A novel homozygous nonsense mutation in the LAMC2 gene in patients with the Herlitz junctional epidermolysis bullosa.
A novel homozygous nonsense mutation in the LAMC2 gene in patients with the Herlitz junctional epidermolysis bullosa.
复制标题
Herlitz 交界性大疱性表皮松解症患者 LAMC2 基因中的一种新的纯合无义突变。
DOI：
10.1093/hmg/3.10.1909
复制
发表时间：
1994
影响因子：
3.5
通讯作者：
Meneguzzi,G
中科院分区：
文献类型：
--
作者：
Baudoin,C;Miquel,C;Gagnoux-Palacios,L;Pulkkinen,L;Christiano,AM;Uitto,J;Tadini,G;Ortonne,JP;Meneguzzi,G
遗传性大疱性表皮松解症（EB）是一组皮肤疾病，其特征是轻微创伤后皮肤明显脆弱并出现水疱(1)。连接型EB （JEB）是由真皮-表皮基底膜透明层内的组织分离引起的(2)。一般形式，或Herlitz JEB，表现为广泛的水泡和糜烂，和新生儿死亡。H-JEB与层粘连蛋白-5缺陷有关（3-5），层粘连蛋白-5是锚定丝的一种蛋白质（6,7），由三条链组成，a3，/33和72(8-10)，每条链由一个不同的基因编码（11,12）。最近在H-JEB中发现了编码层粘连蛋白72链的LAMC2基因突变(3)。在这项研究中，我们报告了在LAMC2中与该疾病相关的一种新的纯合无义突变的鉴定。H-JEB皮肤活检分别来自一名18个月时死亡的男性（先证者1）和一名在胎儿镜检查中被诊断为H-JEB的怀孕20周的选择性流产（先证者2）。患者为意大利南部一个家庭(S)的近亲结合产物。HJEB的诊断建立在临床观察的基础上（2,7），表明该疾病的病因与层粘连蛋白-5有关。因此，我们通过免疫荧光分析来自两个先证的H-JEB皮肤冷冻样品来评估每个层粘连蛋白-5链的表达。分别针对层粘连蛋白a3和bbb33链产生的多克隆抗体SE85和单克隆抗体K140显示免疫反应性降低，而针对层粘连蛋白72链产生的多克隆抗体SE144对样品没有染色作用。由于所有这些抗体都与健康供体皮肤的真皮-表皮连接处发生强烈反应，我们得出结论，LAMC2是这个H-JEB家族的候选基因。为了证实这一观点，我们对先显子和健康对照皮肤角质形成细胞原代培养物中纯化的mRNA进行了Northern blot分析，并与32p标记的cdna NA1（12）、KAL 5.5 C（10）和PCR1杂交，以此来评估laminin-5基因的表达水平。3(9)，分别编码层粘连蛋白链ai、j33和72。与cdna NA1和KAL5杂交得到的信号。5C的强度与健康对照组检测到的强度相当。与cDNA PCR1未见杂交。3（图lc，巷1）。因此，我们得出结论，层粘连蛋白72链的基因与该家族的疾病直接相关。由于先证者皮肤中提取的基因组DNA的Southern blot分析没有发现明显的异常、缺失、插入或广泛的重排，我们使用层粘连蛋白72链转录本的RT-PCR获得的cDNA产物来搜索可能的突变。先证子2和
Hereditary epidermolysis bullosa (EB) is a group of skin diseases characterized by marked skin fragility with development of blisters following minor trauma (1). The junctional forms of EB (JEB) result from tissue separation within the lamina lucida of the dermal-epidermal basement membrane (2). The generalized form, or the Herlitz JEB, presents with widespread blistering and erosions, and frequent neonatal death. H-JEB has been associated with defects in laminin-5 (3-5), a protein of the anchoring filaments (6, 7) consisting of three chains, a3,/33 and 72 (8—10), each encoded by a distinct gene (11, 12). A mutation in the LAMC2 gene encoding the laminin 72 chain has recently been identified in H-JEB (3). In this study we report the identification of a novel homozygous nonsense mutation in LAMC2 associated to this disease. H-JEB skin biopsies were obtained from a male (proband 1) deceased at the age of 18 months and from an elective abortion of a 20 week pregnancy diagnosed with H-JEB upon fetoscopy (proband 2). The patients were products of a consanguineous union in a family (S) from Southern Italy. The diagnosis of HJEB, established on the basis of clinical observations (2, 7), suggested involvement of laminin-5 in the etiology of the disease. We therefore assessed the expression of each individual chain of laminin-5 by immunofluorescence analysis of frozen samples of H-JEB skin from both probands. Polyclonal antibody SE85 and monoclonal antibody K140, raised against laminin a3 and| 33 chains, respectively, displayed a reduced immunoreactivity, whereas polyclonal antibody SE144 specific to the laminin 72 chain did not stain the samples. Since all those antibodies strongly reacted with the dermo-epidermal junction of the skin from healthy donors, we concluded that LAMC2 is the candidate gene in this H-JEB kindred. To confirm this suggestion, the level of expression of the genes for laminin-5 was assessed by Northern blot analysis of mRNA purified from primary cultures of skin keratinocytes obtained from both probands and from healthy controls upon hybridization with 32P-labeled cDNAs NA1 (12), KAL 5.5 C (10) and PCR1. 3 (9), which encode laminin chains ai, j33 and 72, respectively. Hybridization signals obtained with cDNAs NA1 and KAL5. 5C had intensities comparable to those detected in healthy controls. No hybridization was seen with cDNA PCR1. 3 (Fig. lc, lane 1). We therefore concluded that the gene for the laminin 72 chain was directly involved in the disease in this family.Since Southern blot analysis of genomic DNA extracted from the probands' skin did not reveal gross abnormalities, deletions, insertions or extensive rearrangements, we searched for possible mutations using cDNA products obtained by RT-PCR of laminin 72 chain transcripts. Ten ng of total RNA from proband 2 and