University of Groningen

Epidermolysis bullosa simplex Bolling, Maria Caroline

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Download date: 10-10-2021 5 Chromosomal microdeletion explains extracutaneous features observed in a patient with the monogenic genodermatosis Kindler syndrome

SJ White1*, MC Bolling2*, GT Spijker2, MP van den Berg3, PC van den Akker4, RG Hislop5, WHI McLean1, MF Jonkman2

1Epithelial Genetics Group, Division of Molecular Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee, DD1 5EH, UK; Departments of 2Dermatology, 3Cardiology, and 4Genetics, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; and 5Human Genetics Unit, NHS Tayside, Ninewells Hospital and Medical School, Dundee, DD1 9SY, UK.

Accepted for publication in the Journal of Investigative Dermatology

* Both authors contributed equally to this work. Chapter 5

Abstract

Kindler syndrome [MIM#173650] is an autosomal recessive genodermatosis that shares some features with epidermolysis bullosa. The molecular basis of the condition is loss-of-function mutations in the FERMT1 (or KIND1) encoding the actin/membrane-associated focal adhesion fermitin-family-homologue-1 (FFH-1, or kindlin-1). Here, we report on the oldest patient yet described with clinical mucocutaneous and intestinal features of Kindler syndrome, such as poikiloderma, recurrent actinic keratoses, cutaneous squamous cell carcinoma, gingival erosions, esophageal and urethral strictures, and intestinal problems. A seemingly homozygous FERMT1 loss-of-function mutation was found ([c.423_435del; c.448_450del], p.Ser142LeufsX14). However, additional clinical features not readily explainable by FFH-1 loss- of-function were present, like cardiac hypertrophy with intraventricular conduction delay, mild dysmorphic features, mental retardation, and joint deformities. Therefore, array comparative genomic hybridization (aCGH) was performed that revealed a heterozygous ~3 megabase microdeletion of 20p12.3 leading to monoallelic loss of several including FERMT1. Additionally, the gene BMP2 was lost which has been associated with a cardiogenetic arrhythmia disorder, facial dysmorphisms, mental retardation and skeletal malformations. Thus our patient has a contiguous gene syndrome. This study underscores the importance of performing aCGH in cases of seemingly homozygous mutations in monogenic disorders with additional unexplainable clinical features, to exclude a contiguous gene syndrome.

112 Contiguous gene defect in Kindler syndrome

Introduction

Kindler syndrome (KS; [MIM#173650]) is a rare autosomal recessive genodermatosis, with principal features of acral blistering and photosensitivity from early childhood.1, 2 Blistering tends to resolve with age, followed by a diffuse, progressive poikiloderma in later life. Many additional symptoms have been reported.3, 4 Loss of fermitin family homologue-1 (FFH-1, or kindlin-1) expression encoded by the gene FERMT1 (or KIND1), causes KS.5, 6 FFH-1 belongs to a family of that additionally includes FFH-2 and FFH-3, which localize to actin-cell matrix adhesion sites (focal adhesions) and are involved in regulating integrin function.7, 8 KS is the first genodermatosis resulting from defects in the transmembrane linking of the actin cytoskeleton to the extracellular matrix. Loss of FFH-1 in keratinocytes results in reduced cell proliferation, loss of cell polarity, decreased adhesion and increased apoptosis.9 To date, 40 different recessive FERMT1 mutations have been identified, the majority of which are predicted to lead to loss-of- function of FFH-1.4-6, 10-22 Here we report on the oldest patient with mucocutaneous features of KS and an unusual, seemingly homozygous FERMT1 mutation who displayed additional clinical features such as mental retardation, cardiac abnormalities, and skeletal malformation not readily explainable by FFH-1 loss of function. Subsequently, array comparative genomic hybridisation (aCGH) showed that this patient has a contiguous gene syndrome.

Materials and Methods

Immunofluorescence and electron microscopic analysis of patient’s skin samples One 4 mm-diameter punch biopsy (for immunofluorescence) and one 2 mm-diameter punch biopsy (for electron microscopy (EM)) were taken from atrophic skin of the dorsal aspect of the hand. The sample for immunofluorescence was snap frozen and the sample for EM was fixed in 2% glutaraldehyde. EM and immunofluorescence analysis were then performed as previously described.23 The following antibodies were used for immunofluorescence analysis: anti- Kindlin-1 C-terminus (rabbit polyclonal, gift from Prof. J.A. McGrath, London, UK), anti-keratin 5 (rabbit polyclonal BL18, gift from Prof. E.B. Lane, Dundee, Scotland), anti-keratin 14 (mouse clone RCK107, Abcam, Cambridge, UK), anti-laminin-332 (mouse clone GB3, Abcam, Cambridge, UK), anti-type VII collagen (mouse clone LH7.2) and anti-α-actinin (mouse clone BM-75.2) (both Sigma Aldrich, St. Louis, MO), anti-α3 integrin (mouse clone J143), anti-α6 integrin (rat clone GOH3), anti-β4 integrin (mouse clone 58XB4) (all gifts from Dr. A. Sonnenberg, Amsterdam, NL), anti-plectin (mouse clone HD121, gift from Dr. K. Owaribe, Nagoya, Japan), anti-actin (rabbit polyclonal, gift from Prof. G. Gabbiani, Geneva, Switzerland).

FERMT1 gene mutation analysis After informed consent, genomic DNA was obtained from the proband. PCR amplification of the FERMT1 gene was performed as described previously with slight modifications.5 Specific

113 Chapter 5 details on primers and amplification conditions can be obtained by contacting the authors. The study was conducted according to the Declaration of Helsinki Principles. The medical ethical committee of the University Medical Center Groningen, the Netherlands, approved all described studies.

Array comparative genomic hybridisation analysis An aCGH was performed on patient genomic DNA and sex-matched reference DNA (Promega, Southampton, UK) by using a CytoChip (version 2) bacterial artificial chromosome (BAC) array (BlueGnome, Cambridge, UK). Other than labelling 600ng (rather than 400ng) of each DNA sample, this test was done according to the manufacturer’s protocol. Briefly, the DNA samples were labelled overnight with Cy3-dCTP or Cy5-dCTP (GE Healthcare, Uppsala, Sweden) using the BioPrime labelling kit (Invitrogen, Paisley, UK). Unincorporated label was removed using Autoseq G50 columns (GE Healthcare). Overnight dye-swap hybridisations and washes were carried-out using a Tecan HS 400 Pro hybridisation station. Array slides were scanned on an Axon 4100A (Molecular Devises, Sunnyvale, CA, USA) and data analysed with BlueFuse (version 3.5) software (BlueGnome, Cambridge, UK).

Results

Mucocutaneous features of Kindler syndrome with additionally mental retardation, cardiac and skeletal abnormalities The patient is a 63-year-old Caucasian male with a history of blistering and photosensitivity (figure 1a; more detailed clinical images available as supplementary figure S1, at the end of the chapter). As far as is known, no family history of skin disorders was present. Blistering mostly affecting trunk and extremities was present from birth and was more pronounced in the summer, on sun exposure and on minor trauma. The tendency to blister decreased later in life. However, skin fragility and easily erosive skin persisted throughout life. The skin was generally very dry with a wrinkled, so-called ‘cigarette paper’ appearance, particularly on sun-exposed sites. There had been widespread progressive atrophy of the skin and ichthyosiform desquamation mainly on the chest, back and extremities. Telangiectases and areas of hyperpigmentation were evident. Although the patient avoided sun exposure when possible, he nonetheless developed recurrent actinic keratoses on the face and a squamous cell carcinoma of the dorsum of the left hand at age 59. Hair appeared normal. The nails, however, were fragile with a tendency to onychoschizia. Bilateral ectropion was present from early age and the patient suffered recurrent conjunctivitis. Recurrent operations were necessary to correct the reappearing ectropion. Desquamative gingivitis occurred from an early age and the patient had worn upper and lower dental prostheses since the age of 18 as a result of severe dental caries. Intestinal problems had been present since early childhood with alternate periods of diarrhoea and constipation, distal proctitis and anal fissures. A history of esophagitis with secondary stenosis and subsequent

114 Contiguous gene defect in Kindler syndrome dysphagia was confirmed. Urethral stenosis was present. The patient also had several skeletal and joint abnormalities that were present as long as the patient could remember (figure 1b), including pectus deformity, mild thoracolumbal scoliosis, general joint hyperextensibility and fixed hyperextension of the proximal interphalangeal joints of all fingers. Cardiac evaluation revealed mild left ventricular hypertrophy (echocardiogram) and an intraventricular conduction delay. The patient had learning difficulties and only finished specialized primary school. He cannot read or write and was never employed. Psychological testing revealed an Intelligence Quotient of 69 that fits the category (mild) “mental retardation” according to the classification from the Diagnostic & Statistical Manual of Mental Disorders (DSM-IV; http://www.psychiatryonline.com).

Atrophic epidermis with abnormal hemidesmosomes, but no lamina densa reduplications Histopathological examination of skin from the left shoulder displayed hyperkeratosis and atrophic changes including markedly flattened rete ridges and prominent dermal capillaries. EM of this skin showed perinuclear tonofilament retraction in basal cells with a poorly developed cytoskeleton. Hemidesmosomes appeared hypoplastic and rupture of the lamina densa with remains of cells in the upper dermis (figure 1c). However, there was no clear evidence of lamina densa reduplication in this biopsy, nor was there a visible cleft at any level.

Widened and interrupted basement membrane zone with altered distribution of laminin-332, type VII collagen, integrins, α-actinin, and actin, but unreduced FFH-1 staining Indirect immunofluorescence of patient skin with a polyclonal antibody against the FFH-1 C-terminus surprisingly was positive in patient’s skin (figure 1d, control in figure 1e). Keratin 14 positive, but keratin 5 negative, apoptotic bodies were observed in the papillary dermis (figure 1f). Staining for laminin-332 (data not shown) and type VII collagen (figure 1g) showed stretches with irregular and broadened staining, analogous with lamina densa duplication, alternated by stretches with normal linear staining, as observed before.12, 24 Ruptures of the basement membrane were observed. An intermittent and reduced pattern was also observed for integrin β4 (figure 1h; control shown in figure 1i), integrin α6 and plectin (data not shown). Staining for α-actinin showed punctate densities of staining close to the keratinocyte cell borders in patient skin (figure 1j), whereas in control skin the staining was more intense and linear along the entire borders of the keratinocytes (figure 1k). Actin appeared to have a more diffuse cytoplasmatic staining in patient skin (figure 1l) compared to the more peripheral staining in control skin (figure 1m). Integrin α3 was also reduced (figure 1n), compared to control skin (figure 1o).

Seemingly homozygous complex deletion in FERMT1 An unusual type of homozygous deletion mutation in exon 4, [423_435del; 448_450del], was found (figure 2) (GenBank accession no. NM_017671.4). This complex double deletion (13 bp and

115 Chapter 5

a b

c

d e

> > > * >

f gg

h i j k

l m n o

116 Contiguous gene defect in Kindler syndrome

 Figure 1 (previous page): Clinical features of Kindler syndrome in an elderly patient and immuno­ fluorescence analysis of skin. (a) Epidermal atrophy and squamous cell carcinoma of the dorsum of the hand. (b) Telangiectases and hyperpigmentation of the skin. Note thoracic scoliosis. More detailed clinical images are available as supplementary figure S1 at the end of the chapter. (c) Electron microscopy of atrophic skin revealed perinuclear tonofilament retraction (arrows) in basal cells with a poorly developed cytoskeleton. Hemidesmosomes appear hypoplastic (open arrowheads). Rupture of the lamina densa (straight line) with remains of cells in the upper dermis is visible (*). No lamina densa reduplication was seen in this biopsy, nor was there a visible cleft at any level. Scale bar = 1 mm. (d-o) Indirect immunofluorescence of patient’s skin (d, f, g, h, j, l n) and aged-matched human control skin (e, i, k, m, o): (d, e) FFH-1, (f) keratin 14, (g) type VII collagen, (h, i) integrin β4, (j, k) α-actinin, (l, m) actin, and (n, o) integrin α3. Scale bars in panels d-o = 20 mm.

3 bp) results in a frameshift and a premature termination codon (PTC) 14 codons downstream (p.Ser142LeufsX14). Nonsense-mediated decay of the mutant mRNA is predicted to occur with subsequent loss of FFH-1 protein expression. Supposing polypeptide synthesis from the mutant mRNA would happen, the resultant protein would be predicted to be non-functional as the overall 16 bp frameshift would lead to truncation of the pleckstrin homology domain, the bipartite FERM domain and the C-terminus, all of which are considered to be functionally critical 9.

Figure 2: Mutation analysis: double deletion in KIND1 exon 4. Upper sequence: a complex homozygous double deletion mutation was identified in the patient, within exon 4 of the KIND1 gene. This 13 bp plus 3 bp mutation is designated [423_435del; 448_450del] and the overall deletion of 16 bp leads to a frameshift within exon 4 (p.Ser142LeufsX14). Lower sequence: normal sequence of the equivalent region shown in the sequence above above. The bases bounded by the linked arrows represent those that are deleted in the patient with KS.

117 Chapter 5

Monoallelic 3 Mb microdeletion of chromosome 20p12.3 Array CGH revealed a deletion of the short (p) arm of . Seven consecutive clones (RP5-967N21 to RP4-654A7) were lost from 20p12.3, resulting in segmental monosomy for an interstitial region of approximately 3 megabases (Mb), including the FERMT1 gene (see figure 3 and table 1).

Table 1. Results aCGH Normal/Deleted Clone Start (bp) End (bp) Normal flanking RP5-828H9 5,222,982 5,443,401 Deleted RP5-967N21 5,835,384 5,974,735 Deleted RP5-959I16 6,209,827 6,351,388 Deleted RP5-944L9 6,769,485 6,907,224 Deleted RP4-764O22 7,012,215 7,110,690 Deleted RP4-611O11 7,800,924 7,945,860 Deleted RP5-1140M3 8,156,595 8,328,068 Deleted RP4-654A7 8,718,928 8,847,043 Normal flanking RP4-811H13 9,197,701 9,340,963

0.80

0.40

Log2 Ratio 0.00 Ch1/Ch2

-0.40

-0.80

Chromosome 20 p12.3 p12.2

0 10 20 30 40 50 60 Mb (hg18)

Mb (hg18) 5.2 Mb 5.5 Mb 6.0 Mb 6.5 Mb 7.0 Mb 7.5 Mb 8.0 Mb 8.5 Mb 9.0 Mb 9.5 Mb

Chromosome band 20p12.3 20p12.2 aCGH BAC clones RP5-828H9 RP5-967N21 RP5-959I16 RP5-994L9 RP4-764O22 RP4-611O11 RP5-1140M3 RP4-654A7 RP4-811H13

RefSeq Genes LOC149837 CHGB KIND1 BMP2 HAO1 PLCB1 PLCB4

RP5-1022F6.2 TRMT6

MCM8

CRLS1 Figure 3: Chromosome 20p12 deletion mapping by BAC array comparative genomic hybridisation (aCGH). A stretch of ~ 3 megabases, including the gene FERMT1 (KIND1) and several other genes, is deleted on the short arm (p) of chromosome 20 as observed by the loss of seven consecutive clones (RP5-967N21 to RP4-654A7, see also table 1) from 20p12.3. In particular, hemizygosity for the bone morphogenetic protein-2 gene (BMP2) may explain the skeletal abnormalities in the proband.

118 Contiguous gene defect in Kindler syndrome

Discussion

Our patient is the oldest patient reported to be definitely diagnosed with KS by genetic analysis and his phenotype is arguably one of the most severe described. This case highlights the importance of an accurate early diagnosis of KS to enable the provision of appropriate preventive medical and dental care. Diagnosis for KS patients will primarily allow screening for further complications, such as squamous cell carcinoma, and give a more accurate prognosis. Gastrointestinal tract involvement is rarely reported in KS, but studies on FERMT1 knockout mice have revealed the importance of FFH-1 for maintaining intestinal integrity.12, 25, 26 The EM and immunofluorescence findings in skin samples of our patient were initially somewhat confusing with absence of lamina densa reduplication and preserved FFH-1 staining. The absence of reduplication of the lamina densa on electron microscopic analysis of patient’s skin has been described before in KS27, and may be sample error (see below). This is supported by the immunofluorescence findings of widened and interrupted type VII collagen and lamini-332 staining. Moreover reduplication is not a specific finding for KS as it is also seen in other poikilodermatous disorders.24 Unfortunately specific antibodies against FFH-1 to provide in a rapid diagnosis by means of immunofluorescence analysis of patient skin are not available, as is also indicated by the preserved FFH-1 expression in our patient despite a heterozygous FERMT1 frameshift mutation compound heterozygous with complete deletion of FERMT1.28 The antibody used here is a rabbit polyclonal antibody to the FFH-1 C-terminal.5 The persistent positive staining in our patient may be due to read-through or exon-skipping of the FERMT1 transcript leading to an altered FFH-1 protein still containing the epitope(s) for the antibody. Alternatively, cross reactivity with the highly homologous FFH-2 which is also weakly expressed in the epidermis may exist (also known as Mig2).5 Positive indirect immunofluorescence for FFH-1 therefore cannot exclude a diagnosis of KS and molecular screening is necessary. The observed aggregation of α-actinin and loss or retraction of fibrillar actin may be caused by the loss of FFH-1, which is a component of focal adhesions as well.5 Recent co-immunoprecipitation studies indeed implicated α-actinin as an FFH-1 binding partner.22 The epidermal atrophy due to loss of FFH-1 that we also observed in our patient may result from perturbations of the interaction with integrin α3β1.29 Keratinocytes of KS patients show impaired cell adhesion, increased apoptosis and delayed cell spreading.9 Moreover, Has et al. observed reduced activation of Rac1, a Rho GTPase that can be activated by α3β1 integrin and in the epidermis is involved in maintenance of stem cells and cell-cell contacts, in KS cells.22 These data provide a possible mechanism for the FFH-1 loss-of-function-induced epidermal atrophy. Furthermore, perturbations in integrin α3β1 regulation upon FFH-1 loss may be the mechanism behind an increased risk of squamous cell carcinoma in KS. The mechanisms by which FFH-1 regulates integrin functioning are not yet fully understood. The findings by aCGH and the FERMT1 mutation analysis together revealed a monoallelic deletion of the complete FERMT1 gene and a complex mutation leading to a PTC in this gene on the other allele thus explaining the mucocutaneous features of KS. The heterozygous

119 Chapter 5 complex FERMT1 deletion is expected to affect all FFH-1 isoforms and indeed, the presence of intestinal symptoms in this patient may indicate that the colon-specific isoform is affected.5, 25 Many recessive diseases have revealed that heterozygous loss of gene expression does not have to result in disease. However, for other genes haploinsufficiency leading to disease has been reported. The gene FERMT1 encoding the protein FFH-1 is mainly expressed in the epithelia and only in low amounts in brain and skeletal muscle, while no expression in bone, cartilage or heart has been reported.5 The present case is monoallelic for several genes on 20p12.3 and showed clinical features that could not readily be explained by loss of FERMT1 expression, such as mental retardation, and cardiac and skeletal/joint abnormalities, suggesting that the heterozygous loss of the additional genes on chromosome 20p12 are involved. Interestingly, in a recent study five cases were reported with heterozygous microdeletions involving 20p12.3.30 These patients did not show the mucocutaneous features of KS confirming the recessive nature of the disorder. However, they showed mental retardation, as well as features of Wolff-Parkinson-White syndrome (WPW, four out of five), a cardiac conduction disorder due to abnormal electrical connections between atria and ventricles, structural cardiac abnormalities, and dysmorphic features. One case revealed a deletion only involving the gene BMP2, and clinically showed WPW, neurocognitive delay, pectus deformity, small stature, and slight dysmorphic features similar as in the present case. The data on the present case do not confirm the associations of heterozygous BMP2 gene deletions with WPW, as no pre-excitation (short PR-interval and delta-waves) was observed on the patient’s ECG. However, ECG abnormalities were clearly present (intraventricular conduction delay), as was left ventricular hypertrophy on the echocardiogram, indicating the presence of an as yet unspecified form of cardiomyopathy. The data on the previously reported cases and the present case indicate that microdeletions on 20p12 involving BMP2 are additionally associated with features like facial dismorphias, short stature, pectus deformities, and other bone and cartilage abnormalities. BMP2 encodes a bone morphogenic protein-2 (BMP-2), which is expressed in various tissues and thought to be involved in myocardial patterning, as well as bone and cartilage development. BMP2-/- mice showed embryonic lethality, whereas no information concerning above mentioned features was given for BMP2+/- mice. Of note, these mice failed to show a WPW phenotype.30 Additional genetic studies in patients with WPW will have to reveal the role of BMP2 gene abnormalities in this syndrome. The other genes involved in the deletion are CHGB encoding chromogranin-B, TRMT6 encoding tRNA methyltransferase 6 homolog (TRMT6), MCM8 encoding minichromosome maintenance protein 8 , CRLS1 encoding cardiolipin synthase-1 (CRLS1), HAO1 encoding hydroxyacid oxidase-1 (HAO1), and PLCB1 encoding phospholipase C beta-1 (PLCB1). Chromogranin B is a tyrosine-sulphated secretory protein that is expressed in endocrine and neuron cells.31 TRMT6 is a protein thought to be involved in tRNA processing, but the exact function and expression of TRMT6 is unknown.32 MCM8 is mainly expressed in lung, pancreas and lung, and at low levels in brain and heart.33 MCM8 belongs to the family of MCM proteins involved in DNA replication. CRLS is an enzyme involved in cardiolipin synthesis in the mitochondrial

120 Contiguous gene defect in Kindler syndrome membrane. Of note, cardiolipin deficiency is a key factor in Barth syndrome, an X-linked disorder characterized by severe cardiomyopathy, skeletal myopathy and mitochondrial dysfunction.34, 35 This protein was detected in heart, skeletal muscle, and liver.36 HAO1 is expressed in the liver, kidney and pancreas.37 PLCB1 expression was shown in human fetal brain.38 PLCB1 is an enzyme that catalyses the formation of inositol 1,4,5-triphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphanate. This enzyme reaction is important for intracellular transduction of many extracellular signals. As the function of many of these genes and their encoded proteins are not well characterized yet, the effect of the heterozygous loss of expression of these genes on the phenotype in our patient cannot be fully excluded, especially as some of them are also expressed in heart and brain. The present case illustrates that a contiguous gene syndrome should be considered when patients with monogenic disorders (and seemingly homozygous mutations) show clinical features that cannot be explained by the expression profile of the gene involved. Another candidate is for example case 1 with KS, skeletal deformities and neurocognitive delay reported by Nofal et al.39 We recommend performing additional aCGH analysis in those cases to provide a more accurate prognosis and improve genetic counselling. This is also important to prevent confusion and unnecessary concern for other patients with the same monogenic syndrome. Furthermore, this study yields information on the natural history of KS in later life, with obvious prognostic implications for younger patients.

Acknowledgements

We thank Dr. Marinus van Praag for his excellent support in obtaining additional clinical data and G.G. van der Haagen for the psychological tests. SJW is funded by a Clinical Research Training Fellowship from the Medical Research Council. MB is funded by the J.P. Nater Fund. The McLean laboratory is supported by grants from The Dystrophic Epidermolysis Bullosa Research Association, The Pachyonychia Congenita Project, the British Skin Foundation/National Eczema Society (WHIM and Frances J. D. Smith) and the Medical Research Council.

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References

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15. Has C, Bruckner-Tuderman L. A novel nonsense mutation in Kindler syndrome. J Invest Dermatol 2004;122(1):84-6. 16. Has C, Wessagowit V, Pascucci M, Baer C, Didona B, Wilhelm C, et al. Molecular basis of Kindler syndrome in Italy: novel and recurrent Alu/Alu recombination, splice site, nonsense, and frameshift mutations in the KIND1 gene. J Invest Dermatol 2006;126(8):1776-83. 17. Has C, Yordanova I, Balabanova M, Kazandjieva J, Herz C, Kohlhase J, et al. A novel large FERMT1 (KIND1) gene deletion in Kindler syndrome. J Dermatol Sci 2008;52(3):209-12. 18. Fassihi H, Wessagowit V, Jones C, Dopping-Hepenstal P, Denyer J, Mellerio JE, et al. Neonatal diagnosis of Kindler syndrome. J Dermatol Sci 2005;39(3):183-5. 19. Sethuraman G, Fassihi H, Ashton GH, Bansal A, Kabra M, Sharma VK, et al. An Indian child with Kindler syndrome resulting from a new homozygous nonsense mutation (C468X) in the KIND1 gene. Clin Exp Dermatol 2005;30(3):286-8. 20. Burch JM, Fassihi H, Jones CA, Mengshol SC, Fitzpatrick JE, McGrath JA. Kindler syndrome: a new mutation and new diagnostic possibilities. Arch Dermatol 2006;142(5):620-4. 21. Zhou C, Song S, Zhang J. A novel 3017-bp deletion mutation in the FERMT1 (KIND1) gene in a Chinese family with Kindler syndrome. Br J Dermatol 2009;160(5):1119-22. 22. Has C, Herz C, Zimina E, Qu HY, He Y, Zhang ZG, et al. Kindlin-1 Is Required for RhoGTPase- Mediated Lamellipodia Formation in Keratinocytes. Am J Pathol 2009. 23. Jonkman MF, de Jong MC, Heeres K, Pas HH, van der Meer JB, Owaribe K, et al. 180- kD bullous pemphigoid antigen (BP180) is deficient in generalized atrophic benign epidermolysis bullosa. J Clin Invest 1995;95(3):1345-52. 24. Lanschuetzer CM, Muss WH, Emberger M, Pohla-Gubo G, Klausegger A, Bauer JW, et al. Characteristic immunohistochemical and ultrastructural findings indicate that Kindler’s syndrome is an apoptotic skin disorder. J Cutan Pathol 2003;30(9):553-60. 25. Ussar S, Moser M, Widmaier M, Rognoni E, Harrer C, Genzel-Boroviczeny O, et al. Loss of Kindlin-1 causes skin atrophy and lethal neonatal intestinal epithelial dysfunction. PLoS Genet 2008;4(12):e1000289. 26. Kern JS, Herz C, Haan E, Moore D, Nottelmann S, von Lilien T, et al. Chronic colitis due to an epithelial barrier defect: the role of kindlin-1 isoforms. J Pathol 2007;213(4):462-70. 27. Senturk N, Usubutun A, Sahin S, Bukulmez G, Erkek E, Topaloglu R, et al. Kindler syndrome: absence of definite ultrastructural feature. J Am Acad Dermatol 1999;40(2 Pt 2):335-7. 28. Lai-Cheong JE, Ussar S, Arita K, Hart IR, McGrath JA. Colocalization of kindlin-1, kindlin-2, and migfilin at keratinocyte focal adhesion and relevance to the pathophysiology of Kindler syndrome. J Invest Dermatol 2008;128(9):2156-65. 29. Kloeker S, Major MB, Calderwood DA, Ginsberg MH, Jones DA, Beckerle MC. The Kindler syndrome protein is regulated by transforming growth factor-beta and involved in integrin-mediated adhesion. J Biol Chem 2004;279(8):6824-33.

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30. Lalani SR, Thakuria JV, Cox GF, Wang X, Bi W, Bray MS, et al. 20p12.3 microdeletion predisposes to Wolff-Parkinson-White syndrome with variable neurocognitive deficits. J Med Genet 2009;46:168-75. 31. Mahapatra NR, Mahata M, Ghosh S, Gayen JR, O’Connor DT, Mahata SK. Molecular basis of neuroendocrine cell type-specific expression of the chromogranin B gene: Crucial role of the transcription factors CREB, AP-2, Egr-1 and Sp1. J Neurochem 2006;99(1):119-33. 32. Ozanick S, Krecic A, Andersland J, Anderson JT. The bipartite structure of the tRNA m1A58 methyltransferase from S. cerevisiae is conserved in humans. Rna 2005;11(8):1281-90. 33. Johnson EM, Kinoshita Y, Daniel DC. A new member of the MCM protein family encoded by the human MCM8 gene, located contrapodal to GCD10 at chromosome band 20p12.3-13. Nucleic Acids Res 2003;31(11):2915-25. 34. Bolhuis PA, Hensels GW, Hulsebos TJ, Baas F, Barth PG. Mapping of the locus for X-linked cardioskeletal myopathy with neutropenia and abnormal mitochondria (Barth syndrome) to Xq28. Am J Hum Genet 1991;48(3):481-5. 35. Xu Y, Condell M, Plesken H, Edelman-Novemsky I, Ma J, Ren M, et al. A Drosophila model of Barth syndrome. Proc Natl Acad Sci U S A 2006;103(31):11584-8. 36. Lu B, Xu YX, Jiang JJ, Choy PC, Hatch GM, Grunfeld C, et al. Cloning and chracterization of a cDNA encoding human cardiolipin sythase (hCLS1). J Lipid Res 2006;47:1140-5. 37. Jones JM, Morrell JC, Gould SJ. Identification and characterization of HAOX1, HAOX2, and HAOX3, three human peroxismal 2-hydroxy acid oxidases. J Biol Chem 2000;275:12590-7. 38. Caricasole A, Sala C, Roncarati R, Formenti E, Terstappen GC. Cloning and characterization of the human phosphoinositide-specific phospholipase C-beta 1 (PLC beta 1). Biochim Biophys Acta 2000;1517(1):63-72. 39. Nofal E, Assaf M, Elmosalamy K. Kindler syndrome: a study of five Egyptian cases with evaluation of severity. Int J Dermatol 2008;47(7):658-62.

124 Contiguous gene defect in Kindler syndrome

Figure S1. Additional clinical pictures of the patient with a contiguous gene syndrome consisting of Kindler syndrome with cardiac hypertrophy, skeletal/joint abnormalities, mild dysmorphic features and mental retardation, due to a complex FERMT1 out-of-frame deletion on one allele and a microdeletion on the other allele involving the complete FERMT1 gene and several other genes.

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