Mutations in the Keratin 85 (KRT85/Hhb5) Gene Underlie Pure Hair and Nail Ectodermal Dysplasia

Y Shimomura et al. Mutations in the KRT85 Gene

Mutations in the Keratin 85 (KRT85/hHb5) Gene Underlie Pure Hair and Nail Ectodermal Dysplasia

Journal of Investigative Dermatology (2010) 130, 892–895; doi:10.1038/jid.2009.341; published online 29 October 2009

TO THE EDITOR monilethrix hair (Ito et al., 1990). Never- ence sequences (NCBI accession num- Pure hair and nail ectodermal dysplasia theless, it suggests abnormal keratiniza- ber: NG_008353). We identified (PHNED; OMIM 602032) is a con- tion of the hair shaft. As compared with homozygous mutations in the KRT85 in genital disorder characterized by Family A, affected individuals in Family both families. All affected individuals in hypotrichosis and nail dystrophy. The B show a more severe phenotype. They Family A are homozygous for a deletion disease can show either an autosomal display complete alopecia with diffuse mutation c.1448_1449delCT in exon 9 dominant (Barbareschi et al., 1997) or follicular papules on the scalp (Figure of the KRT85 gene, which was not autosomal recessive (Calzavara-Pinton 1g). Facial and body hairs are also reported previously and is predicted to et al., 1991) inheritance pattern. The absent. Furthermore, the tips of nails result in a frameshift at codon 483 and a autosomal recessive form has been show severe deformities (Figure 1h). premature termination codon 18 amino mapped to chromosome 17p12–q21.2 Affected individuals in both families acid residues downstream of the muta- (Naeem et al., 2006a) and 12p11.1– show normal facial features, teeth, and tion (p.Pro483ArgfsX18) (Figure 2a, b). q21.1 (Naeem et al., 2006b, 2007), sweating, and do not show palmoplantar Screening assays with the restriction which contain the type I and type II hyperkeratosis. enzyme NlaIV confirmed that the muta- keratin clusters, respectively. One After obtaining informed consent, tion completely co-segregates with the homozygous mutation in a type II hair we collected peripheral blood samples disease phenotype, and 100 healthy keratin gene KRT85, previously known from members of both families and 100 control individuals from Pakistan do as hHb5, was recently identified in population-matched unrelated healthy not carry the mutation (data not shown). a consanguineous Pakistani family with control individuals in EDTA-containing Since the mutation is located within the autosomal recessive PHNED (Naeem tubes (under institutional approval last exon of the KRT85 gene, aberrant et al., 2006b). However, the clinical and in adherence to the Declaration transcripts with the PTC may be ex- characteristics and the molecular basis of Helsinki Principles). Genomic DNA pressed, and a truncated K85 protein of the disease have been poorly under- was isolated from these samples accor- would be generated. The altered amino stood to date. In this study, we identi- ding to standard techniques. On the acid sequences in C-terminus of the fied two consanguineous Pakistani basis of the clinical features, we diag- mutant K85 protein are largely different families with PHNED and found two nosed both families as PHNED. We first from those of the wild-type K85 protein. distinct homozygous mutations in the performed genotyping using micro- In particular, the mutant protein lacks KRT85 gene in both families. satellite markers around the type I two cysteine residues at amino acid We analyzed two consanguineous keratin cluster on chromosome 17q21 positions 489 and 498 (Figure 2b). Pakistani families, designated as Families and type II keratin cluster on chromo- Therefore, the mutant protein with an A and B, with several affected indivi- some 12q13. The results excluded abnormal tail domain would deleter- duals. Both families show autosomal linkage to the type I keratin cluster in iously affect heterodimer formation. recessive inheritance of the disease both families (data not shown), and The affected individual in Family B trait (Figure 1a). All affected individuals suggested linkage of both families is homozygous for a G-to-A substitu- in both families have shown hypotricho- to the type II keratin cluster (Figure tion at position 233 in exon 1 of the sis and nail dystrophy since birth, but 1a). In particular, critical recombina- KRT85 gene (c.233G4A), which leads there is a difference in severity between tion events were detected between to the transition from arginine to the two families. In Family A, all affected markers D12S398 and D12S1618 in histidine at amino acid position 78 individuals show sparse hair and affected individual IV-1 of family A, as (p.Arg78His) (Figure 2c). This mutation diffuse follicular papules on the scalp well as between markers D12S368 and was previously identified in a Pakistani (Figure 1b-d). Facial and body hair is D12S398 in unaffected individual III-2 family with PHNED, which shows less dense overall. In addition, distal of family A, which allowed the interval complete alopecia and severe nail ends of nails are irregularly shaped of linkage to be narrowed to 1.26 Mb dystrophy (Naeem et al., 2006b). and break easily (Figure 1e). It is flanked by markers D12S368 and It is noteworthy that Family B also noteworthy that the patients’ scalp hair D12S1618 (Figure 1a). The KRT85 gene exhibits severe hair and nail pheno- shows fragility. Scanning electron micro- resides in this region. types, which may suggest that the scopy showed that the thickness of We next sequenced the KRT85 mutation p.Arg78His would disrupt the hair shaft was inconsistent (Figure gene in affected individuals from both the function of the K85 protein 1f). The variation in hair diameter did not families using gene-specific primers, more severely than the mutation show a periodic pattern as observed in which were designed based on refer- p.Pro483ArgfsX18. Including the two

892 Journal of Investigative Dermatology (2010), Volume 130 Y Shimomura et al. Mutations in the KRT85 Gene

a Family A Family B

I I II II III

III IV

Type II keratin gene cluster

bcd

e f

98.2 µm 78.2 µm 89.1 µm

f g

Figure 1. Pedigrees and clinical appearance of two Pakistani families with PHNED. (a) Pedigrees and results of haplotype analysis. The linked haplotypes are indicated in red, and critical recombination events are indicated by arrows. (b–e) Clinical features of affected individuals in Family A. (f) SEM observation of hair of an affected individual in Family A. Bar ¼ 100 mm. (g, h) Clinical features of affected individuals in Family B. Note that affected individuals in Family B show a more severe phenotype than those in Family A. families in this report, only three cases with KRT85 mutations will be The K85 protein is abundantly families with two distinct KRT85 muta- required to determine clear genotype–- expressed in the matrix, precortex, tions have been identified. Additional phenotype correlations. cortex, and cuticle of the hair shaft

www.jidonline.org 893 Y Shimomura et al. Mutations in the KRT85 Gene

481 482 483 484 485 V A R L L 481 482 483 484 485 G T CCC GGG A CCCTTG 481 482 483 484 485 VALLRA V PPSSDPAV TTGGGG A CCCCCC TT G G T GGCCCC GGAACCCTTTGGCCCCGG CCCTT

Patient Carrier Control individual

wild type 481 VAPDSCAPCQPRSSSFSCGSSRSVRFA* 507 mutant 481 VARLLCPLPASFLQLQLRE* 499

76 77 78 79 80 76 77 78 79 80 C GHSF SRGC F TTC GGG AAACC G CCTTTGGGCAA CCCC G G T

A/A homozygous G/G homozygous patient control individual

Figure 2. Identification of mutations in the KRT85 gene. (a) A novel homozygous mutation c.1448_1449delCT (p.Pro483ArgfsX18) in the KRT85 gene in Family A. The deleted nucleotides are boxed. (b) Comparison of amino acid sequences between wild-type and the mutant K85 proteins with the mutation p.Pro483ArgfsX18. Amino acid position 483, where the frameshift occurs, is indicated by an arrow. Cysteine residues at positions 489 and 498 in wild-type protein are in blue. Asterisks, stop codon. (c) A homozygous mutation c.233G4A (p.Arg78His) in the KRT85 gene in Family B.

(Langbein et al., 2001), as well as the crucial role of K85 in the (AMC). YS is supported by a Research Career Development Award from the Dermatology in the keratogenous zone of the nail keratinization of hair and nails in Foundation. matrix (Perrin et al., 2004). The keratins humans. form the intermediate filaments that Yutaka Shimomura1, Muhammad 1 1 anchor to desmosomal components CONFLICT OF INTEREST Wajid , Mazen Kurban , 2 and maintain the tissue integrity The authors state no conflict of interest. Nobuyuki Sato and (Coulombe and Omary, 2002). We Angela M. Christiano1,3 postulate that dysfunction of K85 1 ACKNOWLEDGMENTS Department of Dermatology, Columbia protein would lead to disruption of University, New York, New York, USA; We gratefully acknowledge the family members 2 keratin intermediate filament formation for having participated in this study. We thank Department of Dermatology, Niigata and abnormal desmosomal assembly Ha Mut Lam for excellent technical assistance. University School of Medicine, Niigata, Japan and 3Department of Genetics and in hair and nails. Our findings not This work was supported by US Public Health Service National Institutes of Health grant Development, Columbia University, only expand the limited spectrum of R01AR44924 from the National Institute of New York, New York, USA KRT85 mutations but also underscore Arthritis and Musculoskeletal and Skin Diseases E-mail: [email protected]

894 Journal of Investigative Dermatology (2010), Volume 130 S Lopez et al. Specific Immunological Response to Budesonide

Ito M, Hashimoto K, Katsuumi K, Sato Y (1990) Naeem M, Wajid M, Lee K, Leal SM, Ahmad W REFERENCES Pathogenesis of monilethrix: computer (2006b) A mutation in the hair matrix and Barbareschi M, Cambiaghi S, Crupi AC, Tadini G stereography and electron microscopy. cuticle keratin KRTHB5 gene causes ecto- (1997) Family with ‘‘pure’’ hair–nail ectoder- J Invest Dermatol 95:186–94 dermal dysplasia of hair and nail type. J Med mal dysplasia. Am J Med Genet 72:91–3 Langbein L, Rogers MA, Winter H, Praetzel S, Genet 43:274–9 Calzavara-Pinton P, Carlino A, Benetti A, De Schweizer J (2001) The catalog of human hair Naeem M, John P, Ali G, Ahmad W (2007) Pure Panfilis G (1991) Pili torti and onycho- keratins. II. Expression of the six type II hair-nail ectodermal dysplasia maps to chro- dysplasia: report of a previously undescribed members in the hair follicle and the com- mosome 12p11.1–q21.1 in a consanguineous hidrotic ectodermal dysplasia. Dermato- bined catalog of human type I and II keratins. Pakistani family. Clin Exp Dermatol 32:502–5 logica 182:184–7 J Biol Chem 276:35123–32 Perrin C, Langbein L, Schweizer J (2004) Expres- Coulombe PA, Omary MB (2002) ‘Hard’ and ‘soft’ Naeem M, Jelani M, Lee K et al. (2006a) sion of hair keratins in the adult nail unit: an principles defining the structure, function Ectodermal dysplasia of hair and nail type: immunohistochemical analysis of the ony- and regulation of keratin intermediate fila- mapping of a novel locus to chromosome chogenesis in the proximal nail fold, matrix ments. Curr Opin Cell Biol 14:110–22 17p12–q21.2. Br J Dermatol 155:1184–90 and nail bed. Br J Dermatol 151:362–71

Specific Immunological Response to Budesonide in a Patient with Delayed-Type Hypersensitivity Reaction

Journal of Investigative Dermatology (2010) 130, 895–897; doi:10.1038/jid.2009.308; published online 8 October 2009

TO THE EDITOR Astra, Lund, Sweden) and 8 hours after complete medium supplemented with Although different authors have con- the last inhalation. A patch test was GM-CSF (R&D System, Minneapolis, firmed the involvement of T cells in positive with budesonide and the skin MN) (800 U ml1) and IL-4 (R&D delayed-type hypersensitivity reactions biopsy showed a perivascular mono- System) (500 U ml1) for 4 days to obtain to glucocorticosteroids (GCs) in skin nuclear cell infiltrate, with the presence mo-DCs. biopsies (Padial et al., 2005) or by of CD4 þ and, to a lesser degree, For proliferation assays, lympho- specific lymphocyte responses (Buettiker CD8 þ cells, detected by immunohisto- cytes were cultured in 96-well plates et al., 2006), the underlying mechanisms chemistry. These cells were memory in triplicate, with or without mo-DCs are complex, because both the antiin- cells (CD45RO þ ), with a moderate (150,000 lymphocytes and 15,000 DCs flammatory and pro-allergic effects may expression of the activation marker per well), and with budesonide, take place at the same time. CD25. Patch and drug provocation tests methylprednisolone, and hydrocorti- Dendritic cells (DCs) are central to were negative with dexamethasone, sone at 10 mgml1, 0.1 mgml1, and the development of specific effector beclomethasone, methylprednisolone, 0.001 mgml1, respectively. After 6 and tolerant immunological responses and hydrocortisone. days, 3H-Thymidine incorporation was (Steinman et al., 2003). Low-molecular- In this patient and in five healthy evaluated in a b-counter as counts per weight compounds, including drugs, nonallergic controls who tolerated the minute. The stimulation index (SI) was are able to induce DC maturation drug, we analyzed the in vitro immuno- calculated as the ratio of the mean (Manome et al., 1999; Rodriguez-Pena logical response to different GCs by counts per minute of a drug-stimulated et al., 2006), which could be respon- culturing autologous lymphocytes with sample to the mean counts per minute sible for the loss of tolerance to these and without monocyte-derived DCs of the sample alone. An SI43 was molecules. However, some drugs, such (mo-DCs) and evaluated the specific considered positive. as GCs, can inhibit the generation of lymphocyte proliferation, their activa- For phenotype analysis, a panel mature DCs (Pan et al., 2001), thereby tion markers, and the changes in the of mAbs (HLA-DR-FITC, CD86-FITC, hindering the evaluation of the way this maturational phenotype of DCs. CD80-PE CD40-PE, and CD83-PE drug is involved in delayed-type hyper- Peripheral blood mononuclear cells for mo-DC, and CD4-FITC, CD25-PE, sensitivity reactions. were isolated and cultivated in complete HLA-DR-PE, CD3-PerCP, and CD8-APC We report a 40-year-old man medium (RPMI-1640 medium with 10% for lymphocytes) and an FACSCalibur with rhinoconjunctivitis sensitized to autologous serum). The nonadherent cytometer were used. pollens, who developed a generalized cells containing lymphocytes were fro- In the patient, lymphocyte prolifera- exanthema 5 days after starting treat- zen for future use, and the adherent cells tion tests were positive only with ment with nasal budesonide (Rhinocort, (monocytes) were cultured in 2 ml of budesonide (SI ¼ 3.4), although higher in the presence of mo-DCs (SI ¼ 6.8), Abbreviations: DC, dendritic cell; GC, glucocorticosteroid; mo-DC, monocyte-derived dendritic cell; SI, and in both cases, only at the lowest 1 stimulation index drug concentration (0.001 mgml ).