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An unreported mutation within protein Z gene is associated with very low protein levels in women with fetal loss
An unreported mutation within protein Z gene is associated with very low protein levels in women with fetal loss Gene variant intron C G-42A of protein Z is significantly associated with the occurrence of fetal loss. A previously unreported sporadic missense mutation within exon 8 is described in a patient with very low protein Z levels. (Fertil Steril 2008;90:864–5. 2008 by American Society for Reproductive Medicine.)
Protein Z (PZ) is a vitamin K–dependent glycoprotein regulating the coagulation cascade because of PZ-dependent protease inhibitor (1). Reduced circulating levels of PZ have been suggested to play a role in the occurrence of bleeding (2), deep vein thrombosis (3, 4), and in early (5) as well as late fetal losses (6), although data about the latter are conflicting (7). In a cohort of patients with documented venous thrombosis, we recently showed that the prevalence of PZ levels below the 5.0 (0.52 mg/mL) or the 2.5 percentile of controls (0.47 mg/mL) was higher in these patients (10.2% and 8.7%, respectively) than in controls (4.1%; odds ratio [OR] 2.7 [95% confidence interval (CI) 1.2–7.3] and 2.0%; OR 4.6 [95% CI 1.5–13.9], respectively) (8). A series of variants naturally occurring within the PZ gene locus were investigated in that setting of patients, and PZ levels were found to be associated with the intron C G-42A and the intron F G79A polymorphisms. Among a group of women previously (7) investigated for otherwise unexplained fetal losses, 7 of 124 (5.6%) showed PZ levels under the 5th percentile (i.e., 0.52 mg/mL), calculated in a control group of 104 parous women with at least one uneventful pregnancy and no fetal loss. Five (4.8%; ns) of the 104 controls showed PZ levels under the 5th percentile. Therefore, we decided to investigate, by direct sequencing, whether PZ gene polymorphisms or sporadic mutations could be present in these 7 cases (mean age SD: 33.1 4.3 years) compared with 104 controls (aged 37 5.8 years). Informed consent was obtained from all the subjects. Institutional review board approval was obtained from the local ethics committee. Women with known causes of thrombophilia (factor V Leiden FII A20210 mutations, natural anticoagulant deficiency, or antiphospholipid antibodies) were not considered, as previously described (7). Received January 10, 2007; revised June 6, 2007; accepted July 14, 2007. Reprint requests: Elvira Grandone, M.D., Atherosclerosis and Thrombosis Unit, IRCCS Casa Sollievo della Sofferenza, Viale Padre Pio, 71013 S. Giovanni R, Foggia, Italy (FAX: 39-882-416273; E-mail: [email protected]).
864
Blood samples were collected into vacuum plastic tubes containing 3.8% trisodium citrate and centrifuged at 2,000 g for 15 minutes to obtain platelet-poor plasma. It was frozen and stored in small aliquots at 70 C until tested. Protein Z plasma levels were evaluated by means of ELISA (Asserachrom Protein Z; Diagnostica Stago, Asnieres, France). Deoxyribonucleic acid was extracted from peripheral blood leukocytes according to standard protocols (8). Amplifications of regions of PZ gene containing the intron A g-103a (intron 1; single nucleotide polymorphisms database [dbSNP] no. rs17880587), intron F g79a (intron 6; dbSNP no. rs17882561), and e intron C 42bp (g-42a) polymorphisms, were achieved as previously described (8). The significance of the difference in observed genotypes between the groups was tested using c2 analysis. Odds ratios and 95% CIs were calculated. Two cases (28.6%) and 18 controls (17.3%) were heterozygous for the intron A G -103A mutation; AA genotype was not observed in cases, whereas it was present in 3 controls (2.9%; P>.05). Genotype AG for the Intron F G79A gene variant was observed in 3 cases (42.9%) and 32 controls (30.8%; P>.05); AA genotype was not observed in cases and was present in 5 controls (4.8%). As far as the intron C G-42A gene variant is concerned, 5 cases (71.4%) and 21 controls (20.2%, Fisher exact test P¼.008; OR 9.88 [95% CI 2.03–47.1]) showed genotype AG, whereas no case and 5 controls (4.8%) carried the AA genotype. In addition, 2 patients carried an unreported missense mutation within the exon 8 (T14928C, accession number AF 440358), causing the substitution of a leucine with a proline in the trypsin-like serine protease domain of the protein. Protein Z levels in these patients were 0.25 mg/mL and 0.40 mg/mL, respectively, which means below the 2.5 percentile (0.49 mg/mL) of the controls. It is known that there is a relationship between the structure and function of proteins. The a-helix segments are solidified by having a hydrophobic amino acid in every third to fourth position of the primary structure. When an a-helix structure is formed, all these amino acids (e.g., isoleucine, alanine, valine) line up on one side of the helix. Proline is
Fertility and Sterility Vol. 90, No. 3, September 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.07.1318
FIGURE 1 Conservation of Leu 264 residue in the trypsin-like domain of PZ. The position of Leu264Pro in PZ is shown in bold. Multiple alignment of PZ from different species in the region containing the T T14928C mutation (accession numbers P22891, Q9CQW3, XP_001143484, P00744, XP_416945, XP_001087321, XP_001075483, and XP_693457, respectively). Numbering includes the signal peptide and refers to the human sequence.
In addition, the intron C G-42A gene variant was associated with the occurrence of pregnancy losses in our small sample of patients. Further studies are needed. Elvira Grandone, M.D.a Donatella Colaizzo, Biol.Sc.a Filomena Cappucci, Biol.Sc.a Rosa Lucia D’Ambrosio, Biol.Sc.a Gennaro Vecchione, Biol.Sc.a Maurizio Margaglione, M.D.a,b a Atherosclerosis and Thrombosis Unit, I.R.C.C.S. Casa Sollievo della Sofferenza, S. Giovanni Rotondo; and b Department of Medical Genetics, University of Foggia, Foggia, Italy
REFERENCES
Grandone. Protein Z gene mutations and fetal loss. Fertil Steril 2008.
an amino acid and does not have a side chain. Thus, the presence of proline disrupts the a-helix. For this reason, it is very suggestive to believe that nonconservative substitution of leucine with proline would affect PZ structure. As shown in Figure 1, this residue is highly conserved among different species. Among the controls, no woman carried this mutation. More interestingly, this mutation was not found in 197 patients with deep vein thrombosis or in the 197 age-matched controls previously investigated (8). This unreported sporadic mutation within the PZ gene could explain the very low PZ levels in our patients.
Fertility and Sterility
1. Broze GJ Jr. Protein Z dependent regulation of coagulation. Thromb Haemost 2001;86:8–13. 2. Kemkes-Matthes B, Matthes KJ. Protein Z deficiency: a new cause of bleeding tendency. Thromb Res 1995;79:49–55. 3. Yin ZF, Huang ZF, Cui J, Fiehler R, Lasky N, Ginsburg D, et al. Prothrombotic phenotype of protein Z deficiency. Proc Natl Acad Sci U S A 2000;97:6734–8. 4. Kemkes-Matthes B, Nees M, Kuhnel G, Matzdorff A, Matthes KJ. Protein Z influences prothrombotic phenotype of factor V Leiden in humans. Thromb Res 2002;106:183–5. 5. Gris JC, Quere I, Dechaud H, Mercier E, Pincon C, Hoffet M, et al. High frequency of protein Z deficiency in patients with unexplained early fetal loss. Blood 2002;99:2606–8. 6. Bretelle F, Arnoux D, Shojai R, D’Ercole C, Sampol J, Dignat F, et al. Protein Z in patients with pregnancy complications. Am J Obstet Gynecol 2005;193:1698–702. 7. Grandone E, Colaizzo D, Cappucci F, Cocomazzi N, Margaglione M. Protein Z levels and unexplained fetal losses. Fertil Steril 2004;82: 982–3. 8. Santacroce R, Sarno M, Cappucci F, Sessa F, Colaizzo D, Brancaccio V, et al. Low protein Z levels and risk of occurrence of deep vein thrombosis. J Thromb Haemost 2006;4:2417–22.
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Protein Z (PZ) is a vitamin K–dependent glycoprotein regulating the coagulation cascade because of PZ-dependent protease inhibitor (1). Reduced circulating levels of PZ have been suggested to play a role in the occurrence of bleeding (2), deep vein thrombosis (3, 4), and in early (5) as well as late fetal losses (6), although data about the latter are conflicting (7). In a cohort of patients with documented venous thrombosis, we recently showed that the prevalence of PZ levels below the 5.0 (0.52 mg/mL) or the 2.5 percentile of controls (0.47 mg/mL) was higher in these patients (10.2% and 8.7%, respectively) than in controls (4.1%; odds ratio [OR] 2.7 [95% confidence interval (CI) 1.2–7.3] and 2.0%; OR 4.6 [95% CI 1.5–13.9], respectively) (8). A series of variants naturally occurring within the PZ gene locus were investigated in that setting of patients, and PZ levels were found to be associated with the intron C G-42A and the intron F G79A polymorphisms. Among a group of women previously (7) investigated for otherwise unexplained fetal losses, 7 of 124 (5.6%) showed PZ levels under the 5th percentile (i.e., 0.52 mg/mL), calculated in a control group of 104 parous women with at least one uneventful pregnancy and no fetal loss. Five (4.8%; ns) of the 104 controls showed PZ levels under the 5th percentile. Therefore, we decided to investigate, by direct sequencing, whether PZ gene polymorphisms or sporadic mutations could be present in these 7 cases (mean age SD: 33.1 4.3 years) compared with 104 controls (aged 37 5.8 years). Informed consent was obtained from all the subjects. Institutional review board approval was obtained from the local ethics committee. Women with known causes of thrombophilia (factor V Leiden FII A20210 mutations, natural anticoagulant deficiency, or antiphospholipid antibodies) were not considered, as previously described (7). Received January 10, 2007; revised June 6, 2007; accepted July 14, 2007. Reprint requests: Elvira Grandone, M.D., Atherosclerosis and Thrombosis Unit, IRCCS Casa Sollievo della Sofferenza, Viale Padre Pio, 71013 S. Giovanni R, Foggia, Italy (FAX: 39-882-416273; E-mail: [email protected]).
864
Blood samples were collected into vacuum plastic tubes containing 3.8% trisodium citrate and centrifuged at 2,000 g for 15 minutes to obtain platelet-poor plasma. It was frozen and stored in small aliquots at 70 C until tested. Protein Z plasma levels were evaluated by means of ELISA (Asserachrom Protein Z; Diagnostica Stago, Asnieres, France). Deoxyribonucleic acid was extracted from peripheral blood leukocytes according to standard protocols (8). Amplifications of regions of PZ gene containing the intron A g-103a (intron 1; single nucleotide polymorphisms database [dbSNP] no. rs17880587), intron F g79a (intron 6; dbSNP no. rs17882561), and e intron C 42bp (g-42a) polymorphisms, were achieved as previously described (8). The significance of the difference in observed genotypes between the groups was tested using c2 analysis. Odds ratios and 95% CIs were calculated. Two cases (28.6%) and 18 controls (17.3%) were heterozygous for the intron A G -103A mutation; AA genotype was not observed in cases, whereas it was present in 3 controls (2.9%; P>.05). Genotype AG for the Intron F G79A gene variant was observed in 3 cases (42.9%) and 32 controls (30.8%; P>.05); AA genotype was not observed in cases and was present in 5 controls (4.8%). As far as the intron C G-42A gene variant is concerned, 5 cases (71.4%) and 21 controls (20.2%, Fisher exact test P¼.008; OR 9.88 [95% CI 2.03–47.1]) showed genotype AG, whereas no case and 5 controls (4.8%) carried the AA genotype. In addition, 2 patients carried an unreported missense mutation within the exon 8 (T14928C, accession number AF 440358), causing the substitution of a leucine with a proline in the trypsin-like serine protease domain of the protein. Protein Z levels in these patients were 0.25 mg/mL and 0.40 mg/mL, respectively, which means below the 2.5 percentile (0.49 mg/mL) of the controls. It is known that there is a relationship between the structure and function of proteins. The a-helix segments are solidified by having a hydrophobic amino acid in every third to fourth position of the primary structure. When an a-helix structure is formed, all these amino acids (e.g., isoleucine, alanine, valine) line up on one side of the helix. Proline is
Fertility and Sterility Vol. 90, No. 3, September 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.07.1318
FIGURE 1 Conservation of Leu 264 residue in the trypsin-like domain of PZ. The position of Leu264Pro in PZ is shown in bold. Multiple alignment of PZ from different species in the region containing the T T14928C mutation (accession numbers P22891, Q9CQW3, XP_001143484, P00744, XP_416945, XP_001087321, XP_001075483, and XP_693457, respectively). Numbering includes the signal peptide and refers to the human sequence.
In addition, the intron C G-42A gene variant was associated with the occurrence of pregnancy losses in our small sample of patients. Further studies are needed. Elvira Grandone, M.D.a Donatella Colaizzo, Biol.Sc.a Filomena Cappucci, Biol.Sc.a Rosa Lucia D’Ambrosio, Biol.Sc.a Gennaro Vecchione, Biol.Sc.a Maurizio Margaglione, M.D.a,b a Atherosclerosis and Thrombosis Unit, I.R.C.C.S. Casa Sollievo della Sofferenza, S. Giovanni Rotondo; and b Department of Medical Genetics, University of Foggia, Foggia, Italy
REFERENCES
Grandone. Protein Z gene mutations and fetal loss. Fertil Steril 2008.
an amino acid and does not have a side chain. Thus, the presence of proline disrupts the a-helix. For this reason, it is very suggestive to believe that nonconservative substitution of leucine with proline would affect PZ structure. As shown in Figure 1, this residue is highly conserved among different species. Among the controls, no woman carried this mutation. More interestingly, this mutation was not found in 197 patients with deep vein thrombosis or in the 197 age-matched controls previously investigated (8). This unreported sporadic mutation within the PZ gene could explain the very low PZ levels in our patients.
Fertility and Sterility
1. Broze GJ Jr. Protein Z dependent regulation of coagulation. Thromb Haemost 2001;86:8–13. 2. Kemkes-Matthes B, Matthes KJ. Protein Z deficiency: a new cause of bleeding tendency. Thromb Res 1995;79:49–55. 3. Yin ZF, Huang ZF, Cui J, Fiehler R, Lasky N, Ginsburg D, et al. Prothrombotic phenotype of protein Z deficiency. Proc Natl Acad Sci U S A 2000;97:6734–8. 4. Kemkes-Matthes B, Nees M, Kuhnel G, Matzdorff A, Matthes KJ. Protein Z influences prothrombotic phenotype of factor V Leiden in humans. Thromb Res 2002;106:183–5. 5. Gris JC, Quere I, Dechaud H, Mercier E, Pincon C, Hoffet M, et al. High frequency of protein Z deficiency in patients with unexplained early fetal loss. Blood 2002;99:2606–8. 6. Bretelle F, Arnoux D, Shojai R, D’Ercole C, Sampol J, Dignat F, et al. Protein Z in patients with pregnancy complications. Am J Obstet Gynecol 2005;193:1698–702. 7. Grandone E, Colaizzo D, Cappucci F, Cocomazzi N, Margaglione M. Protein Z levels and unexplained fetal losses. Fertil Steril 2004;82: 982–3. 8. Santacroce R, Sarno M, Cappucci F, Sessa F, Colaizzo D, Brancaccio V, et al. Low protein Z levels and risk of occurrence of deep vein thrombosis. J Thromb Haemost 2006;4:2417–22.
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