Microsurgical reconstruction of lower extremity in homozygosity of C677T MTHFR gene mutation: case report and review of the literature

The presence of hypercoagulable states, either inherited or acquired, has been recognized as a risk factor for free flaps failure. Nevertheless, the actual risk of microvascular failure related to these conditions is yet to be determined. We present and discuss the case of a successful free tissue transfer in a 27-year-old man, with known homozygosity for the C677T polymorphism for the MTHFR gene mutation and anti-erythrocyte panagglutinin with a review of the literature.

INTRODUCTION

Hypercoagulable states represent a risk factor for microvascular thrombosis and failure of microsurgical flaps ^1,2. While technical errors were once considered the most common cause of microvascular failure, to date the widespread use of microsurgical techniques has significantly reduced the rate of flap loss attributable to the surgical procedure ³. Nevertheless, even under the best surgical circumstances, inherited or acquired individual factors, may compromise the outcome of a free flap reconstruction. While acquired conditions may be ascertained with a detailed patient history, inherited factors predisposing to blood clotting are commonly unknown to both patient and surgeon prior to a thrombotic complication. In literature, free flap failure has been related to factor V Leiden mutation, prothrombin A20210G gene mutation, MTHFR gene mutation, PAI-1 gene mutation, protein C and S activity deficiency, antithrombin III deficiency, lupus anticoagulant, plasma homocysteine level, anticardiolipin antibody, antiphospholipid antibody and ß2-glycoprotein antibody ^4,5. To date, there are no standardised guidelines for the preoperative screening of hypercoagulable states. The multitude and the heterogenicity of genetic factors predisposing to blood clotting makes the identification of a comprehensive panel of preoperative tests difficult, and its cost-effectiveness may not be favourable ^6,7. Furthermore, even in the presence of a known diagnosis of thrombophilia, scant literature data does not enable to define the actual risk of microvascular complications. Here we present the details and perioperative management of a successful microsurgical reconstruction in a patient diagnosed with homozygosity for the C677T polymorphism of the MTHFR gene and anti-erythrocyte pan agglutinizing antibody. A review of the literature on peri-operative thrombotic complications in patients with MTHFR gene mutations, undergoing free flap surgery was performed.

CASE REPORT

We report the case of a 27-year-old healthy male who sustained severe polytrauma following a car accident. At admission at our institution, the patient presented with a subamputation of the right leg, open fracture of the left femoral condyle, open fractures of the right radial and ulnar bones and fracture of the right scaphoid bone. For the present report, the authors will provide only details pertaining the management of the right leg injury. In the emergency room, clinical evaluation revealed a Gustilo-Anderson IIIB open fracture of both tibia and fibula (Fig. 1A-B). CT angiography scan showed no major vascular lesions, with integrity of the anterior tibial, posterior tibial and peroneal vessels. Primary management, led by the orthopaedic team, included intravenous antibiotics, irrigation of the wound and thorough debridement of contaminated and devitalized tissue. The tibial fracture was reduced and stabilized with an external fixator. The instability of patient’s conditions did not enable to perform an immediate reconstruction of the soft tissue defect, thereby treatment with VAC therapy was initiated. The patient underwent multiple wound debridement procedures, and regular change of VAC therapy over the first three months following the trauma. Wound swabs were taken and sent for microbiological examination at regular intervals. During hospitalisation, Klebsiella Aerogenes and Pseudomonas Aeruginosa infections of the wound were diagnosed and managed with adjustment of the intravenous antibiotic therapy based on susceptibility testing. At three months from the injury, despite significant growth of granulation tissue was observed at the wound, adequate coverage of the tibial shaft was not attained. After evaluation of the residual defect by the plastic surgeon (B.L.), a reconstruction with Latissimus Dorsi free flap was planned to provide adequate soft tissue coverage of the exposed bone and optimize its vascular supply. Preoperative blood group typing revealed the presence of a pan agglutinizing anti-erythrocyte antibody, previously unknown. The activity of the anti-E alloantibody was found until a 1:4 dilution of the patient serum. Given the incidental finding, a coagulation screen was performed, unveiling patient’s homozygosity for the C677T polymorphism of the MTHFR gene mutation, associated with high blood homocysteine. The patient referred no known personal nor familiar history of arterial or venous thrombosis. Routine coagulation analyses were not altered. Prior to surgery, the patient underwent normovolemic haemodilution, through combined crystalloid and colloid intravenous fluid infusion at a rate of 1.5 mL/kg per hour. During surgery, the Latissimus Dorsi muscle flap was harvested with the patient in left lateral decubitus. The muscle was dissected from its origin at the iliac crest to its tendinous insertion, preserving the thoracolumbar fascia. The thoracodorsal vessels were identified and isolated until their origin. During dissection, the vascular branch to the Serratus Anterior muscle and the thoracodorsal nerve were identified and divided. At the recipient site, a curettage of the wound was performed. The anterior tibial artery with its comitant veins were identified and isolated, with care to preserve the deep peroneal nerve. After preparation of the recipient site, the Latissimus Dorsi pedicle was divided, and the flap transferred from the donor to the recipient area. The thoracodorsal vessels were tunnelled at the posterior aspect of the tibialis anterior muscle to protect the flap pedicle and the microvascular anastomoses. End-to-end anastomoses between the thoracodorsal artery and the anterior tibial artery, and between the thoracodorsal vein and one of the venae comitantes of the anterior tibial pedicle were performed. No signs of intraoperative thromboses were detected. Wound edges were undermined to improve flap’s contour (Fig. 2). Two closed suction drains were placed, one at the donor and one at the recipient site. A meshed split-thickness skin graft, harvested from the anterior aspect of the ipsilateral thigh was used to provide cutaneous coverage of the muscular flap (Fig. 3). At the end of surgery, total amount of blood loss was 180 mL with a value of haemoglobin of 8 g/dL (preoperative value was 9.5 g/dL) and no transfusions of red blood cells were needed. Postoperatively a fluid therapy protocol was initiated. Intravenous infusion of 2500 mL of crystalloids daily (1500 mL Ringer’s lactate solution and 1000 mL electrolyte solution) was started following the surgery to maintain blood haematocrit at 22-25% range, ensuring a urinary output of 1 ml/kg/24h, for the first five postoperative days and titrated over the following days. Thromboembolic prophylaxis with 4000 UI of low molecular weight heparin daily started 6 hours after surgery. The patient was monitored with hand-held doppler ultrasound for 10 days, with no evidence of postoperative vascular impairment. The patient recovered well with no major complications inherent to the microsurgical reconstruction, although definitive bone fixation was delayed due to chronic osteomyelitis of the tibial bone (Figs. 4-5). Blood tests for the search of anti-erythrocyte agglutinin were repeated, with confirmed positivity for anti-E alloantibodies.

DISCUSSION

Hypercoagulable states, either inherited or acquired, may have a role in free flap failure. Polymorphisms of the MTHFR gene (C677T and A1298C) are associated with a reduced activity of the Methylenetetrahydrofolate reductase enzyme, resulting in impaired folate metabolism and hyperhomocisteinemia ⁸. High blood levels of homocysteine determine an increased risk of venous thromboembolism, and may have detrimental effect on the vessel endothelium, through oxidative damage, favouring platelet adhesion and thrombosis ^9,10. There are only few reports in literature addressing the occurrence of microvascular complications in patients with thrombophilia and MTHFR gene mutations (Tab. I) ^11-13.Davison et al. reported four cases of free flap failure in patients undergoing head and neck reconstruction, caused by undiagnosed thrombophilia. Three among these patients were diagnosed with MTHFR gene mutations, two being compound heterozygous for the C677T and A1298C polymorphisms, and one homozygous for the C677T polymorphism. Except one patient with compound heterozygosity, the other two patients presented additional pro-coagulant alterations, including elevated factor VIII activity, elevated PAI-1 activity, factor V Leiden mutation (R506Q polymorphism), and elevated anti-phosphatidyl IgM and IgG antibodies. In all three patients flap surgery was complicated by intraoperative arterial thrombosis. Furthermore, one patient experienced deep venous thrombosis, and one patient had ischemic complications unrelated to the flap ¹². De Fazio et al. presented a case series of patients with thrombophilia undergoing lower limb free tissue transfer. Among the cohort of ten patients who experienced perioperative thrombotic complications, seven were diagnosed with MTHFR gene mutations; of these, two patients had postoperative venous thrombosis, and five patients experienced intraoperative arterial thrombosis. MTHFR gene mutation and high homocysteine levels were detected in five of the six patients with failed free flaps ¹³. Vekris et al. presented the case of a young patient who underwent two consecutive unsuccessful reconstructions for a forearm defect, with the right and left gracilis muscle free flaps, both complicated by postoperative thrombosis and flap failure. Following the two failures, genetic analysis revealed that the patient was homozygous for the MTHFR C677T mutation and heterozygous for the mutations of factor V, G1691A-Leiden. Besides flap failures, the patient had deep venous thrombosis following the first reconstructive attempt, and pulmonary embolism after the second surgery ¹⁴.

Among the cohort of cases retrieved from literature, most patients were diagnosed with multiple genetically inherited alterations, that comprehensively account for the high number of flap vessel thrombosis and systemic thrombotic complications. However, the presence of multiple factors predisposing to clot formation, makes any corelation of single blood alterations with microvascular complications of difficult interpretation.

Beyond the presented reports focusing on MTHFR gene mutations, there is more literature data on microvascular complications occurring in patients with common inherited thrombophilia, including Factor V Leiden, prothrombin gene mutation, protein C and protein S deficiencies, elevated factor VIII, and antithrombin III deficiency ^15,16. Nevertheless, for all these hypercoagulable states, the absence of a control group of patients who have not experienced thrombotic complications after free flap surgery, prevents an estimation of the actual risk.

Our patient was found to be homozygous for the C677T polymorphism of the MTHFR mutation gene. Furthermore, preoperative blood tests revealed the presence of a pan agglutinizing anti-E Rh antibody, also confirmed at 6 months postoperatively. Anti-erythrocytes alloantibodies are often associated with pregnancy-induced alloimmunization and history of chronic blood transfusions in patients with haemolytic disorders as occurred in our patient. However natural anti-E autoantibodies may appear in patients with no known history of transfusion, nor pregnancy ¹⁷. Given the severity and the extent of the defect, a free flap reconstruction was indicated ^18-21. The patient was informed on the risks inherent to the procedure and his hypercoagulable state and accepted to undergo the surgery. A free Latissimus Dorsi muscle flap reconstruction was planned to provide a large soft tissue coverage of the exposed bone and prevent complications. Given the integrity of the anterior tibial, posterior tibial, and peroneal vessels, end-to-end anastomoses between the pedicle vessels and the anterior tibial vessels were performed to maximize the blood flow and decrease the risk of vascular thrombosis. Prior to surgery, the patient underwent normovolemic haemodilution to optimize blood perfusion to the microsurgical flap and reduce the risk of perioperative thrombosis. The efficacy of normovolemic and hypervolemic haemodilution in microsurgical reconstruction is still debated, and in this regard, literature lacks randomized, prospective studies ²². Nonetheless, experimental studies have proven the benefits of haemodilution in reducing the rate of thrombotic complications in free flap surgery, suggesting its potential benefits on free flap survival ^23-26. As regards fluid therapy protocols, crystalloids or a combination of colloidal and crystalloid solutions are commonly utilized for hemodilution. Some literature evidence shows that the integration of colloids in the fluid therapy guarantees the maintainance of adequate flap perfusion, reducing tissue oedema and pressure on the microcirculation ^27,28. A target haematocrit between 22-25% was set preoperatively and maintained for the first five postoperative days to reduce blood viscosity and shear stress on the endothelium.

CONCLUSIONS

Although hypercoagulable states pose a risk for microvascular thrombosis and perioperative systemic thrombotic complications, in our opinion these should not preclude the attempt of a microsurgical reconstruction. The presented case is an example of successful free flap reconstruction in a patient diagnosed with homozygosity for the C677T polymorphism of the MTHFR mutation gene and pan-agglutinizing anti-erythrocyte alloantibody. The perioperative management of high-risk patients undergoing free flap surgery might require haematological and anaesthesiologic referral, with the aim of preparing the patient to the surgery and tailoring preoperative and postoperative prophylactic measures to minimize the risk of complications. Perioperative fluid management is key in the control of flap perfusion, tissue oedema and pressure on the vascular anastomoses. In our patient the adoption of a normovolemic haemodilution protocol and the maintenance of low haematocrit values were successful in securing adequate flap flow, with no need for transfusions and no reported complications. Literature evidence on microsurgical reconstruction outcomes in patients with thrombophilia is scant, and further investigation is required to better define the relative risk of complications in this cohort of patients.

CONFLICT OF INTEREST STATEMENT

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

FUNDING

The authors received no financial support for the research, authorship, and/or publication of this article.

AUTHOR CONTRIBUTIONS

BL: A, W

MG: D, W

GD'O: D, W

EG: D, DT

LV: D, DT

AF: D, DT

RI: D, DT

UT: D, DT

VC: A, W

Abbreviations

A: conceived and designed the analysis

D: collected the data

DT: contributed data or analysis tool

S: performed the analysis

W: wrote the paper

O: other contribution (specify contribution in more detail)

Figures and tables

Figure 1. A) Photograph at patient’s arrival at the hospital: sub-amputation of the right leg, with injury at its middle third, and fracture of the tibial and peroneal bones. The distal part of the leg is displaced and flexed and cannot be seen in this photograph; B) Photograph at patient’s arrival at the hospital: repositioning of the distal segment of the lower limb reveals the partial amputation of the limb, with exposed bones and extensive soft tissue defect.

Figure 2. Intraoperative photograph: right leg soft tissue defect prior to reconstruction. The presence of granulation tissue formed at the wound site after 3-month of VAC therapy can be observed. The tibial shaft is exposed with no adequate soft tissue coverage.

Figure 3. Intraoperative photograph: reconstruction of right soft tissue defect with free Latissimus Dorsi muscle flap. A meshed split-thickness skin graft harvested from the anterior aspect of the ipsilateral thigh was used to provide coverage for the muscle flap.

Figure 4. A) Postoperative photograph: outcome at 6 months from surgery, medial view; B) Postoperative photograph: outcome at 6 months from surgery, frontal view; C) Postoperative photograph: outcome at 6 months from surgery, lateral view.

Figure 5. Postoperative x-rays: bone fixation at 6 months from surgery. A-B) Medial views; C) Posterior view.

Table I. Free flap failure in patients with MTHFR gene mutation.

Author	Patient age, sex	Coagulation alteration	Microsurgical reconstruction	Flap complications	Other complications
Vekris et al., 2007 9	18, M	Heterozygosity for Factor V Leiden, A4070G	I: Gracilis flap (right)	I: flap venous thrombosis	I: Deep venous thrombosis
		Homozygosity (C677T) MTHFR gene	II: Gracilis flap (left)	II: thrombosis graft vessels	II: Pulmonary embolism
Davison et al., 2009 10	43, F	Elevated PAI-1 activity	I: SIEA flap after oncologic resection	I: Intraoperative arterial thrombosis	N/A
		Elevated Factor VIII activity
		Elevated anti-phosphatidyl IgM and IgG antibodies	II: DIEP salvage flap	II: Postoperatory thrombosis (12 h) at arterial anastomosis of DIEP salvage flap
		Compound heterozygosity (C677T, A1298C) MTHFR gene.
		Heterozygosity for factor V Leiden gene (R506Q polymorphism)
	29, F	Elevated Factor VIII activity	I: RFFF flap after oncologic resection	I: Intermittent flow reductions	Donor site ischemia following flap harvest
		Homozygosity (C677T) MTHFR gene	II: RFFF after oncologic resection	II: Intra-operative thrombosis	Deep vein thrombosis at CVC
			III: DIEP salvage flap
	42, F	Compound heterozygosity (C6777T/A1298C) MTHFR gene	I: RFFF after oncologic resection	I: Intraoperative arterial thrombosis of flap artery	Toe gangrene distal to cannulated artery
			II: ALT flap	II: Postoperative recurrent thrombosis of flap artery; partial necrosis of the flap
De Fazio et al. 11	N/A	Heterozygosity (G20210A) Prothrombin gene	I: ALT flap	I: Postoperatory venous thrombosis (POD2) (Failed)	N/A
		Compound heterozygosity (C6777T/A1298C) MTHFR gene
		Hyperhomocysteinemia
		Lupus Anticoagulant
	N/A	Compound heterozygosity (C6777T/A1298C) MTHFR gene	I: Gracilis flap	I: Intraoperative arterial thrombosis, mixed postoperatory thrombosis (POD7) (Failed)	N/A
		Hyperhomocysteinemia
		PAI-1 45/5G
		Lupus Anticoagulant
		Elevated Factor VIII
	N/A	Compound heterozygosity (C6777T/A1298C) MTHFR gene	I: ALT flap	I: Postoperatory venous thrombosis (POD2, POD11) (Failed)	N/A
		Hyperhomocysteinemia
		Compound heterozygosity
	N/A	(C6777T/A1298C) MTHFR gene	I: Rectus Femoris flap	I: Intraoperative arterial thrombosis, postoperative mixed thrombosis (Failed)	N/A
		Hyperhomocysteinemia
		PAI-1 45/5G
		Heterozygosity (G1691A) Factor V Leiden
	N/A	Compound heterozygosity (C6777T/A1298C) MTHFR gene	I: ALT flap (distal third of the leg)	I: Intraoperative arterial thrombosis and postoperative arterial thrombosis (POD 4) (Failed)	N/A
		Hyperhomocysteinemia
De Fazio et al. 11	N/A	Heterozygosity (A1298C) MTHFR gene	I: ALT flap (distal third of the leg)	Intraoperative arterial thrombosis (Salvaged)	N/A
		PAI-1 45/5G
	N/A	Heterozygosity (A1298C) MTHFR gene	I: Medial femoral condyle flap (forefoot)	Intraoperative arterial thrombosis (Salvaged)	N/A
		PAI-1 45/5G
		Hyperhomocysteinemia