Lipasin/betatrophin is differentially expressed in liver and white adipose tissue without association with insulin resistance in Wistar and Goto-Kakizaki rats.

Lipasin is a recently identified lipokine expressed predominantly in liver and in adipose tissue. It was linked to insulin resistance in mice and to type 1 and type 2 diabetes (T1D, T2D) in humans. No metabolic studies concerning lipasin were performed yet in rats. Therefore, we used rat model of T2D and insulin resistance, Goto-Kakizaki (GK) rats, to determine changes of lipasin expression in liver and in white adipose tissue (WAT) over 52 weeks in the relation to glucose tolerance, peripheral tissue insulin sensitivity and adiposity. GK rats were grossly glucose intolerant since the age of 6 weeks and developed peripheral insulin resistance at the age of 20 weeks. Expression of lipasin in the liver did not differ between GK and Wistar rats, declining with age, and it was not related to hepatic triacylglycerol content. In WAT, the lipasin expression was significantly higher in Wistar rats where it correlated positively with adiposity. No such correlation was found in GK rats. In conclusion, lipasin expression was associated neither with a mild age-related insulin resistance (Wistar), nor with severe genetically-based insulin resistance (GK).

Detection of pancreatic islet allograft impairment in advance of functional failure using magnetic resonance imaging.

This study evaluated the ability of magnetic resonance imaging (MRI) to predict failure of pancreatic islets (PI) transplanted into the hepatic portal vein. Brown-Norway (n = 18) and Lewis (n = 6) rats received islets isolated from Lewis donors. The rejection process in Brown-Norway recipients was mitigated by two different immunosuppressive regimens [tacrolimus + hydrocortisone for 3 months (n = 6) or tacrolimus for 12 days (n = 12)]. Longitudinal MRI monitoring of recipients at post-transplantation weeks 1, 2, 3, 4, 6, 8, 10, and 12 confirmed the ability to detect SPIO labeled PI after transplantation into the liver. The relative number of MRI signals related to PI isografts remained stable up to study completion. Recipients of PI allografts were normoglycemic until the end of study; signals declined gradually to 44 ± 17% in these animals. In animals with islets failure during post-transplant week 12, the number of signals decreased to 25 ± 10% of initial values. The difference between groups (islet function/failed) became significant post-transplant week 3. Our data demonstrate that the MRI changes attributable to rejection become apparent within 3 weeks after transplantation, i.e. at least 8 weeks before functional allograft failure.

Expression of 14 von Willebrand factor mutations identified in patients with type 1 von Willebrand disease from the MCMDM-1VWD study.

BACKGROUND: Candidate von Willebrand factor (VWF) mutations were identified in 70% of index cases in the European study 'Molecular and Clinical Markers for the Diagnosis and Management of type 1 von Willebrand Disease'. The majority of these were missense mutations. OBJECTIVES: To assess whether 14 representative missense mutations are the cause of the phenotype observed in the patients and to examine their mode of pathogenicity. METHODS: Transfection experiments were performed with full-length wild-type or mutant VWF cDNA for these 14 missense mutations. VWF antigen levels were measured, and VWF multimer analysis was performed on secreted and intracellular VWF. RESULTS: For seven of the missense mutations (G160W, N166I, L2207P, C2257S, C2304Y, G2441C, and C2477Y), we found marked intracellular retention and impaired secretion of VWF, major loss of high molecular weight multimers in transfections of mutant constructs alone, and virtually normal multimers in cotransfections with wild-type VWF, establishing the pathogenicity of these mutations. Four of the mutations (R2287W, R2464C, G2518S, and Q2520P) were established as being very probably causative, on the basis of a mild reduction in the secreted VWF or on characteristic faster-running multimeric bands. For three candidate changes (G19R, P2063S, and R2313H), the transfection results were indistinguishable from wild-type recombinant VWF and we could not prove these changes to be pathogenic. Other mechanisms not explored using this in vitro expression system may be responsible for pathogenicity. CONCLUSIONS: The pathogenic nature of 11 of 14 candidate missense mutations identified in patients with type 1 VWD was confirmed. Intracellular retention of mutant VWF is the predominant responsible mechanism.

Detailed von Willebrand factor multimer analysis in patients with von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease (MCMDM-1VWD).

BACKGROUND: Type 1 von Willebrand disease (VWD) is a congenital bleeding disorder characterized by a partial quantitative deficiency of plasma von Willebrand factor (VWF) in the absence of structural and/or functional VWF defects. Accurate assessment of the quantity and quality of plasma VWF is difficult but is a prerequisite for correct classification. OBJECTIVE: To evaluate the proportion of misclassification of patients historically diagnosed with type 1 VWD using detailed analysis of the VWF multimer structure. PATIENTS AND METHODS: Previously diagnosed type 1 VWD families and healthy controls were recruited by 12 expert centers in nine European countries. Phenotypic characterization comprised plasma VWF parameters and multimer analysis using low- and intermediate-resolution gels combined with an optimized visualization system. VWF genotyping was performed in all index cases (ICs). RESULTS: Abnormal multimers were present in 57 out of 150 ICs; however, only 29 out of these 57 (51%) had VWF ristocetin cofactor to antigen ratio below 0.7. In most cases multimer abnormalities were subtle, and only two cases had a significant loss of the largest multimers. CONCLUSIONS: Of the cases previously diagnosed as type 1 VWD, 38% showed abnormal multimers. Depending on the classification criteria used, 22 out of these 57 cases (15% of the total cohort) may be reclassified as type 2, emphasizing the requirement for multimer analysis compared with a mere ratio of VWF functional parameters and VWF:Ag. This is further supported by the finding that even slightly aberrant multimers are highly predictive for the presence of VWF mutations.

Impact of plasma von Willebrand factor levels in the diagnosis of type 1 von Willebrand disease: results from a multicenter European study (MCMDM-1VWD).

BACKGROUND: Presence of bleeding symptoms, inheritance and reduced von Willebrand factor (VWF) contribute to the diagnosis of type 1 von Willebrand disease (VWD). However, quantitative analysis of the importance of VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo) levels in the diagnosis is lacking. OBJECTIVES: To evaluate the relative contribution of VWF measurement to the diagnosis of VWD. PATIENTS AND METHODS: From the MCMDM-1VWD study cohort, 204 subjects (considered as affected by VWD based on the enrolling Center diagnoses and the presence of linkage with the VWF locus) were compared with 1155 normal individuals. Sensitivity, specificity and diagnostic positive likelihood ratios (LR) of VWF:Ag and VWF:RCo were computed. RESULTS: ABO blood group was the variable most influencing VWF levels, but adjustment of the lower reference limit for the ABO group did not improve sensitivity and specificity of VWF:Ag or VWF:RCo. The lower reference limit (2.5th percentile) was 47 IU dL(-1) for both VWF:Ag and VWF:RCo and showed similar diagnostic performance [receiver-operator curve area: 0.962 and 0.961 for VWF:Ag and VWF:RCo, respectively; P = 0.81]. The probability of VWD was markedly increased only for values below 40 IU dL(-1) (positive LR: 95.1 for VWF:Ag), whereas intermediate values (40 to 60 IU dL(-1)) of VWF only marginally indicated the probability of VWD. CONCLUSIONS: Although the conventional 2.5 lower percentile has good sensitivity and specificity, only VWF:Ag or VWF:RCo values below 40 IU dL(-1) appear to significantly indicate the likelihood of type 1 VWD. The LR profile of VWF level could be used in a diagnostic algorithm.

[Evaluation of DHPLC analysis for mutation detection in haemophilia A]. / Testování DHPLC analýzy pro detekci mutací u hemofilie A.

BACKGROUND: Haemophilia A is one of the most prevalent inherited bleeding disorders. Causal mutations in the factor VIII gene are detected to facilitate the genetic counselling and to estimate the risk of serious complication associated with standard treatment (factor VIII inhibitor). Wide range of mutations located across the entire length of the factor VIII gene underlies the factor VIII deficiency of variable severity. The only two common recurrent mutations in the factor VIII gene are intron 22 and intron I inversions. In the remaining cases it is necessary to screen all 26 exons encoding 9kb mRNA together with adjacent nonncoding sequences. In order to speed up genotyping in haemophilia A families in the Czech Republic we evaluated DHPLC-based screening technique. METHODS AND RESULTS: We tested sensitivity of the analysis on a panel of DNA samples containing 49 different sequence variations distributed over 21 exons. All the genetic alterations were readily detected. Analysis of family members has shown good reproducibility of the respective elution patterns. DHPLC analysis detected mutations in 4 out of 5 samples from apparently unrelated haemophilia patients, where previously applied multiplex CSGE was not successful. CONCLUSIONS: Establishing of DHPLC analysis will substantially speed up the genotyping of haemophilia A families in the Czech Republic.

A quantitative analysis of bleeding symptoms in type 1 von Willebrand disease: results from a multicenter European study (MCMDM-1 VWD).

BACKGROUND: A quantitative description of bleeding symptoms in type 1 von Willebrand disease (VWD) has never been reported. OBJECTIVES: The aim was to quantitatively evaluate the severity of bleeding symptoms in type 1 VWD and its correlation with clinical and laboratory features. PATIENTS AND METHODS: Bleeding symptoms were retrospectively recorded in a European cohort of VWD type 1 families, and for each subject a quantitative bleeding score (BS) was obtained together with phenotypic tests. RESULTS: A total of 712 subjects belonging to 144 families and 195 controls were available for analysis. The BS was higher in index cases than in affected family members (BS 9 vs. 5, P < 0.0001) and in unaffected family members than in controls (BS 0 vs. -1, P < 0.0001). There was no effect of ABO blood group. BS showed a strong significant inverse relation with either von Willebrand ristocetin cofactor (VWF:RCo), von Willebrand antigen (VWF:Ag) or factor VIII procoagulant activity (FVIII:C) measured at time of enrollment, even after adjustment for age, sex and blood group (P < 0.001 for all the four upper quintiles of BS vs. the first quintile, for either VWF:RCo, VWF:Ag or FVIII:C). Higher BS was related with increasing likelihood of VWD, and a mucocutaneous BS (computed from spontaneous, mucocutaneous symptoms) was strongly associated with bleeding after surgery or tooth extraction. CONCLUSIONS: Quantitative analysis of bleeding symptoms is potentially useful for a more accurate diagnosis of type 1 VWD and to develop guidelines for its optimal treatment.

Linkage analysis in families diagnosed with type 1 von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 VWD.

BACKGROUND: von Willebrand disease (VWD) type 1 is a congenital bleeding disorder caused by genetic defects in the von Willebrand factor (VWF) gene and characterized by a reduction of structurally normal VWF. The diagnosis of type 1 VWD is difficult because of clinical and laboratory variability. Furthermore, inconsistency of linkage between type 1 VWD and the VWF locus has been reported. OBJECTIVES: To estimate the proportion of type 1 VWD that is linked to the VWF gene. PATIENTS AND METHODS: Type 1 VWD families and healthy control individuals were recruited. An extensive questionnaire on bleeding symptoms was completed and phenotypic tests were performed. Linkage between VWF gene haplotypes and the diagnosis of type 1 VWD, the plasma levels of VWF and the severity of bleeding symptoms was analyzed. RESULTS: Segregation analysis in 143 families diagnosed with type 1 VWD fitted a model of autosomal dominant inheritance. Linkage analysis under heterogeneity resulted in a summed lod score of 23.2 with an estimated proportion of linkage of 0.70. After exclusion of families with abnormal multimer patterns the linkage proportion was 0.46. LOD scores and linkage proportions were higher in families with more severe phenotypes and with phenotypes suggestive of qualitative VWF defects. About 40% of the total variation of VWF antigen could be attributed to the VWF gene. CONCLUSIONS: We conclude that the diagnosis of type 1 VWD is linked to the VWF gene in about 70% of families, however after exclusion of qualitative defects this is about 50%.

[New approaches to haemophilia treatment--recombinant and transgenic concentrates, gene therapy and engineered coagulation factors]. / Nové obzory v lécbe hemofilie--rekombinantní a transgenní koncentráty, genová lécba a modifikované koagulacní faktory.

Plasma-derived concentrates of coagulation factors VIII and IX allow for effective treatment of haemophilia A and B. Current problems associated with this therapy include induction of inhibitory antibody directed towards coagulation factor (inhibitor), requirement of frequent intravenous application since childhood, very high price precluding utilisation of the treatment in developing countries and very low risk of transmission of known human infections. Cloning of factors VIII and IX allowed for application of recombinant technologies to address these problems. Small and large animal models have become available to test effectiveness and safety of novel treatments. The third generation recombinant concentrates of factors VIII and IX have recently been approved, which overcome the risk of transmission of a human infection. However, the problem of the inhibitory antibody response remains. Recombinant activated factor VIIa has become effective, safe but expensive alternative for treatment of patients with the inhibitor. High price of recombinant concentrates precludes their use in developing countries. Transgenic animals may in future allow for production of cheaper concentrates. Concluded clinical trials with therapeutic gene transfer in haemophilia A and B have shown that the approach is relatively safe, but not yet effective. Ongoing studies in vitro and in vivo are trying to improve the effectiveness. Engineered molecules of factors VIII and IX already tested in vitro and in vivo, on animal models may in future improve both classical and gene therapies.

[Genetic databases. Though we were not the first ones, at least let us not to be the last]. / Genetické databáze. Kdyz uz nemuzeme být první, at' nejsme alespon poslední.

One of the most necessary tools of today's medical care is the well working database of personal genotypes. It could effectively reduce the increasing expenses because many of the genetic testing could be done only once a life. Any delay in establishing such database would bring not only internal, but also across-border complications due to internationalization of the genetic services. The most modem approach has been applied by the laws which put stress more against abuse than on collecting data. European laws including the Czech Republic seem to be much less progressive. General rules for data storage, and gene banking are still missing in the Czech Republic.

[Molecular pathogenesis of haemophilia A]. / Molekulární patogeneze hemofilie A.

Haemophilia A is one of the most common inherited bleeding disorders. Bleeding phenotype is caused by mutations within factor VIII gene, which result in deficient activity of the factor VIII in plasma. Various genetic defects have been described to affect factor VIII transcription, translation or its amino acid composition. Utilisation of 3D-model of the factor VIII helped to identify amino acid substitutions which adversely affect the protein folding and those which disturb various factor VIII interactions. Genetic defects have been stratified with respect to the relative risk of factor VIII inhibitor formation and a mechanism by which the antibody inhibits its function has been described. The knowledge has been utilised in current diagnostic practice and developement of future therapeutic strategies.

[Molecular diagnosis of haemophilia A in clinical practice]. / Molekulární diagnostika hemofilie A v klinické praxi.

Understanding the pathogenesis of haemophilia A has allowed for detailed diagnosis of the condition at molecular level. Evaluation of interaction between factor VIII and von Willebrand factor has been utilised to distinguish mild forms of haemophilia A from von Willebrand disease. Discovery of wide spectrum of mutations in the factor VIII gene and their association with different severity of the disease allowed for development of a rational strategy for mutation detection in clinical settings. Characterisation of the genetic defects is required for carrier detection and antenatal testing and it also helps to predict risk of factor VIII inhibitor development. Research is ongoing to establish less invasive prenatal testing and to move the testing to pre-gravid period.

Laboratory tests for precise classification and genetic analysis of von Willebrand disease.

von Willebrand disease (vWD) is the most common inherited bleeding disorder, which is highly heterogeneous ranging from asymptomatic laboratory abnormality to a life threatening bleeding. The condition is caused by a quantitative or qualitative deficiency of von Willebrand factor (vWF). Since 1994 it has been classified into six subgroups based on evaluation of the vWF level and function. Correct classification of vWD is required for its optimal management. vWD is inherited as a dominant or recessive trait linked to the vWF gene. However, the inheritance of type 1 vWD is not always linked to the vWF gene and novel modifying genes are expected to play a significant role in the type 1 pathophysiology. Laboratory tests required for correct type classification and for genetic analysis have long been awaited in Czech Republic. The purpose of this report is to inform about discriminatory and genetic tests recently made available at the Institute of Haematology and Blood transfusion.

[Molecular study of type 2 von Willebrand disease]. / Molekulární studie von Willebrandovy choroby typu 2.

BACKGROUND: von Willebrand disease is an inherited bleeding disorders caused by mutations in the von Willebrand factor gene. We attempted to characterise the phenotype and the genotype in the first five families in Czech Republic affected by this heterogeneous disorder. METHODS AND RESULTS: The level of FVIII was measured by the one stage assay, the vWF:Ag by the immunoelectrophoresis, vWF:RiCo by aggregometry. For the vWF multimer analysis a western blot based technique was used. The vWF binding to FVIII was evaluated by the ELISA method. Two families were classified as the type 2A, one as the type 2B and two as the combined type 1/2N. Based on that knowledge, parts of the vWF gene were selected for genetic analysis. The previously described mutations Arg1374His and Gly1579Arg were identified in two families with the type 2A. In the family with type 2B a substitution Arg1308Cys was detected. In one family with the type 1/2N, two different previously described defects were found on the separate alleles of the vWF gene: a deletion of cytosine 2435 and a polymorphism Arg854Gln. Compound heterzygotes had the type 1/2N phenotype, while a carriers of the deletion had type 1 phenotype. In the second type 1/2N family, only the amino acid substitutions Thr791Me was found explaining the qualitative defect. A mutation underlying the quantitative deficiency needs to be searched for throughout the entire vWF gene. CONCLUSIONS: Based on the characterisation of the phenotype and genotype, five apparently unrelated families with the von Willebrand disease were diagnosed according to the revised classification. Our work represents laboratory basis for further studies into von Willebrand disease in Czech Republic.

Thirty-four novel mutations detected in factor VIII gene by multiplex CSGE: modeling of 13 novel amino acid substitutions.

Detection of causal mutations is required for genetic counseling. Molecular modeling combined with patients' phenotype provides significant insight into structure-function relationship of factor (F)VIII molecule. Our objective was to identify defects in the gene of FVIII by a sensitive and simple scanning technique with high throughput in order to study molecular mechanisms by which novel amino acid substitutions may lead to hemophilia A. A cohort of 81 families with mild, moderate and severe hemophilia A negative in intron 22 inversion was studied. For detection of mutations in the FVIII gene a conformation sensitive gel electrophoresis (CSGE) was modified by multiplexing. Thirteen novel amino acid substitutions were studied by molecular modeling and a correlation with the cross-reactive material (CRM) phenotype was performed. In 74 families, 59 different mutations were detected. Six different mutations were recurrent in 21 unrelated families. Thirty-four novel mutations included 19 point mutations, four small insertions, nine small deletions and two complex mutations. Thirteen novel amino acid substitutions occurred at residues conserved in FVIII orthologs. Five of them were associated with a discrepancy between FVIII activity and antigen; another five with CRM reduced phenotype and one with undetectable FVIII antigen. Multiplexing of the CSGE significantly increased its throughput without substantial loss of sensitivity. Molecular modeling suggested mechanisms by which substitutions at residues 382 and 569, located outside the proposed FIXa-binding region, may influence FVIII/FIXa interaction. His2155 was predicted to participate in FVIII/VFW binding.

[Molecular basis of hereditary antithrombin defects in 10 Czech families]. / Molekulární podstata vrozeného defektu antitrombinu u deseti ceských rodin.

BACKGROUND: Molecular basis of antithrombin deficiency has not yet been studied in Czech Republic. We looked for the causal mutations throughout the antithrombin gene in 26 patients from 10 unrelated families with antithrombin defect. METHODS AND RESULTS: We screened the gene by conformation sensitive gel electrophoresis and sequenced the mismatched regions using fluorescence technology to characterise mutations and polymorphisms. Mutations were detected in all ten families. Four novel mutations were identified in four families with type I antithrombin defect: Trp-6Arg, 5386-5387delCT, Glu163Stop, and 13246-13248del TGA causing deletion of Glu377 with change of Asn376 to Lys. In other three type I families we found following mutations: splicing site mutation G2777C, Arg197Stop and entire gene deletion. In the family carrying Trp-6Arg mutation antithrombin Vienna (Gln118Pro) was also detected. Leu99Phe recurrent in south-eastern Europe was identified in three families with type II defect. Only the homozygous carries of the mutation were symptomatic, although the heterozygous carries had decreased functional levels. CONCLUSIONS: Four novel mutations in families with type I antithrombin deficiency were characterised. In one family two different genetic defects were identified to be responsible for type I and II phenotypes. Altogether our data agree with the expected heterogeneity of the AT genetic defect.