Genotype and phenotype relationships in 10 Pakistani unrelated patients with inherited factor VII deficiency.

Inherited factor VII (FVII) deficiency is one of the commonest rare bleeding disorders. It is characterized by a wide molecular and clinical heterogeneity and an autosomal recessive pattern of inheritance. Factor VII-deficient patients are still scarcely explored in Pakistan although rare bleeding disorders became quite common as a result of traditional consanguineous marriages. The aim of the study was to give a first insight of F7 gene mutations in Pakistani population. Ten unrelated FVII-deficient patients living in Pakistan were investigated (median FVII:C = 2%; range = 2-37%). A clinical questionnaire was filled out for each patient and direct sequencing was performed on the coding regions, intron/exon boundaries and 5' and 3' untranslated regions of the F7 gene. Nine different mutations (eight missense mutations and one located within the F7 promoter) were identified on the F7 gene. Five of them were novel (p.Cys82Tyr, p.Cys322Ser, p.Leu357Phe, p.Thr410Ala, c-57C>T, the last being predicted to alter the binding site of transcription factor HNF-4). Half of the patients had single mutations in Cys residues involved in disulfide bridges. The p.Cys82Arg mutation was the most frequent in our series. Six of seven patients with FVII:C levels below 10% were homozygous in connection with the high percentage of consanguinity in our series. In addition, we graded the 10 patients according to three previously published classifications for rare bleeding disorders. The use of the bleeding score proposed by Tosetto and co-workers in 2006 appears to well qualify the bleeding tendency in our series.

[Carriers of haemophilia: Experience of a French university hospital]. / Conductrices d'hémophilie: expérience d'un CHRU en France.

OBJECTIVES: To report the management of carriers of haemophilia in a French university hospital and assess different issues of these patients. PATIENTS AND METHODS: Retrospective study of the carriers of haemophilia who consulted at the university hospital of Montpellier, France, between 1995 and 2011. Information were obtained from medical records and from a questionnaire sent to carriers. We recorded data about biological characteristics, bleeding tendency and management of pregnancies. RESULTS: Sixty-four carriers of haemophilia A or B were included. Their median FVIII or FIX level was 52 % (range, 15-137 %). Menstrual bleeding lasted more than 7 days in 31 % of carriers. A total of 142 pregnancies started in 54 carriers, and 101 resulted in live births with 26 boys with haemophilia. Sixty-two prenatal diagnoses carried out, 15 have terminated their pregnancy because of a hemophiliac male fetus. Seventy-six percent of deliveries were vaginal delivery and 49 % took place in a level-3 maternity. There were 10.8 % and 8.5 % primary and secondary post-partum hemorrhage, respectively. CONCLUSION: The risk of bleeding among carriers of haemophilia is associated with their antihemophilic factor level. To improve the management of carriers, a multidisciplinary and standardized medical record, with a specific questionnaire to evaluate bleedings, could be considered. A regional register that lists all carriers, regardless of their antihemophilic factor level, would also be useful.

Analysis of the genotypes and phenotypes of 37 unrelated patients with inherited factor VII deficiency.

Severe inherited factor VII (FVII) deficiency is a rare autosomal recessive disorder with a poor relationship between FVII coagulant activity and bleeding tendency. Both clinical expression and mutational spectrum are highly variable. We have screened for mutations the FVII gene of 37 unrelated patients with a FVII coagulant activity less than 5% of normal pooled plasmas. The nine exons with boundaries and the 5' flanking region of the FVII gene were explored using a combination of denaturing gradient gel electrophoresis and direct DNA sequencing. This strategy allowed us to characterise 68 out of the 74 predicted FVII mutated alleles. They corresponded to a large panel of 40 different mutations. Among these, 18 were not already reported. Genotypes of the severely affected patients comprised, on both alleles, deleterious mutations which appeared to be related to a total absence of activated FVII. We suggest that this absence of functional FVII can explain the severe clinical expression. Whether a small release of FVII is sufficient to initiate the coagulation cascade and to prevent the expression of a severe phenotype, requires further investigations.

Impact of smoking, physical training and weight reduction on FVII, PAI-1 and hemostatic markers in sedentary men.

The variations of FVII, PAI-1, TAT complexes, fibrinopeptide A, D-Dimers and beta thromboglobulin plasma levels were studied on 30 sedentary men, smokers and non-smokers, who were admitted to a 6 months' program of physical training and smoking cessation. After 3 months of intervention, sustained physical training was associated with the decrease of FVII and PAI-1 levels. Mild exercise performed during a second 3-month period could maintain normal FVII and PAI-1 activities but participants who stopped the training increased their FVII and PAI-1 plasma levels. FVII was not influenced by smoking habits. Smoking cessation seemed to slightly potentiate the decrease of PAI-1 levels associated with mild exercise. Overweight, FVII and PAI-1 levels were correlated and the weight reduction induced by training was related to the changes in the factors. In smokers, physical exercise was associated with a significant increase of hemostatic markers. This exercise-induced variation disappeared after 3 months of intervention in participants who stopped smoking and reappeared in those who smoked again after 6 months of intervention. This finding was not influenced by the physical training program.

Impaired fibrinolytic capacity in patients with inflammatory bowel disease.

The venous occlusion test was applied to 17 patients with inflammatory bowel disease (IBD; 7 cases of Crohn's disease, 10 cases of ulcerative colitis). Results were compared to those obtained in 20 healthy matched control subjects. Patients with IBD had significantly decreased t-PA Ag release (p less than 0.001) and had no significant vWF Ag release. Residual PAI activity was evidenced after venous stasis in the IBD group but not in the control group. Hypofibrinolysis was more important in patients with an evolutive IBD than in patients with IBD in remission. Impaired systemic fibrinolytic capacity might contribute to an increased risk for thromboembolic complications and to the pathogenesis of inflammatory bowel disease.

Hemostasis parameters during ovarian stimulation for in vitro fertilization: results of a prospective study.

OBJECTIVE: To evaluate the effect of IVF-ET on the hemostatic system. DESIGN: Prospective clinical study. SETTING: Apparently healthy age-matched women of the hospital staff at various stage of the menstrual cycle. PATIENT(S): Twenty-five women involved in a IVF-ET program at the Department of Obstetrics and Gynecology, Montpellier University Hospital. INTERVENTION(S): Twenty-six hemostasis parameters evaluated repeatedly in patients undergoing IVF-ET. MAIN OUTCOME MEASURE(S): Blood cell-dependent hemostasis parameters and plasmatic coagulation factors, determined at pituitary desensitization, maximal E2 level, and P plateau. RESULT(S): Activation of the hemostatic system is evidenced at the P plateau, when D-dimers and fragments 1 + 2 of the prothrombin levels rose dramatically. At E2 peak, no significant modification of hemostasis markers was noted. CONCLUSION(S): The present results indicate that ovarian hyperstimulation may induce hemostasis activation at the P plateau. The role of supraphysiologic sex hormone levels on the hemostatic system requires further investigation.

Autoantibody to plasma fibrinopeptide A in a patient with a severe acquired haemorrhagic syndrome.

We describe a 50-year-old man with a severe acquired haemorrhagic syndrome. He had slightly prolonged clotting times using bovine thrombin, human thrombin and reptilase. His plasma contained a polyclonal IgG which interfered with the generation of fibrin monomers without inhibiting the aggregation of preformed monomers. The inhibitor delayed thrombin-induced fibrinopeptide A release. The IgG bound to insolubilized synthetic fibrinopeptide A (one binding site per molecule) and, with higher affinity, to fibrinogen (two binding sites per molecule). It did not bind to insolubilized fibrin monomers. The IgG did not impair the catalytic activity of thrombin toward a small synthetic substrate but inhibited the binding of thrombin to fibrinogen without binding to thrombin. The binding of the anti-fibrinopeptide A autoantibody to fibrinogen might have impaired thrombin-induced fibrinogen to fibrin conversion in vivo. This may have favoured the reported haemorrhagic syndrome which was associated with severe chronic renal insufficiency.

Direct carrier detection for severe haemophilia A: application to families with no available affected male.

Haemophiliae A is a common hereditary disorder of blood coagulation resulting from deficiency of factor VIII. Mutation analysis is the factor VIII gene has been hampered by the large size of the gene and the heterogeneity of molecular defects. In severe haemophiliae A, the most efficient methods of screening for point mutations can detect the lesions in 50 percent of cases only; this is explained by the recent finding (5) of an intragenic inversion that disrupts the factor VIII gene. Since this anomaly could not be characterized by these methods, Lakich et al. have also described a Southern blotting assay that allows a direct determination of the mutation. The use of this assay should greatly increase the feasibility and accuracy with which carrier detection and prenatal diagnosis can be made, as illustrated by the analysis of families with no available affected male that we present here.

DNA analysis of haemophilia A families from southern France. Experience of a hospital laboratory.

A sample of 101 individuals from 19 unrelated families from Southern France affected with haemophilia A was studied in our laboratory from 1990 to 1992. The aim of the analysis was to define the carrier status of women related to a haemophiliac, or to find an informative DNA marker for further prenatal diagnosis in obligate carriers. Three intragenic (BclT/intron 18, XbaI/intron 22, AlwNI/intron x7) and two extragenic polymorphisms (TaqI/St14, BglII/DX13) were used for this study. The tested population exhibited some original characteristics, including a lower rate of heterozygosity for the FVIII BclI polymorphism and a number of specificities for the St14 RFLP. We also compared the different methodologies available for each RFLP in a routine diagnostic service, and determined a strategy for linkage analysis in our population.

[Male pseudo-hermaphroditism. Prenatal factors]. / Pseudo-hermaphrodismes masculins. Facteurs anténatals.

The authors report on nine cases of male pseudo-hermaphroditism (MPH) diagnosed at birth. They had a karyotype 46 XY and their external genital organs were ambiguous. Analysing the conditions under which the pregnancies occurred we found that there was primary infertility before the pregnancy in 4 out of 9 cases. There was raised maternal blood pressure in 3 out of 9 cases and one of them led to eclampsia. Three babies were preterm and 7 showed intra-uterine growth retardation (below the 10th centile). In 3 cases there were other birth defects and difficulties in the neonatal stage in 6 (2 with respiratory distress, 1 with birth asphyxia, 1 with infection and 2 with jaundice). The diagnosis was based on cytogenetic analysis, x-ray of the urinary tract, x-ray of the genital organs, plasma testosterone levels and the binding capacity of dihydrotestosterone receptors on fibroblasts of the sexual skin areas (fentomoles/mg DNA). The partial androgen insensitivity syndrome was proven in two cases. These mothers had taken drugs that could potentially cause external genital ambiguity (demegestone and chlorpromazine). In 7 cases the diagnosis could not be arrived at. It is known to be very difficult to find an aetiology for MPH but the abnormalities of pregnancy and the frequent occurrence of intra-uterine growth retardation suggest that there are metabolic and endocrine upsets so that fetal malnutrition could play a role in poor external genitalia development. It should be possible to set up a large multicentric study in order to acquire more data and work out the factors that lead to MPH and their relationship to one another.

[Recurrent venous thromboembolism caused by heparin cofactor II deficiency. A case]. / Maladie thrombo-embolique veineuse récidivante liée à un déficit en second cofacteur de l'héparine. Une observation.

We report the case of a 28-year-old man who was admitted in an emergency because of severe abdominal pain with gastrointestinal haemorrhage and shock. Laparotomy showed infarction of the small intestine with mesenteric veins thrombosis. Severe thromboembolic complications occurred during the post-operative period: bilateral femoral deep vein thrombosis with pulmonary embolism, axillary and subclavian vein thrombosis associated with an intravenous catheter, portal hypertension related to portal vein thrombosis and cavernoma, thrombosis of the superior longitudinal sinus. Laboratory investigations performed after thrombotic episodes and repeated 5 years later evidenced a type 1 Heparin Cofactor II deficiency (HCII Ag by EID: 40 percent; functional Tollefsen's method: 60 percent). This heterozygous deficiency was also found in one of the patient's sons. This is the first reported case of HCII deficiency associated with mesenteric infarction and cerebral thrombophlebitis. The relationship between these severe venous thrombotic episodes and the HCII deficiency is discussed in relation to the dermatan sulphate-HCII couple physiology. Vascular injury may act as a triggering factor in patients with HCII deficiency.

[Endothelial mediators regulating vascular tonus]. / Les médiateurs endothéliaux régulant le tonus vasculaire.

Vascular endothelium is the unavoidable interface between blood and tissues. Its role as regulator of vascular tone is discussed here. The endothelium possesses different receptors for vasoactive mediators. These receptors activate the endothelial cells which in turn may produce secondary mediators acting on smooth muscle cells by activating or inhibiting their contraction. Such mediators are the endothelins which have a vasoconstrictor effect, and prostacyclin or endothelium-derived relaxation factors (EDRF) which have a vasodilator effect. Thus, the vascular endothelium and the mediators it secretes play a crucial role in vascular tone physiology.

Denaturing gradient gel electrophoresis screening for mutations in the hereditary hyperferritinaemia cataract syndrome.

Hereditary hyperferritinaemia cataract syndrome (HHCS) is characterized by hyperferritinaemia without iron overload. It is essential to differentiate true iron accumulation from HHCS as these patients rapidly develop iron-deficient anaemia when subjected to phlebotomies. The diagnosis of HHCS relies on the identification of point mutations or deletions present in the iron-responsive element of the first exon of the L-ferritin gene. However, many samples referred for diagnosis of putative HHCS are normal. To avoid unnecessary DNA sequencing, we have developed a diagnosis strategy based on the screening of the target DNA region by denaturing gradient gel electrophoresis. This method enabled the accurate identification of 11 different previously known mutations. This strategy will be of interest for family studies or for the screening of large series of patients.

Rapid detection of single nucleotide deletions: application to the beta 6 (-A) mutation of the beta-globin gene and to cystic fibrosis.

The formation of heteroduplexes from the amplified products of homologous alleles has been shown to be useful in the identification of heterozygotes carrying deletion or insertion mutations. Here, we describe an improved procedure that allows the detection of single base pair (bp) deletions on nondenaturing polyacrylamide gels. Carriers for a common Mediterranean beta-thalassemic mutation, beta6 (-A), could be easily detected by use of this method, as could carriers of a 1-bp deletion in the cystic fibrosis gene.

Factor IX gene mutations causing haemophilia B: comparison of SSC screening versus systematic DNA sequencing and diagnostic applications.

The search for mutations of the factor IX gene responsible for haemophilia B should nowadays be used routinely for the molecular diagnosis of this inherited disorder, i.e. carrier detection and prenatal diagnosis. A number of methodologies have been proposed, most of them being delicate or expensive. We have used a simple strategy based on a preliminary screening of eight factor IX gene fragments by single-strand conformation analysis (SSCA), followed by direct sequencing of fragments displaying an abnormal migration pattern. Carrier testing is then performed by use of an enzyme restriction site altered by the mutation or by the SSCA itself. By using this strategy we were able readily to identify the factor IX molecular defect of nine unrelated haemophilia B patients from southern France. We validated the efficiency and reliability of the SSC-based detection of mutations by sequencing all the polymerase chain reaction (PCR) fragments studied in the haemophilic patients. No other sequence alteration could be found except the one detected by SSC analysis. We conclude that this method can be advantageously used for diagnosis purposes in a routine laboratory involved in haemophilia B diagnosis and report nine previously undescribed haemophilia B families with their factor IX mutation.

[Prevalence of minor beta-thalassemia in the region of Gard]. / Prévalence des bêta-thalassémies mineures dans le département du Gard.

We analyzed the hemoglobin patterns of all the adult patients whose hemograms, performed in the hematology laboratory of our hospital, indicated microcytosis (MCV < 82 fl). During a 6 weeks period, 43 cases of microcytosis were identified out of 1827 hemograms and, among them, 16 cases of beta-thalassemia trait were diagnosed, i.e., 0.88%. This rate can probably be extrapolated to the general population of this region. It is in agreement with results obtained by other authors from southeastern France. This frequency of heterozygous beta-thalassemia is non-negligible and strongly suggests that screening for the beta-thalassemia trait in the at risk populations should be undertaken to prevent homozygous forms.

Comparison of the protein and DNA approaches for the characterization of a beta-globin chain variant, hemoglobin Cocody [beta 21 (B3) Asp--->Asn], in a Caucasian patient.

Over the past few years, the methodologies used for the identification of hemoglobin A variants have been greatly improved. Both the protein- and DNA-based strategies have their own advantages and limitations. In this report we illustrate the use of both assays for the characterization of a hemoglobin Cocody variant in a women of Spanish descent. After evaluating the mobility value matrix of the abnormal hemoglobin, the amino acid transition was determined by HPLC and micro-sequencing of the protein. The beta 21 Asp was shown to be substituted by an Asn. At the DNA level, the only nucleotide replacement responsible for this amino acid substitution is GAT--->AAT at codon 21. The analysis of the beta-globin gene by denaturing gradient gel electrophoresis (DGGE) method showed that the mutation was situated in a fragment including exon 1. The hemoglobin variant was then identified to be hemoglobin Cocody by DNA sequencing of this fragment.

Transferrin receptor-2 gene and non-C282Y homozygous patients with hemochromatosis.

More than 80% of the patients affected by hereditary hemochromatosis, a common inherited iron disorder, are homozygotes for the 845G --> A (C282Y) mutation of the HFE gene. However, depending on the population, 10-20% of hereditary hemochromatosis can be linked either to other HFE genotypes, particularly the compound heterozygous state for C282Y and the 187 C --> G (H63D) mutation, or to mutations of new other genes. Recently, Camaschella et al. (Nat. Genet. 25, 14-15, 2000) identified a stop mutation (exon 6 nt 750 C --> T, Y250X) on the transferrin receptor-2 (TFR2) gene in two unrelated Sicilian families with hereditary hemochromatosis. The TFR2 gene is a transferrin receptor gene homologue that seems to be involved in iron metabolism. Moreover, one of the patients described by Camaschella et al. was a H63D homozygote. H63D homozygosity can be associated with various phenotypes from asymptomatic subjects to patients with a typical form of hereditary hemochromatosis. Thus, the Y250X mutation could be the molecular defect responsible for hereditary hemochromatosis in subjects with atypical HFE genotypes. We have searched for the Y250X mutation in 63 unrelated French subjects. Forty-three had a diagnosis of hereditary hemochromatosis based on classical criteria. This group included 12 H63D homozygotes, 3 C282Y heterozygotes, and 3 patients with none of the two most prevalent HFE mutants. These 18 patients had no other HFE sequence change and were subsequently subjected to DNA sequencing of the 15 last exons and flanking sequences of the TFR2 gene. The 25 remaining hereditary hemochromatosis patients who were tested for the Y250X mutant were compound heterozygotes for the C282Y and H63D mutations. Finally, we also tested for this TFR2 mutation 20 H63D homozygotes with milder manifestations of iron overload and no acquired cause of iron overload. None of the 63 tested subjects had the Y250X mutation. Concurrently, none of the 18 hereditary hemochromatosis patients who had their TFR2 gene sequenced had any deleterious mutation. Thus, TFR2 mutations are not responsible for hemochromatosis in non-C282Y homozygous patients of our area.

HFE mutations and transferrin receptor polymorphism analysis in porphyria cutanea tarda: a prospective study of 36 cases from southern France.

BACKGROUND: Porphyria cutanea tarda (PCT) is associated in most cases with iron overload, which may participate in decreased activity of uroporphyrinogen decarboxylase in the liver. The aetiology of this iron overload remains unknown; however, it has been demonstrated that mutations of HFE, the genetic haemochromatosis gene, might be present in a significant proportion of Anglo-Saxon and Italian patients. Furthermore, transferrin receptor polymorphism may influence the affinity of this receptor to its ligand with a subsequent increase of cellular iron absorption and storage. OBJECTIVES: To evaluate the incidence and spectrum of HFE mutations and the relative frequency of the two main alleles of transferrin receptor in patients with PCT originating from southern France, and to evaluate the relationship of these genetic data with iron status, and with hepatitis B and C and human immunodeficiency virus (HIV) infections. METHODS: Thirty-six consecutive patients with either sporadic or familial PCT were prospectively included between 1997 and 2000. Search for the presence of the three main mutations of the HFE gene and identification of the transferrin receptor alleles were performed using polymerase chain reaction followed by enzymatic digestion. Iron parameters and viral status for hepatitis B and C viruses and HIV were determined. RESULTS: Seven patients (19%) showed heterozygous C282Y mutation, but no C282Y homozygote was present; five patients (14%) carried homozygous H63D mutation, while eight (22%) were heterozygous for this mutation. One patient was heterozygous for the S65C mutation (3%). Iron parameters demonstrated overload in all patients, without a clear difference between patients with and without deleterious mutations of the HFE gene. Infection by hepatitis C virus was documented in 20 patients (56%), and was significantly less frequent in patients with deleterious HFE mutations. The profile of transferrin receptor alleles in PCT patients did not show significant variation compared with the general population. CONCLUSIONS: This study confirms the high frequency of HFE mutations in patients with PCT and supports the hypothesis that HFE gene abnormalities might play a significant part in the PCT pathomechanism, probably through iron overload; by contrast, transferrin receptor polymorphisms do not appear to play a significant part in iron overload in PCT.