类脂质蛋白沉积症家系ECM1基因突变检测

[摘要] 目的 检测一类脂蛋白沉积症家系细胞外基质蛋白1（ECM1）基因的突变,并探讨中国类脂蛋白沉积症家系ECM1基因的突变特点。方法 PCR扩增家系成员ECM1基因的全部外显子,并行DNA测序。以100例无关正常人作对照。结果 先证者及其胞兄的ECM1基因第六外显子均检测出第658位碱基发生T G纯合性突变,使220位的半胱氨酸转变为甘氨酸（p.C220G）。其父母均为该突变的杂合性携带者,该突变在100例无关正常对照中未被检测出。结论 p.C220G为引起本家系临床症状的特异性突变。     

Compound heterozygosity for novel splice site mutations of ITGA6 in lethal junctional epidermolysis bullosa with pyloric atresia

Junctional epidermolysis bullosa with pyloric atresia (PA‐JEB) is a rare congenital bullous disease with gastrointestinal disturbance that has been associated with mutations in ITGA6 or ITGB4 encoding the α6 or β4 subunit of integrin, respectively. Only six ITGA6 mutations in PA‐JEB have been reported while many ITGB4 mutations have been identified, and all the ITGA6 mutations were homozygous. Here, we report a case of lethal type PA‐JEB, in which immunofluorescence showed the lack of both α6 and β4 integrins resulting from compound heterozygous splice site mutation in ITGA6, c.387G>T and c.2506‐1G>C. Maternal c.387G>T induced the skipping of the entire exon 3 and both exons 3 and 4, resulting in premature termination codon and in‐frame deletion, respectively. Paternal c.2506‐1G>C caused the skipping of the exon 20 and resulted in in‐frame deletion. As a reason why the present case showed lethal phenotype despite the in‐frame deletion mutation, rapid degradation of neo‐synthesized α6 protein and/or impaired transport of integrin were suggested from previous reports, and the lack of localization of integrin α6β4 to the epidermal basement membrane resulted in skin fragility. Our case expands the variety of integrin α6 mutations in PA‐JEB.     

Progress in epidermolysis bullosa: the phenotypic spectrum of plectin mutations

Plectin, a large multidomain adhesive protein with versatile binding functions, is expressed in a number of tissues and cell types. In the skin, plectin is a critical component of hemidesmosomes, interacting with keratin intermediate filaments and beta4 integrin. Mutations in the plectin gene (PLEC1) result in fragility of skin, demonstrating blister formation at the level of hemidesmosomes. These blistering disorders belong to the spectrum of epidermolysis bullosa (EB) phenotypes, and three distinct variants because of plectin mutations have been identified. First, EB with muscular dystrophy, an autosomal recessive syndrome, is frequently caused by premature termination codon-causing mutations leading to the absence of plectin both in the skin and in the muscle. Second, a heterozygous missense mutation (R2110W) in PLEC1 has been documented in patients with EB simplex of the Ogna type, a rare autosomal dominant disorder. Finally, recent studies have disclosed plectin mutations in patients with EB with pyloric atresia, an autosomal recessive syndrome, frequently with lethal consequences. Collectively, these observations attest to the phenotypic spectrum of plectin mutations, and provide the basis for accurate genetic counselling with prognostic implications, as well as for prenatal diagnosis in families at the risk of recurrence of the 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. OBJECTIVES: To assess the clinical features and the molecular basis of LiP in patients from the Namaqualand area of the Northern Cape province of South Africa and to examine molecular evidence for a founder effect. SUBJECTS AND METHODS: The LiP patient cohort consisted of 29 Coloured patients from Namaqualand and a further seven Caucasoid patients from other areas of South Africa. The control group included 100 healthy geographically and ethnically matched individuals from Namaqualand. Samples were collected after informed consent and with ethics committee approval from the University of the Witwatersrand. LiP patients were examined clinically and a structured recording sheet was completed. A brief neurological evaluation was also performed. The LiP founder effect was investigated at the molecular level by ECM1 mutation detection and haplotype analysis. RESULTS: The most consistent clinical signs for a diagnosis of LiP in this group were a hoarse voice and thickened sublingual frenulum leading to restricted tongue movement. Homozygosity for a nonsense mutation in exon 7 of the ECM1 gene, Q276X, was identified in all patients (Coloured and Caucasoid). Despite this genetic homogeneity, considerable clinical variability in skin presentation and psychiatric involvement was observed. Haplotype analysis using markers from a 9.98-Mb region around the ECM1 locus confirmed the founder effect with a founder core haplotype, 19-Q276X-12 (ND1-ECM1-D1S2343), in all but four LiP-associated alleles (n = 58). A LiP carrier rate of 1 in 9 was observed among the 100 Namaqualand controls, predicting a LiP incidence of 1 in 324 in this community. CONCLUSIONS: Although several consistent clinical features in LiP patients homozygous for the Q276X mutation in the ECM1 gene were observed, there remains considerable clinical variability. This suggests the action of genetic and environmental modifiers of disease severity. Strong molecular evidence supports a single founder effect for the high prevalence of LiP in South Africans, both Coloured and Caucasoid.     

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.     

Interaction of extracellular matrix protein 1 with extracellular matrix components: ECM1 is a basement membrane protein of the skin

The extracellular matrix protein 1 (ECM1) is a secreted glycoprotein, which plays an important role in the structural and functional biology of the skin as demonstrated by the identification of loss-of-function mutations in ECM1 as cause of the genodermatosis lipoid proteinosis, characterized by reduplication of the skin basement membrane and hyalinization of the underlying dermis. To search for binding partner(s) of ECM1, we tested the in vitro binding activity of ECM1a, a major isoform of four ECM1 splice variants, to different skin extracellular matrix proteins (such as laminin 332, collagen type IV, and fibronectin) and polysaccharides (such as hyaluronan, heparin, and chondroitin sulfate A) with solid-phase binding assay. We demonstrated that ECM1a utilizes different regions to bind to a variety of extracellular matrix components. Ultrastructurally, ECM1 is a basement membrane protein in human skin and is part of network-like suprastructures containing perlecan, collagen type IV, and laminin 332 as constituents. Furthermore, ECM1a enhanced the binding of collagen IV to laminin 332 dose-dependently, showing its involvement in the dermal-epidermal junction and interstitial dermis and making the functional link to the pathophysiology of lipoid proteinosis. To our knowledge, this is previously unreported.     

类脂质蛋白沉积症两家系调查及ECM1基因突变检测

目的:检测类脂质蛋白沉积症二家系中细胞外基质蛋白(ECM1)基因突变位点。方法:提取1号家系先证者及其母亲,2号家系先证者、父母、配偶及儿子外周血DNA。PCR技术扩增ECM1基因编码序列,采用一代Sanger法对PCR扩增产物进行测序。结果:1号家系先证者在7号外显子发现已知突变(纯合突变c.960GA),其母亲为杂合携带者;2号家系先证者为遗传复合体,是上述突变位点的杂合携带者,此外在3号外显子上存在1个插入突变c.142insC。结论:类脂质蛋白沉积症存在遗传异质性。     

组织工程化人工皮肤的细胞外基质功能研究

目的：观察组织工程化人工皮肤的细胞外基质(ECM)功能。方法：脯氨酸掺人实验，天狼星红染色，AB—PAS染色。结果：组织工程化人工皮肤天狼星红染色和AB—PAS染色阳性，^3H-脯氨酸掺人值增高。结论：这种组织工程化人工皮肤可以合成、分泌细胞外基质。     

Basement membrane zone and dermal extracellular matrix of the vulva, vagina and amnion: An immunohistochemical study with comparison with non-reproductive epithelium

Background/Objectives: The basement membrane zone (BMZ) is an anatomically defined region present in all types of skin and mucosa, linking the epithelium to the mesenchyme with a complex structure to provide adhesion. Altered antigenic expression of the BMZ is implicated in interface dermatoses, and the BMZ is targeted by autoantibodies in subepidermal immunobullous dermatoses. This study aims to compare the antigenic expression of the BMZ and the dermal extracellular matrix in female genital skin and mucosa and amnion, with non‐reproductive skin and mucosa. Methods: An indirect immunofluorescence technique was used to compare the antigenic expression of hemidesmosome, lamina lucida, anchoring filaments, lamina densa, anchoring fibrils and extracellular matrix in samples of non‐reproductive skin (three), oral mucosa (three), vulval skin (two), vagina (three) and amnion (four). Results: Antigenic expression was similar in the stratified epithelium of reproductive and non‐reproductive skin and mucosa, but differed in the simple cuboidal epithelium of amnion, which had reduced expression of dermal‐associated antigens. Conclusions: The BMZ and dermal extracellular matrix of vagina and vulva are very similar to those of non‐reproductive skin and mucosa despite their various functions, but differs from amnion. Their antigenic expression does not fully account for the anatomical distribution of immunobullous and interface dermatoses.     

Lipoid proteinosis presenting with an unusual nonsense Q32X mutation in exon 2 of the extracellular matrix protein 1 gene

Lipoid proteinosis (LP) is a rare disorder characterized by extensive hyaline‐like deposits on the skin, mucous membranes and various internal organs with varying clinical manifestations. The disorder has been recently shown to result from loss‐of‐function mutations in the extracellular matrix protein 1 gene (ECM1) on 1Q21. The two cases reported here had typical clinical and histological features consistent with LP. Direct sequencing of amplified DNA from the second patient showed a single nucleotide substitution (C > T) at nucleotide 94 within exon 2 of the ECM1 gene, nonsense mutation Q32X. This is the second case reported of LP with involvement of exon 2 of ECM1.     

A case of subepidermal blister disease associated with autoantibody against 450 kD protein.

We report a case of subepidermal blister disease with eruptions distributed on the trunk and extremities. The blisters regressed after administration of corticosteroid and cyclophosphamide. An immunoblot analysis revealed that the patient's serum reacted exclusively with a 450-kD epidermal polypeptide. It did not react with a 230-kD or a 180-kD bullous pemphigoid antigen. The immunofluorescence study showed the patient's serum reacted with the basement membrane zone of human skin.     

Pseudoxanthoma elasticum: biopsy of clinically normal skin in the investigation of patients with angioid streaks

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a genetic disorder characterized by fragmentation and calcification of elastic fibres with resultant pathological changes in the dermis, Bruch's membrane and blood vessels. Defects in Bruch's membrane produce angioid streaks on the retina but this appearance is not pathognomonic of PXE. Biopsy of clinically normal skin or scar tissue in patients with angioid streaks may show the histological features of PXE. OBJECTIVES: To test the hypothesis that biopsy of clinically normal skin is a useful investigation in patients with angioid streaks. METHODS: This prospective study investigated 18 consecutive patients with angioid streaks. Each patient underwent a full dermatological examination and was investigated for diseases known to be associated with angioid streaks. Axillary skin biopsies were taken from 14 consenting patients. RESULTS: Typical PXE was found in 11 patients. No other diseases associated with angioid streaks were identified. Five patients had angioid streaks in the absence of systemic disease. Two patients had nondiagnostic dermatological features which were not clarified by histology. Two of the 11 patients with PXE showed histological evidence of PXE from clinically normal axillary skin. However, in both cases flexural skin elsewhere showed the typical clinical and histological features of PXE. CONCLUSIONS: This study demonstrates the association between angioid streaks and PXE. However, it does not support the hypothesis that biopsy of normal-looking skin is helpful in the investigation of adult patients with angioid streaks.