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Kindler surprise: mutations in a novel actin-associated protein cause Kindler syndrome
Journal of Dermatological Science (2005) 38, 169—175
www.intl.elsevierhealth.com/journals/jods
REVIEW ARTICLE
Kindler surprise: mutations in a novel actinassociated protein cause Kindler syndrome Sharon J. White *, W.H. Irwin McLean Epithelial Genetics Group, Human Genetics Unit, University of Dundee, Ninewells Medical School, Dundee DD1 9SY, UK Received 29 October 2004; received in revised form 21 December 2004; accepted 22 December 2004
KEYWORDS Kindler syndrome; Genodermatosis; Acral blistering; Photosensitivity; Actin cytoskeleton; Extracellular matrix
Summary Kindler syndrome is an autosomal recessive genodermatosis characterized by acral blistering in neonates and diffuse, progressive poikiloderma in later life. Other clinical features include photosensitivity, premature skin ageing and severe periodontal disease. Two groups have recently shown that the molecular basis of Kindler syndrome is loss of a novel epidermal protein, kindlin-1, encoded by the gene KIND1. Two additional kindlin proteins, kindlin-2 and kindlin-3, have also been described. Kindlin-1 is considered to be a component in the linkage of the actin cytoskeleton to the extracellular matrix and as such is proposed to have both structural and cell-signalling functions. Kindler syndrome is therefore the first skin fragility syndrome due to disruption of the actin—extracellular matrix system. # 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Contents Introduction and clinical features . . . . . . . . . . Histopathology . . . . . . . . . . . . . . . . . . . . . . Molecular basis of Kindler syndrome. . . . . . . . . Kindlin protein structure and predicted function . Kindlin protein family . . . . . . . . . . . . . . . . . . Conclusions and future directions . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction and clinical features
* Corresponding author. Tel.: +44 1382425618; fax: +44 1382425619. E-mail address: [email protected] (S.J. White).
Kindler syndrome (KS; OMIM 173650) was first described in a single patient by Dr. Theresa Kindler in the 1950s [1]. The original patient was a 14-yearold girl who appeared to have a combination of
0923-1811/$30.00 # 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2004.12.026
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features of two previously described conditions, epidermolysis bullosa hereditaria and poikiloderma congenitale. The characteristic clinical features are acral blistering and photosensitivity in infancy followed by progressive poikiloderma (diffuse cutaneous atrophy, telangiectases and reticulate pigmentation) later in life [1,2]. Blistering may be spontaneous or traumatic and usually resolves with age, although there is one reported case where bullae recurred in the fifth decade [3]. Nikolsky’s sign is negative [1,4]. Cutaneous atrophy may be widespread although it is most prominent on the dorsa of the hands and feet and on the knees and elbows, giving rise to the so-called ‘cigarettepaper’ like wrinkling of the skin, as shown in Fig. 1 [1]. Several other features have been reported which vary between cases including: digital webbing; palmoplantar keratoderma; nail dystrophy; alopecia; oesophageal, anal, vaginal and urethral stenosis; loss of dermatoglyphics; and phimosis [1,3,5—7]. A wide range of oral manifestations of the syndrome have also been described including mucosal white patches, atrophy and blistering; gingival bleeding and severe periodontitis; ankyloglossia; restricted mouth opening; and malocclusion [1,2,6—9]. Several of the clinical features of Kindler syndrome resemble those found in various other skin fragility disorders, especially those in dystrophic epidermolysis bullosa (DEB; OMIM 226600). Such overlap means there may be difficulties in making a definitive diagnosis of Kindler syndrome, especially in early childhood. Trauma-induced blistering,
S.J. White, W.H.I. McLean
digital webbing, oesophageal strictures, oral and other mucosal lesions are common to both conditions. However, photosensitivity and reticulate pigmentation are predominant only in KS patients. Also, in contrast to Kindler syndrome, blisters in DEB patients tend to heal with severe scarring and furthermore, the cornea and conjunctiva are often involved. Affected DEB individuals also have a predilection to develop multiple cutaneous squamous cell carcinomas, which usually prove fatal by early adulthood. While there have been suggestions that KS patients have an increased risk of mucocutaneous malignancy, the number of individuals known to be affected is very small. Specifically, two groups have reported KS individuals with mucosal malignancy; one patient developed a squamous cell carcinoma of the palate, the other had a squamous cell carcinoma of the lip and a transitional cell carcinoma of the bladder [5,10].
2. Histopathology Histologically, KS skin lesions show atrophy of the epidermis, vacuolisation of the basal layer with clefting between the epidermis and dermis, telangiectatic vessels and, frequently, pigmentary incontinence of the papillary dermis [1,11]. Electron microscopy reveals reduplication of the lamina densa, collagen lysis within the upper dermis and focal clefting in the lamina lucida, as shown in Fig. 2 [4,11]. Hemidesmosomes and anchoring fibrils appear normal. This is in contrast to DEB skin lesions which show blistering below the lamina densa and a slight reduction in the number of anchoring fibrils or subtle changes in fibril diameter [12].
3. Molecular basis of Kindler syndrome
Fig. 1 Clinical appearance of the hands of 7- and 9-yearold Omani siblings with Kindler syndrome. Poikilodermatous change, with considerable skin wrinkling and atrophy, and partial pseudosyndactyly of the fingers are apparent.
Although Kindler syndrome was distinguished from DEB on immunohistochemical, ultrastructural and molecular grounds in the 1990s [4], the pathological basis of the syndrome remained unknown. This was resolved several years later when genome-wide linkage analysis by two separate groups mapped the KS causative gene to chromosome 20p12.3 [13,14]. This genetic locus is distinct from all 10 known epidermolysis bullosa gene loci. In particular, DEB is associated with mutations in the type VII collagen gene (COL7A1), which has been mapped to chromosome 3p21 [15]. The KS gene locus was defined by markers D20S95 and D20S192, corresponding to 280 kb of genomic DNA, and contained six known or predicted genes. Loss-of-function mutations were identified in the FLJ20116/
Mutations in a novel actin-associated protein cause Kindler syndrome
171
Fig. 2 Typical ultrastructural features of Kindler syndrome. Keratin filaments (KIF) and hemidesmosomes (arrows) appear normal. There is evidence of collagen lysis (CL) in the upper dermis. There is not an obvious level of blistering as would be the case in epidermolysis bullosa. A very characteristic finding in Kindler syndrome is extensive reduplication of the basement membrane throughout the upper reaches of the dermis (arrowheads). K = basal keratinocyte; D = dermis; bar = 1 mm.
C20orf42 gene, renamed KIND1 by Siegel et al., encoding the protein kindlin-1 [13]. The KIND1 gene spans 48.5 kb of genomic DNA and consists of 15 exons, with the initiating methionine in exon 2. Exon sizes range from 47 to 234 bp and the predicted open reading frame is 2034 bp, encoding a protein of 677 amino acids with a calculated molecular weight of 77.3 kDa.
4. Kindlin protein structure and predicted function Siegel and colleagues analysed the primary protein structure of the kindlin-1 polypeptide and discovered several characteristics that suggest kindlin-1 is involved in anchoring the actin cytoskeleton to the
plasma membrane [13]. Kindlin-1 has N-terminal homology to filopodin, a Dictyostelium talin homolog and C-terminal homology to human talin. These proteins are involved in anchoring the actin cytoskeleton in focal adhesion plaques. Focal adhesions are complex, dynamic structures that physically link the actin cytoskeleton to the extracellullar matrix and can be observed in cultured cells [16]. In vivo studies using green fluorescent protein-labelled kindlin-1 have shown that kindlin-1 in cultured epithelial cells associates with vinculin and actin at focal adhesions (Fig. 3) [13]. Kindlin-1 also possesses two regions of homology with the FERM (filopodin and ezrin/radixin/moesin) domain, which is shared by erythrocyte protein 4.1, ERM (ezrin, radixin and moesin) proteins, and several other proteins that are involved in cytoskeleton attachment to the
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Fig. 3 Kindlin-1 associates with vinculin and actin at focal adhesions. (a)—(c) Confocal scanning laser micrographs of PtK2 cells transiently transfected with EGFP-kindlin-1 (green) and stained with Alexa Fluor 594 phalloidin to reveal filamentous actin (red). Panel (a) shows a merged image of panels (b) and (c). Panel (b) shows EGFP-kindlin-1 localization
Mutations in a novel actin-associated protein cause Kindler syndrome
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Fig. 4 Schematic diagram of kindlin-1, showing the bipartite FERM domain, Pleckstrin homology (PH) domain and regions with homology to filopodin and talin. The location of reported frameshift and non-sense mutations are also shown. Splice site mutations 385 + 2T > C and 958-1G > A have also been reported [13,14,23,24].
plasma membrane [17]. Unusually, kindlin-1 has a bipartite FERM domain that is interrupted by a PH (pleckstrin homology) domain, as shown diagrammatically in Fig. 4. PH domains mediate associations with specific phosphatidylinositol phospholipids in the plasma membrane and are a feature of cytoskeletal-associated and/or cell-signalling molecules [18]. All of these features strongly support the suggestion of a structural/signalling function for kindlin-1.
5. Kindlin protein family Kindlin-1 shows closest homology to the Caenorhabditis elegans protein UNC-112. UNC-112 is a membrane-associated structural/signalling protein involved in the assembly of cell-extracellular matrix adhesions in the muscle of C. elegans [19]. Cellextracellular matrix adhesions are required at the embryonic stage of C. elegans development for elongation and motion. As development progresses, the C. elegans embryo lengthens to form a worm. Space is limited within the eggshell so the embryo must fold up twice during elongation: the two- and three-fold stages of embryogenesis. UNC-112 is required for the correct assembly of adhesive structures and loss-of-function mutations in UNC-112 lead to lethal pat (paralysed, arrested elongation at twofold) phenotypes. The pat phenotype is characterized by failure of muscle-extracellular matrix adhesion, which is essentially the nematode equiva-
lent of blistering in humans. Therefore, this scenario mirrors the defect seen in Kindler syndrome whereby loss-of-function mutations in KIND1 lead to a loss of adhesion at the dermal/epidermal interface. The fact that worms only have one UNC-112 yet humans have been found to have three different kindlin proteins, may explain the non-lethality in Kindler syndrome. Two further human proteins, products of the gene MIG-2(mitogen-induced gene 2) and the predicted gene MGC10966, have significant overall sequence homology to UNC-112 and kindlin-1 and have the same FERM/PH domain organization. It has been proposed to rename these proteins/genes as kindlin-2/KIND2 and kindlin-3/KIND3 respectively [13]. MIG-2(KIND2) is located on 14q22 and Mig2/kindlin-2 has been shown to associate with migfilin in cell adhesions with migfilin in turn interacting with the actin binding protein filamin [20]. KIND3 (also referred to as MIG2B) [21] is located on 11q12 and, to date, no specific protein interactions of kindlin-3 have been described. Multiple-tissue cDNA panels have been used to highlight the different expression patterns of the three kindlin genes. KIND1 is highly expressed in keratinocytes, weakly expressed in spleen, thymus, brain and lung and not expressed in peripheral blood leukocytes or skeletal muscle. KIND2 expression is moderate in spleen, liver and colon and weak in thymus, brain, skeletal muscle and keratinocytes. KIND3 however is strongly expressed in spleen, thy-
and panel (c) shows actin localization. The ends of actin stress fibre bundles exhibit co-alignment with EGFP-kindlin-1 to produce orange coloration in the merged image (arrowheads). These structures were reminiscent of focal contacts. The area marked by the arrow is enlarged in the inset panels, where the co-localization is more easily seen. (d)—(f) Confocal scanning laser micrographs of PtK2 cells transiently transfected with EGFP-kindlin (green) and stained for vinculin by indirect immunofluorescence using Alexa Fluor 594 (red) to reveal focal contacts. Panel (d) shows a merged image of panels (e) and (f). Panel (d) shows EGFP-kindlin localization. Panel (e) shows vinculin localization. Focal contacts (arrowheads) are seen to co-align with EGFP-kindlin to produce orange coloration in the merged image. Some faint colocalization with cytoskeletal structures also can be seen. The area marked by the arrow is enlarged in the inset panels, where the co-localization is more easily seen. This data confirmed that kindlin-1 is a component of focal contacts.
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mus and peripheral leukocytes, particularly in B cells [21] with weaker expression in colon, brain and skeletal muscle and no expression in keratinocytes [13]. Weinstein et al. reported over-expression of KIND1 in colon and lung carcinomas with little or no overexpression of KIND2 or KIND3 [22]. The significance of this finding is unclear at present, however it may represent changes in kindlin-1 cell-signalling and/or adhesion consistent with a role in tumorigenesis or metastasis. Similarly, increased expression of KIND3 has been reported in B-cell tumours, especially chronic lymphocytic leukaemia [21]. This may also support the suggestion that kindlin proteins can be involved in malignancies. Obviously a great deal more investigation would be necessary to support this hypothesis. Immunohistochemical analysis of normal skin showed kindlin-1 to be present exclusively within the epidermis, particularly in basal keratinocytes. In Kindler patients however, epidermal staining for kindlin-1 was greatly reduced and was almost completely depleted in basal keratinocytes [13]. As yet there has not been kindlin-1 immunohistochemical evaluation of any of the other epithelia, such as the oral mucosa, that are often involved in Kindler syndrome.
6. Conclusions and future directions The rapid advancement of molecular biological techniques over the past decade has led to a much greater understanding of the genetics of many different inherited skin disorders. With the basic molecular defect in Kindler syndrome now identified, some 50 years after it was first reported, there is a need for clarification of exactly how loss of kindlin1 leads to the multiple phenotypic effects observed in affected individuals. There have been almost twenty loss-of-function mutations identified in KS patients to date (Fig. 4) and as more patients are correctly diagnosed this number is likely to increase. With sufficient numbers genotype/phenotype correlation studies would be feasible and may help provide a clue to the occurrence of the various clinical features of the syndrome. Kindler syndrome is the first demonstration of a defect in the actin—ECM system rather than keratin—ECM linkage causing an inherited skin fragility disorder. The identification of two further kindlin proteins, kindlin-2 and kindlin-3, raises the possibility of pathogenic mutations in their respective genes. The molecular organization of the kindlin proteins suggests they play a role in providing cell structure and/or cell-signalling. Studies to determine protein—protein interactions of all three kin-
S.J. White, W.H.I. McLean
dlins and also the use of siRNA technology may help establish their respective intracellular and tissuespecific functions. Finally, the development of animal models of Kindler syndrome may lead to a fuller understanding of kindlin-1 function in the epidermis and other epithelia. Such models may also be ideal for the assessment of the affects of UV light and could therefore be key in helping explain photosensitivity as a feature of the syndrome. For example, a kindlin-1 null-mutation back-crossed onto the hairless mouse background would allow testing of UV-sensitivity in mice. Successful animal models could also be of great importance in testing gene replacement therapies.
Acknowledgements The many patients and research groups referred to in this review are gratefully acknowledged. Thanks also to J.A. McGrath for kindly donating clinical and ultrastructural pictures. W.H.I.M. is funded by a Wellcome Trust Senior Research Fellowship and S.J.W. is funded by an MRC Clinical Research Training Fellowship. Work by this group is also supported by grants from DEBRA (UK) and the Pachyonychia Congenita Project.
References [1] Kindler T. Congenital poikiloderma with traumatic bulla formation and progressive cutaneous atrophy. Br J Dermatol 1954;66(3):104—11. [2] Hacham-Zadeh S, Garfunkel AA. Kindler syndrome in two related Kurdish families. Am J Med Genet 1985;20(1):43—8. [3] Ban M, et al. Kindler’s syndrome with recurrence of bullae in the fifth decade. Br J Dermatol 1996;135(3):503—4. [4] Shimizu H, et al. Immunohistochemical, ultrastructural, and molecular features of Kindler syndrome distinguish it from dystrophic epidermolysis bullosa. Arch Dermatol 1997;133 (9):1111—7. [5] Alper JC, Baden HP, Goldsmith LA. Kindler’s syndrome. Arch Dermatol 1978;114(3):457. [6] Al Aboud K, et al. Kindler syndrome in a Saudi kindred. Clin Exp Dermatol 2002;27(8):673—6. [7] Sharma RC, et al. Kindler syndrome. Int J Dermatol 2003;42(9):727—32. [8] Ricketts DN, et al. Kindler syndrome: a rare cause of desquamative lesions of the gingiva. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997;84(5):488—91. [9] Suga Y, et al. A Japanese case of Kindler syndrome. Int J Dermatol 2000;39(4):284—6. [10] Lotem M, et al. Kindler syndrome complicated by squamous cell carcinoma of the hard palate: successful treatment with high-dose radiation therapy and granulocyte—macrophage colony-stimulating factor. Br J Dermatol 2001;144(6):1284— 6.
Mutations in a novel actin-associated protein cause Kindler syndrome
[11] Haber RM, Hanna WM. Kindler syndrome. Clinical and ultrastructural findings. Arch Dermatol 1996;132(12): 1487—90. [12] Mallipeddi R, et al. Dilemmas in distinguishing between dominant and recessive forms of dystrophic epidermolysis bullosa. Br J Dermatol 2003;149(4):810—8. [13] Siegel DH, et al. Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin—extracellular-matrix linker protein UNC-112, causes Kindler syndrome. Am J Hum Genet 2003;73(1):174—87. [14] Jobard F, et al. Identification of mutations in a new gene encoding a FERM family protein with a pleckstrin homology domain in Kindler syndrome. Hum Mol Genet 2003;12 (8):925—35. [15] Uitto J, Pulkkinen L, Christiano AM. Molecular basis of the dystrophic and junctional forms of epidermolysis bullosa: mutations in the type VII collagen and kalinin (laminin 5) genes. J Invest Dermatol 1994;103(5 Suppl):39S—46S. [16] Wehrle-Haller B, Imhof B. The inner lives of focal adhesions. Trends Cell Biol 2002;12(8):382—9. [17] Chishti AH, et al. The FERM domain: a unique module involved in the linkage of cytoplasmic proteins to the membrane. Trends Biochem Sci 1998;23(8):281—2. [18] Maffucci T, Falasca M. Specificity in pleckstrin homology (PH) domain membrane targeting: a role for a phosphoinositide— protein co-operative mechanism. FEBS Lett 2001;506 (3):173—9. [19] Rogalski TM, et al. The UNC-112 gene in Caenorhabditis elegans encodes a novel component of cell-matrix adhesion structures required for integrin localization in the muscle cell membrane. J Cell Biol 2000;150(1):253—64. [20] Tu Y, et al. Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation. Cell 2003;113(1):37—47. [21] Boyd RS, et al. Proteomic analysis of the cell-surface membrane in chronic lymphocytic leukemia: identification of two novel proteins, BCNP1 and MIG2B. Leukemia 2003;17 (8):1605—12. [22] Weinstein EJ, et al. URP1: a member of a novel family of PH and FERM domain-containing membrane-associated proteins is significantly over-expressed in lung and colon carcinomas. Biochim Biophys Acta 2003;1637(3):207—16. [23] Ashton GH, et al. Recurrent mutations in kindlin-1, a novel keratinocyte focal contact protein, in the autosomal recessive skin fragility and photosensitivity disorder, Kindler syndrome. J Invest Dermatol 2004;122(1):78—83.
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[24] Ashton GH. Kindler syndrome. Clin Exp Dermatol 2004;29 (2):116—21. Sharon J. White graduated from the University of Dundee, Scotland with a BMSc(Honours) in Anatomy in 1993 and a BDS with Honours in 1996. She completed general professional training in 1998 then worked for 3 years as a general dental practitioner in Glasgow, Scotland. In 2001 she returned to Ninewells Hospital, Dundee as a Senior House Officer in Oral Pathology and in 2002 obtained Membership of the Faculty of Dental Surgery (MFDS) of the Royal College of Physicians and Surgeons of Glasgow. She was awarded an MRC Clinical Research Training Fellowship in 2003 and is currently working towards a PhD in Molecular Genetics in the Epithelial Genetics Group, Ninewells, Dundee. She also holds an Honorary Specialist Registrar post in Oral Pathology. Her main interest is oral and maxillofacial pathology, particularly the oral manifestations of hereditary skin disorders. W.H. Irwin McLean graduated from The Queen’s University of Belfast, Northern Ireland with a BSc(Honours) in Microbiology in 1985 and a PhD in Human Genetics in 1988. He worked as a Research Assistant in Medical Genetics in Belfast from 1985 to 1991 and as a Postdoctoral Fellow in Department of Anatomy and Physiology, University of Dundee, Scotland from 1992 to 1996. He moved to Thomas Jefferson University, Philadelphia, USA in 1996 where he was Associate Professor in Dermatology and Cutaneous Biology until 1998, when he returned to Ninewells Medical School at the University of Dundee as a Wellcome Trust Senior Research Fellow and from 2002, as Professor of Human Genetics. He was awarded a DSc degree from The Queen’s University of Belfast in 1999 for his work on inherited skin diseases. He sits on the Editorial boards of the Journal of Investigative Dermatology and Journal of Dermatological Science. His main research interest is understanding the genetics and molecular pathology of hereditary diseases that cause fragility of the epidermis, its appendages and other epithelial tissues. Recently, the emphasis of his work has begun to move away from gene mapping and gene function towards therapy development for inherited skin disorders.
www.intl.elsevierhealth.com/journals/jods
REVIEW ARTICLE
Kindler surprise: mutations in a novel actinassociated protein cause Kindler syndrome Sharon J. White *, W.H. Irwin McLean Epithelial Genetics Group, Human Genetics Unit, University of Dundee, Ninewells Medical School, Dundee DD1 9SY, UK Received 29 October 2004; received in revised form 21 December 2004; accepted 22 December 2004
KEYWORDS Kindler syndrome; Genodermatosis; Acral blistering; Photosensitivity; Actin cytoskeleton; Extracellular matrix
Summary Kindler syndrome is an autosomal recessive genodermatosis characterized by acral blistering in neonates and diffuse, progressive poikiloderma in later life. Other clinical features include photosensitivity, premature skin ageing and severe periodontal disease. Two groups have recently shown that the molecular basis of Kindler syndrome is loss of a novel epidermal protein, kindlin-1, encoded by the gene KIND1. Two additional kindlin proteins, kindlin-2 and kindlin-3, have also been described. Kindlin-1 is considered to be a component in the linkage of the actin cytoskeleton to the extracellular matrix and as such is proposed to have both structural and cell-signalling functions. Kindler syndrome is therefore the first skin fragility syndrome due to disruption of the actin—extracellular matrix system. # 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Contents Introduction and clinical features . . . . . . . . . . Histopathology . . . . . . . . . . . . . . . . . . . . . . Molecular basis of Kindler syndrome. . . . . . . . . Kindlin protein structure and predicted function . Kindlin protein family . . . . . . . . . . . . . . . . . . Conclusions and future directions . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . .
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169 170 170 171 173 174 174 174
1. Introduction and clinical features
* Corresponding author. Tel.: +44 1382425618; fax: +44 1382425619. E-mail address: [email protected] (S.J. White).
Kindler syndrome (KS; OMIM 173650) was first described in a single patient by Dr. Theresa Kindler in the 1950s [1]. The original patient was a 14-yearold girl who appeared to have a combination of
0923-1811/$30.00 # 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2004.12.026
170
features of two previously described conditions, epidermolysis bullosa hereditaria and poikiloderma congenitale. The characteristic clinical features are acral blistering and photosensitivity in infancy followed by progressive poikiloderma (diffuse cutaneous atrophy, telangiectases and reticulate pigmentation) later in life [1,2]. Blistering may be spontaneous or traumatic and usually resolves with age, although there is one reported case where bullae recurred in the fifth decade [3]. Nikolsky’s sign is negative [1,4]. Cutaneous atrophy may be widespread although it is most prominent on the dorsa of the hands and feet and on the knees and elbows, giving rise to the so-called ‘cigarettepaper’ like wrinkling of the skin, as shown in Fig. 1 [1]. Several other features have been reported which vary between cases including: digital webbing; palmoplantar keratoderma; nail dystrophy; alopecia; oesophageal, anal, vaginal and urethral stenosis; loss of dermatoglyphics; and phimosis [1,3,5—7]. A wide range of oral manifestations of the syndrome have also been described including mucosal white patches, atrophy and blistering; gingival bleeding and severe periodontitis; ankyloglossia; restricted mouth opening; and malocclusion [1,2,6—9]. Several of the clinical features of Kindler syndrome resemble those found in various other skin fragility disorders, especially those in dystrophic epidermolysis bullosa (DEB; OMIM 226600). Such overlap means there may be difficulties in making a definitive diagnosis of Kindler syndrome, especially in early childhood. Trauma-induced blistering,
S.J. White, W.H.I. McLean
digital webbing, oesophageal strictures, oral and other mucosal lesions are common to both conditions. However, photosensitivity and reticulate pigmentation are predominant only in KS patients. Also, in contrast to Kindler syndrome, blisters in DEB patients tend to heal with severe scarring and furthermore, the cornea and conjunctiva are often involved. Affected DEB individuals also have a predilection to develop multiple cutaneous squamous cell carcinomas, which usually prove fatal by early adulthood. While there have been suggestions that KS patients have an increased risk of mucocutaneous malignancy, the number of individuals known to be affected is very small. Specifically, two groups have reported KS individuals with mucosal malignancy; one patient developed a squamous cell carcinoma of the palate, the other had a squamous cell carcinoma of the lip and a transitional cell carcinoma of the bladder [5,10].
2. Histopathology Histologically, KS skin lesions show atrophy of the epidermis, vacuolisation of the basal layer with clefting between the epidermis and dermis, telangiectatic vessels and, frequently, pigmentary incontinence of the papillary dermis [1,11]. Electron microscopy reveals reduplication of the lamina densa, collagen lysis within the upper dermis and focal clefting in the lamina lucida, as shown in Fig. 2 [4,11]. Hemidesmosomes and anchoring fibrils appear normal. This is in contrast to DEB skin lesions which show blistering below the lamina densa and a slight reduction in the number of anchoring fibrils or subtle changes in fibril diameter [12].
3. Molecular basis of Kindler syndrome
Fig. 1 Clinical appearance of the hands of 7- and 9-yearold Omani siblings with Kindler syndrome. Poikilodermatous change, with considerable skin wrinkling and atrophy, and partial pseudosyndactyly of the fingers are apparent.
Although Kindler syndrome was distinguished from DEB on immunohistochemical, ultrastructural and molecular grounds in the 1990s [4], the pathological basis of the syndrome remained unknown. This was resolved several years later when genome-wide linkage analysis by two separate groups mapped the KS causative gene to chromosome 20p12.3 [13,14]. This genetic locus is distinct from all 10 known epidermolysis bullosa gene loci. In particular, DEB is associated with mutations in the type VII collagen gene (COL7A1), which has been mapped to chromosome 3p21 [15]. The KS gene locus was defined by markers D20S95 and D20S192, corresponding to 280 kb of genomic DNA, and contained six known or predicted genes. Loss-of-function mutations were identified in the FLJ20116/
Mutations in a novel actin-associated protein cause Kindler syndrome
171
Fig. 2 Typical ultrastructural features of Kindler syndrome. Keratin filaments (KIF) and hemidesmosomes (arrows) appear normal. There is evidence of collagen lysis (CL) in the upper dermis. There is not an obvious level of blistering as would be the case in epidermolysis bullosa. A very characteristic finding in Kindler syndrome is extensive reduplication of the basement membrane throughout the upper reaches of the dermis (arrowheads). K = basal keratinocyte; D = dermis; bar = 1 mm.
C20orf42 gene, renamed KIND1 by Siegel et al., encoding the protein kindlin-1 [13]. The KIND1 gene spans 48.5 kb of genomic DNA and consists of 15 exons, with the initiating methionine in exon 2. Exon sizes range from 47 to 234 bp and the predicted open reading frame is 2034 bp, encoding a protein of 677 amino acids with a calculated molecular weight of 77.3 kDa.
4. Kindlin protein structure and predicted function Siegel and colleagues analysed the primary protein structure of the kindlin-1 polypeptide and discovered several characteristics that suggest kindlin-1 is involved in anchoring the actin cytoskeleton to the
plasma membrane [13]. Kindlin-1 has N-terminal homology to filopodin, a Dictyostelium talin homolog and C-terminal homology to human talin. These proteins are involved in anchoring the actin cytoskeleton in focal adhesion plaques. Focal adhesions are complex, dynamic structures that physically link the actin cytoskeleton to the extracellullar matrix and can be observed in cultured cells [16]. In vivo studies using green fluorescent protein-labelled kindlin-1 have shown that kindlin-1 in cultured epithelial cells associates with vinculin and actin at focal adhesions (Fig. 3) [13]. Kindlin-1 also possesses two regions of homology with the FERM (filopodin and ezrin/radixin/moesin) domain, which is shared by erythrocyte protein 4.1, ERM (ezrin, radixin and moesin) proteins, and several other proteins that are involved in cytoskeleton attachment to the
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Fig. 3 Kindlin-1 associates with vinculin and actin at focal adhesions. (a)—(c) Confocal scanning laser micrographs of PtK2 cells transiently transfected with EGFP-kindlin-1 (green) and stained with Alexa Fluor 594 phalloidin to reveal filamentous actin (red). Panel (a) shows a merged image of panels (b) and (c). Panel (b) shows EGFP-kindlin-1 localization
Mutations in a novel actin-associated protein cause Kindler syndrome
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Fig. 4 Schematic diagram of kindlin-1, showing the bipartite FERM domain, Pleckstrin homology (PH) domain and regions with homology to filopodin and talin. The location of reported frameshift and non-sense mutations are also shown. Splice site mutations 385 + 2T > C and 958-1G > A have also been reported [13,14,23,24].
plasma membrane [17]. Unusually, kindlin-1 has a bipartite FERM domain that is interrupted by a PH (pleckstrin homology) domain, as shown diagrammatically in Fig. 4. PH domains mediate associations with specific phosphatidylinositol phospholipids in the plasma membrane and are a feature of cytoskeletal-associated and/or cell-signalling molecules [18]. All of these features strongly support the suggestion of a structural/signalling function for kindlin-1.
5. Kindlin protein family Kindlin-1 shows closest homology to the Caenorhabditis elegans protein UNC-112. UNC-112 is a membrane-associated structural/signalling protein involved in the assembly of cell-extracellular matrix adhesions in the muscle of C. elegans [19]. Cellextracellular matrix adhesions are required at the embryonic stage of C. elegans development for elongation and motion. As development progresses, the C. elegans embryo lengthens to form a worm. Space is limited within the eggshell so the embryo must fold up twice during elongation: the two- and three-fold stages of embryogenesis. UNC-112 is required for the correct assembly of adhesive structures and loss-of-function mutations in UNC-112 lead to lethal pat (paralysed, arrested elongation at twofold) phenotypes. The pat phenotype is characterized by failure of muscle-extracellular matrix adhesion, which is essentially the nematode equiva-
lent of blistering in humans. Therefore, this scenario mirrors the defect seen in Kindler syndrome whereby loss-of-function mutations in KIND1 lead to a loss of adhesion at the dermal/epidermal interface. The fact that worms only have one UNC-112 yet humans have been found to have three different kindlin proteins, may explain the non-lethality in Kindler syndrome. Two further human proteins, products of the gene MIG-2(mitogen-induced gene 2) and the predicted gene MGC10966, have significant overall sequence homology to UNC-112 and kindlin-1 and have the same FERM/PH domain organization. It has been proposed to rename these proteins/genes as kindlin-2/KIND2 and kindlin-3/KIND3 respectively [13]. MIG-2(KIND2) is located on 14q22 and Mig2/kindlin-2 has been shown to associate with migfilin in cell adhesions with migfilin in turn interacting with the actin binding protein filamin [20]. KIND3 (also referred to as MIG2B) [21] is located on 11q12 and, to date, no specific protein interactions of kindlin-3 have been described. Multiple-tissue cDNA panels have been used to highlight the different expression patterns of the three kindlin genes. KIND1 is highly expressed in keratinocytes, weakly expressed in spleen, thymus, brain and lung and not expressed in peripheral blood leukocytes or skeletal muscle. KIND2 expression is moderate in spleen, liver and colon and weak in thymus, brain, skeletal muscle and keratinocytes. KIND3 however is strongly expressed in spleen, thy-
and panel (c) shows actin localization. The ends of actin stress fibre bundles exhibit co-alignment with EGFP-kindlin-1 to produce orange coloration in the merged image (arrowheads). These structures were reminiscent of focal contacts. The area marked by the arrow is enlarged in the inset panels, where the co-localization is more easily seen. (d)—(f) Confocal scanning laser micrographs of PtK2 cells transiently transfected with EGFP-kindlin (green) and stained for vinculin by indirect immunofluorescence using Alexa Fluor 594 (red) to reveal focal contacts. Panel (d) shows a merged image of panels (e) and (f). Panel (d) shows EGFP-kindlin localization. Panel (e) shows vinculin localization. Focal contacts (arrowheads) are seen to co-align with EGFP-kindlin to produce orange coloration in the merged image. Some faint colocalization with cytoskeletal structures also can be seen. The area marked by the arrow is enlarged in the inset panels, where the co-localization is more easily seen. This data confirmed that kindlin-1 is a component of focal contacts.
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mus and peripheral leukocytes, particularly in B cells [21] with weaker expression in colon, brain and skeletal muscle and no expression in keratinocytes [13]. Weinstein et al. reported over-expression of KIND1 in colon and lung carcinomas with little or no overexpression of KIND2 or KIND3 [22]. The significance of this finding is unclear at present, however it may represent changes in kindlin-1 cell-signalling and/or adhesion consistent with a role in tumorigenesis or metastasis. Similarly, increased expression of KIND3 has been reported in B-cell tumours, especially chronic lymphocytic leukaemia [21]. This may also support the suggestion that kindlin proteins can be involved in malignancies. Obviously a great deal more investigation would be necessary to support this hypothesis. Immunohistochemical analysis of normal skin showed kindlin-1 to be present exclusively within the epidermis, particularly in basal keratinocytes. In Kindler patients however, epidermal staining for kindlin-1 was greatly reduced and was almost completely depleted in basal keratinocytes [13]. As yet there has not been kindlin-1 immunohistochemical evaluation of any of the other epithelia, such as the oral mucosa, that are often involved in Kindler syndrome.
6. Conclusions and future directions The rapid advancement of molecular biological techniques over the past decade has led to a much greater understanding of the genetics of many different inherited skin disorders. With the basic molecular defect in Kindler syndrome now identified, some 50 years after it was first reported, there is a need for clarification of exactly how loss of kindlin1 leads to the multiple phenotypic effects observed in affected individuals. There have been almost twenty loss-of-function mutations identified in KS patients to date (Fig. 4) and as more patients are correctly diagnosed this number is likely to increase. With sufficient numbers genotype/phenotype correlation studies would be feasible and may help provide a clue to the occurrence of the various clinical features of the syndrome. Kindler syndrome is the first demonstration of a defect in the actin—ECM system rather than keratin—ECM linkage causing an inherited skin fragility disorder. The identification of two further kindlin proteins, kindlin-2 and kindlin-3, raises the possibility of pathogenic mutations in their respective genes. The molecular organization of the kindlin proteins suggests they play a role in providing cell structure and/or cell-signalling. Studies to determine protein—protein interactions of all three kin-
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dlins and also the use of siRNA technology may help establish their respective intracellular and tissuespecific functions. Finally, the development of animal models of Kindler syndrome may lead to a fuller understanding of kindlin-1 function in the epidermis and other epithelia. Such models may also be ideal for the assessment of the affects of UV light and could therefore be key in helping explain photosensitivity as a feature of the syndrome. For example, a kindlin-1 null-mutation back-crossed onto the hairless mouse background would allow testing of UV-sensitivity in mice. Successful animal models could also be of great importance in testing gene replacement therapies.
Acknowledgements The many patients and research groups referred to in this review are gratefully acknowledged. Thanks also to J.A. McGrath for kindly donating clinical and ultrastructural pictures. W.H.I.M. is funded by a Wellcome Trust Senior Research Fellowship and S.J.W. is funded by an MRC Clinical Research Training Fellowship. Work by this group is also supported by grants from DEBRA (UK) and the Pachyonychia Congenita Project.
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[11] Haber RM, Hanna WM. Kindler syndrome. Clinical and ultrastructural findings. Arch Dermatol 1996;132(12): 1487—90. [12] Mallipeddi R, et al. Dilemmas in distinguishing between dominant and recessive forms of dystrophic epidermolysis bullosa. Br J Dermatol 2003;149(4):810—8. [13] Siegel DH, et al. Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin—extracellular-matrix linker protein UNC-112, causes Kindler syndrome. Am J Hum Genet 2003;73(1):174—87. [14] Jobard F, et al. Identification of mutations in a new gene encoding a FERM family protein with a pleckstrin homology domain in Kindler syndrome. Hum Mol Genet 2003;12 (8):925—35. [15] Uitto J, Pulkkinen L, Christiano AM. Molecular basis of the dystrophic and junctional forms of epidermolysis bullosa: mutations in the type VII collagen and kalinin (laminin 5) genes. J Invest Dermatol 1994;103(5 Suppl):39S—46S. [16] Wehrle-Haller B, Imhof B. The inner lives of focal adhesions. Trends Cell Biol 2002;12(8):382—9. [17] Chishti AH, et al. The FERM domain: a unique module involved in the linkage of cytoplasmic proteins to the membrane. Trends Biochem Sci 1998;23(8):281—2. [18] Maffucci T, Falasca M. Specificity in pleckstrin homology (PH) domain membrane targeting: a role for a phosphoinositide— protein co-operative mechanism. FEBS Lett 2001;506 (3):173—9. [19] Rogalski TM, et al. The UNC-112 gene in Caenorhabditis elegans encodes a novel component of cell-matrix adhesion structures required for integrin localization in the muscle cell membrane. J Cell Biol 2000;150(1):253—64. [20] Tu Y, et al. Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation. Cell 2003;113(1):37—47. [21] Boyd RS, et al. Proteomic analysis of the cell-surface membrane in chronic lymphocytic leukemia: identification of two novel proteins, BCNP1 and MIG2B. Leukemia 2003;17 (8):1605—12. [22] Weinstein EJ, et al. URP1: a member of a novel family of PH and FERM domain-containing membrane-associated proteins is significantly over-expressed in lung and colon carcinomas. Biochim Biophys Acta 2003;1637(3):207—16. [23] Ashton GH, et al. Recurrent mutations in kindlin-1, a novel keratinocyte focal contact protein, in the autosomal recessive skin fragility and photosensitivity disorder, Kindler syndrome. J Invest Dermatol 2004;122(1):78—83.
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[24] Ashton GH. Kindler syndrome. Clin Exp Dermatol 2004;29 (2):116—21. Sharon J. White graduated from the University of Dundee, Scotland with a BMSc(Honours) in Anatomy in 1993 and a BDS with Honours in 1996. She completed general professional training in 1998 then worked for 3 years as a general dental practitioner in Glasgow, Scotland. In 2001 she returned to Ninewells Hospital, Dundee as a Senior House Officer in Oral Pathology and in 2002 obtained Membership of the Faculty of Dental Surgery (MFDS) of the Royal College of Physicians and Surgeons of Glasgow. She was awarded an MRC Clinical Research Training Fellowship in 2003 and is currently working towards a PhD in Molecular Genetics in the Epithelial Genetics Group, Ninewells, Dundee. She also holds an Honorary Specialist Registrar post in Oral Pathology. Her main interest is oral and maxillofacial pathology, particularly the oral manifestations of hereditary skin disorders. W.H. Irwin McLean graduated from The Queen’s University of Belfast, Northern Ireland with a BSc(Honours) in Microbiology in 1985 and a PhD in Human Genetics in 1988. He worked as a Research Assistant in Medical Genetics in Belfast from 1985 to 1991 and as a Postdoctoral Fellow in Department of Anatomy and Physiology, University of Dundee, Scotland from 1992 to 1996. He moved to Thomas Jefferson University, Philadelphia, USA in 1996 where he was Associate Professor in Dermatology and Cutaneous Biology until 1998, when he returned to Ninewells Medical School at the University of Dundee as a Wellcome Trust Senior Research Fellow and from 2002, as Professor of Human Genetics. He was awarded a DSc degree from The Queen’s University of Belfast in 1999 for his work on inherited skin diseases. He sits on the Editorial boards of the Journal of Investigative Dermatology and Journal of Dermatological Science. His main research interest is understanding the genetics and molecular pathology of hereditary diseases that cause fragility of the epidermis, its appendages and other epithelial tissues. Recently, the emphasis of his work has begun to move away from gene mapping and gene function towards therapy development for inherited skin disorders.