Zhang Yan-Ping, Lin Bin, Ji Yuan-Yuan, Hu Ya-Nan, Lin Xin-Fu, Tang Yi, Zhang Jian-Hui, Wu Shao-Jie, Cai Sen-Lin, Zhou Yan-Feng, Chen Ting, Fang Zhu-Ting, Luo Jie-Wei
Shengli Clinical Medical College of Fujian Medical University, Fuzhou, 350001, China.
Department of Interventional Radiology, Fujian Provincial Hospital, Fuzhou, 350001, China.
Thromb J. 2021 Sep 8;19(1):64. doi: 10.1186/s12959-021-00316-4.
Protein S deficiency (PSD) is an autosomal dominant hereditary disease. In 1984, familial PSD was reported to be prone to recurrent thrombosis. Follow-up studies have shown that heterozygous protein S (PROS1) mutations increase the risk of thrombosis. More than 300 PROS1 mutations have been identified; among them, only a small number of mutations have been reported its possible mechanism to reduce plasma protein S (PS) levels. However, whether PROS1 mutations affect protein structure and why it can induce PSD remains unknown.
The clinical phenotypes of the members of a family with thrombosis were collected. Their PS activity was measured using the coagulation method, whereas their protein C and antithrombin III activities were measured using methods such as the chromogenic substrate method. The proband and her parents were screened for the responsible mutation using second-generation whole exon sequencing, and the members of the family were verified for suspected mutations using Sanger sequencing. Mutant and wild type plasmids were constructed and transfected into HEK293T cells to detect the mRNA and protein expression of PROS1.
In this family, the proband with venous thrombosis of both lower extremities, the proband's mother with pulmonary embolism and venous thrombosis of both lower extremities, and the proband's younger brother had significantly lower PS activity and carried a PROS1 c. 1820 T > C:p.Leu607Ser heterozygous mutation (NM_000313.3). However, no such mutations were found in family members with normal PS activity. The PS expression in the cell lysate and supernatant of the Leu607Ser mutant cells decreased, while mRNA expression increased. Immunofluorescence localization showed that there was no significant difference in protein localization before and after mutation.
The analysis of family phenotype, gene association, and cell function tests suggest that the PROS1 Leu607Ser heterozygous mutation may be a pathogenic mutation. Serine substitution causes structural instability of the entire protein. These data indicate that impaired PS translation and synthesis or possible secretion impairment is the main pathogenesis of this family with hereditary PSD and thrombophilia.
蛋白S缺乏症（PSD）是一种常染色体显性遗传病。1984年，有报道称家族性PSD易发生反复血栓形成。后续研究表明，杂合性蛋白S（PROS1）突变会增加血栓形成风险。已鉴定出300多种PROS1突变；其中，只有少数突变报道了其降低血浆蛋白S（PS）水平的可能机制。然而，PROS1突变是否影响蛋白质结构以及为何会诱发PSD仍不清楚。
收集一个血栓形成家族成员的临床表型。采用凝血法测定其PS活性，采用发色底物法等方法测定其蛋白C和抗凝血酶III活性。先证者及其父母通过二代全外显子测序筛查致病突变，家族成员通过Sanger测序验证可疑突变。构建突变型和野生型质粒并转染至HEK293T细胞，检测PROS1的mRNA和蛋白表达。
在这个家族中，患有双下肢静脉血栓形成的先证者、患有肺栓塞和双下肢静脉血栓形成的先证者母亲以及先证者弟弟的PS活性显著降低，并携带PROS1 c.1820 T>C:p.Leu607Ser杂合突变（NM_000313.3）。然而，PS活性正常的家族成员未发现此类突变。Leu607Ser突变细胞的细胞裂解液和上清液中PS表达降低，而mRNA表达增加。免疫荧光定位显示突变前后蛋白定位无显著差异。
家族表型分析、基因关联分析和细胞功能检测表明，PROS1 Leu607Ser杂合突变可能是致病突变。丝氨酸替代导致整个蛋白质结构不稳定。这些数据表明，PS翻译和合成受损或可能的分泌受损是这个遗传性PSD和血栓形成倾向家族的主要发病机制。
