A novel mutation in a patient with congenital coagulation factor XII deficiency

Field: Medicine (Health Sciences)

Human coagulation factor XII (FXII), also called Hageman factor, is a plasma plycoprotein that is functionally deficient in individuals with Hageman trait; which is an inherited trait discovered by chance during preoperative blood coagulation screening tests. FXII is a single-chain 596-amino-acid zymogen of a serine protease with an approximate molecular weight of 80 000. FXII appears to play an important role in blood coagulation, fibrinolysis and kinin generation. Contact of FXII with negatively charged surfaces leads to proteolytic cleavage and activation of the FXII molecule by plasma kallikrein at Arg353 − Val354. The generated FXIIa can initiate the rapid intrinsic coagulation pathway by activation of Factor XI and also seems to participate in the conversion of plasminogen to plasmin and bradykinin production pathways. Severe deficiency of FXII does not lead to an increased bleeding tendency, but has been associated with an increased risk for venous, as well as for arterial, thromboembolism.1 We report here the genetic analysis of the FXII gene obtained from a patient with congenital FXII deficiency who died of recurrent, lethal cerebral thrombosis. After gene sequencing, we found that the present case of congenital FXII deficiency was the result of the homozygous C46T polymorphism site in the 5′ untranslated region combined with a novel C73S heterozygous amino acid substitution in FXII protein. CASE REPORT Patient The subject was a 73-year-old Chinese woman. The case history showed that she had been diagnosed with “coronary heart disease complicated with atrial fibrillation” and “type 2 diabetes” since 1997. She complained of dizziness, left limbs weakness, dysphasia, and right hemiplegia seven years ago and was admitted to and diagnosed with a “stroke” in the local hospital. She didn’t recover well after treatment. Four years ago, she was admitted to the Department of Neurology of our hospital complaining of headache, dizziness and right limbs weakness. The CT scan showed cerebral infarction of the right temporal lobe. After appropriate treatment she recovered well and was discharged. One year ago the patient was admitted to the same department again complaining of aggravated right hemiplegia, dysphasia and stupor and was diagnosed with an “infarction of left middle cerebral artery, coronary disease complicated with arrhythmia, hypertension and diabetes.” Although the coagulation test showed that her activated partial thromboplastin time (APTT) was severely longer than normal (120 seconds), the patient didn’t have any hemorrhagic signs and recovered well after treatment. After discharge from the hospital, she was given continuous oral treatment including calcium channel blocker, β blocker, α-glucosidase inhibitor and warfarin (1.25 mg/d). She was admitted into our hospital on October 4, 2005 complaining of hemi-weakness of the right limbs and a choking cough for 3 days. Physical examination showed that her blood pressure was 165/78 mmHg and she her heart rate was 76 beats/minute with atrial fibrillation in feature. Neurological examination showed that she had motor dysphasia and reciprocal paralysis of the left face and right limbs. CT scan revealed multiple ischemic infarction foci in her right temporal lobe, frontal lobe and occipital lobe together with multiple encephalomalacia foci at the basal ganglions of both sides and the left cerebellum. Her routine blood test results were normal. Despite vigorous treatment, the patient's condition kept on worsening and she fell into a deep coma. Finally, she died of infectious shock 12 days later. Sample collection, preparation and extraction of DNA Informed consent for venipuncture was obtained from the patient and her family. Blood samples were obtained by clean venipuncture from the patient and her family members in sodium citrate vacutainers. Poor-platelet plasma samples were obtained by centrifuging at 4000 rpm/min under 4°C for 15 minutes and stored under −80°C to be tested for coagulation parameters and factor activity determinations. Genomic DNA was extracted by the phenol-chloroform method as described previously1 from the peripheral blood buffy coat cells for PCR. Coagulation tests and factor activity determinations Determination of APTT, prothrombin times (PT), fibrinogen concentration and factors VIII, IX, XI, XII coagulation activity were performed using the test kits and STA-R Automated Coagulation Laboratory Analyzer bought from the STAGO (France) according to the operation manual. Designation of the primers Seven pairs of primers covering all the exons and their flanking regions of the factor XII gene were designed according to factor XII gene sequence from Pubmed (GenBank AF538691) and from referring to the literature.1 All the primers were synthesized with the 391DNA synthesizer bought from Applied Biosystems Co. (USA). PCR PCR for all the exons and their flanking regions were performed in 100 μl reaction system containing 1 × PCR buffer, 1.5 mmol/L of MgCl2, 0.25 mmol/L of each primer, 0.25 mmol/L of dNTPs, 2.5 U of Taq DNA polymerase (All bought from Shennengbocai Inc., Shanghai), and approximately 500 ng of genomic DNA. DNA samples were denatured at 95°C for 5 minutes followed by 30 cycles, each of 95°C for 30 seconds, respective melting temperatures for 30 seconds and 72°C for 30 seconds and then extended at 72°C for 10 minutes. After PCR, 5 μl of each product was put in electrophoresis in 1.5% agarose and compared with DL2000 molecular marker. Purification and sequencing of PCR product The PCR products were purified from the excised agarose gel slices using the Gel Purification Kit bought from Shennengbocai Inc. to be prepared for sequencing. Sequencing was performed with double deoxygenation chain termination method using an ABI PRISM 377 DNA Sequencer (Applied Biosystems, CA). The results were matched with the published factor XII gene sequence (GeneBank AF538691) using the Chromas software to look for mutation sites. Sequencing results with gene mutation were then reverse sequenced to confirm the mutation. Controls Blood samples drawn from 100 healthy blood donors aged 18 to 53 years old, average age was 38 years old. Among them 61 were male, 39 were female. The Blood Center of Zhejiang Province provided all the samples. The genomic DNA obtained from these samples were used as controls for FXII gene analysis. Coagulation test during the two hospitalization periods During the hospitalization period of the year 2004, the patient's coagulation test results showed that her APTT was longer than 120 seconds and the thrombin time (TT) was 16.6 seconds. But her PT and fibrinogen concentrations (Fib) were normal and her antithrombin activity (AT) and D-Dimer concentrations (D-D) were elevated. During the hospitalization period of the year 2005, her coagulation test results showed that she still had a very long APTT (175.9 seconds), which could be corrected with normal fresh plasma and a long PT (18.75 seconds). The Fib level was 4.78 g/L. Her plasma AT level, factor VIII coagulation activity (FVIII:C), factor IX coagulation activity (FIX:C) and VWF antigen level (VWF:Ag) were all above normal. We also detected weak positive (1:5) antiphospholipid antibodies (ACA) in her blood (Table 1).Table 1: Coagulation tests of the proband during two hospitalization periodsCoagulation test of the patient and her family members We did coagulation tests for all her children (her husband had died previously) and found that one of her sons also had elongated APTT. Both the patient and this son had abnormal factor XII coagulation activity (FXII:A) (Table 2). Her son's abnormal FXII:A is obviously inherited from her (Figure 1).Table 2: Coagulation test results of the patient and her family membersFigure 1.: The pedigree of congenital FXII deficiency. The arrow indicates the propositus.Gene analysis Sequence analysis of all exons and flanking sequences of the FXII gene revealed that the patient was homozygous for a polymorphism mutation of C to T in the 5′ untranslated region near the exon 1 coding region and heterozygous for a one-base substitution of G to C at nucleotide position 3774 in exon 4 (Figure 2), which resulted in the substitution Cys73 (TGT) to Ser (TCT) of the FXII protein. The latter substitution is a novel gene mutation in the FXII gene sequence which has never been previously reported. Sequencing of the FXII gene of the patient's sons also showed the change in the 5′ untranslated region and the same change in exon 4 of the FXII gene sequence was also found in one of her sons who had prolonged APTT and lower plasma FXII:C level. We did not find the same changes in FXII gene sequence in any of the normal controls.Figure 2.: A partial sequence of exon 4 of the patient's FXII gene. Arrow indicates the site of nuceotide 3774 with a heterozygous G→C point mutation.DISCUSSION Through the present pedigree study, we found a homozygous C46T polymorphism site in the 5′ untranslated region and a novel heterozygous G3774C point mutation in exon 4 of the FXII gene of the patient who had significantly decreased plasma FXII level and extremely prolonged APTT. The latter mutation could also be found in one of her son who presents with long APTT and low plasma FXII levels. It is unfortunate that the patient's husband had died long before and we could not get the consent from the patient's brother, so we could not detect the changes in FXII:C level or the gene sequences of these two persons. We can only deduce that her son's Hageman trait was inherited from her. Congenital FXII deficiency is an autosomal recessive (in some cases, dominant) disorder. Several natural mutations have been reported in the human FXII gene, which include missense mutations, deletions, splice site mutations and nucleotide substitutions in the 5′-untranslated region.2-8 Based on the published literature we found that the mutation identified in this pedigree with congenital FXII deficiency has never been reported before. Patients with a FXII deficiency usually present with prolonged APTT. Although this normally is indicative of a bleeding tendency hemorrhage is not observed clinically; as was the case in our report. Instead, a thrombotic phenotype is more frequent in human FXII-deficiencies.9 The patient seemed to be another example of this phenotype. During a period of seven years she had experienced 4 thrombotic events, all diagnosed as cerebral infarctions of different foci, that finally progressed to a lethal cerebral thrombotic event causing diffusive ischemic infarction of the cerebral and cerebellar areas despite continuous use of an anticoagulant (warfarin) and a prolonged PT. The C46T 5′ untranslated region polymorphism found in the patient is a well defined polymorphism related with decreased plasma FXII level. According to Kanaji's study10 the allele frequency of C46/T is relatively common, estimated to be 0.27/0.73 in Orientals. Patients with homozygous a C46T polymorphism of the FXII gene had significantly lower plasma FXII levels than patients with the heterozygous C/T allele type and the wild 46C type. They suggested that this may be due to the influence on the translation capacity of the FXII gene due to the polymorphism mutation. There have been reports on the association of the FXII C46T polymorphism with thrombotic events. But other reports did not support it.11 On the other hand, the patient's son with the wild type of 46C at the 5′ untranslated region also had lower plasma FXII levels, which could not be explained by the mutation of the 5′ untranslated region of his FXII gene. We suggest that the lower plasma FXII activity in the patient's son may be associated with the novel heterozygous point mutation in exon 4 of the FXII gene. The synergetic interaction between the two mutations may have caused the further decrease in the FXII activity in the patient and, finally, the lethal thrombotic events. There are several well-characterized genetic defects that lead to increased thrombotic risk; such as the inheritable defects in genes encoding fibrinogen, prothrombin, antithrombin, protein C, coagulation factor V, factor XII, etc. According to the report of Souto et al,12 among the clotting factors studied, factor XII (FXII) levels exhibited one of the highest heritabilities (67%) and a significant positive genetic correlation (0.351) with thrombotic disease. This is likely due to defective contact activation-stimulated fibrinolysis. Recent work showed that, while FXII rarely contributes to initiation of thrombus formation at sites of vascular lesions, FXIIa on the exposed surface of the thrombus contributes to thrombin generation via FXI activation.13 Evidence shows that the physiological role of FXII may be related to fibrinolysis. FXII structurally resembles the fibrinolytic proteins plasminogen, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). FXIIa indirectly triggers fibrinolysis by activating prekallikrein, which in turn cleaves single-chain u-PA into active two-chain u-PA, and, following activation of prekallikrein, the kallikrein generated can liberate bradykinin from high molecular weight kininogen. Both kallikrein and bradykinin liberate t-PA in vivo. In addition, study results have demonstrated that FXII can also activate plasminogen, a process potentiated by negatively charged surfaces and zinc ions. It is suggested that FXII not only induces fibrinolysis in plasma by activating other plasminogen activators, but also directly induces fibrinolysis by activating plasminogen. So a relationship has been described between depressed FXII-dependent fibrinolysis and cardiovascular diseases. However, other studies suggest that FXII plays no role in ischemic vascular disease.14 And recent work by Kleinschnitz et al15 demonstrated that deficiency or inhibition of FXII protects mice from ischemic brain injury. They suggested that FXII contributes to pathologic clotting through the intrinsic pathway and that some processes involved in pathologic thrombus formation are distinct from those required for normal hemostasis. Therefore, it is unclear from existing literature whether FXII deficiency leads to thrombophilia or is directly related to any other disorder of hemostasis. This patient also presents with other precipitating factors for thrombophilia, including a history of hypertension, coronary heart disease complicated with atrial fibrillation and with diabetes. Atrial fibrillation itself has been overtly accepted as a precipitating factor of recurrent and diffusive cerebral ischemic infarction. Although she had been administered warfarin after the third thrombotic event and her PT was indeed prolonged during this admission to our hospital, there was still a possibility that the PT is not long enough to protect the patient from recurrence of another infarction. Besides, we had only excluded the presence of AT deficiency in the patient and we did not detect the other thrombophilic coagulation parameters; such as congenital protein C or protein S deficiency. In conclusion, we have found a novel heterozygous point mutation in the FXII gene in a patient with inherited FXII deficiency who died of recurrent, lethal cerebral thrombosis. Further study in the molecular mechanism of the novel mutation should be conducted to disclose the relationship between the mutation and FXII deficiency and the occurrence of thrombotic diseases.