Department of Pathology, College of Medicine and Heath Sciences, United Arab Emirates University, P,O, Box 17666 Al Ain, United Arab Emirates.
BMC Med Genet. 2014 Apr 11;15:42. doi: 10.1186/1471-2350-15-42.
The rare autosomal genetic disorder, Spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL), is reported to be caused by missense or splice site mutations in the human discoidin domain receptor 2 (DDR2) gene. Previously our group has established that trafficking defects and loss of ligand binding are the underlying cellular mechanisms of several SMED-SL causing mutations. Here we report the clinical characteristics of two siblings of consanguineous marriage with suspected SMED-SL and identification of a novel disease-causing mutation in the DDR2 gene.
Clinical evaluation and radiography were performed to evaluate the patients. All the coding exons and splice sites of the DDR2 gene were sequenced by Sanger sequencing. Subcellular localization of the mutated DDR2 protein was determined by confocal microscopy, deglycosylation assay and Western blotting. DDR2 activity was measured by collagen activation and Western analysis.
In addition to the typical features of SMED-SL, one of the patients has an eye phenotype including visual impairment due to optic atrophy. DNA sequencing revealed a novel homozygous dinucleotide deletion mutation (c.2468_2469delCT) on exon 18 of the DDR2 gene in both patients. The mutation resulted in a frameshift leading to an amino acid change at position S823 and a predicted premature termination of translation (p.S823Cfs*2). Subcellular localization of the mutant protein was analyzed in mammalian cell lines, and it was found to be largely retained in the endoplasmic reticulum (ER), which was further supported by its N-glycosylation profile. In keeping with its cellular mis-localization, the mutant protein was found to be deficient in collagen-induced receptor activation, suggesting protein trafficking defects as the major cellular mechanism underlying the loss of DDR2 function in our patients.
Our results indicate that the novel mutation results in defective trafficking of the DDR2 protein leading to loss of function and disease. This confirms our previous findings that DDR2 missense mutations occurring at the kinase domain result in retention of the mutant protein in the ER.
罕见的常染色体遗传性疾病，短肢伴异常钙化的脊椎干骺端发育不良（SMED-SL），据报道是由人类盘状结构域受体 2（DDR2）基因突变引起的错义或剪接位点突变。以前我们的研究小组已经证实，几种导致 SMED-SL 的突变的潜在细胞机制是运输缺陷和配体结合丧失。在这里，我们报告了一对近亲结婚的疑似 SMED-SL 患者的临床特征，以及 DDR2 基因中一种新的致病突变的鉴定。
对患者进行临床评估和放射学检查。通过 Sanger 测序对 DDR2 基因的所有编码外显子和剪接位点进行测序。通过共聚焦显微镜、去糖基化分析和 Western blot 确定突变 DDR2 蛋白的亚细胞定位。通过胶原蛋白激活和 Western 分析测量 DDR2 活性。
除了 SMED-SL 的典型特征外，其中一名患者还有眼部表型，包括视神经萎缩引起的视力障碍。DNA 测序显示，两名患者的 DDR2 基因第 18 外显子上均存在一个新的二核苷酸缺失突变（c.2468_2469delCT）。该突变导致移码，导致第 823 位氨基酸发生变化，预测翻译提前终止（p.S823Cfs*2）。突变蛋白的亚细胞定位在哺乳动物细胞系中进行了分析，结果发现其主要保留在内质网（ER）中，这进一步得到其 N-糖基化谱的支持。与细胞内定位错误一致，该突变蛋白在胶原蛋白诱导的受体激活中缺乏活性，表明蛋白运输缺陷是导致我们患者 DDR2 功能丧失的主要细胞机制。
我们的结果表明，该新突变导致 DDR2 蛋白的运输缺陷，导致功能丧失和疾病。这证实了我们之前的发现，即发生在激酶结构域的 DDR2 错义突变导致突变蛋白在内质网中滞留。
