| Background:Hereditary thrombophilia mainly involves children and young adults.Venous thrombosis often occurs in rare sites(such as intracranial venous sinus thrombosis,intraperitoneal deep venous thrombosis and pulmonary embolism).Its severity,harmfulness and sequelae can not be ignored.Genetic factors are known to play an important role in the pathogenesis of venous thromboembolism(VTE).By consulting the relevant literature,we found that the domestic research in this field is still relatively weak,and in-depth research will contribute to the accurate diagnosis and treatment and individualized management of thrombotic diseases.In the past,the first generation of gene detection technology could only explain the etiology of about5～10%.In recent years,the development of second-generation sequencing technology has opened up a new situation for the in-depth study of hereditary thrombophilia.At present,the research on clinical and molecular genetic characteristics of patients with thrombotic diseases in China,especially in South China,is still very limited.Objective:To study the clinical characteristics and genetic related factors of patients with hereditary thrombophilia,accumulate data for the study of hereditary thrombophilia,and explore the application of second generation sequencing in the diagnosis of thrombophilia.Methods:The clinical data and gene sequencing results of 40 patients with thrombotic diseases with gene abnormality were collected,and the related clinical features and gene abnormalities were analyzed.Results:1.General information and clinical manifestationsIn this study,40 unrelated cases of thrombotic diseases were included.The median age of onset was 29.5 years(12-64 years),36 cases(90.0%)were younger than 50 years old for the first time,and 4 cases(10.0%)were older than 50 years old.There were 28 male patients(70.0%)and 12 female patients(30.0%).The ratio of male to female was 2.3:1.The median age of female patients was 30.5 years old,and all of them were of childbearing age(20-43 years old).There were 40 patients with or without obvious predisposing factors.15 patients(37.5%)could identify 1 to 2 risk factors related to thrombosis.Among them,7patients(17.5%)were induced by pregnancy or childbirth,accounting for 58.3%(7 /12)of female patients;The other predisposing factors included 2 cases(5.0%)of tumor related(1 case was diagnosed with ovarian malignant tumor 4 years after onset,and 1 case was diagnosed with acute B-cell leukemia at the same time),1 case(2.5%)of antiphospholipid syndrome,4 cases(10.0%)of long-distance flight / sedentary /braking,1 case(2.5%)of oral estrogen,2 cases(5.0%)of venipuncture.25 patients(62.5%)had no definite cause of onset.All 40 patients were diagnosed with different types of thrombotic diseases by imaging examination.The clinical manifestations of hereditary thrombophlebitis are diverse.It can be seen from simple single site venous thrombosis to complex,rare and severe multiple site thrombosis.Among them,36 cases(90.0%)had venous thrombosis,3 cases(7.5%)had arterial thrombosis(cerebral infarction,myocardial infarction,renal infarction,limb arterial thrombosis),and 1 case(2.5%)had mixed arteriovenous thrombosis(middle cerebral artery,carotid artery,deep veins of both lower limbs).At the first onset,29(72.5%)of 40 patients developed thrombosis in a single site,and 11(27.5%)patients developed thrombosis in two to four sites at the same time.25 patients(62.5%)first presented with DVT of lower limbs,among which 7 patients(28.0%)were complicated with pulmonary embolism;9 cases(22.5%)had intracranial venous sinus thrombosis as the first site,and 2 cases had cerebral hemorrhage;Four patients(10.0%)had intra-abdominal venous thrombosis(including hepatic vein,portal vein,mesenteric vein,splenic vein and renal vein);Two patients(5.0%)had the upper extremity deep vein as the first site.A total of 55 independent thrombotic events occurred in 40 patients,with an average of 1.35 times per patient.Thrombotic events occurred from 1 to 4 times.Among them,29 patients(72.5%)had single thrombosis,11 patients(27.5%)had recurrent thrombosis,9patients(81.8%)had venous thrombosis recurrence,and 2 patients(18.2%)had arterial thrombosis recurrence.There was no significant difference in recurrence rate(p=0.600).2.Analysis of second generation sequencing resultsThe second generation sequencing of the common coagulation related gene panel was performed in 64 patients.At least one gene mutation was detected in 40 patients(62.5%),of which more than one mutation was detected in 9 patients(14.1%),and no mutation was detected in 24 patients.A total of 50 gene mutations were detected in 40 patients,involving 40 loci of 12 genes related to hemorrhagic and coagulation diseases,of which heterozygous variation accounted for 98.0%(49 / 50)and homozygous variation accounted for 2%(1 / 50).In the nature of variation,missense mutation accounted for 74.0%(37 / 50),nonsense mutation accounted for 12.0%(6 /50),small deletion / duplication mutation accounted for 4.0%(2 / 50),frameshift mutation accounted for 4.0%(2 / 50),and splice region mutation accounted for 4%(2/ 50).Among the 50 gene mutations detected,SERPINC1,PROC and PROS1 accounted for 72%(36 / 50),THBD gene mutation accounted for 6%(3 / 50),and coagulation factor gene mutation accounted for 12%(6 / 50).Among them,1 case of F2 G1787 T and 3 cases of F5 gene mutation were detected,which were F5 c.1059 C >G,c.1000 A > G and c.5378 G > T,respectively;FV Leiden mutation was not detected.FGA and FGB gene mutations were found in 1 case respectively.The PLAT gene mutation of tissue plasminogen activator(t-PA)encoding gene was 4%(2/50).The remaining COL3A1 c.3775G>A and CYP4V2 c.802-8＿810delins GC variants were detected in 1 cases.In this group,31 patients(77.5%)were detected with single gene mutation,and 9patients(22.5%)were detected with compound gene mutation involving two or more loci.The compound gene abnormalities were mainly anticoagulant protein complex or anticoagulant protein combined with other gene defects(78.9%,15/19).Among the 50 gene variants,58%(29 / 50)were identified as pathogenic or suspected pathogenic according to the classification guidelines of ACMG,combined with clinical phenotypes and pedigrees of the patients,and 42%(21 / 50)were identified as having unknown significance.3.Comparison of gene test and phenotype testIn this group,30 times of patients with three kinds of anticoagulant protein gene defects were detected,23 times of patients with abnormal phenotype test results,the ratio of phenotype reflecting gene abnormality was 76.7%,and some patients detected anticoagulant protein coding gene abnormality,but the phenotype test results were in the reference range(7 / 30,23.3%).There were 6 patients with abnormal serpinc gene(at activity 54.8±28.4%),including 5 patients with decreased at activity and 1 patient with normal at activity.The ratio of phenotype consistent with gene results was 83.3%(5 / 6);There were 16 cases of PROC gene mutation(PC activity 56.8±35.1%),of which PC activity was reduced in 12 cases,and the ratio of phenotypic coincidence to gene result was 75%(12/16).There were 8 patients with Pros1 gene mutation(PS activity 37.0±27.3%),6 patients with protein S activity decreased,and the proportion of phenotype consistent with gene results was 75%(6 / 8).4.Variation and significance of common thrombus related genesSecond generation sequencing provides molecular level diagnosis for thrombotic diseases and is of great value for further analysis of the molecular mechanism of thrombosis.In this study,13 mutations were detected in the PROC gene,including 7mutations of PROC R189 W,6 heterozygous mutations and 1 homozygous mutation.It shows that the mutation is very common in this group,which is consistent with previous studies,that is,R189 W mutation is the "hot spot" of PROC gene mutation in China.In this study,a p.R596 L mutation was detected in F2 gene.The proband was a28-year-old female with repeated multiple site thrombosis,including acute pulmonary embolism,inferior vena cava,bilateral iliac vein,left renal vein,and bilateral lower extremity vein thrombosis.In the family,her mother and sister had lower extremity deep venous thrombosis,and the first generation verification found that she carried the same site mutation.According to foreign literature,antithrombin resistance may be the pathogenesis,but the mutation has not been reported in China.In this study,we found one case of SERPINC1 double mutation and PROS1 mutation.SERPINC1 M313 V has not been reported previously.The complex gene mutation may be the molecular pathogenesis of thrombosis in this patient,and the mutation that plays a major role in the pathogenesis needs further study.In this study,we found that one case of C205 W mutation in PROS1 gene may be associated with cerebral venous sinus thrombosis,DVT and PE of right lower limb,and the PS activity decreased slightly by 51%.The mutation was not previously reported at home and abroad.Conclusion:1.The main manifestation of hereditary thrombophilia is VTE.In this group of data,intracranial venous sinus thrombosis is more common than acute pulmonary embolism.VTE induced by pregnancy related factors accounts for more than half of women of childbearing age,which should be paid great attention to clinically.A small number of patients may present with single or combined arterial thrombosis.2.Anticoagulant protein gene deficiency was the main cause of hereditary thrombophilia(72%),and proc gene mutation was the most common.PROC C565 T mutation may be an important mutation hotspot of PROC gene,suggesting that patients with protein C deficiency should be screened for this locus.3.Second generation sequencing is of great value in the etiological diagnosis of thrombotic diseases.It is a powerful supplement to phenotypic detection.It can accurately confirm the gene mutation sites and molecular pathogenesis of thrombophilia.It is helpful to guide the clinical development of individualized treatment plans,and provide epidemiological data and drug treatment targets for the prevention and treatment of thrombophilia in China.4.In this study,1 cases of prothrombin defects induced by prothrombin G1787 T mutation may be related to recurrent multiple site venous thrombosis.The pathogenesis may be antithrombin resistance.5.It is found that the AT M313 V and PS C205 W mutation has not been reported before.It is necessary to further study their relationship with thrombotic diseases. |
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