Qualitative and quantitative analysis of FBN1 mRNA from 16 patients with Marfan Syndrome.

BACKGROUND: Pathogenic mutations in FBN1, encoding the glycoprotein, fibrillin-1, cause Marfan syndrome (MFS) and related connective tissue disorders. In the present study, qualitative and quantitative effects of 16 mutations, identified in FBN1 in MFS patients with systematically described phenotypes, were investigated in vitro. METHODS: Qualitative analysis was performed with reverse transcription-PCR (RT-PCR) and gel electrophoresis, and quantitative analysis to determine the FBN1 mRNA levels in fibroblasts from the 16 patients with MFS was performed with real-time PCR. RESULTS: Qualitative analysis documented that the mutations c.4817-2delA and c.A4925G led to aberrant FBN1 mRNA splicing leading to in frame deletion of exon 39 and in exon 39, respectively. No difference in the mean FBN1 mRNA level was observed between the entire group of cases and controls, nor between the group of patients with missense mutations and controls. The mean expression levels associated with premature termination codon (PTC) and splice site mutations were significantly lower than the levels in patients with missense mutations. A high level of FBN1 mRNA in the patient with the missense mutation c.G2447T did not segregate with the mutation in three of his first degree relatives. No association was indicated between the FBN1 transcript level and specific phenotypic manifestations. CONCLUSIONS: Abnormal FBN1 transcripts were indicated in fibroblasts from patients with the splice site mutation c.4817-2delA and the missense mutation c.A4925G. While the mean FBN1 mRNA expression level in fibroblasts from patients with splice site and PTC mutations were lower than the mean level in patients with missense mutations and controls, inter-individual variability was high. The observation that high level of FBN1 mRNA in the patient with the missense mutation c.G2447T did not segregate with the mutation in the family suggests that variable expression of the normal FBN1 allele may contribute to explain the variability in FBN1 mRNA level.

Management of Wrong Blood Transfusion to an RhD Negative Woman in Labor.

Blood transfusion is life-saving in massive hemorrhage. Before pre-transfusion tests with ABO and RhD typing results are available, O RhD negative packed red blood cell (PRBC) units are used without cross-matching in emergency. RhD negative girls and women of child-bearing age should always receive RhD negative blood transfusions to prevent RhD-alloimmunization because anti-D-related hemolytic disease of fetus and newborn (HDFN) can result in mild to severe anemia, and in a worst-case scenario death of an RhD positive fetus and/or newborn. However, "wrong blood to wrong patient" happens unintentionally. Here we report an emergency blood transfusion with one unit of RhD positive PRBCs to an RhD negative young woman when estimated blood loss was 2500 mL during delivery and surgical removal of retained placenta. Realizing the mistake, management with high dose anti-D immunoglobulin (Ig) was initiated to remove the RhD positive red blood cells (RBCs) from the patient's circulation. Such mitigation is recommended only for girls and women of child-bearing age. Follow-up was performed by flow cytometry until RhD positive RBCs were no longer detected. Ten months after the delivery, antibody screening was negative. However, we still do not know whether we managed to prevent RhD-alloimmunization.

Functional characterization of the protein C A267T mutation: evidence for impaired secretion due to defective intracellular transport.

BACKGROUND: Activated protein C (PC) is a serine protease that regulates blood coagulation by inactivating coagulation factors Va and VIIIa. PC deficiency is an autosomally inherited disorder associated with a high risk of recurrent venous thrombosis. The aim of the study was to explore the mechanisms responsible for severe PC deficiency in a patient with the protein C A267T mutation by in-vitro expression studies. RESULTS: Huh7 and CHO-K1 cells were transiently transfected with expression vectors containing wild-type (WT PC) and mutated PC (A267T PC) cDNAs. PC mRNA levels were assessed by qRT-PCR and the PC protein levels were measured by ELISA. The mRNA levels of WT PC and A267T PC were similar, while the intracellular protein level of A267T PC was moderately decreased compared to WT PC. The secretion of A267T PC into the medium was severely impaired. No differences in molecular weights were observed between WT and A267T PC before and after treatment with endo-ß-N-acetylglucosaminidase. Proteasomal and lysosomal degradations were examined using lactacystin and bafilomycin, respectively, and revealed that A267T PC was slightly more susceptible for proteasomal degradation than WT PC. Intracellular co-localization analysis indicated that A267T PC was mainly located in the endoplasmic reticulum (ER), whereas WT PC was observed in both ER and Golgi. CONCLUSIONS: In contrast to what has been reported for other PC mutants, intracellular degradation of A267T PC was not the main/dominant mechanism underlying the reduced intracellular and secretion levels of PC. Our results indicate that the A267T mutation most likely caused misfolding of PC, which might lead to increased retention of the mutated PC in ER.

Rapid and efficient FBN1 mutation detection using automated sample preparation and direct sequencing as the primary strategy.

Mutations in the fibrillin-1 (FBN1) gene cause Marfan syndrome (MFS) and the other type-1 fibrillinopathies. Finding these mutations is a major challenge considering that the FBN1 gene has a coding region of 8,600 base pairs divided into 65 exons. Most of the more than 600 known mutations have been identified using a mutation scanning method prior to sequencing of fragments with a suspected mutation. However, it is not obvious that these screening methods are ideal, considering cost, efficiency, and sensitivity. We have sequenced the entire FBN1 coding sequence and flanking intronic sequences in samples from 105 patients with suspected MFS, taking advantage of robotic devices, which reduce the cost of supplies and the quantity of manual work. In addition, automation avoids many tedious steps, thus reducing the opportunity for human error. Automated assembling of PCR, purification of PCR products, and assembly of sequencing reactions resulted in completion of the FBN1 sequence in half of the time needed for the manual protocol. Mutations were identified in 69 individuals. The mutation detection rate (76%), types, and genetic distribution of mutations resemble the findings in other MFS populations. We conclude that automated sequencing using the robotic systems is well suited as a primary strategy for diagnostic mutation identification in FBN1.

Protein C mutation (A267T) results in ER retention and unfolded protein response activation.

BACKGROUND: Protein C (PC) deficiency is associated with a high risk of venous thrombosis. Recently, we identified the PC-A267T mutation in a patient with PC deficiency and revealed by in vitro studies decreased intracellular and secreted levels of the mutant. The aim of the present study was to characterize the underlying mechanism(s). METHODOLOGY/PRINCIPAL FINDINGS: CHO-K1 cells stably expressing the wild-type (PC-wt) or the PC mutant were generated. In order to examine whether the PC mutant was subjected to increased intracellular degradation, the cells were treated with several inhibitors of various degradation pathways and pulse-chase experiments were performed. Protein-chaperone complexes were analyzed by treating the cells with a cross-linker followed by Western blotting (WB). Expression levels of the immunoglobulin-binding protein (BiP) and the phosphorylated eukaryotic initiation factor 2α (P-eIF2α), both common ER stress markers, were determined by WB to examine if the mutation induced ER stress and unfolded protein response (UPR) activation. We found no major differences in the intracellular degradation between the PC variants. The PC mutant was retained in the endoplasmic reticulum (ER) and had increased association with the Grp-94 and calreticulin chaperones. Retention of the PC-A267T in ER resulted in UPR activation demonstrated by increased expression levels of the ER stress markers BiP and P-eIF2α and caused also increased apoptotic activity in CHO-K1 cells as evidenced by elevated levels of DNA fragmentation. CONCLUSIONS/SIGNIFICANCE: The reduced intracellular level and impaired secretion of the PC mutant were due to retention in ER. In contrast to other PC mutations, retention of the PC-A267T in ER resulted in minor increased proteasomal degradation, rather it induced ER stress, UPR activation and apoptosis.

Hereditary protein C deficiency caused by the Ala267Thr mutation in the protein C gene is associated with symptomatic and asymptomatic venous thrombosis.

INTRODUCTION: Protein C (PC) is a key anticoagulant that regulates hemostasis, and inherited deficiency of PC is an established risk factor for venous thrombosis (VT). The factor V Leiden mutation causing activated PC (APC) resistance is an additional risk factor for VT. Reduced PC levels in the circulation and/or APC resistance do not necessarily lead to thrombotic disease. In the present study we describe and characterize an ethnic Lebanese family in which individuals with reduced PC levels and APC resistance have various clinical symptoms. METHODS: PC activity and antigen levels and APC resistance in the family members were quantified with commercial kits. Sequencing of PC DNA and mRNA was performed with BigDye Terminator Cycle Sequencing kit on the ABI 3730 Genetic Analyzer. RESULTS: PC antigen and anticoagulant activity in the plasma of the proband and family members ranged from 9% to 69% and 3% to 63%, respectively, compared to levels measured in pooled normal plasma. Sequencing analysis of the PC gene of family members revealed that they were either homozygous or heterozygous for the Ala267Thr mutation. In addition, three of them exhibited APC resistance. None of the family members, except the proband, have had a history of VT despite that two of them have two genetic risk factors for thrombosis.

Prevalence data on all Ghent features in a cross-sectional study of 87 adults with proven Marfan syndrome.

The prevalence of each single feature in the Ghent criteria in patients with Marfan syndrome (MFS) is not known. To elucidate this, a cross-sectional study of 105 adults with presumed MFS was carried out. All patients were examined by the same group of investigators with standardized and complete assessment of all features in the Ghent criteria. Eighty-seven (83%) fulfilled the criteria in 56 different variants. The most prevalent major criterion in Ghent-positive persons was dural ectasia (91%), followed by major genetic criterion (89%) and ectopic lenses (62 %). In 14 persons (16%), the diagnosis was dependent on the dural findings. In all, 79% fulfilled both major dural and major genetic (positive family history and/or FBN1 mutation) criteria, suggesting that most patients with MFS might be identified by investigating these criteria. A history or finding of ascending aortic disease was present in 46 patients (53%). This low prevalence might partly reflect a high number of diagnosed patients encompassing the whole spectrum of the syndrome. The study confirms the need to examine for the complete set of features in the Ghent criteria to identify all patients with MFS. The majority of persons with MFS might be identified by the combined assessment of dura mater and family history, supplemented with DNA analysis in family-negative cases. The low prevalence of ascending aortic disease might indicate better future prospects in an adult population than those traditionally considered.

Search for correlations between FBN1 genotype and complete Ghent phenotype in 44 unrelated Norwegian patients with Marfan syndrome.

In monogenic disorders, correlation between genotype and phenotype is a premise for predicting prognosis in affected patients. Predictive genetic testing may enable prophylaxis and promote clinical follow-up. Although Marfan syndrome (MFS) is known as a monogenic disorder, according to the present diagnostic criteria a mutation in the gene FBN1 is not sufficient for the diagnosis, which also depends on the presence of a number of clinical, radiological, and other findings. The fact that MFS patient cohorts only infrequently have been examined for all relevant phenotypic manifestations may have contributed to inconsistent reports of genotype-phenotype correlations. In the Norwegian Study of Marfan syndrome, all participants were examined for all findings contained in the Ghent nosology by the same investigators. Mutation identification was carried out by robot-assisted direct sequencing of the entire FBN1 coding sequence and MLPA analysis. A total of 46 mutations were identified in 44 unrelated patients, all fulfilling Ghent criteria. Although no statistically significant correlation could be obtained, the data indicate associations between missense or splice site mutations and ocular manifestations. While mutations in TGF-domains were associated with the fulfillment of few major criteria, severe affection was indicated in two cases with C-terminal mutations. Intrafamilial phenotypic variation among carriers of the same mutation, suggesting the influence of epigenetic facors, complicates genetic counseling. The usefulness of predictive genetic testing in FBN1 mutations requires further investigation.