Protective effect of diltiazem on myocardial ischemic injury associated with .OH generation

Two patients from two unrelated families with a history of thrombosis showed severe plasma activated protein C (APC) resistance. However, genotypic analysis demonstrated that the patients were heterozygous for factor V (FV) Leiden mutation. Coagulation studies revealed that FV clotting activity and antigen were similarly reduced at about 50% of normal in the patients. One brother of propositus A also showed the same abnormalities. Genetic analysis showed that, in addition to FV Leiden mutation in exon 10 of the FV gene (G1691A), these patients had a transition in exon 13 of the FV gene (A4070G; R2 allele) predicting His1299Arg substitution in the mature FV. Study by RT-PCR of platelet FV mRNA indicated that the mRNA produced by the FV gene, marked by the R2 allele, was reduced in amount in both pseudohomozygous patients of family A. The R2 allele has previously been demonstrated to be significantly associated with plasma FV deficiency in the Italian population. The presence of FV deficiency did not protect the propositi from thrombosis. These data confirm that genotypic analysis is mandatory in patients with phenotypic severe APC resistance before these patients are definitely classified as homozygotes for FV Leiden and that further genotypic analysis is advisable.     

Resistance to activated protein C caused by the factor VR506Q mutation is a common risk factor for venous thrombosis

In 1993, inherited resistance to activated protein C (APC) was described as a novel risk factor for venous thrombosis. APC-resistance is present in 20-60% of venous thrombosis cases. It is caused by a single point mutation in the factor V gene which substitutes arginine (R) at position 506 with a glutamine (Q). The mutation is common in Caucasians with up to 15% prevalence in the population, whereas it is not found among other human races. Mutated factor V (FVR506Q, FV:Q506 or FV Leiden) is partially resistant to APC which results in a hypercoagulable state conferring a life-long increased risk of thrombosis. Individuals having FV:Q506 combined with other anticoagulant defects have a high risk of thrombosis, and it is now generally accepted that severe thrombophilia is a multigenetic disease. Easy functional and genetic tests for inherited APC-resistance will profoundly influence the development of prophylactic regimens and hopefully result in a decreased incidence of thrombosis.     

Immunomodulating activity of allopurinol in experimental lens-induced uveitis

In order to define the thrombophilic conditions related to activated protein C resistance (APC-R) and protein S (PS) deficiency and to detect the possible combination of these defects, we studied nine unrelated patients selected because of low anticoagulant response to APC and reduced PS activity with at least one first degree relative having the same coagulation feature. The mean APC ratio was 0.64 (normal values > 0.80) and range 0.35-0.80. Three of the patients were heterozygous and two were homozygous for the Leiden mutation (FVR506Q); in the remaining four there was no mutation. The mean PS activity was 41.6% and range 32-54 (normal 65-150%). Five of the patients had low PS activity despite normal total and free antigenic levels, and normal activity when measured at higher plasma dilution; these were carrying at least one gene for the Leiden mutation. In the remaining four patients the crossed-immunoelectrophoresis and the Western-blotting analysis showed a type I PS deficiency confirmed by the familial restriction fragment length polymorphism analysis. Thus, four families were diagnosed with the type I PS defect and five with congenital APC-R. No combined PS/FV Leiden or type II PS defect was found. The only defect found was in the anticoagulant PC pathway. We therefore designed a procedure to diagnose thrombophilic conditions related to this pathway. This study indicates that a specific methodological approach must be used to accurately characterize APC-R and PS deficiency and that care is necessary to avoid the possibility of misdiagnosis.     

Five novel mutations of the protein S active gene (PROS 1) in 8 Norman families

To further elucidate the molecular basis for hereditary thrombophilia, we screened the protein S active gene in 11 families with type I deficiency, using a strategy based on denaturing gradient gel electrophoresis (DGGE) of all the coding sequences. Fragments with an abnormal DGGE pattern were sequenced, and 5 novel mutations were identified in 8 families. The mutations were a 7-nucleotide deletion in exon II, a 4-nucleotide deletion in exon III, a T insertion in exon VII, a C to T transition transforming Leu 259 into Pro and a T to C transition transforming Cys 625 into Arg in 4 families. These mutations were the only sequence variations found in the propositus' gene exons and co-segregated with the plasma phenotype. A total of 28 members of these 8 families were heterozygous for one of the 5 mutations. Twenty-four (58,5%) of the 41 deficient subjects over 18 years of age had clinical thrombophilia, whereas the 13 subjects under 18 were asymptomatic. Of the 28 subjects, 6 (21,5%) were also found to bear the factor V Arg 506 Gln mutation.     

Axonal regeneration through muscle autografts submitted to local anaesthetic pretreatment

Over a 3 year period the R506Q mutation in the factor V (FV) FV:Q506 gene, FV, factor XII (FXII), prothrombin, protein C, protein S, antithrombin, heparin cofactor II, anticardiolipin antibodies and lipoprotein (a) (Lp(a)) were measured in 32 infants and children with sinus thrombosis. Heterozygous FV:Q506 (n=5), homozygous FV:Q506 (n=2), homozygous FXII deficiency (n=1), protein C deficiency type I (n=5), protein C deficiency type II (n=1), antithrombin deficiency type I (n=1) increased Lp (a) (n=5), activated protein C-resistance without mutation in the FV gene (n=2), and increased anticardiolipin IgG antibodies (n=2) were diagnosed in the children investigated. In a further two patients we found combinations of increased Lp(a) with moderate hyperhomocystinaemia and heterozygous plasminogen deficiency with heterozygous FXII deficiency. In addition, increased anticardiolipin IgG antibodies were found in combination with heterozygous FV:Q506 (n=1) and protein C type I deficiency (n=2) respectively. Out of 32 patients with venous sinus thrombosis, 3 showed additional peripheral venous vascular occlusion. Contributing factors were present in 31 out of 32 patients investigated. Family members of 10 affected children had suffered from venous thrombo-embolism prior to the study. CONCLUSION: Our data suggest that additional contributing factors may promote manifestation of cerebral venous sinus thrombosis in infants and children with an inherited prothrombotic state. Further prospective studies are required to evaluate their potential role as "triggering" agents.     

A novel missense mutation in patients from a retinoblastoma pedigree showing only mild expression of the tumor phenotype

We have used single strand conformation polymorphism analysis to study the 27 exons of the RB1 gene in individuals from a family showing 'mild' expression of the retinoblastoma phenotype. In this family affected individuals developed unilateral tumors and, as a result of linkage analysis, unaffected mutation carriers were also identified within the pedigree. A single band shift using SSCP was identified in exon 21 which resulted in a missense mutation converting a cys-->arg at nucleotide position 28 in the exon. The mutation destroyed an NdeI restriction enzyme site. Analysis of all family members demonstrated that the missense mutation co-segregated with patients with tumors or who, as a result of linkage analysis had been predicted to carry the predisposing mutation. These observations point to another region of the RB1 gene where mutations only modify the function of the gene and raise important questions for genetic counseling in families with these distinctive phenotypes.     

Rapid multiplex analysis for the factor V Leiden and prothrombin G20210A mutations associated with hereditary thrombophilia

Thromboembolic episodes are common events and affect approximately one in 1,000 persons annually. Pulmonary embolism alone accounts for 50,000 to 100,000 deaths per year in the United States with > 50% of those being elderly persons. Resistance to activated protein C is the most common inherited disorder associated with hereditary thrombophilia. A missense mutation has been identified in the gene coding for coagulation factor V (codon 506) which renders this procoagulant factor resistant to inactivation by activated protein C resulting in an increased risk for venous thrombosis. Recently, a second polymorphism was identified in the prothrombin gene (factor II) which is also associated with increased risk for venous thrombosis. Because of the high prevalence of these two mutations in the general population as well as in specific patient populations, the ability readily to detect these two mutations must be feasible. In this study, we evaluated 303 patients for the prothrombin mutatin (G20210A) which were previously tested for the factor V mutation using established polymerase chain reaction-mediated restriction fragment length polymorphism assays. In these patients, 30 (9.9%) were found to be heterozygous for the factor V Leiden mutation with no homozygous mutants identified. Twenty individuals (6.6%) were heterozygous for the prothrombin G20210A mutation, and we identified two individuals (0.66%) who were homozygous for the 20210A allele. Of the total 303 individuals screened, two were double heterozygotes for both the factor V Leiden and the prothrombin gene mutations. We also describe a multiplex polymerase chain reaction-mediated restriction fragment length polymorphism assay for detecting both mutations in a single-tube double-enzyme digestion reaction making identification of these two mutations easily achievable.     

CT pulmonary angiography. Stay tuned

It is well established that genetic disorders interact with environmental factors to cause thrombotic diseases. Therefore, antithrombin, protein C, protein S deficiencies and the more recently described factor V Leiden and prothrombin mutations are currently been investigated to explain some thrombophilic states. We report the case of a 63-year-old man who developed two transient ischemic attacks and two years later an extensive femoro-iliac venous thrombosis. He was genotyped as FV R506Q negative and FII G20210A positive in homozygous state (FII 20210AA).     

A novel mutation of presenilin 1 in familial Alzheimer's disease in Israel detected by denaturing gradient gel electrophoresis

Germline mutations in the presenilin 1 (PS1) gene apparently account for the majority of early-onset, familial Alzheimer's disease (AD). Using a mutation-screening strategy (denaturing gradient gel electrophoresis; DGGE), we analyzed a large family with early onset AD and seizures. The patients in this family showed a novel missense mutation in exon 5 of the PS1 gene (A to T change in codon 120, altering glutamine to aspartic acid). This novel mutation is located within the second hydrophilic domain of the molecule, a region not particularly involved in previously described germline mutations, and is of unknown biological significance. These results also demonstrate that DGGE can be used effectively to screen for mutations within this gene.     

Noninvasive postoperative ventilation using a buccal mask

Two families with type I plasminogen deficiency and APC resistance are reported. The proposita of family A suffered from ischemic stroke when taking estrogen-progesterone-containing oral contraceptive. Several hemostatic challenges in the past (ovariectomy, appendectomy, and two pregnancies) were without thrombosis. Plasminogen activity and antigen (60 and 58%, normal range 72-136 and 69-135%, respectively) were reduced, and an increased APC resistance (APC-SR= 1.55; normal range 1.8-3.00) associated with G --> A change at 1,691 nucleotide position in exon 10 of FV gene (FV Leiden) was observed. The asymptomatic son had isolated plasminogen deficiency (activity 57% and antigen 60%) whereas the asymptomatic daughter had isolated APC resistance (APC-SR = 1.61) and FV Leiden mutation. The proposita of family B, referred for superficial thrombophlebitis, had low plasminogen levels (activity 55% and antigen 53%) and APC resistance (APC-SR = 1.5) whereas the asymptomatic mother and the brother had isolated APC resistance (APC-SR = 1.62 and 1.8, respectively) and the asymptomatic father isolated plasminogen deficiency (activity 61% and antigen 62%). These data suggest that the combination of plasminogen deficiency and APC resistance probably does not significantly increase the risk of venous thrombosis. However, larger experience with additional cases is needed to definitely assess the magnitude of thrombotic risk in these families.     

Activated protein C resistance--a recently discovered hereditary thrombophilia

Activated protein C resistance is an inherited thrombophilia caused by a point mutation in the factor V gene (G to A transition in nucleotide 1691 in the factor V gene with replacement of arginine (R) 506 by glutamine (Q) in the factor V molecule). The mutation is commonly named factor V R506Q or factor V Leiden. The mutation results in a poor anticoagulant response to activated protein C. APC resistance is inherited autosomally, and approximately 5-10% of the Norwegian population are carriers of the mutation. It is present in 20-50% of all cases of venous thromboembolism. Among asymptomatic heterozygous family members of affected individuals there is a five to eight-fold increase in the risk of venous thromboembolism, whereas there may be a 100-fold increased risk among homozygous individuals. The risk for asymptomatic carriers without a family history is yet not known. Activated protein C resistance is a major risk factor for venous thromboembolism, and the detection of activated protein C resistance is vital for proper prophylaxis and treatment of this disorder. It is essential therefore that as many medical specialists as possible acquire knowledge of activated protein C resistance. This report describes a family with activated protein C resistance and the main indications for screening for inherited thrombophilia.     

Chemotherapy of metastatic endometrial cancer

Hereditary fructose intolerance (HFI) is a potentially fatal autosomal recessive disease of carbohydrate metabolism. HFI patients are deficient in aldolase B, the isozyme expressed in fructose-metabolizing tissues. The eight protein coding exons, including splicing signals, of the aldolase B gene from one American HFI patient were amplified by the polymerase chain reaction (PCR). Single-strand conformational polymorphism (SSCP) analysis and direct sequence determination were applied to the amplified fragments. The mutations in the patient's alleles were identified as a nonsense mutation (R59op) in exon 3 and a missense mutation (C134R) in exon 5. These mutations were confirmed by sequence determination of cloned PCR-amplified exons 3 and 5 from the patient. Allele specific oligonucleotide (ASO) hybridizations of amplified exons 3 and 5 showed the Mendelian inheritance of both mutations. Site-directed mutagenesis was used to generate an expression plasmid for the C134R mutation, and the mutant enzyme was expressed in bacteria. Assays of partially purified enzyme preparations showed that this missense mutation results in an apparently unstable enzyme that retains partial activity. This is the first evidence for a partially active aldolase B from an HFI individual with an identified mutation, and supports the hypothesis that adequate gluconeogenesis/glycolysis is maintained in HFI patients by the presence of partially active enzymes.     

The prevalence of factor V Leiden (1691 G-->A) mutation in Turkey

Resistance to activated protein C (APC), which is caused by a single point mutation in the gene for factor V, is a common risk factor for thrombosis. In this study, we screened factor V (FV) Leiden mutation in 81 subjects. The mutation in the heterozygous form was found in 7.1 percent of our normal population. This high frequency suggests that screening for the FV mutation should be considered in patients with a family history of thrombosis.     

Mutations in the gene for cardiac myosin-binding protein C and late-onset familial hypertrophic cardiomyopathy

BACKGROUND: Mutations in the gene for cardiac myosin-binding protein C account for approximately 15 percent of cases of familial hypertrophic cardiomyopathy. The spectrum of disease-causing mutations and the associated clinical features of these gene defects are unknown. METHODS: DNA sequences encoding cardiac myosin-binding protein C were determined in unrelated patients with familial hypertrophic cardiomyopathy. Mutations were found in 16 probands, who had 574 family members at risk of inheriting these defects. The genotypes of these family members were determined, and the clinical status of 212 family members with mutations in the gene for cardiac myosin-binding protein C was assessed. RESULTS: Twelve novel mutations were identified in probands from 16 families. Four were missense mutations; eight defects (insertions, deletions, and splice mutations) were predicted to truncate cardiac myosin-binding protein C. The clinical expression of either missense or truncation mutations was similar to that observed for other genetic causes of hypertrophic cardiomyopathy, but the age at onset of the disease differed markedly. Only 58 percent of adults under the age of 50 years who had a mutation in the cardiac myosin-binding protein C gene (68 of 117 patients) had cardiac hypertrophy; disease penetrance remained incomplete through the age of 60 years. Survival was generally better than that observed among patients with hypertrophic cardiomyopathy caused by other mutations in the genes for sarcomere proteins. Most deaths due to cardiac causes in these families occurred suddenly. CONCLUSIONS: The clinical expression of mutations in the gene for cardiac myosin-binding protein C is often delayed until middle age or old age. Delayed expression of cardiac hypertrophy and a favorable clinical course may hinder recognition of the heritable nature of mutations in the cardiac myosin-binding protein C gene. Clinical screening in adult life may be warranted for members of families characterized by hypertrophic cardiomyopathy.     

Familial adenomatous polyposis coli in the Czech population. I. Detection of an additional 3 mutations out of a total of 7 in exon 15 of the APC gene

BACKGROUND: Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease characterized by multiple adenomatous polyps in the colon which progress to carcinoma. FAP is caused by germ-line mutation of the tumor-suppressor adenomatous polyposis coli (APC) gene, the structure and coding sequence of which have been known from 1991. The diagnosis of FAP has classically been based on the detection of multiple colorectal adenomas, often after carcinoma development. Presymptomatic genetic testing for the presence of an allele carrying the FAP mutation is now possible using a variety of techniques. METHODS AND RESULTS: The present paper is the first part of an analysis of 37 different Czech families with 83 members affected by FAP. Our goal is to identify the mutation characteristic for each family for early diagnosis of FAP. We screened clinically manifest representatives of nine families for mutations in exon 15 of the APC gene. First, we searched for the mutation hot spots (codons 1061 and 1309, respectively) and later for the entire exon 15. Denatured gradient gel electrophoresis (DGGE) of amplified regions ov exon 15 has been used to identify DNA sequence variations followed by sequencing verification. In seven patients, seven different mutations in exon 15 of APC gene, four deletion mutants (5-base deletions in codons 1061 and 1309, 1-base deletion in codons 759 and 7-base deletion combined with a 2-base insertion in codon 712), one insertion mutation (1-base/A/insertion in codon 1554) and two point mutations (C to T and C to A substitutions in codons 737 and 935, respectively, in both cases leading to formation of stop codons) have been found. CONCLUSIONS: From seven different mutations found, 4 mutations have been previously described (mutations in codons 935, 1061, 1309 and 1554), 3 mutations in the APC gene are described here for the first time, namely the mutations in codons 712, 759 and 767.     

Study of the prothrombin gene 20201 GA variant in FV:Q506 carriers in relationship to the presence or absence of juvenile venous thromboembolism

The G20210A transition of the prothrombin gene has been identified as a common but probably mild hereditary risk factor for venous thromboembolism (VTE). However, the prothrombin gene variant might contribute to the penetrance of thromboembolic disease in many patients with other prothrombotic defects, such as the FV:R506Q mutation. In this investigation, the A20210 allele was found in 9 of 450 healthy controls (2%). Among 89 asymptomatic FV:Q506 carriers, 3 subjects were doubly affected (3.4%). In contrast, of 263 unrelated carriers of the FV:Q506 mutant with a history of juvenile VTE, 30 also had the prothrombin gene G20210A variant (11.4%), including 25 of 220 patients who were heterozygous (11.4%) and 5 of 43 homozygous (11.6%) for FV:Q506. Thus, the A20210 allele of the prothrombin gene is significantly overrepresented in symptomatic FV:Q506 carriers compared with healthy controls (P<0.0001) and asymptomatic relatives carrying the FV mutant (P=0.02). Persons homozygous for the 20210A allele were not found. A statistically significant increase in the prevalence of more unusual sites of venous thrombosis at clinical onset was found in doubly affected patients (9 of 30; 30%) compared with patients without the prothrombin gene variant (26 of 233; 11. 1%) (P=0.004). First VTE occurred spontaneously in 53.3% of all doubly affected patients (16 of 30) and in 28.3% of all simply affected patients (66 of 233) (P=0.005). Among patients with VTE preceded by circumstantial risk factors, the A20210 allele was found in 7.7% (14 of 181). We conclude that the A20210 allele of the prothrombin gene is frequently coinherited in symptomatic FV:Q506 carriers and possibly influences age, site, and type of thrombotic onset manifestation in these patients.     

Analysis of TNFB and TNFA NcoI RFLP in colorectal cancer

The R506Q mutation ("Factor V Leiden") is responsible for the resistance to activated Protein C (aPCR), that is evaluated by coagulation tests. Such tests cannot be used in patients with lupus anticoagulants (LAs), due to the interfering effect exerted by these antibodies on "in vitro" phospholipid-dependent coagulation tests. For this reason, assays have been developed to evaluate aPCR that are insensitive to the presence of LA antibodies. We evaluated two such coagulation tests in the plasma of 82 consecutive patients with LAs. By polymerase chain reaction 3 patients (3.6%) were found heterozygous for the R506Q mutation. aPCR was evaluated by two clotting assays, proposed to be "insensitive" to the presence of LAs: 1. aPCR-tissue factor-based assay, using Factor V deficient plasma and 1:40 diluted test plasma; 2. aPCR-dRVVT-based assay with highly concentrated phospholipids. Their interassay coefficient of variation was 28% and 6.2%, respectively. Compared to the polymerase chain reaction analysis, the 2 tests displayed the following characteristics: sensitivity 67% vs 100%, specificity 92% vs 96%, positive predictive value 25% vs 50%, negative predictive value 99% vs 100%. respectively. Among LA patients without the R506Q mutation, 5 scored positive in the aPCR-tissue factor-based assay, 2 in the aPCR-dRVVT-based assay and another one in both assays. Our findings suggest that the aPCR-dRVVT-based test is more reliable and sensitive than the aPCR-tissue factor-based one to the R506Q mutation in patients with LAs. Both assays, when negative, make unlikely the presence of the R506Q mutation. Polymerase chain reaction analysis remains, however, to be performed when either test is positive.     

Characterization of the genetic defects in recessive type 1 and type 3 von Willebrand disease patients of Italian origin

The genetic defects causing recessive type 1 and type 3 von Willebrand disease (VWD) in eight families from the northern part of Italy have been investigated. Mutations were identified in 14 of the 16 disease-associated von Willebrand factor (VWF) genes. Only one mutation, a stop codon in exon 45, was previously reported. Several new mutations were identified: one cytosine insertion in exon 42, one guanine deletion in exon 28, one probably complete VWF gene deletion, one substitution in the 3' splice site of intron 13, one possible gene conversion, and three candidate missense mutations. One missense mutation, the substitution of a cysteine in exon 42, was identified in all type 3 VWD patients that were previously characterized as a subgroup with significant increase of factor VIII procoagulant activity after desmopressin infusion. This paper demonstrates again that the molecular defects of quantitative VWD are diverse and located throughout the entire VWF gene.     

Germline mutations in the MEN1 gene: basis for predictive genetic screening and clinical management of multiple endocrine neoplasia type 1 (MEN1) families

BACKGROUND AND OBJECTIVE: Mutations in the MEN 1 gene were recently discovered as the causative genetic defect of the autosomal dominantly inherited multiple endocrine neoplasia type 1. It was the aim of this study to evaluate the spectrum of MEN 1 mutations in our own series of patients in order to obtain a basis for predictive family screening. PATIENTS AND METHODS: Genomic DNA from peripheral blood of 21 patients with MEN 1, members of 14 non-related MEN 1 families, was examined for MEN 1 germ-line mutations by means of single-strand conformation variant analysis (SSCP) and direct DNA sequencing. In addition, blood from 20 asymptomatic family members of five families was tested for its predictive value. RESULTS: Eleven different heterozygotic germ-line mutations, among them eight frameshift, two missense and one nonsense mutations, were identified. In four of the 20 asymptomatic members from five MEN 1 families who had been tested after appropriate genetic counselling, the MEN 1 mutation characteristic for the particular family was found. Clinical screening programme in three mutation carriers revealed abnormal findings in all three: one primary hyperparathyroidism, one prolactinoma and one nonfunctioning pancreatic tumour each. The 16 family members without MEN 1 mutation were spared further unnecessary screening investigations. CONCLUSION: Although the function of the MEN 1 gene is not yet known, molecular genetic tests provide a basis for genetic counselling, predictive genetic screening and clinical management of MEN 1 families.