Karin Wulff

Twenty two novel mutations of the factor VII gene in factor VII deficiency

Factor VII is a vitamin K‐dependent coagulation protease essential for the initiation phase of normal hemostasis. The human factor VII gene (FVII, also known as F7) spans 13 kb and is located on chromosome 13, 2.8 kb upstream of the factor X gene. In the Greifswald FVII deficiency study the molecular basis for inherited factor VII was investigated. All exons, exon‐intron boundaries and the promotor of the FVII gene were amplified by PCR and directly sequenced. 87 unrelated probands with reduced or low FVII activities were investigated. Thirty‐four different FVII gene lesions were analyzed in 101 FVII alleles of 77 unrelated probands. Twenty‐two of these FVII gene lesions are novel FVII variations. The 34 different lesions comprise 31 point mutations and three small deletions. A transition in the CpG doublet accounted for 12 of the 34 different mutants. Sixteen mutations were noted only once. The missense mutation A294V and the double mutation A294V; 11128delC in exon 8 were by far the most common mutations found in this study. The haplotype of the different mutant FVII alleles were analyzed using six polymorphisms of the FVII gene. The haplotypes were identified in 29 mutant FVII alleles. Five different haplotypes are linked to the mutant FVII alleles. Except for one, the same haplotype was detected in FVII genes with an identical FVII gene mutation. Different haplotypes were identified in two patients with the mutant allele A206T. It is likely that identical mutant FVII alleles with the same haplotype share the same origin. Hum Mutat 15:489–496, 2000.

Lithuanian haemophilia A and B registry comprising phenotypic and genotypic data.

Haemophilia represents the most common hereditary severe bleeding disorder in humans. About 100 families with this condition live in Lithuania, one of the Baltic states with a population of 3·7 million. Haemophilia care and genetic counselling are still rendered difficult owing to limited availability of clotting factor concentrate and molecular genetic diagnosis. In the present study, a haemophilia registry, comprising phenotypic and genotypic data of the majority of Lithuanian haemophilia A and B patients, was established. The phenotype includes the degree of severity, factor VIII:C, factor VIII:Ag, factor IX:C, von Willebrand factor and antigen (VWF:RiCoF, vWF:Ag) and inhibitor status. Genotyping of the factor VIII and IX genes was performed using mutation screening methods and direct sequencing. In 61 out of 63 patients with haemophilia A (96·8%) and all eight patients with haemophilia B (100%), the causative mutations could be detected. Nineteen of the factor VIII gene defects and two of the factor IX gene mutations are reported for the first time. Identified mutations allowed direct carrier diagnosis in 83 female relatives revealing 44 carriers, 38 non‐carriers and one somatic mosaicism. The information provided by this registry will be helpful for monitoring the treatment of Lithuanian haemophilia patients and also for reliable genetic counselling of the affected families in the future.

Large-Scale Screening for Factor V Leiden Mutation in a North-Eastern German Population

Preliminary epidemiological data showed a high but varying prevalence of factor V Leiden mutation in various European populations. To analyze population differences statistically and generate reliable evaluation criteria for morbidity estimates, large numbers of unselected probands from different populations have to be tested. A convenient, efficient, reliable and cost efficient method for large-scale screening of factor V Leiden mutation has been developed using capillary blood samples soaked onto filter paper cards for the detection of mutations by heteroduplex analysis. Screening 1,628 alleles of a north-eastern German population by this procedure revealed an allele frequency of 3.56% (carrier rate 7.12%) which is significantly higher than those published for Italy and the Netherlands. Differences in allele frequencies compared to other European populations could statistically not be proved based on the small size of the published samples.

Severe clinical expression in X-linked Emery–Dreifuss muscular dystrophy

X-linked Emery-Dreifuss muscular dystrophy (EDMD) is a relatively rare benign neuromuscular disorder which can vary remarkably in onset, course and severity. In the present study, a TCTAC deletion spanning the nucleotides 631-635 of the emerin gene caused an unusually severe disease phenotype including loss of ambulation and severe muscle wasting in two affected brothers. The same mutation has been reported previously in an unrelated family showing a significantly milder phenotype. The interfamilial heterogeneity in distribution and in severity of the features in the two families point to environmental or genetic modification as the cause of clinical variability in Emery-Dreifuss muscular dystrophy.

Influence of Factor V HR2 on Thrombin Generation and Clinical Manifestation in Rare Bleeding Disorders

In this study we investigated the influence of the presence of the factor V HR2 haplotype, defined by the factor V gene mutation H1299R (FVHR2), on thrombin generation. Measurements were performed in platelet-poor plasma of individuals with factor VHR2 or factor VLeiden in comparison to a control group carrying none of these mutations. Coagulation was triggered by low concentrations of recombinant tissue factor in the presence of activated protein C. Thrombin generation was monitored by a fluorogenic substrate. The endogenous thrombin potential was calculated from the obtained curves. As a result we observed an increased thrombin generation both for individuals heterozygous and homozygous for FVHR2. The level of endogenous thrombin potential is in the same range as in samples of patients heterozygous or homozygous for FVLeiden. The results indicate that FVHR2 plays a role as a risk factor for venous thrombosis in homozygous patients through an increased thrombin generation. The association between different clinical manifestations in individuals with FVII deficiency and endogenous thrombin potential and the presence of FVHR2 was studied.

Early onset of cardiomyopathy in two brothers with X-linked Emery–Dreifuss muscular dystrophy

Two brothers are reported suffering from X-linked Emery-Dreifuss muscular dystrophy caused by a 59bp deletion eliminating nucleotides 329-388 of the STA gene. Besides the typical findings for Emery-Dreifuss muscular dystrophy, both patients showed an unusual early onset of cardiac symptoms at age 6 and 9 years, respectively, coinciding with unusual high creatine kinase. A cardiological follow up showed worsening of the cardiac condition in the beginning of the second decade. The two boys described here belong to the very few Emery-Dreifuss muscular dystrophy patients with early onset of cardiac involvement and contribute to the variability of cardiac symptoms in Emery-Dreifuss muscular dystrophy.

Variability of clinical manifestations of factor VII-deficiency in subjects homozygous or heterozygous for the F7 gene mutation A294V

Inherited factor VII deficiency (FVIID) is a rare autosomal bleeding disorder. Subjects with reduced FVII activity and identified F7 gene mutation are registered in the International Greifswald registry of FVIID.[1][1] The mutation A294V is the most frequent among 717 FVIID patients in Central and

Faktor-XII-Mangel, APC-Resistenz, Hyperhomocysteinämie und von-Willebrand-Erkrankung Typ 1 in einer Familie

ZusammenfassungEine junge Frau mit verstärkten Regelblutungen und länger anhaltenden Blutungen aus nur geringgradigen Verletzungen wurde untersucht, wobei eine sehr stark verlängerte PTT ( >200 s) auffiel. Die Messung der Faktor-XII-Aktivität ergab mit weniger als 2% einen hochgradigen Mangel. Die angegebenen Blutungssymptome sind jedoch auf eine gleichzeitig bestehende von-Willebrand-Erkrankung Typ 1 zurückzuführen. Zusätzlich waren die APC-Resistenz pathologisch und die Homocysteinkonzentration im Plasma der Patientin erhöht. Auf genomischer Ebene konnten die Mutationen G1691:A im Faktor-V-Gen und C677:T im Methylentetrahydrofolatreduktase(MTHFR)-Gen heterozygot nachgewiesen werden. Die Analyse des Faktor-XII-Gens ergab eine Punktmutation in der Promotorregion (G:C-Transversion, nt–8), die auch im Faktor-XII-Gen des Vaters nachzuweisen ist. Die niedrige Faktor-XII-Aktivität bei der jungen Frau deutet auf die Anwesenheit einer 2. Mutation hin (Compound-Heterozygotie), die sie von ihrer Mutter geerbt haben muß. Diese 2. Mutation ist bis jetzt nicht gefunden worden. Die 15jährige Schwester der Patientin berichtet ebenfalls über verstärkte Regelblutungen. Die Gerinnungsanalyse ergab eine verminderte Faktor-XII-Aktivität und eine von-Willebrand-Erkrankung Typ 1. Die verminderte Faktor-XII-Aktivität ist mit dem heterozygoten Status für diesen Defekt vereinbar. Die erhöht gefundene Resistenz gegenüber APC konnte durch den Nachweis der Mutation G 1691 A im Faktor-V-Gen in heterozygoter Form bestätigt werden. Mutter und Vater der Schwestern sind für den Faktor-XII-Mangel und die Mutation im MTHFR-Gen heterozygot. Bei der Mutter besteht Homozygotie für die Mutation im Faktor-V-Gen (1691 AA). Alle untersuchten Familienmitglieder zeigten erhöhte D-Dimer-Konzentrationen im Plasma. Diskussion: Mit diesem Fallbericht soll auf die Bedeutung der Menorrhagie als häufiges Symptom einer von-Willebrand-Erkrankung hingewiesen werden. Da zusätzlich Risikofaktoren für eine Thrombophilie bestehen, wird auf die Probleme, die den Patientinnen aus der Anwendung oraler Kontrazeptiva entstehen können, ebenso hingewiesen wie auf mögliche Komplikationen im Verlauf einer Schwangerschaft.SummaryA young woman presenting with marked menstrual blood loss and prolonged bleeding after only minimal injuries was investigated. Because of an extremely prolonged PTT (more than 200 s) we measured the factor XII activity and found a severe deficiency. The bleeding symptoms however could be ascribed to the existence of von Willebrands disease type 1. In addition, the patient had an elevated resistance against activated protein C (APC-resistance) and a moderately elevated concentration of homocysteine in her blood plasma. At the genomic level we found heterozygosity for the factor V mutation G 1691 A and the mutation C 677 T in the methylenetetrahydrofolate reductase (MTHFR)-gene. The analysis of the factor XII gene revealed the existence of a point mutation in the promoter region (G→C transversion, nt–8). The same mutation could be found in the paternal factor XII gene. The low factor XII activity in the woman seems to be the result of a second mutation originating from the maternal factor XII gene (compound heterozygosity). However, this second mutation could not be found up to now. In the sister of the women, a 15 year old girl, there is also a marked menstrual blood loss. The coagulation analysis revealed reduced activities of the factor XII and also von Willebrands disease type 1. The reduced factor XII activity seems to be compatible with the heterozygous state of factor XII deficiency. In the girl there was also an increased APC resistance, corresponding with heterozygosity for the factor V mutation G 1691 A. Mother and father are heterozygous for the factor XII deficiency and the mutation C 677 T in the MTHFR gene. In the mother homozygosity for the mutation in the factor V gene (1691 AA) could be found. All family members investigated exhibited elevated D-dimer concentrations in the plasma. Discussion: It is the aim of this report to give attention to menorrhagias as predominant features of von Willebrand’s disease in females. Furthermore in women with von Willebrand’s disease in combination with inherited risk factors for thrombophilia problems could arise from the use of oral contraceptives and also during pregnancy. In our opinion, physicians, especially paediatricians, who take care of adolescents, should be familiar with such problems.