003
Letter to the Editor Dorfman-Chanarin syndrome without mental retardation caused by a homozygous ABHD5 splice site mutation that skips exon 6
Dorfman-Chanarin syndrome (DCS) is a rare autosomal recessive form of congenital ichthyosis, characterized by the presence of intracellular lipid droplets in multiple organs [1,2] (OMIM: 275630). Extra-cutaneous manifestations variably include fatty liver, myopathy, cataracts, and a variety of neurologic symptoms, such as mental retardation [3]. DCS patients often have mutations in ABHD5, which encodes abhydrolase domaincontaining 5 (ABHD5), an activator of adipose triglyceride lipase, leading to accumulation of triglycerides [4,5]. Herein, we report a case of DCS without mental retardation, caused by a homozygous ABDH5 splicing site mutation, which resulted in the skipping of the entire exon 6. Moreover, we suggest a genotype–phenotype correlation of this type of ABDH5 mutation and DCS without mental retardation. A 36-year-old Tunisian man with fine, gray to brown scales on his body (that were apparent since birth), came to our clinic (Fig. 1A). His parents were non-consanguineous. He had three siblings; one sister also had congenital ichthyosis, but no mental retardation. Our patient showed pruritus and hypohidrosis, but did not experience mental retardation, growth retardation, muscle weakness, microtia, hearing involvement, or cataracts. A laboratory test revealed the following parameters: WBC, 3400/ mL;PLT, 76,000/ mL;AST, 118 IU/L;ALT, 86 IU/L;LDH, 353 IU/L;g-GTP, 157 IU/ L;T-Bil, 1.7 mg/dL;D-Bil, 0.7 mg/dL; and CK, 663 IU/L. Computer tomography revealed low density in the whole liver and splenomegaly. The results of a liver biopsy previously performed at a different hospital revealed lipid deposits in the hepatocytes. Light microscopy of a skin lesion sample taken from the patient's trunk showed marked hyperkeratosis, with only a small number of parakeratotic cells (Fig. 1B). There were large cytoplasmic vacuoles containing amorphous material in the basal layer cells of the epidermis (Fig. 1C). A peripheral blood smear showed leucocytes containing prominent lipid vacuoles (Jordan's anomaly) (Fig. 1D), which is the pathognomonic finding in DCS. From these findings, the patient was diagnosed as DCS. Following ethical approval, informed consent was obtained in compliance with the Declaration of Helsinki guidelines. The entire coding regions of ABHD5, including the exon/intron boundaries, were sequenced using genomic DNA samples from the patient [3].
Abbreviations: ABHD5, abhydrolase domain-containing 5; ALT, alanine2oxoglutarate aminotransferase; AST, aspartate2-oxoglutarate aminotransferase; CK, creatine kinase; D-Bil, direct bilirubin; DCS, Dorfman-Chanarin syndrome; g-GTP, g-glutamyl transpeptidase; LDH, lactate dehydrogenase; PLT, platelets; T-Bil, total bilirubin; WBC, white blood cells.
The patient had a homozygous ABHD5 mutation c.773 1 G > A (Fig. 2A). c.773 1 G > A was previously reported as a pathogenic ABHD5 splice site mutation found in two Tunisian families [4]. However, the ABHD5 mRNA products derived from the mutant allele were not analyzed in detail. We therefore examined the consequences of this ABHD5 mutation. Reverse transcription polymerase chain reaction (RT-PCR) analysis using total RNA samples from the patient's plucked scalp hair showed two aberrantly-spliced mRNA products, but did not show any normally spliced mRNA products (Fig. 2B) (Supplementary Table S1) [6,7]. Sequencing of the aberrant cDNA fragments revealed that the major product skipped the entire exon 6 and the minor product skipped the 50 –95 bp of exon 6 (Fig. 2B). Thus, the splice-site mutation resulted predominantly in the skipping of the entire exon 6, leading to premature translation termination. This caused a truncation of ABHD5 into p.Ser258ArgfsX21. The minor product from the aberrant splicing, in which the 50 –95 bp of exon 6 was skipped, resulted into the formation of p.Gly259PhefsX65, although the amount of this minor product appeared to be very small (Fig. 2B). Interestingly, as shown in Fig. 2B, RT-PCR of the scalp hair of a normal subject also produced the alternatively spliced mRNA product, in which the entire exon 6 was skipped. This finding was consistent with a previous report [8]. Supplementry material related to this article found, in the online version, at 10.1016/j.jdermsci.2014.05.009. In order to confirm that the alternatively spliced mRNA product (in which the entire exon 6 was skipped) exists in normal fetal and adult brains as well as in normal adult livers, RT-PCR was conducted using total RNA derived from human tissue that was purchased from Takara Bio Inc. (Otsu, Japan). We found that the alternatively spliced ABHD5 mRNA product, in which the entire exon 6 was skipped, existed at higher levels in the normal fetal brain than in the normal adult brain, liver, or scalp hair (Fig. 2C). ABHD5 has been reported to colocalize with perilipin [9]. The truncated ABHD5 p.Ser258ArgfsX21 possibly dissociates from perilipin because the truncated ABHD5 lacks p.260Glu, which is a critical residue for binding to perilipin [9]. The clinical manifestations of one of the Tunisian patients with the c.773 1 G > A homozygous mutation, were previously described in detail, and they were found to be identical to those of the patient currently under investigation (Supplementary Table S2) [4]. The patient had bilateral cataracts and mild deafness, in addition to hepatomegaly, liver steatosis, and ichthyosis. However, this patient did not exhibit any signs of mental retardation. The laboratory parameters of the previously reported patient were similar to those of the patient currently under investigation [4]. Another DCS patient with a homozygous c.959 + 6 A > T mutation, leading predominantly to the skipping of the entire exon 6 of ABHD5, had ichthyosis and hepatomegaly, but also showed no signs of mental retardation (Supplementary Table S2) [8]. There is a notion that genotype– phenotype correlations are not possible in DCS [10]. However, we suggest that the ABHD5 splicing site mutation, which results into the skipping of the entire exon 6, is associated with a DCS phenotype that is not associated with mental retardation. Based on
