Bengt Zöller

Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C.

Resistance to activated protein C (APC) is the most prevalent inherited cause of venous thrombosis. The APC resistance phenotype is associated with a single point mutation in the factor V gene, changing Arg506 in the APC cleavage site to a Gln. We have investigated 50 Swedish families with inherited APC resistance for this mutation and found it to be present in 47 of them. Perfect cosegregation between a low APC ratio and the presence of mutation was seen in 40 families. In seven families, the co-segregation was not perfect as 12 out of 57 APC-resistant family members were found to lack the mutation. Moreover, in three families with APC resistance, the factor V gene mutation was not found, suggesting another still unidentified cause of inherited APC resistance. Of 308 investigated families members, 146 were normal, 144 heterozygotes, and 18 homozygotes for the factor V gene mutation and there were significant differences in thrombosis-free survival curves between these groups. By age 33 yr, 8% of normals, 20% of heterozygotes, and 40% of homozygotes had had manifestation of venous thrombosis.

Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis

Resistance to activated protein C (APC) is a major cause of familial thrombophilia, and can be corrected by an anticoagulant activity expressed by purified factor V. We investigated linkage between APC resistance and the factor V gene in a large kindred with familial thrombophilia. Restriction fragment length polymorphisms in exon 13 of the factor V gene were informative in 14 family members. The 100% linkage between factor V gene polymorphism and APC resistance strongly suggested a factor V gene mutation as a cause of APC resistance. A point mutation changing Arg506 in the APC cleavage site to a Gln was found in APC resistant individuals. These results suggest factor V gene mutation to be the most common genetic cause of thrombophilia.

Risk of subsequent ischemic and hemorrhagic stroke in patients hospitalized for immune-mediated diseases: a nationwide follow-up study from Sweden

BackgroundCertain immune-mediated diseases (IMDs) have been associated with increased risk for cardiovascular disorders. The aim of the present study was to examine whether there is an association between 32 different IMDs and first hospitalization for ischemic or hemorrhagic stroke.MethodsAll individuals in Sweden hospitalized with a main diagnosis of IMD (without previous or coexisting stroke), between January 1, 1987 and December 31, 2008 (n = 216,291), were followed for first hospitalization for ischemic or hemorrhagic stroke. The reference population was the total population of Sweden. Adjusted standardized incidence ratios (SIRs) for ischemic and hemorrhagic stroke were calculated.ResultsTotally 20 and 15 of the 32 IMDs studied, respectively, were associated with an increased risk of ischemic and hemorrhagic stroke during the follow-up. The overall risks of ischemic and hemorrhagic stroke during the first year after hospitalization for IMD were 2.02 (95% CI 1.90–2.14) and 2.65 (95% CI 2.27–3.08), respectively. The overall risk of ischemic or hemorrhagic stroke decreased over time, to 1.50 (95% CI 1.46–1.55) and 1.83 (95% CI 1.69–1.98), respectively, after 1–5 years, and 1.29 (95% CI 1.23–1.35) and 1.47 (95% CI 1.31–1.65), respectively, after 10+ years. The risk of hemorrhagic stroke was ≥2 during the first year after hospitalization for seven IMDs: ankylosing spondylitis (SIR = 8.11), immune thrombocytopenic purpura (SIR = 8.60), polymyalgia rheumatica (SIR = 2.06), psoriasis (SIR = 2.88), rheumatoid arthritis (SIR = 3.27), systemic lupus erythematosus (SIR = 8.65), and Wegener´s granulomatosis (SIR = 5.83). The risk of ischemic stroke was ≥2 during the first year after hospitalization for twelve IMDs: Addison’s disease (SIR = 2.71), Crohn´s disease (SIR = 2.15), Grave´s disease (SIR = 2.15), Hashimoto´s thyroiditis (SIR = 2.99), immune thrombocytopenic purpura (SIR = 2.35), multiple sclerosis (SIR = 3.05), polymyositis/dermatomyositis (SIR = 3.46), rheumatic fever (SIR = 3.91), rheumatoid arthritis (SIR = 2.08), Sjögren’s syndrome (SIR = 2.57), systemic lupus erythematosus (SIR = 2.21), and ulcerative colitis (SIR = 2.15).ConclusionsHospitalization for many IMDs is associated with increased risk of ischemic or hemorrhagic stroke. The findings suggest that several IMDs are linked to cerebrovascular disease.

Clarification of the Risk for Venous Thrombosis Associated with Hereditary Protein S Deficiency by Investigation of a Large Kindred with a Characterized Gene Defect

Vitamin K-dependent protein S plays an important role in the regulation of the coagulation cascade [1]. This protein increases the rate of degradation of activated factors V and VIII by acting as a cofactor to activated protein C, thereby limiting thrombin production. The presence of factor V, with which protein S acts in synergy, amplifies the function of protein S as a cofactor to activated protein C in factor VIII proteolysis. Protein S has recently been found to have anticoagulant functions independent of activated protein C: It directly inhibits procoagulant enzyme complexes [2, 3]. The relative importance of these different functions in maintaining intravascular fluidity is still unknown. However, heterozygous deficiency of protein S was described as a cause of venous thrombosis in 1984 [4] and has subsequently been identified in numerous clinically affected families, in which it is inherited as an autosomal dominant trait. This deficiency has been found in 1.5% to 7% of selected groups of thrombophilic patients [5-8], although an estimate of the prevalence in the general population awaits a sufficiently large study. Homozygous protein S deficiency is an extremely rare and life-threatening disorder associated with severe neonatal purpura fulminans [9]. Unlike other coagulation inhibitors, protein S has some functions that are affected by C4b-binding protein, a component of the complement cascade, to which 60% to 70% of protein S is bound in vivo [10]. Once bound, protein S can no longer act as a cofactor to activated protein C but retains some of its inhibitory properties. Because of the difference in function between the bound and unbound forms of protein S and uncertainty over which function is the most important, levels of both total and free protein S antigen are usually measured in patient plasma samples. The function of protein S as a cofactor to activated protein C may also be assessed. Protein S deficiency is diagnosed if one or more of these measurements is found to be below the lower limit of a laboratory reference range. Several problems are encountered in the diagnosis of protein S deficiency, including the large overlap in antigen levels between normal and heterozygous persons [11]. This overlap may be due to the effect of sex and hormones on total protein S levels [12, 13]. We recently described an age-related increase in total protein S antigen, independent of the influence of sex, in both normal and protein S-deficient persons [14]. This phenomenon also complicates diagnosis made on the basis of total protein S measurement and causes phenotypic variation within the same kindred. Furthermore, some assays for protein S activity are influenced by a mutation in the gene for factor V (Arg506Gln), which causes resistance to activated protein C and is a common, if relatively mild, risk factor for thrombosis. Heterozygosity for this mutation can result in apparent reduction of protein S cofactor activity to activated protein C in the laboratory assessment of normal persons [15]. Despite these problems with diagnosis, some studies have attempted to compute the risk for thrombosis associated with phenotypic protein S deficiency [11, 16-18]. In deficient families, the probability that affected family members remain thrombosis-free at 45 years of age has been reported to be 0.35 to 0.50 [11, 16]. However, the incidence of thrombosis varies among different families; this suggests problems with precise diagnosis or the presence of other genetic risk factors. Of note, no study has examined the risk associated with genetically confirmed protein S deficiency, which would remove the diagnostic uncertainties. The identification of gene mutations that cause protein S deficiency is complicated by the size of the gene (>80 kilobase-pairs) that encodes protein S [19-21] and by the presence of a pseudogene. However, an increasing number of studies have identified such mutations in probands or small family groups. The first database of protein S gene mutations was recently published by the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis [22]. Mutations in the coding region or at intron-exon boundaries have generally been identified in approximately 50% of protein S-deficient probands. We recently identified a single causative mutation (which results in a Gly295Val substitution) in a large protein S-deficient kindred [14]. This mutation is not thought to be common in the general population. The availability of comprehensive phenotypic, genotypic, and clinical data has enabled the interrelations among these data to be investigated and has thereby provided quantitative information on the risk for thrombosis associated with a mutation in the protein S gene and the value of different assays in predicting clinical events. Methods Participants The manifestations of thrombosis in the 122-member family under investigation (most family members live in northern Sweden) were attributed to protein S deficiency in 1993 [23]. This kindred has also been part of a larger study that involved 18 families with phenotypic protein S deficiency [16, 24] and provided an explanation for phenotypic variation in familial protein S deficiency [14]. All participants gave informed consent, and the medical ethics committee at the University of Lund approved all of these studies, including the present one. Study participants answered a questionnaire about their medical history, with emphasis on manifestations of deep venous thrombosis, pulmonary embolism, superficial thrombophlebitis, and arterial thrombosis. Symptomatic family members were also interviewed by a physician or had their medical records reviewed. The term deep venous thrombosis includes deep venous thrombosis of the leg and thrombosis in such unusual locations as the axillary, mesenteric, and cerebral veins. Thrombotic event refers to deep venous thrombosis, pulmonary embolism, or superficial thrombophlebitis diagnosed by a physician on the basis of physical examination. Laboratory Methods Blood sampling and routine coagulation were performed as described elsewhere [25]. Total and free protein S antigen levels were measured by doing radioimmunoassay [26]. Protein S levels were compared with laboratory reference ranges for levels of both free (reference range, 56 to 182 nmol/L) and total (reference range, 219 to 407 nmol/L) antigen. Persons receiving anticoagulation were compared with an anticoagulated control group. The control groups that we used have been described elsewhere [24]. Of the 122 family members, 44 had free protein S antigen levels below the lower limit of the reference range; 13 of the 44 were receiving oral anticoagulants at the time of sampling. Molecular Genetic Investigation The methods used to identify and detect the novel protein S gene mutation, Gly295Val, in 122 genomic DNA samples have been reported elsewhere [14, 27]. All 44 family members with reduced free protein S antigen levels were heterozygous for the mutation; the remaining 78 relatives who had normal free protein S antigen levels were normal at this site. This finding confirmed that the Gly295Val mutation was the cause of protein S deficiency in this family. A single asymptomatic family member with normal free protein S antigen levels had previously been found to be heterozygous for the factor V Arg506Gln mutation. Statistical Analysis Thrombosis-free survival curves were constructed according to the method of Kaplan and Meier [28]. Two curves were compared by using the log-rank test, which results in a test statistic with chi-squared distribution and one degree of freedom [29]. This analysis was performed by using Statistica software (Statsoft, Inc., Tulsa, Oklahoma). Univariate and multivariate Cox regression analyses [30] were performed by using Statistica software or SPS (SPS, Inc., Chicago, Illinois). All 122 family members were included in the analysis for risk for thrombosis. Results Demographic and Clinical Data Samples of plasma and genomic DNA were available for 122 germline family members (60 men and 62 women; mean age SD, 36 17 years [range, 7 to 82 years]) spanning five generations. The distribution of patient samples was 1, 8, 41, 57, and 15 from the first, second, third, fourth, and fifth generations, respectively. A histogram of the current ages of the study participants is shown in Figure 1. Twenty-five (57%) of the 44 family members with the Gly295Val mutation had one or more venous thrombotic events (mean age at first event, 31 years [range, 11 to 71 years]) compared with 5 (6%) of the 78 family members who lacked the mutation (mean age at first event, 29 years [range, 16 to 43 years]). The clinical manifestations in symptomatic family members are summarized in Table 1. First thrombotic events were associated with one or more circumstantial risk factors in 12 symptomatic relatives (48%) with the Gly295Val mutation and 2 family members (40%) who lacked the mutation (Table 2). Three carriers of the mutation (6.8%) and none of the normal family members had arterial thrombotic events (postoperative bilateral arterial thrombosis requiring bilateral above-the-knee amputation, myocardial infarction, and embolization requiring below-the-knee amputation). Figure 1. Histogram of the current ages of the investigated persons in the study family. Table 1. Clinical Manifestations of Venous Thrombosis in Symptomatic Family Members with and without the Gly295Val Mutation Table 2. Circumstantial Risk Factors Associated with First Thrombotic Episodes in 25 Symptomatic Relatives with the Gly295Val Mutation and 5 Symptomatic Relatives without the Mutation According to Kaplan-Meier analysis of thrombosis-free survival, the probability that a family member who carries the Gly295Val mutation would remain free of venous thrombosis at 30 years of age is 0.5 (95% CI, 0.33 to 0.66) compared with 0.97 (CI, 0.93 to 1.0) for normal family members (Figure 2)

Age- and Gender-Specific Familial Risks for Venous Thromboembolism A Nationwide Epidemiological Study Based on Hospitalizations in Sweden

Background— This nationwide study sought to determine age- and gender-specific familial risks in siblings hospitalized for venous thromboembolism (VTE). Methods and Results— The Swedish Multigeneration Register on 0- to 75-year-old subjects was linked to the Hospital Discharge Register for the years 1987–2007. Standardized incidence ratios were calculated for individuals whose siblings were hospitalized for VTE compared with those whose siblings were not affected. Among a total of 45 362 hospitalized cases with VTE, 2393 affected siblings were identified, with a familial standardized incidence ratio of 2.45 (95% confidence interval [CI], 1.66 to 3.61). Gender-specific differences in incidence rates were observed. The familial risks were significantly increased from the age of 10 to 69 years, with a familial standardized incidence ratio of 4.77 (95% CI, 1.96 to 10.83) at ages 10 to 19 years, which decreased to 2.08 (95% CI, 1.35 to 3.20) at ages 60 to 69 years, although the absolute risk increased with age. The familial standardized incidence ratios for siblings with 2 and ≥3 affected probands were 51.87 (95% CI, 31.47 to 85.00) and 53.69 (95% CI, 25.59 to 108.50), respectively. Spouses had low familial risks (standardized incidence ratio=1.07; 95% CI, 1.04 to 1.10; observed spouse cases=3900). Conclusions— Familial factors, although influenced by age and gender, are important risk factors for VTE. The present study shows that VTE is aggregated in families and suggests that uncovering the sources of familial aggregation (genetic and nongenetic) may be worthwhile. Moreover, in a small fraction of siblings, the familial risk was very high, suggesting segregation of rare but strong genetic risk factors.Background— This nationwide study sought to determine age- and gender-specific familial risks in siblings hospitalized for venous thromboembolism (VTE). Methods and Results— The Swedish Multigeneration Register on 0- to 75-year-old subjects was linked to the Hospital Discharge Register for the years 1987–2007. Standardized incidence ratios were calculated for individuals whose siblings were hospitalized for VTE compared with those whose siblings were not affected. Among a total of 45 362 hospitalized cases with VTE, 2393 affected siblings were identified, with a familial standardized incidence ratio of 2.45 (95% confidence interval [CI], 1.66 to 3.61). Gender-specific differences in incidence rates were observed. The familial risks were significantly increased from the age of 10 to 69 years, with a familial standardized incidence ratio of 4.77 (95% CI, 1.96 to 10.83) at ages 10 to 19 years, which decreased to 2.08 (95% CI, 1.35 to 3.20) at ages 60 to 69 years, although the absolute risk increased with age. The familial standardized incidence ratios for siblings with 2 and ≥3 affected probands were 51.87 (95% CI, 31.47 to 85.00) and 53.69 (95% CI, 25.59 to 108.50), respectively. Spouses had low familial risks (standardized incidence ratio=1.07; 95% CI, 1.04 to 1.10; observed spouse cases=3900). Conclusions— Familial factors, although influenced by age and gender, are important risk factors for VTE. The present study shows that VTE is aggregated in families and suggests that uncovering the sources of familial aggregation (genetic and nongenetic) may be worthwhile. Moreover, in a small fraction of siblings, the familial risk was very high, suggesting segregation of rare but strong genetic risk factors. # Clinical Perspective {#article-title-47}

Risk of Subsequent Coronary Heart Disease in Patients Hospitalized for Immune-Mediated Diseases: A Nationwide Follow-Up Study from Sweden

Background Certain immune-mediated diseases (IMDs), such as rheumatoid arthritis and systemic lupus erythematosus, have been linked to cardiovascular disorders. We examined whether there is an association between 32 different IMDs and risk of subsequent hospitalization for coronary heart disease (CHD) related to coronary atherosclerosis in a nationwide follow up study in Sweden. Methods and Findings All individuals in Sweden hospitalized with a main diagnosis of an IMD (n = 336,479) without previous or coexisting CHD, between January 1, 1964 and December 31 2008, were followed for first hospitalization for CHD. The reference population was the total population of Sweden. Standardized incidence ratios (SIRs) for CHD were calculated. Overall risk of CHD during the first year after hospitalization for an IMD was 2.92 (95% CI 2.84–2.99). Twenty-seven of the 32 IMDs studied were associated with an increased risk of CHD during the first year after hospitalization. The overall risk of CHD decreased over time, from 1.75 after 1–5 years (95% CI 1.73–1.78), to 1.43 after 5–10 years (95% CI 1.41–1.46) and 1.28 after 10+ years (95% CI 1.26–1.30). Females generally had higher SIRs than males. The IMDs for which the SIRs of CDH were highest during the first year after hospitalization included chorea minor 6.98 (95% CI 1.32–20.65), systemic lupus erythematosus 4.94 (95% CI 4.15–5.83), rheumatic fever 4.65 (95% CI 3.53–6.01), Hashimotos thyroiditis 4.30 (95% CI 3.87–4.75), polymyositis/dermatomyositis 3.81 (95% CI 2.62–5.35), polyarteritis nodosa 3.81 (95% CI 2.72–5.19), rheumatoid arthritis 3.72 (95% CI 3.56–3.88), systemic sclerosis 3.44 (95% CI 2.86–4.09), primary biliary cirrhosis 3.32 (95% CI 2.34–4.58), and autoimmune hemolytic anemia 3.17 (95% CI 2.16–4.47). Conclusions Most IMDs are associated with increased risk of CHD in the first year after hospital admission. Our findings suggest that many hospitalized IMDs are tightly linked to coronary atherosclerosis.

Prevalence of factor V gene mutation amongst myocardial infarction patients and healthy controls is higher in Sweden than in other countries

Objective. Haemostatic imbalance may be an aetiological factor in the development of acute coronary syndromes. Inherited resistance to activated protein C (APC) is a common disorder associated with hypercoagulability and lifelong risk of venous thrombosis. APC resistance is due to a single mutation in the gene coding for coagulation factor V (FV:Q506). To test the importance of the FV:Q506 mutation in premature myocardial infarction (MI), its prevalence was investigated in Swedish patients with MI before the age of 50 years.

Resistance to activated protein C, the FV:Q506 allele, and venous thrombosis

Abstract Vitamin K-dependent protein C is an important regulator of blood coagulation. After its activation on the endothelial cell surface by thrombin bound to thrombomodulin, it cleaves and inactivates procoagulant cofactors Va and VIIIa, protein S and intact factor V working as cofactors. Until recently, genetic defects of protein C or protein S were, together with antithrombin III deficiency, the established major causes of familial venous thromboembolism, but they were found in fewer than 5–10% of patients with thrombosis. In 1993, inherited resistance to activated protein C (APC) was described as a major risk factor for venous thrombosis. It is found in up to 60% of patients with venous thrombosis. In more than 90% of cases, the molecular background for the APC resistance is a single point mutation in the factor V gene, which predicts substitution of an arginine (R) at position 506 by a glutamine (Q). Mutated factor V (FV : Q506) is activated by thrombin or factor Xa in normal way, but impaired inactivation of mutated factor Va by APC results in life-long hypercoagulability. The prevalence of the FV : Q506 allele in the general population of Western countries varies between 2 and 15%, whereas it is not found in several other populations with different ethnic backgrounds. Owing to the high prevalence of FV : Q506 in Western populations, it occasionally occurs in patients with deficiency of protein S, protein C, or antithrombin III. Individuals with combined defects suffer more severely from thrombosis, and often at a younger age, than those with single defects, suggesting severe thrombophilia to be a multigenetic disease.

The factor VR506Q mutation causing APC resistance is highly prevalent amongst unselected outpatients with clinically suspected deep venous thrombosis

Objective. Resistance to activated protein C (APC resistance), caused by a single point mutation in the factor V gene (FV:R506Q), is a major risk factor for venous thrombosis. As the significance of this mutation among unselected outpatients with deep‐vein thrombosis (DVT) is not established, we have studied its prevalence among consecutive outpatients attending the emergency room due to a clinically suspected DVT.

Determination of 14 Circulating microRNAs in Swedes and Iraqis with and without Diabetes Mellitus Type 2

Background Recent reports suggest that immigrants from Middle Eastern countries are a high-risk group for type 2 diabetes (T2D) compared with Swedes, and that the pathogenesis of T2D may be ethnicity-specific. Deregulation of microRNA (miRNA) expression has been demonstrated to be associated with T2D but ethnic differences in miRNA have not been investigated. The aim of this study was to explore the ethnic specific expression (Swedish and Iraqi) of a panel of 14 previously identified miRNAs in patients without T2D (including those with prediabetes) and T2D. Methods A total of 152 individuals were included in the study (84 Iraqis and 68 Swedes). Nineteen Iraqis and 14 Swedes were diagnosed with T2D. Expression of the 14 selected miRNAs (miR-15a, miR-20, miR-21, miR-24, miR-29b, miR-126, miR-144, miR-150, miR-197, miR-223, miR-191, miR-320a, miR-486-5p, and miR-28-3p) in plasma samples was measured by real-time PCR. Results In the whole study population, the expression of miR-24 and miR-29b was significantly different between T2D patients and controls after adjustment for age, sex, waist circumference, family history of T2D, and a sedentary lifestyle. Interestingly, when stratifying the study population according to country of birth, we found that higher expression of miR-144 was significantly associated with T2D in Swedes (OR = 2.43, p = 0.035), but not in Iraqis (OR = 0.54, p = 0.169). The interaction test was significant (p = 0.017). Conclusion This study suggests that the association between plasma miR-144 expression and T2D differs between Swedes and Iraqis.