蛋白S缺乏致肺栓塞患者的PROS1基因突變
發(fā)布時(shí)間：2018-09-01 16:09
【摘要】：背景:靜脈血栓栓塞(VTE)是由血管壁的損傷,血流速度減緩或停止,以及血液的高凝狀態(tài)引起的。包括下肢深靜脈血栓(DVT)和肺栓塞(PE)。近年來,肺栓塞因其發(fā)病急,臨床表現(xiàn)不典型,死亡率高等特點(diǎn)越發(fā)被人們重視。目前遺傳性蛋白質(zhì)S缺乏(PSD)是靜脈血栓栓塞的已確立的危險(xiǎn)因素。PSD是具有不完全外顯率的常染色體顯性遺傳疾病,與編碼蛋白S基因(PROS1)突變或多態(tài)性相關(guān),該蛋白S基因共16個(gè)外顯子,跨越101kb,位于3q11.1。目前國內(nèi)外已經(jīng)鑒別出大約200余種PROS1突變引起PS合成或功能的改變。但是,關(guān)于蛋白S缺陷癥導(dǎo)致肺栓塞的報(bào)道并不多見,對(duì)符合條件的患者行PROS1基因檢測(cè)的報(bào)道較少。目的:通過對(duì)蛋白S缺乏合并肺動(dòng)脈栓塞的患者行PROS1的基因檢測(cè)和臨床表型診斷,找到突變位點(diǎn),歸納突變類型。方法:選取吉林大學(xué)白求恩第一臨床醫(yī)院自2011年6月至2016年12月期間,行肺動(dòng)脈CTA檢查明確診斷為肺栓塞的患者中,其游離蛋白S活性低于50%的患者40例,同時(shí)選取游離蛋白S活性為正常的患者2例。收集他們的外周血10ml,室溫下離心分離血細(xì)胞及血漿,血漿應(yīng)用原理凝固方法檢測(cè)游離蛋白S活性,應(yīng)用ELISA方法檢測(cè)游離蛋白S定量,總蛋白S定量;血細(xì)胞提取DNA,用于PCR擴(kuò)增和測(cè)序。將患者蛋白S缺乏的情況分為3種類型。I型的特征在于患者血液中總PS、游離PS抗原水平及PS活性同等降低(量的減少);II型的特征在于患者血液中PS的功能活性降低,而總的及游離的PS抗原水平仍正常(質(zhì)的減少);III型的特征在于總PS抗原水平在正常范圍內(nèi),而游離PS抗原的水平減少。定位測(cè)得的PROS1序列的突變位點(diǎn),并歸納總結(jié)。結(jié)果：游離蛋白S活性低于50%的患者40例,其中男性18人(45%),女性22人(55%),男性年齡為(54.88±20.42)歲,女性年齡為(56.59±19.09)歲。其中,遺傳性蛋白S缺乏分型:I型患者:22人(55%),II型患者:8人(20%),III型患者:4人(10%),不能分型患者:6人(15%)。行PROS1基因擴(kuò)增和測(cè)序,我們?cè)诰幋a序列上發(fā)現(xiàn)了21種不同位點(diǎn)的突變,主要分布在2、5、8、10、12、14、16號(hào)外顯子上。其中15種突變發(fā)生氨基酸的改變,5種突變氨基酸未改變,1種突變成終止密碼。在非編碼序列上我們同樣找到了15種不同位點(diǎn)的突變,主要分布在16號(hào)外顯子上。蛋白S活性正常的患者2例,其中男性1人,年齡為60歲,女性1名,年齡為59歲。在編碼序列上發(fā)現(xiàn)了15種不同位點(diǎn)的突變,主要分布在2、5、10號(hào)外顯子上。其中12種突變發(fā)生氨基酸的改變,2種突變氨基酸未改變,1種突變成終止密碼。在非編碼序列上我們同樣找到了10種不同位點(diǎn)的突變,主要分布在16號(hào)外顯子上。2例PS活性正常的患者檢測(cè)的結(jié)果一致,并且在40例PSD患者中均能找到。40例PSD患者與其進(jìn)行結(jié)果相比:8號(hào)外顯子第86位C全部發(fā)生了c.783 CT(pro261 pro)突變,但是轉(zhuǎn)錄的氨基酸仍為脯氨酸(pro)未變。16號(hào)外顯子第131位A發(fā)生了c.2097 AG(pro 699 pro)突變,同樣為同義突變,但是這種突變并未在40例患者中都發(fā)生(36/40)。有3例患者12號(hào)外顯子第32位的G發(fā)生了c.1283 GA(ser 428 asn)突變,導(dǎo)致氨基酸由絲氨酸(ser)突變成天冬酰胺(asn);1例患者12號(hào)外顯子第140位G發(fā)生c.1391 GA(arg 464 gin)突變,導(dǎo)致氨基酸由精氨酸(arg)突變成谷氨酰胺(gin);1例患者12號(hào)外顯子第146位T發(fā)生c.1397 TC(val 466 ala)突變,導(dǎo)致氨基酸由纈氨酸(val)突變成丙氨酸(ala)。2例患者14號(hào)外顯子第2位T發(fā)生了c.1590 TC(asn 530 asn)突變;但是轉(zhuǎn)錄的氨基酸仍為天冬酰胺(asn)未變。結(jié)論:1、在40例遺傳性蛋白S缺乏導(dǎo)致肺栓塞的患者的編碼序列上發(fā)現(xiàn)了2種不同位點(diǎn)的同義突變,位于8號(hào)外顯子第86位C全部發(fā)生了c.783 CT(pro 261 pro)突變(40/40),16號(hào)外顯子第131位A發(fā)生了c.2097 AG(pro 699 pro)突變(36/40),這些突變可能引起PSD的發(fā)生。2、我們發(fā)現(xiàn)了3例患者發(fā)生了c.1283 GA(ser 428 asn)突變(3/40),1例患者發(fā)生c.1391 GA(arg 464 gin)突變(1/40),1例患者發(fā)生c.1397 TC(val466 ala)突變(1/40),2例患者發(fā)生了c.1590 TC(asn 530 asn)突變(2/40)。
[Abstract]:BACKGROUND: Venous thromboembolism (VTE) is caused by vascular wall injury, slowing or stopping of blood flow, and hypercoagulable state of blood, including deep venous thrombosis (DVT) and pulmonary embolism (PE). Recently, pulmonary embolism (PE) has attracted more and more attention because of its acute onset, atypical clinical manifestations and high mortality. Poor (PSD) is an established risk factor for venous thromboembolism (VTE). PSD is an autosomal dominant inherited disease with incomplete penetrance and is associated with mutation or polymorphism of the coding protein S gene (PROS1). The protein S gene has 16 exons, spanning 101 kb, and is located in 3q11.1. At present, more than 200 mutations of PROS1 have been identified to cause PS at home and abroad. However, there are few reports about protein S deficiency leading to pulmonary embolism, and few reports about the detection of PROS1 gene in eligible patients. From June 2011 to December 2016, 40 patients with pulmonary embolism diagnosed by CTA were selected from Bethune First Hospital of Jilin University. Their free protein S activity was lower than 50% and 2 patients with normal free protein S activity were selected. Their peripheral blood was collected for 10 ml and centrifuged at room temperature. The free protein S activity was detected by the principle coagulation method, the free protein S and the total protein S were detected by the ELISA method, and the DNA was extracted from the blood cells for PCR amplification and sequencing. Type II was characterized by a decrease in the activity of PS in the blood, while the overall and free PS antigen levels remained normal (qualitative reduction); Type III was characterized by a decrease in total PS antigen levels within the normal range, while free PS antigen levels. Protein S activity was lower than 50% in 40 patients, including 18 males (45%) and 22 females (55%). The male age was (54.88 20.42) and the female age was (56.59 Sequencing, we found 21 different mutations in the coding sequence, mainly distributed in exon 2, 5, 8, 10, 12, 14 and 16. Among them, 15 mutations occurred in amino acids, 5 mutation amino acids remained unchanged, and 1 mutation became a termination code. Exon 2. Two patients with normal protein S activity, one male, 60 years old, one female, 59 years old, were found. 15 mutations were found in the coding sequence, mainly distributed in exons 2, 5 and 10. Among them, 12 mutations occurred in amino acids, 2 mutations in amino acids remained unchanged, and one mutation became a termination code. In the non-coding sequence, we also found 10 different mutations, mainly in exon 16. 2 patients with normal PS activity showed the same results, and 40 patients with PSD were found. 40 patients with PSD compared with the results: all of the 86th C of exon 8 had C. 783 CT (pro261 pro) mutation, but the mutation was reversed. The amino acid in exon 131 A of exon 16 was c.2097 AG (pro 699 pro) mutation, which was also synonymous with the mutation. However, this mutation did not occur in all 40 patients (36/40). In 3 patients, c.1283 GA (ser 428 asn) mutation occurred in exon 12 32 G, resulting in amino acid mutation by ser. Asparagine (asn); c.1391 GA (arg 464 gin) mutation at position 140 of exon 12 in 1 patient, resulting in amino acid mutation from Arg to glutamine (gin); c.1397 TC (val 466 ala) mutation at position 146 of exon 12 in 1 patient, resulting in amino acid mutation from valine (val) to ALA in exon 14 in 2 patients C. 1590 TC (asn 530 asn) mutation occurred in the 2nd T, but the transcriptional amino acid was asparagine (asn) unchanged. Conclusion: 1. Two different mutations were found in the coding sequence of 40 patients with pulmonary embolism caused by inherited protein S deficiency. All of the 86th C in exon 8 had C. 783 CT (pro 261 pro) mutation (40/40). C. 2097 AG (pro 699 pro) mutation (36/40) occurred in 131 A of exon 16. These mutations may cause PSD. 2. We found that 3 patients had C. 1283 GA (ser 428 asn) mutation (3/40), 1 patient had C. 1391 GA (arg 464 gin) mutation (1/40), 1 patient had C. 1397 TC (val466 ala) mutation (1/40), and 2 patients had C. 1397 TC (val466 ala) mutation (1/40). .1590 TC (ASN 530 ASN) mutation (2/40).
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R563.5
本文編號(hào)：2217696
[Abstract]:BACKGROUND: Venous thromboembolism (VTE) is caused by vascular wall injury, slowing or stopping of blood flow, and hypercoagulable state of blood, including deep venous thrombosis (DVT) and pulmonary embolism (PE). Recently, pulmonary embolism (PE) has attracted more and more attention because of its acute onset, atypical clinical manifestations and high mortality. Poor (PSD) is an established risk factor for venous thromboembolism (VTE). PSD is an autosomal dominant inherited disease with incomplete penetrance and is associated with mutation or polymorphism of the coding protein S gene (PROS1). The protein S gene has 16 exons, spanning 101 kb, and is located in 3q11.1. At present, more than 200 mutations of PROS1 have been identified to cause PS at home and abroad. However, there are few reports about protein S deficiency leading to pulmonary embolism, and few reports about the detection of PROS1 gene in eligible patients. From June 2011 to December 2016, 40 patients with pulmonary embolism diagnosed by CTA were selected from Bethune First Hospital of Jilin University. Their free protein S activity was lower than 50% and 2 patients with normal free protein S activity were selected. Their peripheral blood was collected for 10 ml and centrifuged at room temperature. The free protein S activity was detected by the principle coagulation method, the free protein S and the total protein S were detected by the ELISA method, and the DNA was extracted from the blood cells for PCR amplification and sequencing. Type II was characterized by a decrease in the activity of PS in the blood, while the overall and free PS antigen levels remained normal (qualitative reduction); Type III was characterized by a decrease in total PS antigen levels within the normal range, while free PS antigen levels. Protein S activity was lower than 50% in 40 patients, including 18 males (45%) and 22 females (55%). The male age was (54.88 20.42) and the female age was (56.59 Sequencing, we found 21 different mutations in the coding sequence, mainly distributed in exon 2, 5, 8, 10, 12, 14 and 16. Among them, 15 mutations occurred in amino acids, 5 mutation amino acids remained unchanged, and 1 mutation became a termination code. Exon 2. Two patients with normal protein S activity, one male, 60 years old, one female, 59 years old, were found. 15 mutations were found in the coding sequence, mainly distributed in exons 2, 5 and 10. Among them, 12 mutations occurred in amino acids, 2 mutations in amino acids remained unchanged, and one mutation became a termination code. In the non-coding sequence, we also found 10 different mutations, mainly in exon 16. 2 patients with normal PS activity showed the same results, and 40 patients with PSD were found. 40 patients with PSD compared with the results: all of the 86th C of exon 8 had C. 783 CT (pro261 pro) mutation, but the mutation was reversed. The amino acid in exon 131 A of exon 16 was c.2097 AG (pro 699 pro) mutation, which was also synonymous with the mutation. However, this mutation did not occur in all 40 patients (36/40). In 3 patients, c.1283 GA (ser 428 asn) mutation occurred in exon 12 32 G, resulting in amino acid mutation by ser. Asparagine (asn); c.1391 GA (arg 464 gin) mutation at position 140 of exon 12 in 1 patient, resulting in amino acid mutation from Arg to glutamine (gin); c.1397 TC (val 466 ala) mutation at position 146 of exon 12 in 1 patient, resulting in amino acid mutation from valine (val) to ALA in exon 14 in 2 patients C. 1590 TC (asn 530 asn) mutation occurred in the 2nd T, but the transcriptional amino acid was asparagine (asn) unchanged. Conclusion: 1. Two different mutations were found in the coding sequence of 40 patients with pulmonary embolism caused by inherited protein S deficiency. All of the 86th C in exon 8 had C. 783 CT (pro 261 pro) mutation (40/40). C. 2097 AG (pro 699 pro) mutation (36/40) occurred in 131 A of exon 16. These mutations may cause PSD. 2. We found that 3 patients had C. 1283 GA (ser 428 asn) mutation (3/40), 1 patient had C. 1391 GA (arg 464 gin) mutation (1/40), 1 patient had C. 1397 TC (val466 ala) mutation (1/40), and 2 patients had C. 1397 TC (val466 ala) mutation (1/40). .1590 TC (ASN 530 ASN) mutation (2/40).
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R563.5
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