- Email: [email protected]
The effect of the Val34Leu polymorphism in the factor XIII gene in infants with a birth weight below 1500 g
The effect of the Val34Leu polymorphism in the factor XIII gene in infants with a birth weight below 1500 g Wolfgang Göpel, MD, Evelyn Kattner, MD, Jürgen Seidenberg, MD, Thomas Kohlmann, MD, Hugo Segerer, and Jens Möller, MD, for the Genetic Factors In Neonatology Study Group Objectives: The 34Leu polymorphism of the factor XIII gene is associated with a low rate of brain infarction and a higher incidence of primary intracerebral hemorrhage in adults. We evaluated the effect of the polymorphism on the subsequent development of isolated intracranial hemorrhage and white matter disease in preterm infants with a birth weight <1500 g (very low birth weight [VLBW] infants). Study design: We studied 531 VLBW infants and 301 control infants born at term. The factor XIII 34Leu polymorphism was detected by polymerase chain reaction and restriction enzyme digestion. Results: Allele frequencies were not different from term and VLBW infants (Val/Val, 53.1% and 57.8%; Val/Leu, 38.8% and 37.6%; Leu/Leu, 8.0% and 4.5%, respectively). VLBW infants carrying the Leu/Val or Leu/Leu allele had a significant reduced risk of the development of white matter disease (3.6% vs 10.4% in infants without the polymorphism, P = .003). In a multivariate logistic regression analysis, only gestational age <28 weeks (odds ratio, 3.8; 95% confidence interval, 1.9-7.5; P < .001), and the factor XIII 34Leu allele (odds ratio, 0.3; 95% confidence interval, 0.1-0.7; P = .005) had significant prognostic value in predicting subsequent white matter disease. However, VLBW infants who carried the factor XIII 34Leu allele also had a moderately increased risk of the subsequent development of isolated intraventricular hemorrhage (14.3% vs 10.1% in infants without the mutation, P = .17). Conclusions: VLBW infants carrying the factor XIII 34Leu polymorphism had a decreased risk for white matter disorders. (J Pediatr 2002;140:688-92) Factor XIII, on activation by thrombin, cross-links fibrin, thus stabilizing the fibrin clot and increasing its resis-
tance to fibrinolysis. A point mutation in codon 34 of exon 2 of the factor XIII gene has been described, leading
From the Department of Pediatrics and Department of Social Medicine, University of Lübeck, Childrens Hospital Auf der Bult, Hannover, Elisabeth Kinderkrankenhaus, Oldenburg, and Childrens Hospital St Hedwig, Regensburg, Germany.
Supported by Deutsche Forschungsgemeinschaft grant No. Go 955/1-1. Submitted for publication Sept 7, 2001; revision received Jan 17, 2002; accepted Feb 5, 2002. Reprint requests: W. Göpel, MD, Department of Pediatrics, University of Lübeck, 23538 Lübeck, Germany. Copyright © 2002, Mosby, Inc. All rights reserved. 0022-3476/2002/$35.00 + 0 9/21/123666 doi:10.1067/mpd.2002.123666
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to a Valine-Leucine change (factor XIII valine [Val] 34leucine [Leu]) in the α-subunit of the factor XIII gene, near the thrombin activation site. 1 The factor XIII 34Leu polymorphism accelerates activation of factor XIII by thrombin and affects the structure of the cross-linked fibrin clot. Clots prepared from plasma samples homozygous for the factor XIII 34Leu allele showed a finer fibrin meshwork, with thinner fibers and reduced space between the fibers. 2 In adults, homozygous or heterozygous carrier state of the factor XIII 34Leu polymorphism is associated with a lower rate of ischemic events such as venous thrombosis, myocardial infarction, and brain infarction.3-5 However, the polymorphism is also associated with an increased rate of bleeding events such as primary intracerebral hemorrhage.6
IVH Leu PCR Val VLBW
Intraventricular hemorrhage Leucine Polymerase chain reaction Valine Very low birth weight
Intracranial hemorrhage and cerebral white matter damage are frequent in preterm infants with a very low birth weight (VLBW) and important with respect to long-term neurodevelopmental outcome.7 We investigated the relation between factor XIII 34Leu genotype and white matter disorders or isolated intraventricular hemorrhage (IVH) in a large cohort of preterm infants with a birth weight <1500 g (VLBW infants).
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METHODS Between December 1999 and June 2001, 334 VLBW infants were prospectively enrolled in an open, multicenter study concerning the influence of genetic factors on the clinical course of VLBW infants. In April 2001, followup was completed in 185 VLBW infants. Furthermore, we retrospectively enrolled 372 VLBW infants who were admitted to the intensive care ward of the Medical University of Lübeck between June 1992 and December 1999. Of the VLBW infants, 14 were excluded. Five infants were excluded because of lethal malformations (Edwards-syndrome, n = 1; body stalk syndrome, n = 1; histologic confirmed lung-hypoplasia, n = 3). Because the allele frequency of the factor XIII Val34Leu polymorphism is lower in African and far Eastern populations,8 infants of such descendence were excluded (n = 6). Three infants were excluded because no cranial ultrasound was done during their stay in the hospital. The remaining 543 VLBW infants formed our study population. A random sample of 301 white, healthy, term singletons served as control infants. Two hundred four healthy term infants were enrolled retrospectively in the Medical University of Lübeck between 1996 and 1999. Ninety-seven term infants were enrolled prospectively between December 1999 and April 2001 in the centers participating in our study. Furthermore, we determined the factor XIII 34Leu mutation in all mothers of VLBW and term infants who were prospectively enrolled in our multicenter study. Every VLBW infant had at least one ultrasound study of the brain. Serial ultrasound studies were done in infants with a high risk of IVH (birth weight <1000 g or gestational age <28 weeks) and in every infant with proven or suspected abnormalities in the first ultrasound scan to document the progression and maximal extension of hemorrhage and white matter disease.
Figure. Percentage of VLBW infants with white matter disease and isolated IVH with regard to their factor XIII 34Leu carrier status.
Table I. Genotype distribution of factor XIII 34Leu allele in VLBW infants and term infants and their mothers
Genotype
VLBW infants (n = 531) Term infants (n = 301) Mothers of VLBW infants (n = 138) Mothers of term infants (n = 97)
Val/Val
Val/Leu
57.8 53.1 55.1 53.6
37.6 38.8 38.4 41.2
Leu/Leu 4.5 8.0 6.5 5.2
P value* .22 .89
All data are given as percentages. *Val/Leu and Leu/Leu genotype vs wild-type (Val/Val) compared with term infants and mothers of term infants, respectively. Fisher exact test.
IVH grade I was defined as blood in the periventricular germinal matrix regions, grade II as blood within the ventricular system without ventricular distension, grade III as blood within and distending the lateral ventricles, and grade IV as blood within the ventricular system and parenchymal involvement with subsequent parenchymal destruction. Periventricular leukomalacia was defined as one or more echodense lesions that eventually cavitate to become a porencephalic cyst. IVH grade I-III without additional white matter damage was summarized as isolated IVH. IVH grade IV and periventricular leukomalacia were summarized as white matter disorders. Follow-up of prospectively enrolled VLBW infants was complete when the
infants had been discharged from the hospital. The parents had given written informed consent. The clinical data of all patients and mothers were documented and coded before mutation analysis. DNA samples of the infants were extracted either from cotton swabs (prospective part of the study and 37 VLBW infants of the retrospective part of the study) or from blood stored on filter paper as part of the neonatal routine screening (retrospective part of the study). All VLBW infants were included irrespective of their survivor status except for a small group of 37 VLBW infants born in Lübeck between January 1998 and December 1999. In this retrospective subgroup, we were only able to study survivors. DNA was extracted with a commer689
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Table II. Clinical data of VLBW infants with respect to factor XIII 34Leu carrier status
Mean birth weight (SD) Mean gestational age (SD) Male sex Antenatal treatment with steroids Need of mechanical ventilation Death rate Infants with isolated IVH Infants with white matter disease
Val/Val (n = 307)
Val/Leu or Leu/Leu (n = 224)
Val/Leu (n = 200)
Leu/Leu (n = 24)
P value
1079 (279) 28.6 (2.7) 48.9 74.5 66 4.2 10.1 10.4
1093 (292) 28.9 (2.6) 48.2 74.4 62 4.5 14.3 3.6
1100 (292) 28.9 (2.5) 49 73.5 63.5 5 13.5 4
1036 (296) 28.8 (3.1) 41.6 82.6 50.0 0.0 20.8 0.0
.47 .17 .93 1.0 .36 1.0 .17 .003
Mean birth weight is given as grams and mean gestational age as weeks. All other data are given as percentages. All P values are 2-sided and given for infants with Val/Val genotype vs infants with Val/Leu or Leu/Leu genotype. Birth weight and gestational age are compared by means of Wilcoxon rank-sum test; all other values are compared by Fisher exact test.
Table III. Multivariate analysis: Gestational age and factor XIII 34Leu carrier status as independent predictors of white matter disease and isolated IVH
Dependent variable White matter disease Independent variable Factor V Leiden (heterozygous) Male sex Retrospective inclusion Multiple birth Prothrombin 20210A (heterozygous) Antenatal treatment with steroids Factor XIII 34Leu (heterozygous or homozygous) Gestational age < 28 wk
Isolated IVH
OR
95% CI
P value
OR
95% CI
P value
0.8 0.9 1.6 1.7 2.2 0.5 0.3 3.8
0.2–3.6 0.4–1.7 0.7–3.5 0.9–3.5 0.7–7.2 0.2–1.1 0.1–0.7 1.9–7.5
.79 .67 .22 .12 .20 .066 .005 < .001
1.1 0.7 1.1 1.0 0.24 0.6 1.5 4.9
0.4–3.0 0.4–1.2 0.6–2.0 0.6–1.9 0.03–1.9 0.3–1.1 0.9–2.7 2.7–8.6
.87 .23 .85 .92 .17 .10 .13 < .001
OR, Odds ratio; CI, confidence interval.
cially available kit (Qiamp tissue kit, Qiagen, Hilden, Germany). The factor XIII 34Leu polymorphism was detected by polymerase chain reaction (PCR) and restriction enzyme digestion with Dde I as previously described.9 Furthermore, we analyzed the factor V Leiden mutation and the prothrombin 20210A mutation to rule out possible confounding effects of these prothrombotic mutations. Factor V Leiden was detected by PCR and restriction enzyme digestion and the prothrombin G20210A mutation by allele-specific PCR, as previously described.10,11 The results of allelespecific PCR were confirmed by an690
other PCR and restriction enzyme digestion in positive samples and a random number of wild-type samples.12 All parts of the study were approved by the local committee on research in human subjects of the Medical University of Lübeck. Hypotheses were evaluated with the Fisher exact test (2-sided), Wilcoxon rank sum test, and multivariate logistic regression models.
RESULTS The genotype distribution of factor XIII 34Leu in VLBW infants, term infants, and their mothers are given in
Table I. Genotyping of the factor XIII 34Leu allele was not successful in 12 DNA samples from dried blood spots. No differences in the allelic distribution between the groups were observed, and the genotypes were in Hardy-Weinberg equilibrium in all groups. Clinical data of VLBW infants according to their factor XIII 34Leu status are given in Table II. There were no differences with regard to birth weight, gestational age, sex, antenatal treatment with steroids, need of mechanical ventilation, and death rate. The frequencies of the two types of ultrasound abnormalities, isolated IVH
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VOLUME 140, NUMBER 6 and white matter disease, were contradictory with respect to the factor XIII 34Leu carrier status. The frequency of IVH grade IV and periventricular leukomalacia in carriers of the Leu allele was 1.8% and 1.8%, respectively, compared with 5.9% and 4.5% in infants homozygous for the Val allele. Infants who carried the Leu allele had a 3-fold decreased risk of white matter disease (3.6% vs 10.4% in infants homozygous for factor XIII 34Val, P = .003). This trend was more pronounced in infants who were homozygous for the 34Leu allele (Table II and the Figure). The frequency of IVH grade I/II/III in infants carrying the Leu allele was 8%/3.1%/3.1% compared with 3.9%/3.6%/2.6% in infants homozygous for the Val allele. Because of the increased rate of IVH grade I in carriers of the Leu allele, there was a nonsignificant trend to a higher frequency of isolated hemorrhage (IVH grade IIII) in these infants (14.3% vs 10.1% in infants homozygous for factor XIII 34Val, P = .17). Again, this trend was more pronounced in 34Leu homozygotes (Table II and the Figure). To rule out possible confounding factors, we did a multivariate analysis including gestational age <28 weeks, sex, multiple birth, antenatal steroid treatment, retrospective or prospective inclusion of the infant, carrier status of the factor V Leiden mutation, the prothrombin 20210A mutation, and the factor XIII 34Leu polymorphism as independent variables and the development of white matter disease or isolated IVH as dependent variables. Only gestational age and factor XIII 34Leu carrier status were independent predictors for the subsequent development of white matter disease and isolated hemorrhage (Table III). As expected, low gestational age was a risk factor for the development of both IVH grade I-III and white matter disease, respectively. However, the heterozygous or homozygous carrier state of the factor XIII 34Leu allele was a protective factor against white matter disease (odds
ratio, 0.3; 95% confidence interval, 0.1 to 0.7; P = .005).
DISCUSSION Neonatal cranial ultrasound is a routine method to detect intracranial abnormalities in preterm infants with a high risk of the subsequent development of motor dysfunction and mental retardation. On the basis of neuropathology, ultrasound abnormalities in VLBW infants can be categorized into two groups: isolated bleeding into the germinal matrix or the ventricle (IVH grade I-III) and white matter disease (with or without IVH) reflecting ischemic or hemorrhagic infarction.13,14 In long-term follow-up studies of VLBW infants, white matter disease was found to be a better predictor of poor neurologic outcome than isolated IVH.7,15,16 Coagulation is essential in the control of intraventricular hemorrhage, but it may be a risk factor for the occurrence of white matter disease, which is due to or augmented by venous or arterial infarction. Serial coagulation tests are usually not possible in VLBW infants because of the relatively large amount of blood needed for testing. Furthermore, reference values for coagulation parameters in VLBW infants are scarce, and coagulation disturbances are common, even in healthy VLBW infants. Therefore, it is not surprising that an independent pathogenetic role of coagulation disturbances in the etiologic factors of IVH or white matter disease has been difficult to establish.17 Our group studies the association between frequent genetic polymorphisms that influence coagulation and the development of IVH and white matter disease in VLBW infants. The factor XIII 34Leu polymorphism, whose effects in VLBW infants are reported here, is of particular interest because factor XIII activity is strongly influenced by heredity18 and the factor XIII 34Leu allele is associated with a higher rate of primary
intracerebral hemorrhage and a lower rate of brain infarction in adults.5,6 In our large group of VLBW infants, heterozygous or homozygous carrier state of the factor XIII 34Leu allele was associated with a 3-fold decreased risk of white matter disease. This result is important because white matter disease is a very strong predictor of long-term neurologic disability of VLBW infants. Since ischemic and hemorrhagic infarction is relevant in the pathogenesis of parenchymal destruction in VLBW infants,14 the reduced risk of white matter disease in factor XIII 34Leu carriers might be due to the finer and more delicate structure of their fibrin clots.2 This view is furthermore supported by our result of an increased rate of isolated intracranial hemorrhage in VLBW infants carrying the factor XIII 34Leu allele. The pathogenesis of IVH and white matter disease is obviously influenced by multiple factors.17 Our data support the view that the relative importance of coagulation in the development and course of these diseases may have been underestimated. Furthermore, the negative association of the factor XIII 34Leu allele with white matter disease indicates that drugs that influence factor XIII activity such as the 34Leu allele might be useful in the prevention of white matter disease in VLBW infants. We thank Ritva Schröder and Lynn Ellenberg for excellent laboratory assistance, all doctors and nurses of the participating hospitals, Birgit Roenspiess for skillful data collection, and especially all infants and their parents for their support.
REFERENCES 1. Mikkola H, Syrjälä M, Rasi V, Vathera E, Hamalainen E, Peltonen L, et al. Deficiency in the α-subunit of coagulation factor XIII: two novel point mutations demonstrate different effects on transcript levels. Blood 1994;84:517-25. 2. Ariens RAS, Philippou H, Nagaswami C, Weisel JW, Lane DA, Grant PJ. The factor XIII V34L polymorphism 691
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4.
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6.
7.
8.
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accelerates thrombin activation of factor XIII and affects cross-linked fibrin structure. Blood 2000;96:988-95. Catto AJ, Kohler HP, Coore J, Mansfield MW, Stickland MH, Grant PJ. Association of a common polymorphism in the factor XIII gene with venous thrombosis. Blood 1999;93:906-8. Kohler HP, Stickland MH, OsseiGerning N, Carter A, Mikkola H, Grant PJ. Association of a common polymorphism in the factor XIII gene with myocardial infarction. Thromb Haemost 1998;79:8-13. Elbaz A, Poirier O, Canaple S, Chedru F, Cambien F, Amarenco P. The association between the Val34Leu polymorphism in the factor XIII gene and brain infarction. Blood 2000;95:986-91. Catto AJ, Kohler HP, Bannan S, Stickland M, Carter A, Grant PJ. Factor XIII Val34Leu, a novel association with primary intracerebral hemorrhage. Stroke 1998;29:813-6. Whitaker AH, Feldman JF, Van Rossem R, Schonfeld IS, Pinto-Martin JA, Torre C, et al. Neonatal cranial ultrasound abnormalities in low birth weight infants: relation to cognitive outcomes at six years of age. Pediatrics 1996;98:719-29. Attie-Castro FA, Zago MA, Lavina J, Elion J, Rodrigue-Delfin L, Guerriero JF, et al. Ethnic heterogeneity of the factor XIII Val34Leu polymorphism. Thromb Haemost 2000;84:601-3.
9. Kangsadalampa S, Board PG. The Val34Leu polymorphism in the subunit of coagulation factor XIII contributes to the large normal range in activity and demonstrates that the activation peptide plays a role in catalytic activity. Blood 1998;92:2766-70. 10. Ridker PM, Hennekens CH, Lindpainter K, Stampfer MJ, Eisenberg PR, Miletich JP. Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke and venous thrombosis in apparently healthy men. N Engl J Med 1995;332:912-7. 11. Poort SR, Bertina RM, Vos HL. Rapid detection of the prothrombin 20210A variation by allele specific PCR. Thromb Haemost 1997;78:1157-8. 12. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3’-untranslated region of the prothrombin gene is associated with elevated plasma thrombin levels and an increase in venous thrombosis. Blood 1996;88:3698-703. 13. Paneth N, Rudelli R, Monte W, Rodriguez E, Pinto J, Kairam R, et al. White matter necrosis in very low birth weight infants: neuropathologic and ultrasonographic findings in infants surviving six days or longer. J Pediatr 1990;116:975-84. 14. Gould SJ, Howard S, Hope PL, Reynolds EOR. Periventricular intraparenchymal cerebral haemorrhage in
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preterm infants: the role of venous infarction. J Pathol 1987;151:197-202. Aziz K, Vickar DB, Sauve RS, Etches PC, Pain KS, Robertson CMT. Provincebased study of neurologic disability of children weighing 500 to 1249 grams at birth in relation to neonatal cerebral ultrasound findings. Pediatrics 1995;95:837-44. Pinto-Martin JA, Riolo S, Cnaan A, Holzmann C, Susser MW, Paneth N. Cranial ultrasound prediction of disabling and nondisabling cerebral palsy at age two in a low birth weight population. Pediatrics 1995;95:249-50. Volpe JJ. Neurology of the newborn. Philadelphia (PA): WB Saunders Co; 2001. p. 428-93. de Lange M, Sieder H, Ariens RAS, Spector TD, Grant PJ. The genetics of haemostasis: a twin study. Lancet 2001;357:101-5.
APPENDIX Other Investigators of the Genetic Factors in Neonatology Study Group K. Albrecht (Bremen), R. Jensen (Heide), B. Köhler, A. Leonhardt, M. Raab (Marburg), A. Schwabe (Oldenburg), T. Schaible (Mannheim), A. Warnecke (Oberhausen), H. Wehninger (Kassel), and A v.d. Wense (Hamburg).
FELLOWSHIPS Fellowships available in pediatric subspecialties and those for general academic pediatric training are listed once a year, in January, in The Journal of Pediatrics. Each June, forms for listing fellowships available for the academic year beginning 18 months after publication are sent to the Chairman of the Department of Pediatrics at major hospitals in the United States and Canada. In addition, a copy of the application form appears in the July, August, and September issues of The Journal (please use the current form). Should you desire to list fellowships, a separate application must be made each year for each position. All applications must be returned to Mosby, Inc., by October 15 preceding the listing year to ensure publication. Additional forms will be supplied on request from the Periodical Editing Department, Mosby, Inc, 11830 Westline Industrial Dr, St Louis, MO 63146-3318 (phone: 800-325-4177, ext 2838, or 314-579-2838).
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tance to fibrinolysis. A point mutation in codon 34 of exon 2 of the factor XIII gene has been described, leading
From the Department of Pediatrics and Department of Social Medicine, University of Lübeck, Childrens Hospital Auf der Bult, Hannover, Elisabeth Kinderkrankenhaus, Oldenburg, and Childrens Hospital St Hedwig, Regensburg, Germany.
Supported by Deutsche Forschungsgemeinschaft grant No. Go 955/1-1. Submitted for publication Sept 7, 2001; revision received Jan 17, 2002; accepted Feb 5, 2002. Reprint requests: W. Göpel, MD, Department of Pediatrics, University of Lübeck, 23538 Lübeck, Germany. Copyright © 2002, Mosby, Inc. All rights reserved. 0022-3476/2002/$35.00 + 0 9/21/123666 doi:10.1067/mpd.2002.123666
688
to a Valine-Leucine change (factor XIII valine [Val] 34leucine [Leu]) in the α-subunit of the factor XIII gene, near the thrombin activation site. 1 The factor XIII 34Leu polymorphism accelerates activation of factor XIII by thrombin and affects the structure of the cross-linked fibrin clot. Clots prepared from plasma samples homozygous for the factor XIII 34Leu allele showed a finer fibrin meshwork, with thinner fibers and reduced space between the fibers. 2 In adults, homozygous or heterozygous carrier state of the factor XIII 34Leu polymorphism is associated with a lower rate of ischemic events such as venous thrombosis, myocardial infarction, and brain infarction.3-5 However, the polymorphism is also associated with an increased rate of bleeding events such as primary intracerebral hemorrhage.6
IVH Leu PCR Val VLBW
Intraventricular hemorrhage Leucine Polymerase chain reaction Valine Very low birth weight
Intracranial hemorrhage and cerebral white matter damage are frequent in preterm infants with a very low birth weight (VLBW) and important with respect to long-term neurodevelopmental outcome.7 We investigated the relation between factor XIII 34Leu genotype and white matter disorders or isolated intraventricular hemorrhage (IVH) in a large cohort of preterm infants with a birth weight <1500 g (VLBW infants).
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METHODS Between December 1999 and June 2001, 334 VLBW infants were prospectively enrolled in an open, multicenter study concerning the influence of genetic factors on the clinical course of VLBW infants. In April 2001, followup was completed in 185 VLBW infants. Furthermore, we retrospectively enrolled 372 VLBW infants who were admitted to the intensive care ward of the Medical University of Lübeck between June 1992 and December 1999. Of the VLBW infants, 14 were excluded. Five infants were excluded because of lethal malformations (Edwards-syndrome, n = 1; body stalk syndrome, n = 1; histologic confirmed lung-hypoplasia, n = 3). Because the allele frequency of the factor XIII Val34Leu polymorphism is lower in African and far Eastern populations,8 infants of such descendence were excluded (n = 6). Three infants were excluded because no cranial ultrasound was done during their stay in the hospital. The remaining 543 VLBW infants formed our study population. A random sample of 301 white, healthy, term singletons served as control infants. Two hundred four healthy term infants were enrolled retrospectively in the Medical University of Lübeck between 1996 and 1999. Ninety-seven term infants were enrolled prospectively between December 1999 and April 2001 in the centers participating in our study. Furthermore, we determined the factor XIII 34Leu mutation in all mothers of VLBW and term infants who were prospectively enrolled in our multicenter study. Every VLBW infant had at least one ultrasound study of the brain. Serial ultrasound studies were done in infants with a high risk of IVH (birth weight <1000 g or gestational age <28 weeks) and in every infant with proven or suspected abnormalities in the first ultrasound scan to document the progression and maximal extension of hemorrhage and white matter disease.
Figure. Percentage of VLBW infants with white matter disease and isolated IVH with regard to their factor XIII 34Leu carrier status.
Table I. Genotype distribution of factor XIII 34Leu allele in VLBW infants and term infants and their mothers
Genotype
VLBW infants (n = 531) Term infants (n = 301) Mothers of VLBW infants (n = 138) Mothers of term infants (n = 97)
Val/Val
Val/Leu
57.8 53.1 55.1 53.6
37.6 38.8 38.4 41.2
Leu/Leu 4.5 8.0 6.5 5.2
P value* .22 .89
All data are given as percentages. *Val/Leu and Leu/Leu genotype vs wild-type (Val/Val) compared with term infants and mothers of term infants, respectively. Fisher exact test.
IVH grade I was defined as blood in the periventricular germinal matrix regions, grade II as blood within the ventricular system without ventricular distension, grade III as blood within and distending the lateral ventricles, and grade IV as blood within the ventricular system and parenchymal involvement with subsequent parenchymal destruction. Periventricular leukomalacia was defined as one or more echodense lesions that eventually cavitate to become a porencephalic cyst. IVH grade I-III without additional white matter damage was summarized as isolated IVH. IVH grade IV and periventricular leukomalacia were summarized as white matter disorders. Follow-up of prospectively enrolled VLBW infants was complete when the
infants had been discharged from the hospital. The parents had given written informed consent. The clinical data of all patients and mothers were documented and coded before mutation analysis. DNA samples of the infants were extracted either from cotton swabs (prospective part of the study and 37 VLBW infants of the retrospective part of the study) or from blood stored on filter paper as part of the neonatal routine screening (retrospective part of the study). All VLBW infants were included irrespective of their survivor status except for a small group of 37 VLBW infants born in Lübeck between January 1998 and December 1999. In this retrospective subgroup, we were only able to study survivors. DNA was extracted with a commer689
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Table II. Clinical data of VLBW infants with respect to factor XIII 34Leu carrier status
Mean birth weight (SD) Mean gestational age (SD) Male sex Antenatal treatment with steroids Need of mechanical ventilation Death rate Infants with isolated IVH Infants with white matter disease
Val/Val (n = 307)
Val/Leu or Leu/Leu (n = 224)
Val/Leu (n = 200)
Leu/Leu (n = 24)
P value
1079 (279) 28.6 (2.7) 48.9 74.5 66 4.2 10.1 10.4
1093 (292) 28.9 (2.6) 48.2 74.4 62 4.5 14.3 3.6
1100 (292) 28.9 (2.5) 49 73.5 63.5 5 13.5 4
1036 (296) 28.8 (3.1) 41.6 82.6 50.0 0.0 20.8 0.0
.47 .17 .93 1.0 .36 1.0 .17 .003
Mean birth weight is given as grams and mean gestational age as weeks. All other data are given as percentages. All P values are 2-sided and given for infants with Val/Val genotype vs infants with Val/Leu or Leu/Leu genotype. Birth weight and gestational age are compared by means of Wilcoxon rank-sum test; all other values are compared by Fisher exact test.
Table III. Multivariate analysis: Gestational age and factor XIII 34Leu carrier status as independent predictors of white matter disease and isolated IVH
Dependent variable White matter disease Independent variable Factor V Leiden (heterozygous) Male sex Retrospective inclusion Multiple birth Prothrombin 20210A (heterozygous) Antenatal treatment with steroids Factor XIII 34Leu (heterozygous or homozygous) Gestational age < 28 wk
Isolated IVH
OR
95% CI
P value
OR
95% CI
P value
0.8 0.9 1.6 1.7 2.2 0.5 0.3 3.8
0.2–3.6 0.4–1.7 0.7–3.5 0.9–3.5 0.7–7.2 0.2–1.1 0.1–0.7 1.9–7.5
.79 .67 .22 .12 .20 .066 .005 < .001
1.1 0.7 1.1 1.0 0.24 0.6 1.5 4.9
0.4–3.0 0.4–1.2 0.6–2.0 0.6–1.9 0.03–1.9 0.3–1.1 0.9–2.7 2.7–8.6
.87 .23 .85 .92 .17 .10 .13 < .001
OR, Odds ratio; CI, confidence interval.
cially available kit (Qiamp tissue kit, Qiagen, Hilden, Germany). The factor XIII 34Leu polymorphism was detected by polymerase chain reaction (PCR) and restriction enzyme digestion with Dde I as previously described.9 Furthermore, we analyzed the factor V Leiden mutation and the prothrombin 20210A mutation to rule out possible confounding effects of these prothrombotic mutations. Factor V Leiden was detected by PCR and restriction enzyme digestion and the prothrombin G20210A mutation by allele-specific PCR, as previously described.10,11 The results of allelespecific PCR were confirmed by an690
other PCR and restriction enzyme digestion in positive samples and a random number of wild-type samples.12 All parts of the study were approved by the local committee on research in human subjects of the Medical University of Lübeck. Hypotheses were evaluated with the Fisher exact test (2-sided), Wilcoxon rank sum test, and multivariate logistic regression models.
RESULTS The genotype distribution of factor XIII 34Leu in VLBW infants, term infants, and their mothers are given in
Table I. Genotyping of the factor XIII 34Leu allele was not successful in 12 DNA samples from dried blood spots. No differences in the allelic distribution between the groups were observed, and the genotypes were in Hardy-Weinberg equilibrium in all groups. Clinical data of VLBW infants according to their factor XIII 34Leu status are given in Table II. There were no differences with regard to birth weight, gestational age, sex, antenatal treatment with steroids, need of mechanical ventilation, and death rate. The frequencies of the two types of ultrasound abnormalities, isolated IVH
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VOLUME 140, NUMBER 6 and white matter disease, were contradictory with respect to the factor XIII 34Leu carrier status. The frequency of IVH grade IV and periventricular leukomalacia in carriers of the Leu allele was 1.8% and 1.8%, respectively, compared with 5.9% and 4.5% in infants homozygous for the Val allele. Infants who carried the Leu allele had a 3-fold decreased risk of white matter disease (3.6% vs 10.4% in infants homozygous for factor XIII 34Val, P = .003). This trend was more pronounced in infants who were homozygous for the 34Leu allele (Table II and the Figure). The frequency of IVH grade I/II/III in infants carrying the Leu allele was 8%/3.1%/3.1% compared with 3.9%/3.6%/2.6% in infants homozygous for the Val allele. Because of the increased rate of IVH grade I in carriers of the Leu allele, there was a nonsignificant trend to a higher frequency of isolated hemorrhage (IVH grade IIII) in these infants (14.3% vs 10.1% in infants homozygous for factor XIII 34Val, P = .17). Again, this trend was more pronounced in 34Leu homozygotes (Table II and the Figure). To rule out possible confounding factors, we did a multivariate analysis including gestational age <28 weeks, sex, multiple birth, antenatal steroid treatment, retrospective or prospective inclusion of the infant, carrier status of the factor V Leiden mutation, the prothrombin 20210A mutation, and the factor XIII 34Leu polymorphism as independent variables and the development of white matter disease or isolated IVH as dependent variables. Only gestational age and factor XIII 34Leu carrier status were independent predictors for the subsequent development of white matter disease and isolated hemorrhage (Table III). As expected, low gestational age was a risk factor for the development of both IVH grade I-III and white matter disease, respectively. However, the heterozygous or homozygous carrier state of the factor XIII 34Leu allele was a protective factor against white matter disease (odds
ratio, 0.3; 95% confidence interval, 0.1 to 0.7; P = .005).
DISCUSSION Neonatal cranial ultrasound is a routine method to detect intracranial abnormalities in preterm infants with a high risk of the subsequent development of motor dysfunction and mental retardation. On the basis of neuropathology, ultrasound abnormalities in VLBW infants can be categorized into two groups: isolated bleeding into the germinal matrix or the ventricle (IVH grade I-III) and white matter disease (with or without IVH) reflecting ischemic or hemorrhagic infarction.13,14 In long-term follow-up studies of VLBW infants, white matter disease was found to be a better predictor of poor neurologic outcome than isolated IVH.7,15,16 Coagulation is essential in the control of intraventricular hemorrhage, but it may be a risk factor for the occurrence of white matter disease, which is due to or augmented by venous or arterial infarction. Serial coagulation tests are usually not possible in VLBW infants because of the relatively large amount of blood needed for testing. Furthermore, reference values for coagulation parameters in VLBW infants are scarce, and coagulation disturbances are common, even in healthy VLBW infants. Therefore, it is not surprising that an independent pathogenetic role of coagulation disturbances in the etiologic factors of IVH or white matter disease has been difficult to establish.17 Our group studies the association between frequent genetic polymorphisms that influence coagulation and the development of IVH and white matter disease in VLBW infants. The factor XIII 34Leu polymorphism, whose effects in VLBW infants are reported here, is of particular interest because factor XIII activity is strongly influenced by heredity18 and the factor XIII 34Leu allele is associated with a higher rate of primary
intracerebral hemorrhage and a lower rate of brain infarction in adults.5,6 In our large group of VLBW infants, heterozygous or homozygous carrier state of the factor XIII 34Leu allele was associated with a 3-fold decreased risk of white matter disease. This result is important because white matter disease is a very strong predictor of long-term neurologic disability of VLBW infants. Since ischemic and hemorrhagic infarction is relevant in the pathogenesis of parenchymal destruction in VLBW infants,14 the reduced risk of white matter disease in factor XIII 34Leu carriers might be due to the finer and more delicate structure of their fibrin clots.2 This view is furthermore supported by our result of an increased rate of isolated intracranial hemorrhage in VLBW infants carrying the factor XIII 34Leu allele. The pathogenesis of IVH and white matter disease is obviously influenced by multiple factors.17 Our data support the view that the relative importance of coagulation in the development and course of these diseases may have been underestimated. Furthermore, the negative association of the factor XIII 34Leu allele with white matter disease indicates that drugs that influence factor XIII activity such as the 34Leu allele might be useful in the prevention of white matter disease in VLBW infants. We thank Ritva Schröder and Lynn Ellenberg for excellent laboratory assistance, all doctors and nurses of the participating hospitals, Birgit Roenspiess for skillful data collection, and especially all infants and their parents for their support.
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APPENDIX Other Investigators of the Genetic Factors in Neonatology Study Group K. Albrecht (Bremen), R. Jensen (Heide), B. Köhler, A. Leonhardt, M. Raab (Marburg), A. Schwabe (Oldenburg), T. Schaible (Mannheim), A. Warnecke (Oberhausen), H. Wehninger (Kassel), and A v.d. Wense (Hamburg).
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