Ien Chan

Autoantibodies to extracellular matrix protein 1 in lichen sclerosus

BACKGROUND Lichen sclerosus is a common acquired inflammatory disorder of skin and mucous membranes. The aetiology is unknown, although HLA-subtype susceptibility and high rates of other autoimmune disorders suggest that autoantibodies to specific mucocutaneous antigens are involved. The clinicopathological similarities between lichen sclerosus and lipoid proteinosis, which results from mutations in extracellular matrix protein 1 (ECM1), suggest this protein as an autoantigen. METHODS We analysed serum autoantibody profiles in 171 individuals (86 with lichen sclerosus, 85 healthy controls) by immunoblotting of extracts from normal human skin and lipoid proteinosis skin (lacking ECM1). We generated a full-length glutathione-S-transferase fusion protein for ECM1 to confirm specific immunoreactivity. We affinity-purified serum from patients with lichen sclerosus and did indirect immunofluorescence microscopy on normal skin with or without preabsorption with recombinant ECM1. FINDINGS By immunoblotting, IgG autoantibodies were found in 20 (67% [95% CI 45-84]) of 30 lichen sclerosus serum samples. The highest titre was 1 in 20. The bands were not detected in ECM1-deficient substrate. These samples, and those from 56 other patients with lichen sclerosus, showed immunoreactivity to the recombinant ECM1 protein (64 of 86 positive; 74% [65-84]). Only six (7% [2-13]) of 85 control serum samples were positive. Affinity-purified IgG from serum of patients with lichen sclerosus labelled skin similarly to a polyclonal antibody to ECM1. The positive staining was blocked by preabsorption with excess recombinant ECM1 protein. INTERPRETATION These findings provide evidence for a specific humoral immune response to ECM1 in lichen sclerosus and offer insight into disease diagnosis, monitoring, and approaches to treatment.

The molecular basis of lipoid proteinosis: mutations in extracellular matrix protein 1

Abstract:  Lipoid proteinosis (OMIM 247100), also known as Urbach–Wiethe disease or hyalinosis cutis et mucosae, is a rare autosomal recessive disorder characterized by generalized thickening and scarring of the skin and mucosae. In 2002, the disorder was mapped to a locus on chromosome 1q21 and pathogenic mutations were identified in the ECM1 gene, which encodes for the glycoprotein extracellular matrix protein 1 (ECM1). ECM1 has since been shown to have several important biological functions. It has a role in the structural organization of the dermis (binding to perlecan, matrix metalloproteinase‐9 and fibulin) as well as being targeted as an autoantigen in the acquired disease lichen sclerosus. ECM1 also shows over‐expression in certain malignancies and is abnormally expressed in chronologically aged and photo‐aged skin. Thus far, 26 different inherited mutations in ECM1 have been reported in lipoid proteinosis. In this article, we provide an update on the molecular pathology of lipoid proteinosis, including the addition of 15 new mutations in ECM1 to the mutation database, and review the biological functions of the ECM1 protein in health and disease.

Clinical and molecular characterization of lipoid proteinosis in Namaqualand, South Africa.

Background  Lipoid proteinosis (LiP) is a rare autosomal recessive disorder characterized by a hoarse voice, warty skin infiltration and scarring. Mutations within the extracellular matrix protein 1 (ECM1) gene cause LiP. Since the early 1970s it has been recognized that South Africa has one of the largest groups of LiP patients worldwide, suggesting a probable founder effect. As LiP patients present with considerable clinical variability, this group of patients offers a unique opportunity for genotype–phenotype correlation.

The role of extracellular matrix protein 1 in human skin

Extracellular matrix protein 1 (ECM1) was first identified in 1994 as an 85‐kDa glycoprotein secreted by a mouse osteogenic stromal cell line. Subsequently, the human homologue has been found to regulate endochondral bone formation, and to stimulate proliferation of endothelial cells and induce angiogenesis. However, a role for ECM1 in skin physiology and homeostasis has also emerged. Specifically, in 2002, loss‐of‐function mutations in the ECM1 gene were discovered to be the cause of the rare autosomal recessive genodermatosis, lipoid proteinosis. This inherited disorder is characterized clinically by skin and mucosal infiltration and scarring and histologically by disruption/duplication of basement membrane and widespread deposition of hyaline material in the dermis. Moreover, other recent studies have identified circulating autoantibodies against the ECM1 protein in most patients with lichen sclerosus, a common chronic inflammatory condition that shares some clinicopathological features with lipoid proteinosis. ECM1 thus serves as a target antigen in both an inherited and an acquired skin disorder. Within the epidermis, ECM1 has a role in the control of keratinocyte differentiation. Within the dermis, ECM1 binds to the major heparan sulphate proteoglycan, perlecan. In this way, ECM1 may act as a ‘biological glue’ in the dermis, helping to regulate basement membrane and interstitial collagen fibril macro‐assembly and growth factor binding. ECM1 may also have a role in other acquired skin disorders and physiological skin changes including scarring, wound healing and skin ageing, although this remains to be determined.

Familial multiple cutaneous and uterine leiomyomas associated with papillary renal cell cancer

Multiple cutaneous and uterine leiomyomas is an autosomal dominant condition that results in benign smooth muscle tumours of the skin and, in females, uterine fibroids. This syndrome overlaps with hereditary leiomyomatosis and renal cell cancer syndrome in which affected individuals may develop the rare type II papillary renal cell cancer, in addition to skin leiomyomas. Recently, heterozygous mutations in the gene encoding fumarate hydratase have been found to underlie both conditions. Fumarate hydratase is an enzyme that catalyses the conversion of fumarate to malate in the Krebs cycle and may also function as a tumour suppressor gene. We report a family with multiple leiomyomas, uterine fibroids and papillary renal cell cancer. The proband is a 77‐year‐old Polish woman who developed multiple cutaneous leiomyomas on her right upper arm in her thirties and subsequently underwent a hysterectomy for uterine fibroids in her forties. She has four offspring: her eldest daughter also has skin and uterine leiomyomas with a similar onset; her son has multiple skin leiomyomas and in addition was diagnosed with metastatic papillary renal cell cancer at the age of 50 years; the two youngest daughters are unaffected. DNA sequencing in all the affected individuals disclosed a heterozygous G→C substitution at nucleotide 173 of the fumarate hydratase gene, that converts an arginine residue (CGA) to proline (CCA). This missense mutation has not been reported previously and is designated R58P. Interestingly, the clinically asymptomatic 20‐year‐old son of the individual with renal cancer was also found to be heterozygous for R58P. It is likely that he will develop skin leiomyomas in the future but the risk of renal cancer is difficult to predict. Nevertheless, detection of this mutation has important implications for screening and genetic counselling in this and other family members.

Characterization of IgG autoantibodies to extracellular matrix protein 1 in lichen sclerosus.

Although the precise aetiology of lichen sclerosus is unknown, evidence for an autoimmune basis to the disorder is emerging. Indeed, circulating IgG autoantibodies to the glycoprotein extracellular matrix protein 1 (ECM1) have been demonstrated in the sera of about 75% of affected individuals. To assess this humoral immune response further, immunoblotting was performed using bacterial recombinant proteins spanning different domains of the ECM1 protein. The aim was to identify autoantibody‐reactive sites recognized by 90 lichen sclerosus sera. The subclass distribution of anti‐ECM1 IgG autoantibodies was also determined in 54 lichen sclerosus sera. Immunoblotting showed that the IgG autoantibodies from lichen sclerosus patients recognize multiple antigenic reactive sites on the ECM1 protein within both the amino terminus (50/90, 55.6%) and the protein loop cysteine‐rich repeat domains (54/90, 60%), although few sera (7/90, 7.8%) had antibodies to the carboxyl terminus of ECM1. IgG subclass analysis revealed that the anti‐ECM1 autoantibodies belong predominantly to the IgG2 subclass (48/54, 88.9%), either IgG2 alone (28/54, 51.9%) or in combination with one or more other IgG subclasses. No correlation was found between the site(s) of the ECM1 epitopes or the anti‐ECM1 IgG profile and any specific clinical parameters. Nevertheless, characterization of anti‐ECM1 antibodies does provide further insight into humoral immune responses and understanding disease mechanisms in lichen sclerosus.

Extracellular matrix protein 1 autoantibodies in male genital lichen sclerosus.

1 Yanaba K, Yoshizaki A, Muroi E et al. CCL13 is a promising diagnostic marker for systemic sclerosis. Br J Dermatol 2010; 162:332–6. 2 LeRoy EC, Black C, Fleischmajer R et al. Scleroderma (systemic sclerosis): classification, subsets and pathogenesis. J Rheumatol 1988; 15:202–5. 3 Gambichler T, Tigges C, Burkert B et al. Absolute count of T and B lymphocyte subsets is decreased in systemic sclerosis. Eur J Med Res 2010; 15:44–6. 4 Schmidt K, Martinez-Gamboa L, Meier S et al. Bronchoalveolar lavage fluid cytokines and chemokines as markers and predictors for the outcome of interstitial lung disease in systemic sclerosis patients. Arthritis Res Ther 2009; 11:R111. 5 Fineschi S, Goffin L, Rezzonico R et al. Antifibroblast antibodies in systemic sclerosis induce fibroblasts to produce profibrotic chemokines, with partial exploitation of toll-like receptor 4. Arthritis Rheum 2008; 58:3913–23. 6 Varga J, Abraham D. Systemic sclerosis: a prototypic multisystem fibrotic disorder. J Clin Invest 2007; 117:557–67. 7 Yanaba K, Muroi E, Yoshizaki A et al. Serum CXCL16 concentrations correlate with the extent of skin sclerosis in patients with systemic sclerosis. J Rheumatol 2009; 36:1917–23. 8 Yanaba K, Yoshizaki A, Muroi E et al. Serum CCL23 levels are increased in patients with systemic sclerosis. Arch Dermatol Res 2011; 303:29–34. 9 Homey B, Meller S. Chemokines and other mediators as therapeutic targets in psoriasis vulgaris. Clin Dermatol 2008; 26:539–45. 10 Pivarcsi A, Homey B. Chemokine networks in atopic dermatitis: traffic signals of disease. Curr Allergy Asthma Rep 2005; 5:284–90.

Molecular basis of lipoid proteinosis in a Libyan family

Summary Lipoid proteinosis is an autosomal recessive condition associated with variable scarring and infiltration of skin and mucosae. The disorder has recently been shown to result from loss‐of‐function mutations in the extracellular matrix protein 1 gene (ECM1) on 1q21. Extracellular matrix protein 1 has important physiological and biological roles in aspects of epidermal differentiation, binding of dermal collagens and proteoglycans, and in regulation of angiogenesis. Thus far pathogenic mutations have been described in 16 different families with lipoid proteinosis throughout the world. In this report, we describe the clinico‐pathological features of a 10‐year‐old boy with lipoid proteinosis from a consanguineous Libyan family. By direct sequencing of the affected individuals genomic DNA, we identified a homozygous nonsense mutation in exon 2 of the ECM1 gene, Q32X. This mutation is the most 5′ of all ECM1 mutations described thus far and is predicted to ablate the ECM1a, ECM1b and ECM1c splice variants of the ECM1 gene and to result in a severe clinical phenotype. Sequencing of DNA from the affected individuals five siblings revealed that four were heterozygous carriers of Q32X, findings that have important implications for genetic counselling given the high frequency of consanguineous marriages in Libya.

Progressive osseous heteroplasia resulting from a new mutation in the GNAS1 gene

Progressive osseous heteroplasia (OMIM 166350) is a rare autosomal dominant condition that presents in childhood as dermal ossification and may progress deeper to involve subcutaneous fat and connective tissue. Recently, paternally inherited inactivating mutations in the GNAS1 gene on chromosome 20q13 have been implicated in the pathogenesis, although sporadic cases have also been reported. We report a 9‐year‐old British Chinese girl with progressive osseous heteroplasia resulting from a de novo missense mutation (W281R) in the GNAS1 gene. She is of small stature (0.4th centile) and started to develop skin lesions at the age of 9 months. These have been confirmed histologically as osteoma cutis. She is of normal intelligence and development and has no dysmorphic features. The GNAS1 gene exhibits imprinting and maternally inherited mutations have previously been shown to result in Albrights hereditary osteodystrophy (OMIM 103580) with pseudohypothyroidism type 1a, whereas paternally inherited mutations result in progressive osseous heteroplasia or the Albrights hereditary osteodystrophy phenotype with pseudopseudohypothyroidism (OMIM 300800). With only nine mutations of the GNAS1 gene reported so far in progressive osseous heteroplasia, this new mutation helps to extend further the genotype–phenotype correlation.

Rapp–Hodgkin syndrome and the tail of p63

We report the clinical and molecular abnormalities in a 19‐year‐old woman with Rapp–Hodgkin ectodermal dysplasia syndrome. The physical features include mid‐facial hypoplasia, uncombable hair, cleft palate and bifid uvula, lacrimal duct obstruction and dry skin. Sequencing of the p63 gene reveals a new heterozygous frameshift mutation, 1787delG, in exon 14. The frameshift results in changes to the tail of p63 with the addition of 68 missense amino acids downstream and a delayed termination codon that extends the protein length by 21 amino acids. These changes are predicted to disrupt the normal repressive function of the transactivation inhibitory domain leading to gain‐of‐function for at least two isoforms of the p63 transcription factor. The expanding p63 mutation database demonstrates that there is overlap between Rapp–Hodgkin syndrome and several other ectodermal dysplasia syndromes, notably Hay–Wells syndrome, and that characterization of the functional consequences of these p63 gene mutations at a molecular and cellular level is likely to provide further insight into the clinical spectrum of these developmental malformation syndromes.