Biomedical subjects
M Makris
Publications and source records attributed to M Makris.
Familial thrombophilia: genetic risk factors and management.
There are now a number of potential candidates for inherited thrombophilia but a definite causal relationship has been established for only a proportion of these. Accepted causes of familial thrombophilia include the factor V Leiden defect and the prothrombin 20210 G > A variant, as well as deficiencies of antithrombin, protein C and protein S. Together these inherited abnormalities account for 30-50% of individuals presenting with venous thromboembolism. Factor V Leiden, which is present in up to 7% of the European population, is the most common cause of familial thrombophilia. On a worldwide basis its prevalence varies greatly with ethnic origin. In common with other types of familial thrombophilia the frequency of factor V Leiden is highly dependent on the population group studied. Venous thromboembolism, present in approximately 55% of individuals with familial coagulation inhibitor deficiencies, is the predominant clinical manifestation of familial thrombophilia. There are indications that the venous thrombotic risk is somewhat less in those with factor V Leiden. The thrombotic risk is markedly increased in those with combined defects and in those who are homozygous for factor V Leiden. Risk factors for thrombosis include pregnancy, including the puerperium, surgery, oral contraceptive usage and prolonged periods of immobilization. A substantial proportion of venous thrombotic events may occur spontaneously, i.e. without an obvious precipitating event. The management of patients with familial thrombophilia comprises counselling, thromboprophylaxis and thrombosis treatment. Although the immediate treatment of an acute thrombotic event is not significantly different from that of patients without recognised abnormalities, detailed patient management is seriously hampered by a lack of appropriate clinical trials. Prospective clinical studies, designed to ascertain individual thrombotic risk and to evaluate different therapeutic strategies are urgently required.
Co-inheritance of the 20210A allele of the prothrombin gene increases the risk of thrombosis in subjects with familial thrombophilia.
The presence of the 20210A allele of the prothrombin (PT) gene has recently been shown to be a risk factor for venous thromboembolism. This is probably mediated through increased plasma prothrombin levels. The aim of this study was to compare the prevalence of the prothrombin 20210A allele in control subjects and in subjects with recognised thrombophilia and to establish whether the additional inheritance of the PT 20210A allele is associated with an increased risk of venous thromboembolism. 101 subjects with a history of venous thromboembolism and diagnosed as having either factor V Leiden (R506Q) or heritable deficiencies of protein C, protein S or antithrombin were studied. The prevalence of the PT 20210A allele in this group was compared with the results obtained for 150 control subjects. In addition, the relationships were examined between genetic status and the number of documented thromboembolic episodes, and between plasma prothrombin levels and possession of the PT 20210A allele. 8 (7.9%) of the 101 patients were also heterozygous for the PT 20210A allele. This compares with 0.7% in the control subjects (p = 0.005). After exclusion of patients on warfarin, the mean plasma prothrombin of 113 subjects without 20210A was 1.09 U/ml, as compared with 1.32 U/ml in 8 with the allele (p = 0.0002). Among the 101 patients with either factor V Leiden, protein S deficiency, protein C deficiency or antithrombin deficiency, the age adjusted mean (SD) number of venous thromboembolic episodes at diagnosis was 3.7 (1.5) in those with the PT 20210A allele, as compared with 1.9 (1.1) in those without (p = 0.0001). We have demonstrated that the prevalence of the PT 20210A allele is significantly greater in subjects with venous thrombosis and characterised heritable thrombophilia than in normal control subjects and that the additional inheritance of PT 20210A is associated with an increased risk of venous thromboembolism. We have also confirmed that plasma prothrombin levels are significantly greater in subjects possessing the PT 20210A compared with those who do not.
Factor V Leiden in Greek thrombophilic patients: relationship with activated protein C resistance test and levels of thrombin-antithrombin complex and prothrombin fragment 1 + 2.
We studied 172 Greek patients (72 men aged 44.0 +/- 16.7 years and 100 women aged 46.5 +/- 14.1 years) with an unexplained thrombophilic tendency. One hundred and four apparently healthy persons (63 men aged 34.2 +/- 10.0 years and 41 women aged 37.1 +/- 13.3 years) were included as a control group. We performed the activated protein C resistance (APC-r) test using a clotting test (Chromogenix kit), detection of factor V Leiden using polymerase chain reaction (PCR)-restriction fragment length polymorphisms and measurement of thrombin-antithrombin complexes (TAT) and prothrombin fragment 1 + 2 (F1 + 2) levels with an immunoenzymatic assay. The normal range for the APC-r test (> 2.12) was determined from the controls. The factor V Leiden mutation was found in 31.9% of all the patients tested, in 28.1% of the unrelated patients with documented thrombophilic tendency of unknown origin and in 4.8% of the healthy controls. The APC-r test had a sensitivity of 0.42 and a specificity of 0.91 for the detection of factor V Leiden. Furthermore, we found no significant difference in levels of TAT and F1 + 2 between patients with and without the mutation and there was no correlation between aPC-r values and levels of TAT and F1 + 2.
Increased fetal loss in women with heritable thrombophilia.
BACKGROUND: A successful outcome of pregnancy requires an efficient uteroplacental vascular system. Since this system may be compromised by disorders of haemostasis associated with a prothrombotic state, we postulated that maternal thrombophilia might be a risk factor for fetal loss. We studied the relation between heritable thrombophilic defects and fetal loss in a cohort of women with factor V Leiden or deficiency of antithrombin, protein C, or protein S. METHODS: We studied 1384 women enrolled in the European Prospective Cohort on Thrombophilia (EPCOT). Of 843 women with thrombophilia 571 had 1524 pregnancies; of 541 control women 395 had 1019 pregnancies. The controls were partners of male members of the EPCOT cohort or acquaintances of cases. We analysed the frequencies of miscarriage (fetal loss at or before 28 weeks of gestation) and stillbirth (fetal loss after 28 weeks of gestation) jointly and separately. FINDINGS: The risk of fetal loss was increased in women with thrombophilia (168/571 vs 93/395; odds ratio 1.35 [95% Cl 1.01-1.82]). The odds ratio was higher for stillbirth than for miscarriage (3.6 [1.4-9.4] vs 1.27 [0.94-1.71]). The highest odds ratio for stillbirth was in women with combined defects (14.3 [2.4-86.0]) compared with 5.2 (1.5-18.1) in antithrombin deficiency, 2.3 (0.6-8.3) in protein-C deficiency, 3.3 (1.0-11.3) in protein-S deficiency, and 2.0 (0.5-7.7) with factor V Leiden. The corresponding odds ratios for miscarriage in these subgroups were 0.8 (0.2-3.6), 1.7 (1.0-2.8), 1.4 (0.9-2.2), 1.2 (0.7-1.9), and 0.9 (0.5-1.5). Significantly more pregnancy terminations had been done in women with thrombophilia than in controls (odds ratio 2.9 [1.8-4.8]); this discrepancy was apparent in nine of 11 participating centres and for all thrombophilia subgroups. INTERPRETATION: Women with familial thrombophilia, especially those with combined defects or antithrombin deficiency, have an increased risk of fetal loss, particularly stillbirth. Our findings have important implications for therapy and provide a rationale for clinical trials of thromboprophylaxis for affected women with recurrent fetal loss.
Molecular basis of protein S deficiency in three families also showing independent inheritance of factor V leiden.
The molecular basis of type I or III Protein S deficiency has been investigated in three kindred also showing independent inheritance of factor V (FV) Leiden. A T to C transition in codon 570 (Met-->Thr) was identified in the propositi and shown to segregate with protein S deficiency in all but one of the affected members of two kindred. This individual was heterozygous for a second transition (C to T) causing substitution of serine 624 by leucine. A second member of the same family, with markedly reduced free protein S levels when compared with affected relatives, was heterozygous for both mutations. Haplotype analysis of individuals with the mutated ATG570ACG allele in the two kindred suggested they may have been related by a common ancestor. A G to A transition resulting in substitution of cysteine 145 by tyrosine was detected in the third kindred. All mutations are believed to interfere with protein S binding to C4b-binding protein resulting in reduced free protein S levels. Of the five individuals studied who had experienced thrombotic events, three had combined protein S deficiency and FV Leiden reemphasising the importance of FV Leiden as an additional risk factor for thrombosis in protein S deficiency.
The natural history of chronic hepatitis C in haemophiliacs.
Most haemophiliacs treated with non-virally-inactivated clotting factor concentrates have been infected with hepatitis C virus (HCV). We have studied the natural history of chronic HCV infection by following all 138 HCV-positive patients from our centre for periods of up to 28 years. As well as the clinical and biochemical characteristics, we studied 116 liver samples from 63 patients obtained at elective biopsy (n = 103) or autopsy (n = 13). 36 (26%) of the patients were HIV positive, and three were chronic carriers of hepatitis B. Evidence of previous exposure to hepatitis A and B was found in 37.2% and 48.1% respectively. Raised transaminase levels were found in 82.6% of patients. 11 of 15 patients with normal transaminases tested by PCR for HCV RNA were positive, indicating that most patients, even in this group, have chronic hepatitis C infection. Cirrhosis was diagnosed by liver histology in 19 patients, and nine patients developed liver failure. The incidence of cirrhosis rose rapidly 15 years after HCV infection to 15.6 per 1000 person-years. Multivariate analysis showed that HIV status, length of time since HCV infection and age at HCV infection were independently associated with both the development of cirrhosis and liver failure. Two patients developed hepatocellular carcinoma: one of these was exposed only to a single batch of FVIII concentrate 11 years earlier. Chronic hepatitis C is increasingly recognized as a major cause for morbidity and mortality in haemophiliacs, especially those who are HIV positive and who were infected at an older age.
Warfarin induced skin necrosis associated with activated protein C resistance.
Explore the source record for details and available documents.
Characterisation of type 2N von Willebrand disease using phenotypic and molecular techniques.
von Willebrand factor (vWF) is a multimeric glycoprotein found in plasma non covalently linked to factor VIII (FVIII). Type 2N von Willebrand disease (vWD) is caused by a mutation in the vWF gene that results in vWF with a normal multimeric pattern, but with reduced binding to FVIII. We have utilised methods for the phenotypic and genotypic detection of type 2N vWD. The binding of FVIII to vWF in 69 patients, 36 with type 1 vWD, 32 with mild haemophilia A and one possible haemophilia A carrier with low FVIII levels was studied. Of these, six were found to have reduced binding (five type 1 vWD, one possible haemophilia A carrier). DNA was extracted from these patients and exons 18-23 of the vWF gene encoding the FVIII binding region of vWF were analysed. After direct sequencing and chemical cleavage mismatch detection, a Thr28Met mutation was detected in two unrelated individuals, one of whom appears to be a compound heterozygote for the mutation and a null allele. No mutations were found in the region of the vWF gene encoding the FVIII binding region of vWF in the other four patients.
Heterogeneity of hepatitis C virus genotypes in hemophilia: relationship with chronic liver disease.
In this study we have determined the hepatitis C virus (HCV) serotype and genotype in a cohort of 96 HCV-infected hemophiliacs and have examined the relationship between HCV genotype and severity of chronic liver disease as determined by liver biopsy. HCV serotype was determined by specific enzyme-linked immunosorbent assays (ELISAs) and genotype by restriction fragment length polymorphism (RFLP) and HCV viral sequencing. The pattern of genotype distribution was quite unlike that of HCV-infected United Kingdom (UK) blood donors in that five of the six known HCV genotypes were represented, 50% were type 1, 13% type 2, and 18% type 3. An unexpected observation was the presence of HCV genotype 4 in four patients and type 5 in two patients. An additional feature was the presence of mixed infection, detected in 14% and 7% by serotype and genotype analysis, respectively. Liver biopsies were available from 51 patients. Cirrhosis was present in five of 27 (19%) of individuals with type 1, in 2 of 9 (22%) with type 2, and 5 of 8 (63%) of those with type 3. The heterogeneous pattern of HCV genotype distribution in this cohort of patients and the observed relationship between the severity of the related liver disease and specific HCV genotype may have important implications with respect to the natural history and treatment of HCV-related chronic liver disease in infected hemophiliacs worldwide.
Long-term management after splenectomy. Lifelong penicillin unproved in trials...
Explore the source record for details and available documents.
Peripheral mononuclear cells of haemophiliacs with chronic liver disease are infected with replicating hepatitis C virus.
Most haemophiliacs treated with non-virally inactivated concentrates have been infected with the hepatitis C virus (HCV). 19/21 (90%) patients with HCV antibody were found to have HCV RNA in their serum and in 16 of these it was also detected in peripheral blood mononuclear cells (PBMC). We used specific primers to detect the negative (replicative) strand of this single positively stranded virus. The negative strand was detected in the serum or PBMC of 16 patients indicating active replication outside the liver. The detection of replicating HCV RNA in the cells of HIV negative haemophiliacs could explain at least some of the immunological abnormalities previously reported in these patients and attributed to clotting factor concentrates.
Increased soluble IL-2 receptor levels in HCV-infected haemophiliacs: a possible indicator of liver disease severity.
We have measured sIL-2R in 60 haemophiliacs and 20 male control subjects. Haemophiliacs were grouped according to their HIV/HCV antibody status. Group 1 (n = 20) comprised HIV + ve/HCV + ve, group 2 (n = 27) HIV - ve/HCV + ve and group 3 (n = 13) HIV - ve/HCV - ve. Group 4 comprised the normal control subjects. We also examined, retrospectively, the relationship between the severity of chronic liver disease, assessed histologically, and sIL-2R levels in selected patients. There was no significant difference between sIL-2R levels of the group 1 and group 2 patients, and the levels for both were significantly greater than those of either the group 3 patients or the control subjects. sIL-2 levels were also higher in selected patients with cirrhosis than in those with chronic active hepatitis (CAH) or chronic persistent hepatitis (CPH). We conclude that in haemophiliacs, chronic HCV-related liver disease is associated with increased plasma levels of sIL-2R and that the degree of elevation may reflect the severity of the associated chronic liver disease.
Further evidence that activated protein C resistance can be misdiagnosed as inherited functional protein S deficiency.
A recent report that activated protein C (APC) resistance interferes with functional protein S (PS) assays prompted us to re-investigate two pedigrees previously diagnosed as having functional PS deficiency. APC resistance was demonstrated in all individuals with apparent functional PS deficiency. The latter diagnosis was shown to be due to the assay being non-linear, functional protein S becoming normal at higher dilutions. This observation, taken in conjunction with results of in vitro recovery studies with purified PS, leads us to conclude that APC resistance was the primary disorder in both pedigrees. The misdiagnosis of APC resistance as functional PS deficiency can be prevented by performing the PS assay at several dilutions, including concentrations lower than those recommended by PS assay manufacturers. Subjects previously diagnosed as having functional PS deficiency should be re-investigated for APC resistance.
The development of antiphospholipid antibodies in haemophilia is linked to infection with hepatitis C.
In haemophilia the presence of antibodies to antiphospholipid has been linked with HIV infection, but other possibilities have not been fully explored and the specificity for various phospholipids not established. In order to investigate further the pathogenesis and clinical significance of these antibodies, we have determined IgG and IgM antibodies to a variety of phospholipids, including cardiolipin, in the serum of 52 haemophiliacs, and related our findings to the presence of antibodies to HIV and hepatitis C virus (HCV), as well as to clotting factor concentrate usage and blood markers of liver biochemistry. Our results demonstrate that the presence of infection with hepatitis C virus is strongly associated with raised serum levels of antiphospholipid antibody even in the absence of HIV infection. They suggest that earlier conclusions on the relationships of antiphospholipid to HIV infection in haemophilia should be revised and that chronic infection with the hepatitis C virus should be added to the list of infective causes for the development of antiphospholipid antibody.
Hepatitis C viral RNA in clotting factor concentrates and the development of hepatitis in recipients.
The polymerase chain reaction (PCR) was used to detect hepatitis C (HCV) viral sequences (HCV-RNA) in clotting factor concentrates that had been stored at 4 degrees C for 1 to 16 years. A total of 43 concentrates were tested, comprising 31 batches of factor VIII, 6 of factor IX, 2 of antithrombin III, 3 of FEIBA, and 1 of factor VII. HCV-RNA was detected in 13 of the 43 batches (30.2%). Concentrates that had not undergone viral inactivation during manufacture were significantly more likely to contain detectable HCV-RNA than concentrates that had been virally inactivated (56.3% v 14.5%, P = .006). HCV sequences were more commonly detected in concentrates made from paid donor plasma than in those made from volunteer donor plasma (44% v 11%, P = .041), and more commonly in virally inactivated concentrates with pre-1989 than with post-1989 expiration dates (50% v 0%, P = .004). Of the four batches of heat-treated products that were HCV-RNA positive, at least three transmitted non-A, non-B hepatitis (NANBH). An association between the presence of HCV-RNA in concentrates and the development of NANBH was demonstrated in nine previously untreated patients on prospective follow-up. HCV-RNA was detected in the concentrates administered to the six patients whose alanine aminotransferase (ALT) abnormalities met the diagnostic criteria for NANBH and who later seroconverted for HCV, but it was not detected in the concentrates administered to the three patients whose ALT abnormalities failed to satisfy the diagnostic criteria and who did not seroconvert. We suggest that the use of this PCR technique to monitor clotting factor concentrates derived from pooled blood may potentially contribute to product safety.
Chronic hepatitis in haemophilia.
Chronic hepatitis affects almost all haemophiliacs treated with non-virally inactivated clotting factor concentrates. The virus responsible is hepatitis C (HCV) and most patients have non-neutralising antibodies with circulating virus. Although the majority also have evidence of past infection with hepatitis B, less than 5% are chronic carriers of HBsAg. Chronic hepatitis C can be associated with severe and progressive liver disease but the development of complications is slow. Treatment with recombinant interferon alpha given subcutaneously normalises the liver function in 50% of patients, but 50% of responders relapse on stopping treatment. Liver transplantation is successful in patients with advanced liver disease and it offers the added advantage of phenotypic cure of the haemophilic state.
Interferon alfa for chronic hepatitis C in haemophiliacs.
Chronic hepatitis C virus (HCV) associated liver disease is an important cause of morbidity and mortality in haemophilia. Recombinant interferon alfa-2b was used in a randomised controlled liver biopsy trial to treat haemophiliacs with chronic HCV. All 18 patients entered had antibodies to HCV. During the first year of the study, 10 patients were randomised on the basis of histology to receive interferon alfa-2b, 3 million units subcutaneously, thrice weekly and eight to receive no treatment (control group). After 12 months, all patients had a second liver biopsy and the control group patients were offered interferon at the same dosage but for only six months. The alanine aminotransferase (ALT) activity had returned to normal in four of 10 patients treated for one year and five of six patients treated for six months, compared with none of the eight patients in the control group (p < 0.01). Although the histological scores of the two groups were similar at entry into the study, after one year the biopsy specimens in the treated group showed significant improvement compared with controls (p < 0.01). It is concluded that interferon alfa-2b is effective in returning ALT values to normal and improving liver histology in at least 50% of patients treated.