Bleeding Tendency in Factor XI Deficiency: Report on two Families and the Detection of a Novel Mutation within the Factor XI Gene

Report on two Families and the Detection of a Novel Mutation within the Factor XI Gene

B. Maak, L. Kochhan, J. Schröder, J. Oldenburg, P. Heuchel, B. Harwardt and U. Budde

The inherited Factor XI deficiency seems to be, with only few exceptions, a rare dis- order worldwide. In patients with factor XI deficiency spontaneous bleedings are uncommon, bleeding events occur mostly after surgical procedures or injuries in persons with a markedly decreased residual factor XI-activity (homozygous or compound-heterozygous factor XI deficiency). However, there are patients with only mildly reduced factor XI activity (factor XI activity between 15 and approxi- mately 60%) and significant bleeding problems. In addition, families with a quite varying bleeding behavior in the individual members exist. The reason for this dif- ference is not fully understood as yet. Therefore, it may be speculated that in such patients an additional defect of the hemostatic system is present. In some studies the coexistence of factor XI deficiency and hemophilia A, von Willebrand’s disease and also platelet function disorders has been described [1, 5, 7], furthermore, in two reports it could be demonstrated that patients with factor XI deficiency and bleed- ing symptoms tended to have lower values of von Willebrand factor in comparison with patients without bleeding symptoms [1, 3].

We describe here the results of our studies of two patients and their family members with mild factor XI deficiency. In both cases a second defect of the hemostatic system could be detected and in our opinion this defect was responsible for the different bleeding tendency of the patients in comparison with the other family members. The factor XI deficiency of the investigated individuals was con- firmed by evidence of mutations within the factor XI gene. In one of the families we identified a novel mutation (Ala 464 (GCC) > Val (GTC), exon 12).

Material and Methods

Patients and Family Members Case 1:

In the patient, a 4 month old boy, a premature synostosis of the skull was surgical- ly treated. A massive bleeding occurred during the procedure and persisted for the next hours. After administration of fresh frozen plasma the bleeding ceased imme- diately. As the cause of the bleeding factor XI deficiency was presumed. Five months later the parents and the boy contacted our hospital. The boy was in good health and without any bleeding signs. The mother of the boy reported easy bruising and

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marked menstrual blood loss before oral contraceptive treatment. The maternal grand-grandmother also reported easy bruising, her menstrual blood loss was not remarkable and no bleeding problems occurred after two deliveries. Dental extrac- tions were also uneventful. After a fracture of the right radius an unusually large hematoma was observed.

The father of the patient reported no bleedings after tonsillectomy and appen- dectomy.

Case 2:

The patient, a 17 year old girl reported a life-long tendency to easy bruising and delayed bleedings after incidental injuries. The menstrual blood loss was strong and decreased after introduction of oral contraceptive treatment.

The father of the girl is also factor XI deficient. He reported frequent epistaxis only during childhood and is currently symptom-free. After multiple dental extrac- tions and two herniotomies no bleeding events occurred.

Coagulation Tests

Blood was taken from an antecubital vein using a commercially available device (BD vacutainer). Nine parts of blood were carefully mixed with one part of 0.129 m trinatrium citrate solution. Thromboplastin time (PT, Quick value) and activated partial thromboplastin time (aPTT) were measured with an automated device. The fibrinogen content was determined as so-called derived fibrinogen during the PT assay. Coagulation factor assays were performed as clotting tests with specifically deficient plasmas according to the instructions given by the manufacturer.

Antithrombin was estimated by a colorimetric assay. The APC-resistance was deter- mined with an aPTT-based reagent kit. All reagents for the coagulation assays were purchased from IL. The concentration of von Willebrand factor (VWF:Ag) was assayed by an ELISA kit (Bio-Merieux). Protein Z and PAI-1 were measured also by ELISA techniques.

Genomic Analysis

Total DNA of all patients was extracted from whole blood using micro spin columns

(Quiagen, Hilden, Germany). Exons of the factor XI gene were amplified by poly-

merase chain reaction (PCR). Cycle sequencing was performed using the Big Dye

Terminator Cycle Sequenzing kit (Perkin Elmer Applied Biosystems, Foster City,

USA) and analyzed using an automated DNA sequencer (ABI PRISM/377 DNA

Sequencer, Applied Biosystems, Weiterstadt, Germany). Primers used for sequen-

cing were the same as those used for PCR amplification. Mutation analysis of the

factor VIII gene was performed as previously described [8].

Results

In our first case the in an other hospital presumed factor XI deficiency was con- firmed (Table 1). We found on different occasions factor XI-activities between 32 and 37%. In his mother the factor XI-activities were in the same magnitude and also in his maternal grand-grandmother a decreased factor XI-activity has been found. The mutation Thr 475 (ACA) > Ile (ATA) in the exon 12 of the factor XI gene is responsible for the reduced factor XI-activities. As an additional defect we found in the patient reduced factor VIII-activities and consequently the mild form of hemophilia A. As the cause of the slight factor VIII deficiency the mutation Gly 22 (GGT) > Cys (TGT) within the exon 1 of the factor VIII gene was identified. The mutation was also present in the patients mother, her factor VIII values could be found within the normal range. In the grand-grandmother the factor VIII activity was also normal.

Table 1

Patient Mother Father Maternal grand- Normal grandmother range

Quick [%] 94 101 117 121 70–120

PTT [sec] 46 31 31 36 25–35

Fibrinogen [g/l] 2.15 2.35 1.66 3.34 1.8–4.50

VWF:Ag [%] 84/84 80/87/96 >120 >120 50–150

VWF:RiCoF [%] 84/110 56/84/56 110 112 60–150

VIII:C [%] 38/40/32 74/81/68 110 97 70–150

IX :C [%] 67 92 110 79 50–150

XI :C [%] 37/32/34 53/34/42 78 36 70–150

XII:C [%] 80 126 101 82 70–150

AT [%] n.d. 93 n.b. 102 80–120

APC-Ratio n.d. 2.35 2.50 2.50 2.0–3.5

Protein C [%] n.d. 114 n.d. 125 70–120

Protein S [%] n.d. 75 n.d. 118 70–130

Protein Z [mg/l] 1.26 1.48 n.d. 2.46 0.8–2.0

PAI-1 [µg/l] 36 7 20 20 4–5

Mutation Thr 475 Thr 475 n.d. Thr 475

XI-Gene (ACA), (ACA), (ACA),

> Ile (ATA) > Ile (ATA) > Ile (ATA)

Exon 12, Exon 12, Exon 12,

heterozygous heterozygous heterozygous

Mutation Gly 22 Gly 22 n.d. n.d.

VIII-Gene (GGT)> (GGT)>

Cys (TGT) Cys (TGT) Exon 1 Exon 1,

heterozygous

n.d. = not determined

In the second family investigated the factor XI-activities were found between 26–35%, father and daughter are hemizygous for the mutation Ala 464 (GCC) > Val (GTC) which is localized within the exon 12 of the factor XI gene.

In both persons the levels of the von Willebrand factor have been found near the lower limit of the normal range. In contrast to the young girl her father offers a distinctly-decreased protein C activity.

Discussion and Conclusions

In the two families presented here the reduced factor XI activity is of hereditary origin. The analysis of the factor XI genes of the first patient and his family mem- bers revealed a base exchange (C > T) within the exon 12. This mutation was de- scribed for the first time in 1995 and is characterized by a reduced stability of the Table 2

Patient Mother Father Normal range

Quick [%] 117 117 107 70–120

PTT [sec] 40/39 32 38 25-35

Fibrinogen [g/l] 3.19 2.88 1.95 1.8–4.50

VWF:Ag [%] 65 134 77 50–150

VWF:RCoF [%] 56 225(CBA)* 56 60–150

VWF-Multi- normal normal n.d.

meric analysis

VIII:C [%] 72 102 81 70–150

IX:C [%] 95 n.d. 72 70–150

XI:C [%] 34/26 85 35 70–150

XII:C [%] 66/72 107 50 70–150

AT [%] 98 n.d. 75 80–120

APC-Ratio 2.35 n.d. 3.0 2.0–3.5

Protein C [%] 99 n.d. 49 70 -120

Protein S [%] 87 n.d. 75 70–130

Protein Z [mg/l] 1.84 n.d. 0.91 0.8–2.0

PAI–1 [µg/l] 6 n.d. 8 4–45

Mutation Ala 464 (GCC) n.d. Ala 464 (GCC)

XI-Gene > Val (GTC) >Val (GTC)

Exon 12, Exon 12

heterozygous heterozygous

*CBA = Collagen binding activity

n.d. = not determined

factor XI-mRNA and therefore a reduced expression of factor XI [4]. The molecular mechanism of the novel mutation (case 2) in the factor XI gene (Ala 464 (GCC)

> Val (GTC)) remains to be established.

In the first family there is a clear-cut difference between the aPTT-value of the patient to the corresponding values of his mother and his grand-grandmother. In our opinion, this difference cannot be explained by the age of the patient alone.

Therefore a second defect was supposed, the results of further coagulation testing revealed a reduced factor VIII activity. The cause of this low factor VIII level is the mutation Gly 22 (GGT) > Cys (TGT) within the exon 1 of the factor VIII gene [2].

The same mutation was found in the patients mother in a heterozygous state. We believe that the massive peri- and postoperative bleeding in the patient could be attributed to the mild hemophilia rather than to the factor XI deficiency. Mother and grand-grandmother of the patient offer a bleeding tendency which may be the result of the mild factor XI deficiency.

In our second patient and also in the father there is a combination of mild factor XI deficiency and low normal values of von Willebrand factor. This may be the reason for the bleeding tendency in the girl. The father shows additionally a protein C deficiency which counteracts to the effects of factor XI deficiency and low von Willebrand factor. The cessation of the bleeding tendency after the childhood may support this assumption.

Finally we emphasize that the aPTT assay is not always suitable for the detection of mild factor XI deficiency [6]. In a great number of patients normal or only slight- ly prolonged aPTT values could be found. Therefore we recommend a specific fac- tor XI assay in patients with bleeding symptoms which can not be explained by other diagnostic measures.

References

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2. HAMSTeRS: http: // europium.csc.mrc.ac.uk

3. Maak B, Bergmann F, Kochhan L, Estel Ch (2003) Factor XI deficiency caused by a hitherto unknown mutation in the factor XI gene. Scharrer I, Schramm W (Ed.) 32

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4. Mc Vey JH, Imanaka Y, Nishimura T, O’Brien DP, Bolton-Maggs PHB, Tuddenham EGD (1995) Identification of a novel mechanism of human genetic disease : a missense mutation causing factor XI deficiency through a change in mRNA stability. Thromb Haemostas 73:

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7. Tavori S, Brenner B, Tatarsky I (1990) The effect of combined factor XI deficiency with von Willebrand factor abnormalities on haemorrhagic diathesis. Thromb Haemostas 63: 36–38 8. Uen C, Klopp N, Oldenburg I, Brackmann HH, Schramm W, Schwaab R, Graw I (2003) 11 novel mutations in the factor VIII encoding gene lead to severe or moderate hemophilia A.

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