Heritability of elevated risk factor VIII antigen levels in factor V Leiden families with thrombophilia

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Bntish Jownal oj Haemalology 2000 109 519-522

Heritability of elevated factor VIII antigen levels in factor V

Leiden families with thrombophilia

P W KAMPHÜISEN,1 R L E N S E N ,2 J. J HdUWING-DuiSTERMAAT,4 J C J ElKENBOOM,1 M H A R V E Y ,3

R M BERTINA1 AND F R ROSENDAAL] 2 1 Haemostasis and Thrombosis Research Centre and the Oepaitments of 2Chmcal Epidemiology and 3Immunohaematology and Blood Bank, Leiden Umversity Medical Centre, and ^Institute of Epidemwlogy and Bwstatistics, Erasmus Medical Centre Rotterdam, The Netherlands

Received l November 1999, accepted for pubhcation 10 February 2000

Summary. Factoi VIII activity (factor VIII C) and factor VIII antigen (factor VIII Ag) levels abovelSO lU/dl are associated with a five- to sixfold increased nsk of venous thrombosis compared with levels < 100 lU/dl These high levels are present in 25% of patients with a first episode of deep-vem thrombosis and in 11% of healthy controls von Willebrand lactor (VWF) and blood group are impoitant determmants of the factor VIII level m plasma and therefoie contnbute to thrombotic nsk, while factor VIII appears to be the final effector Previously, we found famihal clustermg of factor VIII C levels m women which remamed after adjustment for VWF and blood group In the present study, we analysed the famihal influence on factor VIII Ag levels exceeding ISO lU/dl in 12 large families with thrombophilia in which high factor VIII Ag levels contnbute to thrombotic nsk As expected, blood group was a mam determmant of the plasma factor VIII level 58 relatives (32%) had factor

VIII levels above 150 Ιϋ/dl and 50 (86%) of these had blood group non-0 After adjustment for blood group and age, we found an association between factor VIII Ag levels m sister pairs (0 35, P = 0 003), brother pairs (0 35, P = 0 003), brother-sister pairs (0 35, P < 0 001) and in mother-son pairs (0 45 P = 0 02), but not in father-daughter or father-son pairs The famihal aggregation test was strongly positive for factoi VIII Ag levels (P < 0 001) and remamed so after adjustment for the influence of blood group We conclude that high factor VIII Ag levels are a highly prevalent nsk factoi for venous thrombosis and contnbute to risk m families with thrombophilia, and that these high levels are hkely to be genetically determmed by factors other than just blood group

Keywords: factor VIII, famihal clustenng

thrombophilia factoi V Leiden,

Elevated factor VIII activity (factor VIII C) levels are associ-ated with an increased nsk of venous thrombosis (Koster et al, 1995) Factor VIII C levels higher than 150 lU/dl mcrease the thrombosis nsk five- to sixfold compaied with levels below 100 Ιϋ/dl (Koster et al, 1995) von Willebrand factor (VWF) and blood group are well-known determmants of the factor VIII level m plasma and so contnbute to thrombotic nsk, whereas factor VIII itself appears to be the final effector m promotmg thrombosis (Koster et al, 1995) Elevated factor VIII C levels are highly correlated with factor VIII antigen (factor VIII Ag) levels (Kamphmsen etal, 1997, O'Donnell et al, 1997) This suggests that the observed elevation of factor VIII C levels reflects a true mcrease in Correspondence Professor F R Rosendaal Chmcal Epidemiology CO-P, Leiden Umversity Medical Centre PO Box 9600 2300 RC Leiden The Netherlands E-mail f r rosendaal@lumc nl

factor VIII protem and is not the lesult of activation of the coagulation System during the blood collection procedure (Kamphmsen et al, 1997, O'Donnell et al, 1997) Factor VIII Ag levels above 150 lU/dl were, hke factor VIII C levels, also associated with a flvefold increased nsk of venous thrombosis (Kamphuisen et al 1997) We have recently shown that elevated factor VIII levels m thrombosis patients aie not the result of acute phase reactions because elevated factor VIII levels remamed associated with a sixfold increased risk after adjustment for C-reactive protem (CRP), a sensitive marker for acute phase processes (Kamphuisen ei al, 1999) These observations lend further support to a causal relation-ship between high factor VIII levels and venous thrombosis

High factor VIII levels are common, m our study, 25% of the patients with a first episode of venous thrombosis and 11% of the healthy controls had factor VIII levels above 150 lU/dl (Koster et al, 1995) Considering the sixfold

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520 P. W. Kamphuisen et al

increased thrombosis risk and the high prevalence in the Population, factor VIII levels exceeding 150 lU/dl contribute importantly to deep-vein thrombosis.

We previously reported that factor VIII: C levels show a familial clustering, which remains after correction for VWF and blood group (Kamphuisen et al, 1998). That study was performed in female relatives of probands with documented haemophilia A who came to the Leiden haemophilia centre for carriership testing. We designed a study that allowed us to incorporate a large number of families that were seen over a period of more than 10 years for carrier testing. However, the design of this study also had several drawbacks. First, factor VIII:C levels were measured over a 10-year period, which might have led to extra Variation in the plasma factor VIII level. Furthermore, only women were tested, which provides less information on familial clustering than when both women and men are tested. And, fmally, in this group of women, high levels of factor VIII were not äs common äs among patients with thrombosis, and the association between levels and thrombosis was not studied nor was the heritability of high levels (above 150 lU/dl) itself.

In the present study, we have analysed the familial aggregation of factor VIII levels in men and women of 12 large families with thrombophilia who had a proband with both venous thrombosis and factor V Leiden. In these families, factor VIII:Ag levels above 150 lU/dl contribute to the thrombosis risk of factor V Leiden carriers (Lensen et al, 1999). We tested whether clustering of factor VIII:Ag levels higher than 150 lU/dl occurred and the effect of blood group on these high levels.

SUBJECTS AND METHODS

Family data. The 12 probands originated from a larger panel of 28 patients who were referred to our centre for diagnostic work-up for venous thrombophilia, i.e. patients with a positive family history of venous thrombosis (in addition to the proband, at least two symptomatic relatives), and who did not have deficiencies of protein C, protein S or antithrombin. These 28 patients were screened for the presence of the factor V Leiden mutation that was detected in 12 patients. We invited the siblings, parents and children of these 12 probands äs well äs uncles and aunts of the affected parental side and, if these were carriers, their children (first cousins of the proband). Family members under 15 years of age were excluded for practical purposes. Of the 12 probands, 182 family members (93%) participated in the study while 12 did not, three because they lived abroad and nine for reasons unknown.

The probands and family members were not selected on the basis of factor VIII:Ag levels because these levels were not known at that Urne.

Laboratory assays. Blood was collected from the ante-cubital vein in 0-106 M trisodium citrate. ABO phenotypes were deduced from the reactions of plasma isoagglutinins to AI, B, A1B or (äs negative control) 0 test blood cell suspensions (3% v/v) in a Standard spin-tube agglutination technique carried out at room temperature (Walker, 1990). All the subjects were considered to be immune competent and

free from malignancy or infection. The chance of an ABO misphenotyping because of the absence of the appropriate isoagglutinin, anomalous occurrence of anti-A or -B or acquisition of a B-antigen was considered to be unlikely. Factor VIII antigen was measured by a Sandwich type enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies directed against the light chain of factor VIII (Kamphuisen ei al, 1997). Pooled normal plasma, calibrated against the WHO Standard (91/666) for factor VIILAg, was used äs a reference.

Staüstical analysis. The distribution of factor VIII:Ag levels was skewed and was logarithmically transformed for all statistical analyses. The analysis was performed using linear regression, with age entered äs a continuous variable (in years), and blood group dichotomized into two groups (0 for blood group 0, l for non-0).

To investigate genetic effects on factor VIII:Ag levels, the residuale of the multiple regression models were used. Residuais are the differences between the observed level Y, for person i and the predicted value μ, obtained from the multiple regression model. As a first test for familial effects, correlations between the residuale (obtained from the multiple regression model) of pairs of relatives were calculated. Familial aggregation of factor VIIIiAg levels was studied using a recently developed method (Houwing-Duistermaat et al, 1995) that tests the null hypothesis of no correlation within randomly chosen pedigrees. The correla-tion of the genetic effects is tested by the weighted sum of correlations between pairs of relatives within a pedigree 0. The test for familial aggregation is positive when the calculated Q is larger than the expected 0-value under the null hypothesis of no aggregation (for more details, see Houwing-Duistermaat ei al, 1995; Kamphuisen et al, 1998). The familial clustering of factor VIII levels exceeding 150 lU/dl was tested with the individual factor VIII level dichotomized into two groups (Y, = 0 for factor VIII:Ag levels < 150 lU/dl, Y, = l for factor VIII:Ag levels s 150 lU/dl). In this way, the value of 0 will be determined mainly by pairs of relatives who both have factor VIII levels higher than 150 lU/dl.

RESULTS

We studied 182 relatives of 12 probands with factor V Leiden and a positive family history of venous thrombosis. The mean size of the families was 19 members, with a ränge from 3 to 29 members. The mean age at the time of the study was 40 years (ränge 15-88 years); 92 (50%) were men and 90 were women. Of these 182 relatives, 91 were heterozygous for factor V Leiden, one relative was homo-zygous and 90 were non-carriers.

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Table I. Conelation coefficients for factor VIII Ag levels between first-degree relative pairs *

Relationship No of pairs P-value

Father— son Mother-son Father-daughter Molher-daughter Sisters Brothers Brothei-sister 28 26 21 33 70 70 180 0 17 0 4 5 0 14 0 27 0 35 0 35 0 31 0 24 0 02 0 45 0 13 0 003 0 004 < 0 001

*Adjusted for age and blood group

difference 43 lU/dl, 95% CI 29-57 lU/dl) Factor VIII levels were above 150 lU/dl m 58 mdividuals (32%) Of these mdividuals, 50 subjects had blood group non-0 (86%) and eight (14%) had blood group 0

We tested the correlaüons of factor VIII Ag levels between all first-degree relative pairs After adjustment for blood group and age, the factor VIII levels in sibhngs showed a strong association (Table I) In sister pairs, the correlation of factor VIII levels was 0 35 (P = 0 003) and m biother pairs it was 0 3 5 (P = 0 004), whereas m brother-sistei. pairs this correlation was 0 3 1 (P < 0 001) In mother-son pairs, factor VIII Ag levels were also highly correlaled (r = 0 45, P = 0 02) The other first-degree lelationships showed no clear correlaüons (Table I)

We assessed the familial clustering of factor VIII Ag levels with the familial aggregation test Table II shows that when factor VIII Ag levels are tested äs contmuous variables the familial aggregation statistic 0 was clearly higher than the expected Q under the null hypothesis of no correlation, also afler adjustment for age and blood group Unadjusted, Q was 308, whereas the expected value of no correlation was 175 (P < 0 001) Adjusted Q was 301 with an expected Q of 172 (P < 0 001) Table III shows the familial clustering of factor VIII levels above 150 lU/dl äs a dichotomized variable The familial aggregation test was strongly positive agam In this test, Q was l 65 wilh an expected value of no correlation of 0 9 7 (P < 0 001) Adjustment for blood group and age did not essentially change 0 (Table III)

DISCUSSION

Our study showed that within thrombophiha famüies factor

Table H. Familial aggregation of iactor VIII Ag levels Factor VIII Ag Unadjusted Adjusted* Q Exp (Q) P value 308 175 < 0 001 301 172 < 0 001

Familial Clustering of High Factor VIII Levels 521

Table III. Familial aggregation of Iactor VIII Ag levels above 150 lü/dl* Q Exp (Q) P value Factor VIII Ag Unadjusled 1 65 0 97 < 0 001 Adjusted* 1 56 0 97 < 0 001

VIII Ag levels are highly aggregated The families we studied had thrombophiha, in which high factor VIII levels contnbute to the nsk of factor V Leiden carners (Lensen et al, 1999) Familial clustering of factor VIII Ag levels higher than 150 lU/dl was clearly demonstrated by the familial aggregation test, and this remamed after adjust-ment for the effects of blood group and age This suggests a genetic mfluence on high factor VIII levels other than just blood gioup

Familial aggregation was prominent, mdicatmg that factoi VIII levels were strongly influenced by familial factors The support for familial aggregation of factor VIII levels was much stronger for the families studied here than that obtamed with the female relatives of haemophilia A patients tested in oui previous study (Kamphuisen et al, 1998) This might have been the result of the larger size of the famihes which positively influenced the statistic Q of familial aggregation The inclusion of men and women tested in the present study could also affect the outcome because the familial aggregation test uses all possible combmations withm pedigrees Further, factor VIII levels were measured over a much shorter penod than m our previous study (< 4 weeks), which will reduce mterassay vanations m the factor VIII level

These are famihes with thrombophiha who will probably have several nsk factors for thrombosis As high factor VIII levels are associated with venous thrombosis, we may have selected foi high factor VIII levels m choosmg these famihes This is reflected by the higher number of factor VIII levels above 150 lU/dl in the thrombophiha famihes (32%) than m the thrombosis patients m the LETS study (25%) (Koster et al, 1995) The calculated 0 of familial aggregation for high factor VIII levels is mamly the result of concordant pairs with high factoi VIII levels The probabihty that siblmgs have both elevated factor VIII levels is more hkely in famihes that have a high prevalence of elevated factor VIII levels than in famihes m which low factor VIII levels are more common This means that a selection for high factor VIII levels m famihes will underestimate the hentabihty of high factor VIII levels

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522 P. W. Kamphuisen et al

mother-daughter, father-daughter or father-son pairs. Especially the finding that factor VIII levels were not correlated m father-daughter pairs, who share the same X-chromosome, does not support an X-Iinked determinant of factor VIII levels We have to consider the possibility that the correlation of factor VIII between fathers and daughters is lowered by the maternal X-chromosome of the daughter. A daughter who has a father with high factor VIII and a mother with low factor VIII may have an mtermediate factor VIII level This will negatively influence the correla-tion between father and daughter. Very recently, Mansvelt

et al (1998) investigated the promoter and 3' termmus of

the factor VIII gene for vanations associated with high factor VIII:C levels, but found none. It remains possible that a part of the Variation of factor VIII is determmed by genetic factors located on the X-chromosome, outside the factor VIII gene We can also not rule out the possibility that environmental factors and clustering withm famihes also contnbute to the familial aggregation of high factor VIII.

The mean difference m factor VIII Ag level between blood group 0 and non-0 was 43 lU/dl Among subjects with factor VIII levels above ISO lU/dl, 86% had blood group non-0, indicatmg that a substantial part of the elevation in factor VIII is attnbutable to blood group. Most of the effect of blood group on the factor VIII level is mediated through VWF (Koster et al, 1995; Kamphuisen et al, 1998), but the exact mechamsm öl how ABO blood group mfluences VWF is unclear Blood group A, B and H(0) oligosacchande structures have been identified on VWF (Sodetz et al, 1979; Matsui et al, 1992). As modification of carbohydrates has been shown to influence the half-life of VWF m the circu-laüon in animal models (Stoddart et al, 1996; Sodetz et al, 1977), it is possible that ABO blood group determmants affect the clearance of VWF in plasma VWF is an important determinant of the factor VIII level in plasma, which is explamed by VWF bemg the carrier protem for factor VIII (Tuddenham et al, 1982, Bnnkhous et al, 1985) Whether the different types of ABO blood group also influence the factor VIII survival in plasma remains to be determmed.

We conclude that there is strong support that factor VIII levels exceeding 150 lU/dl aggregate in famihes, also after adjuslment for blood group and age. This is further evidence that the high plasma factor VIII levels that previously were found to be associated with a thrombotic risk in case-control and family studies are determmed by genetic factors other than just blood group.

ACKNOWLEDGMENTS

We would like to thank H. de Ronde for determmation of the factor VIII antigen levels. This study was supported by a grant (no. 950-10-629) from The Netherlands Orgamzaüon for Scientific Research (NWO) and by a grant (no. 95 026) from The Netherlands Heart Foundation (NHS)

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