Abstract：
OBJECTIVE: To analyze the genetic variant and molecular pathogenesis in a Chinese pedigree affected with Multiple epiphyseal dysplasia (MED).
METHODS: A MED pedigree which had presented at the Beijing Jishuitan Hospital Affiliated to Capital Medical University on September 13, 2020 was selected as the study subject. Clinical data of the pedigree were collected. Peripheral blood samples were drawn from pedigree members for the extraction of genomic DNA. Whole exome sequencing (WES) was carried out for the pedigree. Candidate variant was verified by Sanger sequencing. Wild type and mutant SLC26A2 expression plasmids were constructed and transfected into human primary chondrocytes. The effect of the variants on the protein localization and cell proliferation was determined by immunofluorescence and CCK8 assays.
RESULTS: WES and Sanger sequencing revealed that the proband has harbored compound heterozygous variants of the SLC26A2 gene, including a paternally derived c.484G>T (p.Val162Leu) missense variant and a maternally derived c.485_486delTG (p.Val162Glyfs*12) frameshifting variant. The SLC26A2WT and its mutant SLC26A2Val162Leu and SLC26A2Val162Glyfs*12 expression plasmids were distributed in the nuclei and cytoplasm of human primary chondrocytes. Compared with SLC26A2WT, the expressions of SLC26A2Val162Leu and SLC26A2Val162Glyfs*12 were decreased, along with reduced proliferation of human primary chondrocytes.
CONCLUSIONS: The c.484G>T and c.485_486delTG compound heterozygous variants of the SLC26A2 gene may affect the proliferation of human primary chondrocytes and underlay the pathogenesis of MED in this pedigree.
METHODS: A MED pedigree which had presented at the Beijing Jishuitan Hospital Affiliated to Capital Medical University on September 13, 2020 was selected as the study subject. Clinical data of the pedigree were collected. Peripheral blood samples were drawn from pedigree members for the extraction of genomic DNA. Whole exome sequencing (WES) was carried out for the pedigree. Candidate variant was verified by Sanger sequencing. Wild type and mutant SLC26A2 expression plasmids were constructed and transfected into human primary chondrocytes. The effect of the variants on the protein localization and cell proliferation was determined by immunofluorescence and CCK8 assays.
RESULTS: WES and Sanger sequencing revealed that the proband has harbored compound heterozygous variants of the SLC26A2 gene, including a paternally derived c.484G>T (p.Val162Leu) missense variant and a maternally derived c.485_486delTG (p.Val162Glyfs*12) frameshifting variant. The SLC26A2WT and its mutant SLC26A2Val162Leu and SLC26A2Val162Glyfs*12 expression plasmids were distributed in the nuclei and cytoplasm of human primary chondrocytes. Compared with SLC26A2WT, the expressions of SLC26A2Val162Leu and SLC26A2Val162Glyfs*12 were decreased, along with reduced proliferation of human primary chondrocytes.
CONCLUSIONS: The c.484G>T and c.485_486delTG compound heterozygous variants of the SLC26A2 gene may affect the proliferation of human primary chondrocytes and underlay the pathogenesis of MED in this pedigree.
摘要：
目的：分析1例多发性骨phy发育不良(MED)家系的遗传变异和分子致病机制。
方法：选择2020年9月13日在首都医科大学附属北京积水潭医院发表的MED家系作为研究对象。收集家系的临床数据。从谱系成员中抽取外周血样品用于提取基因组DNA。对谱系进行全外显子组测序(WES)。通过Sanger测序验证候选变体。构建野生型和突变型SLC26A2表达质粒并转染到人原代软骨细胞中。通过免疫荧光和CCK8测定确定变体对蛋白质定位和细胞增殖的影响。
结果：WES和Sanger测序显示先证者含有SLC26A2基因的复合杂合变体，包括父系衍生的c.484G>T(p.Val162Leu）错义变体和母系衍生的c.485_486delTG（p。Val162Glyfs*12）移码变体。SLC26A2WT及其突变体SLC26A2Val162Leu和SLC26A2Val162Glyfs*12表达质粒分布在人原代软骨细胞的细胞核和细胞质中。与SLC26A2WT相比，SLC26A2Val162Leu和SLC26A2Val162Glyfs*12的表达降低，伴随着人原代软骨细胞的增殖减少。
结论：SLC26A2基因的c.484G>T和c.485_486delTG复合杂合变体可能会影响人原代软骨细胞的增殖，并为该家系MED的发病机理奠定了基础。
方法：选择2020年9月13日在首都医科大学附属北京积水潭医院发表的MED家系作为研究对象。收集家系的临床数据。从谱系成员中抽取外周血样品用于提取基因组DNA。对谱系进行全外显子组测序(WES)。通过Sanger测序验证候选变体。构建野生型和突变型SLC26A2表达质粒并转染到人原代软骨细胞中。通过免疫荧光和CCK8测定确定变体对蛋白质定位和细胞增殖的影响。
结果：WES和Sanger测序显示先证者含有SLC26A2基因的复合杂合变体，包括父系衍生的c.484G>T(p.Val162Leu）错义变体和母系衍生的c.485_486delTG（p。Val162Glyfs*12）移码变体。SLC26A2WT及其突变体SLC26A2Val162Leu和SLC26A2Val162Glyfs*12表达质粒分布在人原代软骨细胞的细胞核和细胞质中。与SLC26A2WT相比，SLC26A2Val162Leu和SLC26A2Val162Glyfs*12的表达降低，伴随着人原代软骨细胞的增殖减少。
结论：SLC26A2基因的c.484G>T和c.485_486delTG复合杂合变体可能会影响人原代软骨细胞的增殖，并为该家系MED的发病机理奠定了基础。
