Successful liver transplantation for homozygous protein <scp>C</scp> deficiency with a type II mutation using a heterozygous living related donor
Authors: Alexander A. Boucher, Lori Luchtman‐Jones, Jaimie D. Nathan, Joseph S. Palumbo
Journal: American Journal of Hematology (2017)

A 29-month-old male with homozygous protein C (PC) deficiency was referred to our institution to be considered for liver transplantation. The patient, born to consanguineous parents in the United Arab Emirates (UAE), was diagnosed at 6 months of age. At that time, he had an open biopsy of a right thigh soft tissue “mass” of unclear etiology. He developed purpura fulminans (PF) within days of the biopsy, requiring escharotomies of both thighs for compartment syndrome complicated by necrotizing fasciitis. Extensive thrombophilia testing was performed, which demonstrated normal levels of factor VIII (fVIII), antithrombin III (ATIII), protein S, and homocysteine. Factor V Leiden (FVL), prothrombin gene polymorphism, and antiphospholipid antibody testing were normal/negative. PC antigen level was normal at 102 IU/dL; however, chromogenic PC activity was 36 IU/dL and clottable activity was 9 IU/dL, consistent with a type II, or functional, PC deficiency. Genetic sequencing of the PROC gene revealed a recently described homozygous mutation c.1198G>A (p.Gly400Ser).1 This mutation involves a highly conservative amino acid which the amino acid substitution prediction programs PolyPhen-2, SIFT, and Mutation Taster predicted to be “probably damaging,” “deleterious,” and “disease causing,” respectively, according to Prevention Genetics Lab (Marshfield, Wisconsin). As expected, both parents were heterozygous for the same mutation. PC is a hepatically synthesized, vitamin K-dependent serine protease with a major role in natural anticoagulation. It is activated via proteolytic cleavage by the thrombin–thrombomodulin complex. Activated PC forms a complex with its cofactor protein S and the endothelial protein C receptor (EPCR), which in turn halts continued thrombin generation by proteolytically inactivating coagulation factors Va and VIIIa.2 Autosomal dominant heterozygous PC deficiency has variable penetrance and is most commonly associated with venous thromboembolism (VTE), but arterial thromboses can also occur.3, 4 Over 370 mutations have been described in the PROC gene, found on chromosome 2q13-q14. The carrier frequency of inherited PC mutations is estimated to be 1:200 to 1:700.5, 6 Heterozygous quantitative or qualitative PC deficiencies increase the risk of thrombotic complications approximately 3–4 fold, but the overall risk often depends on the presence or absence of other inherited and acquired thrombophilic risk factors.3, 7, 8 Severe congenital PC deficiency is a rare autosomal recessive condition (estimated prevalence 1:20 000) resulting from homozygous or compound heterozygous mutations in the PROC gene.3 Most mutations lead to type I deficiencies, where both antigen and activity levels are uniformly decreased. Less common is type II, or functional deficits, which demonstrate normal or mildly reduced antigen levels but significantly diminished function.9, 10 His maternal aunt, a potential liver donor, was also heterozygous for the same PROC mutation. Notably, the mother has five children and no affected family members reported a history of thrombosis or miscarriage. Available PC antigen and activity comparisons among family members are shown in Table 1. Details of initial PC testing in the UAE are unknown. At our institution, chromogenic activity testing was performed using BIOPHEN PC 5 kit (Hyphen Biomed, Neauville-Sur-Oise, France). Clottable activity was tested using STA-StaClot PC kit and antigen levels with ASSERACHROM PC kit (both from Stago, Asnières sur Seine, France). PC activity levels in the mother and aunt were near the lower limit of normal with a modest discordance between the clottable and chromogenic assays, whereas the patient had a pronounced discordance between the activity assays. The mother and aunt both had normal fVIII activity and were negative for FVL and Prothrombin G20210A mutations. When evaluating PC, one must measure both antigen and functional activity and rule out acquired causes of PC deficiency like liver disease or warfarin use.11 Activity can be measured using either a chromogenic or a clottable assay.11 The chromogenic assay evaluates the ability of activated PC to cleave a synthetic substrate, liberating a chromogenic peptide. The clottable assay measures the capacity of activated PC to degrade factors Va and VIIIa. The chromogenic assay is less prone to interference from inhibitors (eg, lupus anticoagulants, fVIII elevations, direct thrombin inhibitors). However, because it does not require multiple critical protein–protein interactions important in PC function, the chromogenic assay may not accurately reflect the functional potential of a given Type II mutation. Factor VIII elevation and FVL can each cause discordant results with lower clottable activity levels and should be tested in these situations. It must also be noted that there are age-specific lower PC ranges throughout childhood.11, 12 Soon after diagnosis, the patient was noted to have atrophic maculae with impaired vision along with hypertension and parenchymal changes on renal ultrasound suggestive of bilateral papillary necrosis. An electroencephalogram showed nonspecific abnormalities with a normal brain MRI at 9 months of age, but a repeat MRI at 30 months demonstrated bilateral asymmetric encephalomalacia. Despite these presumed post-thrombotic morbidities, the child remained high-functioning. At presentation for possible liver transplant, he could ambulate without assistance, use a smart phone by holding it close to his face, communicate effectively in Arabic, and he had learned a few English words. Following his PF episode, he was started on 1 mg/kg enoxaparin twice daily. Once the diagnosis of severe PC deficiency was made, subcutaneous PC concentrate was added at 50 IU/kg every 6 hours to maintain a trough PC activity above 50 IU/dL. Over several months, he transitioned to 1.5 mg/kg daily of enoxaparin and daily subcutaneous administration of PC concentrate (40 IU/kg) that maintained clottable PC activity troughs >20 IU/dL. In preparation for possible liver transplant, he also underwent a short trial of continuous infusion PC concentrate to monitor PC clearance. Baseline testing (obtained as a trough 24 hours after subcutaneous PC concentrate infusion) showed a PC antigen level >101 IU/dL with a low clottable activity level of 23 IU/dL and a normal chromogenic activity of 81 IU/dL. After a 100 IU/kg bolus of PC concentrate followed by 60 IU/kg continuous infusion for 6 hours, his clottable activity normalized to 98 IU/dL 15 minutes after the bolus and remained >100 IU/dL for several hours after the intravenous (IV) infusion finished. Severe PC deficiency most often presents as PF with widespread macrovascular or microvascular thromboses within the first hours to days of life. This morbidity may involve vasculature in the skin along with retinal, renal, and cerebral circulations, potentially culminating in disseminated intravascular coagulation or death.3, 13 Aggressive empiric factor replacement therapy along with anticoagulation can be life-saving, particularly if started after the initial clinical suspicion, as definitive testing can take days to weeks to result. Several weeks after a thromboembolic event, a stable patient with severe PC deficiency generally transitions to maintenance treatment involving anticoagulation with or without PC concentrate.14 Multiple secondary prophylaxis strategies have been described, almost exclusively based on type I deficiencies.3, 14-16 One option is therapeutic anticoagulation with enoxaparin or high-intensity warfarin (goal INR ≥ 3.0) alone. An alternative is using high-dose PC concentrate (∼250–300 IU/kg) every 48 hours with lower-dose anticoagulation. The half-life of subcutaneous PC concentrate has been reported to be close to 16 hours which allows for every-other-day dosing. A third option, as was utilized here, is a combination of anticoagulation and daily lower-dose PC concentrate (40–100 IU/kg). While the goal trough in this situation is not definitive, many hematologists aim for clottable PC activity levels >25 IU/dL. It has also been reported that monitoring d-dimer trends can signal early evidence of thromboembolic recurrence.3 While therapeutic anticoagulation and PC concentrate have markedly reduced morbidity and mortality, they also have risks and are not curative. The likelihood of severe bleeding complications is always present, especially for those on higher doses of enoxaparin or warfarin. Subcutaneous PC concentrate has diminished the need for long-term central access for frequent IV infusions, but IV dosing is still necessary if acute thrombosis recurs. Moreover, regular subcutaneous PC concentrate dosing can be a significant burden on the patient and family. The only available curative therapy is orthotopic liver transplantation. After extensive evaluation at our institution, he underwent a related living donor liver transplant (LDLT) at 32 months of age. His maternal aunt, who had heterozygous PC deficiency as described previously, was selected as donor because she had the most optimal left liver segment allograft anatomy among the available family members. Preoperatively, he received a PC concentrate bolus of 70 IU/kg followed by continuous infusion of 10 IU/kg/hour. He required one 50 IU/kg bolus intraoperatively due to borderline low clottable PC activity levels. He also received a dose of ATIII intraoperatively (ATIII level of 42 IU/dL). He did not suffer any perioperative hematologic complications and received no other PC-containing blood products. Unfractionated heparin (UFH) infusion was started 12 hours postoperatively and aspirin 40.5 mg daily began on postoperative day (POD) 2 per institutional protocol. The UFH infusion was titrated in a range of 16–20 IU/kg based on a goal anti-Xa of 0.3-0.5 IU/mL, rather than a fixed dose of 15 IU/kg × 5 days as would be our institutional standard for LDLT. The clottable PC activity ranged from 68 to 149 IU/dL postoperatively and the PC concentrate infusion was modulated to achieve >60 IU/dL (Figure 1). UFH was stopped on POD 2 due to mild bleeding. His clottable PC activity level was greater than 100 IU/dL at that time. His PC concentrate infusion was weaned off by POD 5. The clottable PC activity reached its perioperative nadir at 68 IU/dL on POD 7, similar to aunt's baseline clottable activity level. At follow-up seven weeks post-transplant, the recipient's clottable activity level remained 63 IU/dL with an antigen level >100 IU/dL. Eight months after transplant, he remains off PC concentrate and anticoagulation and has not experienced any late hemorrhagic or thrombotic complications. Shown is the patient's clottable PC activity relative to the timing of liver transplantation. Note that the baseline (BL, *) PC activity was measured by the referring institution when the patient was 6 months old. The activity measured on day −90 (**) was obtained during a brief hiatus from PC concentrate infusion. The patient was transitioned from daily subcutaneous PC concentrate to a continuous IV infusion on day −1 (arrow) which was weaned off by POD 5 (arrowhead). The dotted line represents the lower limit of the normal range for the clottable PC activity in our reference lab (61 IU/dL) The donor also received thromboprophylaxis that was more aggressive than our institutional standard due to her heterozygous PROC mutation. Given her benign history, she did not receive empiric PC concentrate preoperatively. As with the recipient, the goal clottable activity level was >60 IU/dL. Four hours postoperatively, her clottable activity level dropped to 28 IU/dL and she received 50 IU/kg IV PC concentrate. Her clottable activity level was 62 IU/dL 12 hours after the infusion. Other hemostatic labs were normal or unremarkable. Clottable activity was monitored every 12 hours through POD 2. Additional PC concentrate 50 IU/kg was given at 18 hours (clottable activity 51 IU/dL) and 48 hours postoperatively (clottable activity 48 IU/dL). At 48 hours, an ATIII level of 61 IU/dL prompted a 2000 U antithrombin infusion. Clottable PC activity was monitored daily from POD 3 until POD 8. She required 50 IU/kg PC concentrate (clottable activity 52 IU/dL) on POD 6 and 25 IU/kg PC concentrate on POD 8 (activity 49 IU/dL). The donor was maintained on therapeutic UFH for the first week postoperatively rather than the institutional standard of prophylactic enoxaparin. On POD 8, she was transitioned to therapeutic enoxaparin (every 12 hour dosing) for a total of six weeks. This contrasts with our institutional standard for liver transplant donors in which thromboprophylaxis ends at hospital discharge. She had no hematologic complications and PC levels returned to baseline. The rarity of severe PC deficiency and inherent risks with liver transplantation make this curative therapy exceptional. Only seven case reports of this intervention exist in the literature.17-23 For all but one of these, the transplanted organs, both cadaveric and living, came from otherwise healthy donors to ensure resolution of the PC deficiency. The case report of a donor with a chronic disease described successful serial, or “domino,” liver transplants in which a patient with maple syrup urine disease was the donor for another patient with homozygous PC deficiency.21 The case presented here is novel as it is the first published report of curative liver transplantation for homozygous PC deficiency from a living donor who is heterozygous for the same mutation. The decision to use this donor was based on multiple factors. Importantly, no heterozygous family members had experienced thromboembolic events in their first several decades of life, suggesting a lower long-term risk of thrombosis post-transplant compared to his pre-transplant risk. Also, the wait time to find a suitable cadaveric donor is unpredictable. Use of a living related donor expedited transplant timing, thereby minimizing the risk for further morbidity from his disease. This case highlights several unique facets of care for a patient with homozygous PC deficiency. His PROC mutation declared itself in an atypical fashion in that he did not develop PF until 6 months of age, albeit after a minor surgery. Most patients with homozygous PC deficiency present soon after birth with classic PF, though late onset disease has been reported, even into adulthood.19, 24 The delayed presentation may suggest that this PROC mutation is less critical for stable function with normal hemostasis but could still lead to classic complications with any disturbance of hemostatic balance. The c.1198G>A (p.Gly400Ser) mutation is only two amino acid residues away from Ser402, the active serine in the catalytic triad, corresponding to Ser360 as modeled by Fisher et al.25 The normal antigen levels with incongruous activity levels observed in this family are consistent with the mutation causing impaired proteolytic function rather than a quantitative deficiency. The significant discrepancy observed between the chromogenic and clottable activity levels in the affected individuals, especially in the homozygous state, highlights the importance of using both activity assays for testing. Here, the chromogenic assay may be less sensitive to the particular functional deficit incurred by the patient's mutation. Given the novelty of the mutation, we cannot say with certainty why the patient's results were more discordant than other family members. One hypothesis is that since the other family members were heterozygous for this mutation, enough normal PC was present to remain asymptomatic with less pronounced laboratory abnormalities. Discordant activity levels in which chromogenic assays are less sensitive to functional PC mutations have been described previously.26 The fact that the dysfunctional protein appears to retain some residual function could explain the atypical course for someone with homozygous disease. From a surgical standpoint, two remarkable points can be made. Most importantly, the case demonstrates that using a donor who is heterozygous for PC deficiency is not illogical, and it can be curative. Ultimately, both patients- postoperative courses were uneventful and neither required continuous PC concentrate infusion for greater than five days. This case also reinforces previous findings that the liver's ability to generate PC is not compromised for a long period of time postoperatively. Similar to two previously reported cases, PC synthesis returned quickly post-transplant.20, 21 Also paralleling these previous cases, PC was replaced until synthetic function returned. It is not clear that postoperative PC replacement is necessary but was done so here out of caution and lack of precedence. Transplantation with a liver from a donor heterozygous for PC deficiency will still leave this patient with a higher lifetime risk of VTE, perhaps 3–4 fold. However, most of these events are not life-threatening and occur after the fifth decade of life. Thus, a successful liver transplant in this setting likely provides him many years free of aggressive anticoagulation. Furthermore, most patients with heterozygous PC deficiency never experience thromboembolism. The benign course of this patient's family members is reassuring, as the recipient's post-transplant activity levels mirror those of other family members. However, it must be noted that the success experienced here may not hold true in donors with a more severe heterozygous mutation. In conclusion, our experience using a related donor, despite having heterozygous PC deficiency, broadens the possibilities for curative liver transplant in this rare but highly morbid and often fatal condition. The authors have no financial relationships relevant to this article to disclose. The authors have no conflicts of interest relevant to this article to disclose.