Elsy Sjörin

Platelet Aggregation Induced by I-Desamino-8-D-Arginine Vasopressin (dDAVP) in Type IIb von Willebrand's Disease

Type IIB von Willebrands disease is a distinct form of this disorder, in which there are abnormal factor VIII/von Willebrand factor multimers in plasma (but normal multimers in platelets) and heightened interaction between the von Willebrand factor and platelets in the presence of ristocetin. We have found that infusion of desmopressin acetate (1-desamino-8-D-arginine vasopressin [DDAVP]), an agent used in the treatment of von Willebrands disease, causes platelet aggregation and thrombocytopenia in patients with Type IIB disease. In vitro, platelets in normal plasma and those obtained from patients with Type IIB disease before DDAVP infusion aggregated upon the addition of platelet-poor plasma from Type IIB patients treated with DDAVP. Platelet aggregation was associated with adsorption of multimers of factor VIII/von Willebrand factor onto the platelets and was inhibited by EDTA. We conclude that in Type IIB von Willebrands disease, DDAVP releases an abnormal factor with platelet-aggregating properties. DDAVP should not be used to treat patients with Type IIB disease, since the presence of platelet aggregates in the circulation may be harmful.

Haemophilia B mutations in Sweden: a population‐based study of mutational heterogeneity

The present series comprises all families (n = 77) with haemophilia B in Sweden and may be considered to be representative for the purposes of a population‐based study of mutational heterogeneity. The 77 families (38 severe, 10 moderate, 29 mild) had 51 different mutations in total. Thirteen families had total, partial or small deletions, two had mutations in the promoter, eight families had splice site mutations, 14 had nonsense and the remaining 41 had missense mutations. Ten of the mutations, all C→T or G→A, recurred in 1–6 other families. Using haplotype analysis of seven polymorphisms in the factor IX (FIX) gene, we found that the 77 families carried 65 unique, independent mutations. Of the 48 families with severe or moderate haemophilia, 23 (48%) had a sporadic case of haemophilia compared with 31 families out of 78 (40%) in the whole series. Five of those 23 sporadic cases carried de novo mutations, 11 out of 23 of the mothers were proven carriers and, in the remaining seven families, it was not possible to determine carriership. Eleven of the 48 patients (23%) with severe haemophilia B developed inhibitors and all of them had deletions or nonsense mutations. Thus, 11 out of 37 (30%) patients with severe haemophilia B as a result of deletion/nonsense mutations developed inhibitors compared with 0 out of 11 patients with missense mutations. The ratio of male to female mutation rates was 5·3 and the overall mutation rate was 5·4 × 10−6 per gamete per generation.

Origin of mutation in sporadic cases of haemophilia A

The aim of this study was to define the origin of mutation in sporadic cases of severe haemophilia A. The series was composed of 31 families with sporadic severe haemophilia A in the geographical catchment area of the Malmö haemophilia centre. The mutation was characterized in 29/31 families: inversion type 1 (n = 11), inversion type 2 (n = 3), other inversion (n = 1), small or partial deletion (n = 6), insertion (n = 2), non‐sense mutation (n = 4) and mis‐sense mutation (n = 2). Of 29 probands, eight carried a de novo mutation, whereas the probands mother was found to carry the mutation in 21/29 families. Of the 21 carrier mothers, 16 had de novo mutations (i.e. the probands maternal grandfather and grandmother were non‐carriers). Owing to the lack of samples from the grandparents, origin could not be determined in the remaining five families. Polymorphisms of the FVIII gene were used to determine whether the de novo mutation of the carrier mother was of paternal or maternal origin. In 15/16 cases the mutation was of paternal origin and in 1/16 cases of maternal origin. In the series as a whole, mutation frequency was 6‐fold higher in males than in females, but no differences in the ratio of sex‐specific mutations rates was found among different types of mutation.

Why does the mutation G17736A/Val107Val (silent) in the F9 gene cause mild haemophilia B in five Swedish families?

Summary.  The mutation G17736A/Val107Val (silent) was found in five of a total of 86 families with haemophilia B in Sweden. It is unlikely that five families with analogous clinical expression will have the same polymorphism, which is not found in other patients or normal subjects, or that they will be the only families in the population without any other causative mutation. All affected individuals in the five families were found to have factor IX (F9) coagulation activity 15–20 U dL−1, corresponding F9 protein levels and the same clinical history of mild haemophilia. Lymphocyte mRNA was extracted from one of the haemophiliacs and from a healthy male. RT‐PCR of the mRNA and subsequent PCR amplification produced cDNA fragments of the same length from the patient and the normal subject, indicating no exon skipping or retention of introns. Sequencing of cDNA from codon 68 in exon D to codon 180 in exon F revealed that the patient had the G17736A mutation but no other abnormalities. We conclude that G17736A/Val107Val causes mild haemophilia B. Although, exon skipping and retention of introns can be excluded as pathophysiological mechanisms, it is plausible that the studied mutation has more subtle effects on a splicing site or interferes with a splicing enhancer site. Also, changes to synonymous codons may reduce the translation rate and thereby alter F9 protein folding in vivo, which would explain the phenotype. Confirmation of these assumptions requires methods that are more sensitive than those available today, and our discussion illustrates the existing obstacles.

Moderate haemophilia B in a female carrier caused by preferential inactivation of the paternal X chromosome

Abstract:  The case of a female with moderate haemophilia B is reported. She is the only affected member of her family, and factor IX RFLP analysis shows her to have inherited no maternal markers for polymorphisms located in the first intron and 8 Kb 3′ of the polyadenylation signal (Ddel and Hhal, respectively). This clearly indicates a deletion involving at least the last 7 exons of the factor IX gene. Her other factor IX gene inherited from her healthy father is normal as her son is also healthy. This suggests the patients haemophilia to be due to gross bias in the proportion of factor IX‐producing cells with an inactive paternal X chromosome. Methylation studies on the 5′ region of the PGK gene show that virtually all the patients lymphocytes carry a hypermethylated and presumably an inactive paternal X chromosome. The reason for this bias in the activity of her two X chromosomes is not clear, as no chromosomal alterations were found.

Inversions of the factor VIII gene in Swedish patients with severe haemophilia A

Abstract: The series comprised 49 Swedish patients with severe haemophilia A [belonging to 49 families (21 with known and 28 with sporadic haemophilia)], of whom 12 had developed F. VIII inhibitors. Using Southern blotting, 45% (22/49) were found to have inversions, i.e., intrachromosomal rearrangements of the tip of the X‐chromosome. Twenty patients had one or the other of the two variants of inversions recently published, whereas 2 patients manifested novel band patterns. Inversions were found in 50% of the families with sporadic haemophilia, and in 38% of those with known haemophilia. Fourteen families with sporadic haemophilia A had inversions, the proband carrying the de novo mutation in 4 cases and the probands mother in 10 cases. Six inversions derived from a male and five from a female X‐chromosome meiosis, the origin of the remaining three was not established. Genetic counselling of patients with severe haemophilia A and their families will be considerably improved, as inversions occur in half the severe cases and can be detected by a simple Southern blotting procedure.

Female haemophilia A caused by skewed X inactivation.

According to Lyon s hypothesis, one X chromosome in a female is silent because of random inactivation early in embryonic life. This usually affects the maternally and the paternally derived X chromosome equally often. However, one X chromosome in a female can be more extensively inactivated than the other, and that is called skewed X inactivation. Haemophilia is a recessive X-linked disorder, and thus it almost exclusively affects males, and females are asymptomatic carriers. In about 40% of haemophilia A patients, the disorder is caused by inversions in the F8 gene, and the remaining cases arise as a result of a variety of mutations in F8, including small and large deletions, insertions, and non-sense and missense mutations. Here, we describe two young females with haemophilia A. One of these girls also has incontinentia pigmenti (IP), a dominant X-chromosome-linked disease associated with skewed X inactivation. The clinical presentation of IP varies greatly and includes characteristics such as miscarriage of male foetuses and abnormalities of the skin, hair, teeth, nails, eyes and nervous system in the affected females. The girl with IP inherited, this condition from her mother, and she is also a genetically obligate carrier presenting with the mild haemophilia A that runs in her father s family. The other girl has no family history of severe haemophilia A but exhibits an inversion in the F8 gene and F8 coagulant activity (F8C) <1%, which is extremely unusual. Case 1 is an 8-year-old girl whose mother is afflicted with IP and whose father has mild haemophilia A. The mother s symptoms include blindness in one eye and abnormalities of a few teeth. Our patient has a more severe form of the disease, with the addition of spastic diplegia. She suffered bleeding after a minor surgical procedure and at that time was found to have an activated partial thromboplastin time (APTT) of 62 s (normal 25– 34 s) and F8C about 6% of normal. Repeated testing showed consistent F8 levels, which agrees with the criteria of mild haemophilia A. Genetically, she is an obligate carrier of haemophilia A, since her father has a mild form of the disorder (Fig. 2). He has the point mutation A6372G, which changes Tyr2105 to Cys, and his F8C level is about 10– 12%. Several members of his family have developed antibodies when given F8 concentrate [1]. At present, our patient s only medications are tranexamic acid and desmopressin as needed and F8 concentrate is administered solely in the event of a severe bleed or surgery. Case 2 is an 8-month-old girl was referred to the coagulation department with a large haematoma in the abdominal wall after bladder puncture performed because of suspected urinary tract infection. During the neonatal period, she had a subgaleal haematoma that required transfusion, but no coagulation testing was carried out at that time. Coagulation assays showed prolonged APTT (66 s), and the F8C level was <1%; all other tests were normal. She has no family history of haemophilia. She is now 3 years old and doing well on prophylactic therapy with F8 concentrate three times a week. F8C was measured using a one-stage recalcification assay calibrated with the international standards for F8C (normal range 60–140 IU dL). To investigate F8 gene inversions, DNA was extracted from EDTA anticoagulated blood by standard methods. PCR was performed using genomic DNA and Expand Long Template DNA polymerases (Boehringer Mannheim, Mannheim, Germany). F8 gene mutations were characterized by sequencing genomic DNA by a modified DiDeoxi method. Fluorescence in situ hybridization (FISH) was conducted by selecting three contiguous bacterial Correspondence: Karin E. Knobe, Department of Pediatrics, University Hospital, SE-205 02 Malmö, Sweden. Tel.: +46 40 331000; fax: +46 40 336226; e-mail: karin.knobe@med.lu.se

Investigation of disease-associated factors in haemophilia A patients without detectable mutations.

Summary.  To investigate disease causing mechanism in haemophilia A patients without detectable mutation. Screening for F8 mutations in 307 haemophilia A patients using: re‐sequencing and inversion PCR, reverse transcription (RT‐PCR) of mRNA, MLPA analysis, haplotyping using SNP and microsatellite markers. No F8 mutations were detected in 9 of the 307 patients (2.9%) using re‐sequencing and inversion PCR. MLPA analysis detected duplication in exon 6 in one patient and RT‐PCR showed no products for different regions of mRNA in four other patients, indicating failed transcription. No obvious associations were observed between the phenotypes of the nine patients, their F8 haplotypes and the putative mutations detected. The mutation‐positive patients carrying the same haplotypes as the mutation‐negative patients show a multitude of different mutations, emphasizing the lack of associations at the haplotype level. VWF mutation screening and factor V measurements ruled out type 2N VWD and combined factor V and VIII deficiency respectively. To further investigate a possible role for FVIII interacting factors the haplotypes/diplotypes of F2, F9, F10 and VWF were compared. The nine patients had no specific haplotype/diplotype combination in common that can explain disease. Duplications and faulty transcription contribute to the mutational spectrum of haemophilia A patients where conventional mutation screening fail to identify mutations.

Functional analysis of the EGF-like domain mutations Pro55Ser and Pro55Leu, which cause mild hemophilia B.

Summary.  We studied the functional role of two mutations, Pro55Ser and Pro55Leu, located in the N‐terminal Epidermal Growth Factor‐like domain (EGF1) of coagulation factor (F) IX. Both mutations cause mild hemophilia B with habitual FIX coagulant activities of 10–12% and FIX antigen levels of 50%. We found that activation by FVIIa/TF and FXIa was normal for FIXPro55Ser, but resulted in proteolysis of FIXPro55Leu at Arg318‐Ser319 with a concomitant loss of amidolytic activity, suggesting intramolecular communication between EGF1 and the serine protease domain in FIX. This was further supported by experiments using an anti‐EGF1 monoclonal antibody. Activation of FX by FIXaPro55Ser was impaired in both the presence and the absence of phospholipid or FVIIIa, indicating that Pro55 is not directly involved in binding to FVIIIa. We also studied the effect of the two Pro55 mutations on Ca2+ affinity and found only small changes. Thus, the Pro55Ser mutation causes hemophilia primarily through to an impaired ability to activate FX whereas at least in vitro the Pro55Leu defect interferes with the activation of FIX.

More than half the sporadic cases of hemophilia A in Sweden are due to a recent mutation.

ABSTRACT. The aim of this study was to ascertain how many of the sporadic cases of severe Haemophilia A in Sweden are due to recent mutation, and establish its origin. DNA analysis was performed in 18 randomly selected families with a sporadic case of severe haemophilia A. Restriction fragment length polymorphism (RFLP) patterns were investigated, two intragenic (Bcl I, Xba I/Kpn I) and two extragenic (DX 13, St 14) RFLP being used. In 10/18 families a haemophilia‐linked gene was found to have derived from the healthy maternal grandfather; on the basis of clotting and immunological assay results, the odds were high (> 104:1) for maternal carriership in four of these 10 cases, and for maternal non‐carriership in two, four being indeterminate. In 4/18 families a haemophilia‐linked gene derived from the healthy maternal grandmother; according to clotting and immunological assay results, in two cases the odds were high for maternal non‐carriership. In the remaining 4/18 families no conclusions could be drawn from the RFLP pattern as to the origin of mutation. We conclude that at least 55 % of the sporadic cases of severe hemophilia A in Sweden are due to a recent mutation within the last two genrations.