Abstract

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A heterozygous Arg393His point mutation at the reactive site of antithrombin (AT) gene causing thrombosis in a Vietnamese patient is reported.  The present variant is characterized by a severe reduction of functionally active AT plasma concentration to 42% of normal resulting in multiple severe thrombotic events such as cerebral venous thrombosis, recurrent deep veinous thrombosis (DVT) and the development of kidney cancer.  This is the first report of a patient with kidney cancer in whom mutation of AT gene has been investigated.  The “two-way association” between cancer and thrombosis in which thromboembolism (VTE) can be both a presenting sign and a complication of cancer is discussed.  Efficacy of low-dose wafarin anticoagulant used for preventing DVT and kidney cancer from this patient is also mentioned.  For this variant AT (Arg393His), the name AT Hanoi is proposed.

Introduction

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Antithrombin (AT) is the primary inhibitor of thrombin and other activated serine proteases in the blood coagulation system [1].  AT belongs to a large group of structurally related proteins known as serine protease inhibitors (serpins).  The gene for AT is 13.5 kb in length and is located on human chromosome 1 at q23-25.  It contains 7 exons and 6 introns [2].  AT is synthesized by hepatocytes as a 464-amino acid precursor.  Before secretion into the blood, the 32-amino acid signal peptide is cleaved to yield the mature protein with 432-amino acids.  Plasma AT is a single-chain glycoprotein with a molecular weight of 58.2 kd and three disulfide bonds linking cysteine residues 8-128, 21-95, and 247-430 [3,4].  The reactive site, an Arg 393-Ser 394 bond, is located in the C-terminal domain of the molecule [5].  The heparin binding site located near the NH₂ terminus [6], which interacts with heparin to modify the affinity of AT for its protease [7].  AT deficiency is a rare hereditary disorder that is inherited in an autosomal dominant fashion.  AT deficiency generally comes to light when a patient suffers recurrent venous thromboembolism (VTE), which includes both deep venous thrombosis (DVT) and pulmonary embolism (PE).  DVT usually occurs in the legs and is characterized by pain, swelling and redness of the limb.  VTE may also occur in more unusual places in the brain (cerebral venous thrombosis), liver (portal vein thrombosis and hepatic vein thrombosis), mesenteric vein, kidney (renal vein thrombosis) and possibly the veins of the arms [8].  Arterial thrombosis is uncommon, but has been reported [9].           

In the present study, a heterozygous Agr393His point mutation at the reactive site of AT gene causing thrombosis in a Vietnamese patient is reported. The present variant is characterized by a severe reduction of functionally active AT plasma concentration to 42% of normal resulting in multiple severe thrombotic events such as cerebral venous thrombosis, recurrent deep veinous thrombosis (DVT) and the development of kidney cancer. The “two-way association” between cancer and thrombosis in which thromboembolism (VTE) can be both a presenting sign and a complication of cancer is discussed.  Efficacy of low-dose wafarin anticoagulant used for preventing DVT and kidney cancer from this patient is also mentioned.  For this variant AT (Arg393His), the name AT Hanoi is proposed, for the city in Viet Nam where the proband (II-2) was born.

Case History Of the Proband and his Family History

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The proband (II-2) is a 58-year-old Vietnamese man.  He is non-smoking, non-alcoholic person.  At age 23, he had a history of intracranial hypertension including projectile vomiting without nausea, seizures (epilepsy), visual disturbances, vertigo and tinnitus.  There were no infectious problems.  There were no specific treatments.  The cause of this intracranial hypertension remained unknown.   At age 42, he had suffered of swelling and pain in the left leg and deep venous thrombosis (DVT) was confirmed by Doppler ultrasound.  He was treated with warfarin (1mg/day).   However, just after two weeks of treatment with warfarin (1mg/day), he had suffered of pain in the back and the presence of blood in urine was observed and he was diagnosed with kidney cancer.  After surgical removal of the left kidney affected by cancer at the Regional Haguenau Hospital, Haguenau, France, he was treated with warfarin (1mg/day).  No specific cancer treatment such as chemotherapy, radiotherapy, etc was applied after this surgery.  He remained well but he had recurrent DVT at the same site of the left leg as soon as he stopped the treatment with warfarin.  Apart from this thrombotic event, he was free of cancer.  When he was 52-year-old, AT deficiency was diagnosed at the Medical Center of University of California, San Diego, San Diego, U.S.A. and revealed a reduction of functionally active AT plasma concentration to 42% of normal: 50% compared to 118% of the norm. Since then, he was given long-term treatment with warfarin (4mg/day) to maintain an international normalized ratio (INR) of 2 to 3.  Currently, he is well, free of cancer and thrombotic event.  The family pedigree of the proband (II-2) is shown in Figure 1.  Other family members related to the mother of the proband (II-2) such as maternal uncles, cousins had had symptoms suggesting AT deficiency such as cerebral venous thrombosis, myocardial infarction, retinal vein occlusion but were now dead.

Materials and Methods

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AT assay:

AT assay was performed at the Medical Center of University of California, San Diego, San Diego, U.S.A.  Blood sample was obtained from the patient (proband II-2).  AT assay was performed on the Sysmex CA-6000 (Sysmex Corporation, Kobe, Japan) automated instrument using citrated plasma samples and Berichrom^® Antithrombin-III (A) (Chromogenic method by Dade Behring Berrychrome, Newark, DE, U.S.A.).  The reagents of this kit and the reaction conditions used are according to the manufacturer’s recommendations.

Isolation of genomic DNA:

The RNA-free genomic DNA samples were isolated from whole peripheral blood   or from buccal cells in mouthwash (Original Mint Scope^® Mouthwash, Procter&Gamble) of the patient (proband II-2) and his different family members using the Puregene^® DNA Purification Kit (Gentra System, Minneapolis, Minnesota, U.S.A.).  The DNA concentration was determined by using the ND-1000 spectrophotometer NanoDrop^® device.

Amplification:

In this study, the exons 3a and 3b were designated as exons 3 and 4, respectively.  So that the numbering the exons 1 to 7 more accurately reflects the structure of AT gene than does the nomenclature of Bock, Marrinan, and Radziejewska [10].  For genomic characterization, each of the seven exons and flanking intronic sequences of the human AT gene locus (GenBank X68793) were PCR-amplified by means of primers designed to be specific to the intronic genomic sequences. PCR primers employed and amplicon sizes are shown in Table 1.   Amplification was conducted using a DNA Thermal cycler (MiniCycler^TM, MJ Research).  The reaction was conducted in a total volume of 50µL with 2.5 units of Taq DNA polymerase (Invitrogen, Carlsbad, CA, U.S.A.).  Amplification conditions were as follow:  Denaturation at 94°C for 1 min, annealing at 60°C for 2 min, and elongation at 72°C for 1 min, each for 35 cycles.  The PCR product was analyzed by electrophoresis on a 1.5% agarose gel.  The PCR product was then isolated and purified by using the reagents and conditions according to the manufacturer’s instructions of the QIA quick^® PCR Purification Kit (QIAGEN Sciences, Maryland, U.S.A.).  The obtained purified PCR product was sequenced using the same primers as for PCR and the ABI DNA sequencer (Applied Biosystems).

Results

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In order to confirm the diagnosis of AT deficiency, the sequencing analysis of the complete coding region of AT gene from DNA genomic (isolated from whole peripheral blood and buccal cells) of the proband (II-2) was performed and revealed a heterozygous point mutation (exon 7:  g.13830G>A; c.1274G>A; p.425R>H) (Figure 2).  Depending on the chosen start the position of the nucleotide variant, this p.425R>H (Arg 425His) corresponds to Agr393His variant.  The mutation analysis of AT gene from DNA genomic (isolated from buccal cells) from the parents and different family members of the proband (II-2) was also performed.  The sequencing results showed that the heterozygous Arg393His point mutation was also found from the mother (I-2), the proband’s brother (II-3), and one of the two his children, III-6 (see Figure 1).  The mother (I-2) is carrier of AT deficiency.  In addition, the sequencing results obtained for ten exons and flanking intronic sequences of the p53 gene locus from DNA genomic of the proband (II-2) revealed no mutations (data not shown).

Discussion

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The existence of a deficiency state involving AT was recognized first by Egeberg who described a Norwegian family with recurrent episodes of (VTE) [11].  Since then, families with hereditary AT deficiency have been found in many countries, and the link between AT deficiency and thrombosis has now been clearly established [12,13]. Arg393His point mutation of AT was previously reported in AT Chicago [14], Glasgow [15], Sheffield [16], and Kumamoto [17]. The present Arg393His point mutation is characterized by a functionally active AT plasma concentration so reduced (42% of normal) resulting in multiple severe thrombotic events such as intracranial hypertension probably due to a cecebral venous thrombosis, recurrent DVT and the development of kidney cancer. It is also important to note that mutations and allele loss of the p53 gene have been associated with tumors from a wide variety of human organs (including lung, breast, colon, esophagus, liver, bladder, ovary, and brain) and hematopoietic tissue [18].  Mutations in the p53 gene are now the most frequently (about half) observed genetic lesion in spontaneous human cancers [19].  In the present study, no mutation in the p53 gene from this proband (II-2) was found (data not shown). Since Trousseau reported in 1865 the occurrence of migratory thrombophlebitis in patients with cancer [20], researchers have increasingly recognized the “two-way association” idea of the relationship between cancer and thrombosis in which VTE can be both a presenting sign and a complication of cancer [21].  It has been postulated that clot formation at the tumor periphery may: (1) facilitate attachment of metastasis tumor cells to endothelial cells (tumor cells which fail to adhere do not survive); (2) provide nutrients and/or growth stimulants; (3) serve as structural lattice upon which tumor cells can proliferate; or (4) protect the tumor cells from host defense mechanisms [22]. Also, on the basis of statistical results from cohort studies and clinical trials from a series of patients hospitalized for VTE, it has been suggested that thrombotic episodes may also precede the diagnosis of cancer by months or years thus representing a potential marker for occult malignancy, in some patients [23,24]. The severe reduction in AT plasma concentration associated with this Arg393His point mutation could represent a genetic trait that predisposes to the blood coagulation, beneficial to cancer progression from this patient.  In this case, VTE is the manifestation of an occult malignancy.  Finally, the presence of kidney cancer from this patient was detected at an early stage during the treatment with warfarin (1mg/day) for DVT of the leg at age 42 (via the observation of blood in urine, see case history of the proband II-2). In this case, the increased risk of anticoagulant-associated bleeding (warfarin) at the site of the cancer [25,26] was positively contributed to the early detection of kidney cancer from this patient.  It is important to note that the use of conventional cancer chemotherapeutic agents are usually cytotoxic drugs which, when successful, have a greater toxic effect on the tumor than on the host.  Immuno-therapy and anticoagulant therapy are aimed at enhancing host response to the tumor [22]. Patients with VTE are generally managed with anticoagulant therapy with the aim of treating the acute event and preventing death to PE, in addition to minimizing the risk of recurrent VTE [27]. However, traditional approaches to anticoagulant therapy are often hampered by the presence of malignant disease and its treatment [27]. In addition, cancer patients are at increased risk of recurrent VTE and anticoagulant-associated bleeding [25,26].  Thus, the management of VTE may be complex in patients with cancer, and VTE can further compromise quality of life.  Because of the links between coagulation, cancer biology and prognosis, interest has grown in the potential benefit of anticoagulants such as warfarin and low-molecular-weight heparin (LMWH) for the prevention or treatment of cancer [28]. Apart from warfarin anticoagulant, no specific treatment for cancer such as chemotherapy, radiotherapy, etc was applied to this patient (proband II-2).  Since 16 years under low-dose wafarinanticoagulant treatment and currently, he is well, free of cancer and thrombotic event. It appears that low-dose warfarin is an effective and safe treatment in preventing VTE and cancer from this patient. This observation has supported the “two-way association” idea of the relationship between cancer and thrombosis mentioned above [23,24]. That is, coagulation activation itself may contribute to the progression of some tumors and that anticoagulants may, therefore, have anti-cancer activity [23,24].  It is evident that the association between VTE and cancer cannot be substantiated by a single case description but this is the first report of a genetic trait used for the demonstration of such a relationship. Although some studies have suggested that warfarin may also improve survival in cancer patients and reduce the incidence of cancer [29-31], larger case-control studies are required to confirm the findings of this preliminary observation and more research is needed to further define which cancer type and stage would most benefit from warfarin.

A question arises from this study concerns the highly variable clinical phenotype of hereditary AT deficiency ranging from asymptomatic to severe recurrent VTE or arterial thrombosis, leading to death [8,9]. The age of onset of the first thrombotic episode exhibited by a patient with hereditary AT deficiency varies considerably, and some patients remain asymptomatic for decades [32].  Indeed, based on the case history of the proband (II-2) and his family history (Figure 1), the proband’s mother (I-2), 94-year-old and carrier of AT deficiency, is asymptomatic.  The proband (II-2) had had cerebral venous thrombosis at age 23 and had suffered recurrent DVT of the leg and developed kidney cancer at age 42. The proband’s brother (II-3), showed the presence of heterozygous Arg393His point mutation, suffered repeated DVT of the leg and mesenteric venous thrombosis at age 50. His AT plasma concentration is not available.  One of the two his children (III-6), 25-year-old and showed the presence of heterozygous Agr393His point mutation, is actually no symptoms of AT deficiency. There are probably other yet undiscovered factors other than the genotype at the AT locus such as modifying genes and environmental factors acting in the phenotypical expression of the disease associated with AT deficiency. Another question arises as to whether asymptomatic patients with no additional risk factors should undergo some prophylactic therapy. Long-term anticoagulation carries with it significant risks.

In conclusion, this is the first report of a patient with kidney cancer in whom mutation of AT gene has been investigated.  The use of PCR coupled with direct sequencing performed from DNA isolated from buccal cells in mouthwash will allow the molecular characterization of most AT deficiencies in the simplest manner with no need of blood collection from patients. 

References

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Source(s) of Funding

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University of California, San Diego

Competing Interests

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No

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