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Functional Polymorphism in Gamma-Glutamylcarboxylase is a Risk Factor for Severe Neonatal Hemorrhage
Functional Polymorphism in Gamma-Glutamylcarboxylase is a Risk Factor for Severe Neonatal Hemorrhage Olivier M. Vanakker, MD, PhD, Kris De Coen, MD, Laura Costrop, Paul J. Coucke, PhD, Piet Vanhaesebrouck, MD, PhD, and Anne De Paepe, MD, PhD A neonate who received vitamin K (VK) supplementation then developed severe late-onset bleeding with abnormal prothrombin time and activated partial thromboplastine time. The bleeding was corrected after intravenous VK. Molecular analysis of the gamma-glutamylcarboxylase gene revealed a heterozygous single nucleotide polymorphism, which decreases carboxylase activity and induces VK-dependent coagulation deficiency. (J Pediatr 2011;159:347-9)
H
emorrhagic disease of the newborn (HDN) is characterized by moderate (bruising) to severe (hemorrhagic stroke) bleeding due to vitamin K (VK) deficiency. HDN can be classified according to the time of presentation after birth as early (0-24 hours), classic (2-7 days), and late (1-8 weeks) HDN.1 The pathophysiology of neonatal hemorrhages results from a deficient activation of the VK-dependent coagulation factors. After translation, these proteins have essential posttranslational modification in the VK cycle, where a hepatic endoplasmatic reticulum g-glutamylcarboxylase (GGCX) carboxylates glutamate residues into glutamic acid residues.2 The fully carboxylated coagulation factors then are secreted into the circulation. VK is an essential cofactor in this carboxylation step with VK hydroquinone oxidized to VK epoxide. A second enzyme, VK epoxide reductase (VKORC1) reduces the epoxide back to the hydroquinone isoform (Figure).2 The etiology of VK deficiency in HDN depends on its time of onset, including abnormal maternal VK absorption or medication that alters maternal VK uptake (early onset), breast feeding (classic), or malabsorption syndromes (late onset).3 Lossof-function mutations in the genes that encode the VK-cycle key enzymes, GGCX and VKORC1, also cause a deficiency of the VK-dependent clotting factors.4 Presently, more polymorphisms in GGCX and VKORC1 that influence (either increasing or decreasing) the activity of the VK cycle are being identified. The regulation of anticoagulant therapy should take into account the effect of polymorphisms on VK-dependent coagulation factor activity.5,6
Patient Presentation The second child of healthy, nonconsanguineous parents, was born at term after an uncomplicated pregnancy and delivery. The mother did not take any medication before or
GGCX HDN SNP VK VKORC1
Gamma-glutamylcarboxylase Hemorrhagic disease of the newborn Single nucleotide polymorphism Vitamin K Vitamin K epoxide reductase
during pregnancy. The family history was negative for bleeding diathesis. Immediately after birth, routine VK prophylaxis was given as an intramuscular injection of 1 mg VK.1 Because the mother was breast feeding, oral supplementation of VK (25 mcg daily) was started. At 3 weeks of age, the baby was admitted to the neonatal ward because of hematemesis from gastrointestinal hemorrhage. Clinical examination revealed palatal ecchymosis. Biochemical analysis showed normal platelet count but a significantly prolonged prothrombin time and activated partial thromboplastine time (Table), and a first dose of intravenous VK was administered. Within the next 24 hours, coagulation biochemistry returned to normal (Table). Cranial magnetic resonance imaging revealed a large subdural hematoma and several intracerebral hemorrhages. Because of progressive somnolent behavior, the intracerebral hemorrhage required neurosurgical intervention. Because of the severe bleeding diathesis in spite of adequate oral VK supplementation, additional examinations were performed to exclude the battered child syndrome, liver dysfunction, and malabsorption syndromes, all of which were normal. To further investigate the cause of the bleeding abnormalities, molecular analyses of the genes that encode the VK-cycle enzymes, GGCX and VKORC1, were performed via direct sequencing of the coding region and exon-intron boundaries. Molecular analysis of the VKORC1 gene was normal. Exon 8 of the GGCX gene had a known heterozygous single nucleotide polymorphism (SNP), p.R325Q (c.8016G > A) (Figure). Molecular analysis of the parents revealed the father to be homozygous for p.R325Q, but the mother did not carry this variant.
Discussion An expanding interest in the pharmacogenomics of warfarin therapy has led to increased awareness of the functional
From the Center for Medical Genetics (O.V., L.C., P.C., A.P.), Department of Neonatal Intensive Care (K.C., P.V.), Ghent University Hospital, Ghent, Belgium The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright ª 2011 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2011.04.044
347
THE JOURNAL OF PEDIATRICS
www.jpeds.com
Vol. 159, No. 2
Figure. Left panel: Representation of the vitamin K-cycle. Right panel: A, Electropherogram of the proband, demonstrating a black and green double peak (arrow), compatible with the heterozygous presence c.8016G > A SNP (green peak), together with the wild type allele (black peak); B, Electropherogram, showing a single green peak (arrow) in the father being homozygous for the c.8016G > A SNP. NQO1/2, NAD(P)H:quinone oxidoreductase 1&2.
effects of not only mutations but also of polymorphisms in the genes that encode the VK-cycle enzymes.5,6 However, information on the associations of these polymorphisms with diseases that involve the VK-dependent coagulation factors, such as HDN, are scarce. We present a newborn with severe, late-onset HDN. Molecular screening of the GGCX gene revealed heterozygosity for a previously reported SNP, p.R325Q.6 Functional significance has been assumed because this SNP is predicted by the GGCX topological model to reside within the cytoplasmatic domain of GGCX, within the same cytoplasmatic loop as residues 343 to 345, which mediate enzyme-substrate binding and gamma-carboxylase activity.7 Indeed, decreased carboxylase activity was shown in vitro and in patients who are heterozygous or homozygous for this SNP, and in whom a lower dosage of coumarin for effective anticoagulant therapy is required.7,8 This effect is more pronounced in heterozygous compared with homozygous subjects.7 At the same time, a high Km value (Michaelis Menten constant) of the p.R325Q GGCX was noted, which reflected a high affinity for VK and suggested that a higher
VK intake may counteract the phenotypic effects of this genotype.7 These findings may shed light on the pathophysiology of the VK-cycle deficiency in the patient. Although conventional oral VK supplementation was given, this amount of VK is insufficient because of the decreased gammacarboxylase activity, which leads to inadequate coagulation factor activation and bleeding. High doses of intravenous VK passed the critical threshold required for adequate protein carboxylation and hence normalization of coagulation. Beyond the neonatal physiological decrease in VK, during which these events occurred, VK levels remained sufficiently high to stay above the threshold for normal coagulation. The father, being homozygous for the SNP, did not have a history of neonatal bleeding diathesis. It is noteworthy that the decrease in carboxylase activity is lower in homozygous compared with heterozygous patients.7 A possible explanation for this finding could be positive molecular heterosis. This molecular mechanism occurs when subjects
Table. Biochemical test results at day of admittance and follow-up Platelets (103/mL) Aspartate aminotransferase (U/L) Alanine aminotransferase (U/L) Prothrombin time (%) Activated partial thromboplastine time (sec) Fibrinogen (mg/dL)
T0
T24h
T9mo
Reference T0/24h
Reference T9mo
445 44 35 6 >180 410
462 47 44 115 30,5 332
278 43 24 93 39,5 435
150-450 14-86 13-45 70-120 32-55,2 200-400
150-450 16-61 13-45 70-120 28,9-38,1 200-400
T0, before treatment with intravenous VK; T24h, 24 hours after treatment with intravenous VK; T9mo, 9 months after treatment with intravenous VK.
348
Vanakker et al
August 2011 heterozygous for a specific SNP show a greater effect for a quantitative trait compared with subjects homozygous for the SNP.9 Homozygosity for this SNP in one of the parents, however, does imply that a next child may be at risk for neonatal bleeding diathesis, and preventive parenteral VK supplementation is warranted. The genetic variants described in neonatal hemorrhage that involve VK-dependent proteins are factor X mutations.10 The present case adds to the knowledge that variants in the genes that encode the VK-cycle enzymes are a plausible cause of neonatal bleeding. Their identification is not only relevant from a diagnostic perspective but also with respect to genetic counseling and therapeutic management for the parents and their children. n Submitted for publication Jan 4, 2011; last revision received Mar 28, 2011; accepted Apr 27, 2011.
References 1. Van Winckel M, De Bruyne R, Van De Velde S, Van Biervliet S, Vitamin K, an update for the paediatrician. Eur J Pediatr 2009;186:127-34. 2. Stafford DW. The vitamin K cycle. J Thromb Haemost 2005;3:1873-8.
CLINICAL AND LABORATORY OBSERVATIONS 3. Greer FR. Vitamin K deficiency and hemorrhage in infancy. Clin Perinatol 1995;22:759-77. 4. Weston BW, Monahan PE. Familial deficiency of vitamin K-dependent clotting factors. Haemophilia 2008;14:1209-13. 5. Schalekamp T, de Boer A. Pharmacogenetics of oral anticoagulant therapy. Curr Pharm Des 2010;16:187-203. 6. Kimura R, Miyashita K, Kokubo Y, Akaiwa Y, Otsubo R, Nagatsuka K, et al. Genotypes of vitamin K epoxide reductase gamma-glutamyl carboxylase, and cytochrome P450 2C9 as determinants of daily warfarin dose in Japanese patients. Thromb Res 2007;120:181-6. 7. Kinoshita H, Nakagawa K, Narusawa K, Goseki-Sone M, FukushiIrie M, Mizoi L, et al. A functional single nucleotide polymorphism in the vitamin-K-dependent gamma-glutamyl carboxylase gene (Arg325Gln) is associated with bone mineral density in elderly Japanese women. Bone 2007;40:451-6. 8. Crosier M, Peter I, Booth SL, Bennett G, Dawson-Hughes B, Ordovas JM. Association of sequence variations in vitamin K epoxide reductase and gamma-glutamyl carboxylase genes with biochemical measures of vitamin K status. J Nutr Sci Vitaminol 2009;55:112-9. 9. Comings DE, MacMurray JP. Molecular heterosis: a review. Mol Genet Metab 2000;71:19-31. 10. Herrmann FH, Navarette M, Salazar-Sanchez L, Carillo JM, Auerswald G, Wulff K. Homozygous factor X gene mutations Gly380Arg and Tyr163DelAT are associated with perinatal intracranial hemorrhage. J Pediatr 2005;136:128-30.
Functional Polymorphism in Gamma-Glutamylcarboxylase is a Risk Factor for Severe Neonatal Hemorrhage
349
H
emorrhagic disease of the newborn (HDN) is characterized by moderate (bruising) to severe (hemorrhagic stroke) bleeding due to vitamin K (VK) deficiency. HDN can be classified according to the time of presentation after birth as early (0-24 hours), classic (2-7 days), and late (1-8 weeks) HDN.1 The pathophysiology of neonatal hemorrhages results from a deficient activation of the VK-dependent coagulation factors. After translation, these proteins have essential posttranslational modification in the VK cycle, where a hepatic endoplasmatic reticulum g-glutamylcarboxylase (GGCX) carboxylates glutamate residues into glutamic acid residues.2 The fully carboxylated coagulation factors then are secreted into the circulation. VK is an essential cofactor in this carboxylation step with VK hydroquinone oxidized to VK epoxide. A second enzyme, VK epoxide reductase (VKORC1) reduces the epoxide back to the hydroquinone isoform (Figure).2 The etiology of VK deficiency in HDN depends on its time of onset, including abnormal maternal VK absorption or medication that alters maternal VK uptake (early onset), breast feeding (classic), or malabsorption syndromes (late onset).3 Lossof-function mutations in the genes that encode the VK-cycle key enzymes, GGCX and VKORC1, also cause a deficiency of the VK-dependent clotting factors.4 Presently, more polymorphisms in GGCX and VKORC1 that influence (either increasing or decreasing) the activity of the VK cycle are being identified. The regulation of anticoagulant therapy should take into account the effect of polymorphisms on VK-dependent coagulation factor activity.5,6
Patient Presentation The second child of healthy, nonconsanguineous parents, was born at term after an uncomplicated pregnancy and delivery. The mother did not take any medication before or
GGCX HDN SNP VK VKORC1
Gamma-glutamylcarboxylase Hemorrhagic disease of the newborn Single nucleotide polymorphism Vitamin K Vitamin K epoxide reductase
during pregnancy. The family history was negative for bleeding diathesis. Immediately after birth, routine VK prophylaxis was given as an intramuscular injection of 1 mg VK.1 Because the mother was breast feeding, oral supplementation of VK (25 mcg daily) was started. At 3 weeks of age, the baby was admitted to the neonatal ward because of hematemesis from gastrointestinal hemorrhage. Clinical examination revealed palatal ecchymosis. Biochemical analysis showed normal platelet count but a significantly prolonged prothrombin time and activated partial thromboplastine time (Table), and a first dose of intravenous VK was administered. Within the next 24 hours, coagulation biochemistry returned to normal (Table). Cranial magnetic resonance imaging revealed a large subdural hematoma and several intracerebral hemorrhages. Because of progressive somnolent behavior, the intracerebral hemorrhage required neurosurgical intervention. Because of the severe bleeding diathesis in spite of adequate oral VK supplementation, additional examinations were performed to exclude the battered child syndrome, liver dysfunction, and malabsorption syndromes, all of which were normal. To further investigate the cause of the bleeding abnormalities, molecular analyses of the genes that encode the VK-cycle enzymes, GGCX and VKORC1, were performed via direct sequencing of the coding region and exon-intron boundaries. Molecular analysis of the VKORC1 gene was normal. Exon 8 of the GGCX gene had a known heterozygous single nucleotide polymorphism (SNP), p.R325Q (c.8016G > A) (Figure). Molecular analysis of the parents revealed the father to be homozygous for p.R325Q, but the mother did not carry this variant.
Discussion An expanding interest in the pharmacogenomics of warfarin therapy has led to increased awareness of the functional
From the Center for Medical Genetics (O.V., L.C., P.C., A.P.), Department of Neonatal Intensive Care (K.C., P.V.), Ghent University Hospital, Ghent, Belgium The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright ª 2011 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2011.04.044
347
THE JOURNAL OF PEDIATRICS
www.jpeds.com
Vol. 159, No. 2
Figure. Left panel: Representation of the vitamin K-cycle. Right panel: A, Electropherogram of the proband, demonstrating a black and green double peak (arrow), compatible with the heterozygous presence c.8016G > A SNP (green peak), together with the wild type allele (black peak); B, Electropherogram, showing a single green peak (arrow) in the father being homozygous for the c.8016G > A SNP. NQO1/2, NAD(P)H:quinone oxidoreductase 1&2.
effects of not only mutations but also of polymorphisms in the genes that encode the VK-cycle enzymes.5,6 However, information on the associations of these polymorphisms with diseases that involve the VK-dependent coagulation factors, such as HDN, are scarce. We present a newborn with severe, late-onset HDN. Molecular screening of the GGCX gene revealed heterozygosity for a previously reported SNP, p.R325Q.6 Functional significance has been assumed because this SNP is predicted by the GGCX topological model to reside within the cytoplasmatic domain of GGCX, within the same cytoplasmatic loop as residues 343 to 345, which mediate enzyme-substrate binding and gamma-carboxylase activity.7 Indeed, decreased carboxylase activity was shown in vitro and in patients who are heterozygous or homozygous for this SNP, and in whom a lower dosage of coumarin for effective anticoagulant therapy is required.7,8 This effect is more pronounced in heterozygous compared with homozygous subjects.7 At the same time, a high Km value (Michaelis Menten constant) of the p.R325Q GGCX was noted, which reflected a high affinity for VK and suggested that a higher
VK intake may counteract the phenotypic effects of this genotype.7 These findings may shed light on the pathophysiology of the VK-cycle deficiency in the patient. Although conventional oral VK supplementation was given, this amount of VK is insufficient because of the decreased gammacarboxylase activity, which leads to inadequate coagulation factor activation and bleeding. High doses of intravenous VK passed the critical threshold required for adequate protein carboxylation and hence normalization of coagulation. Beyond the neonatal physiological decrease in VK, during which these events occurred, VK levels remained sufficiently high to stay above the threshold for normal coagulation. The father, being homozygous for the SNP, did not have a history of neonatal bleeding diathesis. It is noteworthy that the decrease in carboxylase activity is lower in homozygous compared with heterozygous patients.7 A possible explanation for this finding could be positive molecular heterosis. This molecular mechanism occurs when subjects
Table. Biochemical test results at day of admittance and follow-up Platelets (103/mL) Aspartate aminotransferase (U/L) Alanine aminotransferase (U/L) Prothrombin time (%) Activated partial thromboplastine time (sec) Fibrinogen (mg/dL)
T0
T24h
T9mo
Reference T0/24h
Reference T9mo
445 44 35 6 >180 410
462 47 44 115 30,5 332
278 43 24 93 39,5 435
150-450 14-86 13-45 70-120 32-55,2 200-400
150-450 16-61 13-45 70-120 28,9-38,1 200-400
T0, before treatment with intravenous VK; T24h, 24 hours after treatment with intravenous VK; T9mo, 9 months after treatment with intravenous VK.
348
Vanakker et al
August 2011 heterozygous for a specific SNP show a greater effect for a quantitative trait compared with subjects homozygous for the SNP.9 Homozygosity for this SNP in one of the parents, however, does imply that a next child may be at risk for neonatal bleeding diathesis, and preventive parenteral VK supplementation is warranted. The genetic variants described in neonatal hemorrhage that involve VK-dependent proteins are factor X mutations.10 The present case adds to the knowledge that variants in the genes that encode the VK-cycle enzymes are a plausible cause of neonatal bleeding. Their identification is not only relevant from a diagnostic perspective but also with respect to genetic counseling and therapeutic management for the parents and their children. n Submitted for publication Jan 4, 2011; last revision received Mar 28, 2011; accepted Apr 27, 2011.
References 1. Van Winckel M, De Bruyne R, Van De Velde S, Van Biervliet S, Vitamin K, an update for the paediatrician. Eur J Pediatr 2009;186:127-34. 2. Stafford DW. The vitamin K cycle. J Thromb Haemost 2005;3:1873-8.
CLINICAL AND LABORATORY OBSERVATIONS 3. Greer FR. Vitamin K deficiency and hemorrhage in infancy. Clin Perinatol 1995;22:759-77. 4. Weston BW, Monahan PE. Familial deficiency of vitamin K-dependent clotting factors. Haemophilia 2008;14:1209-13. 5. Schalekamp T, de Boer A. Pharmacogenetics of oral anticoagulant therapy. Curr Pharm Des 2010;16:187-203. 6. Kimura R, Miyashita K, Kokubo Y, Akaiwa Y, Otsubo R, Nagatsuka K, et al. Genotypes of vitamin K epoxide reductase gamma-glutamyl carboxylase, and cytochrome P450 2C9 as determinants of daily warfarin dose in Japanese patients. Thromb Res 2007;120:181-6. 7. Kinoshita H, Nakagawa K, Narusawa K, Goseki-Sone M, FukushiIrie M, Mizoi L, et al. A functional single nucleotide polymorphism in the vitamin-K-dependent gamma-glutamyl carboxylase gene (Arg325Gln) is associated with bone mineral density in elderly Japanese women. Bone 2007;40:451-6. 8. Crosier M, Peter I, Booth SL, Bennett G, Dawson-Hughes B, Ordovas JM. Association of sequence variations in vitamin K epoxide reductase and gamma-glutamyl carboxylase genes with biochemical measures of vitamin K status. J Nutr Sci Vitaminol 2009;55:112-9. 9. Comings DE, MacMurray JP. Molecular heterosis: a review. Mol Genet Metab 2000;71:19-31. 10. Herrmann FH, Navarette M, Salazar-Sanchez L, Carillo JM, Auerswald G, Wulff K. Homozygous factor X gene mutations Gly380Arg and Tyr163DelAT are associated with perinatal intracranial hemorrhage. J Pediatr 2005;136:128-30.
Functional Polymorphism in Gamma-Glutamylcarboxylase is a Risk Factor for Severe Neonatal Hemorrhage
349