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Background: This study aimed to conduct molecular diagnostics among individuals with hemophilia B (HB) and carriers of hemophilia in Mongolia. Methods: Eight patients (six severe, two mild) with HB and their 12 female relatives were enrolled from eight families. Sanger sequence was performed for mutation identification. The questionnaire survey was conducted to evaluate carrier symptoms in female relatives. Results: Two families had a history of HB. A total of five different variants (c.223C > T; c.344A > G; c.464G > C; c.187_188del; and c.1314_1314delA) were identified in six patients with severe HB. Of these, two (c.187_188del and c.1314_1314delA) were novel. No variant in the entire F9 was found in two patients with mild HB. Nonsense c.223C > T (p.Arg75*) mutation was detected in two unrelated patients. Carrier testing identified five mothers as carriers, while one younger sister was a non‐carrier. The carrier status of six female relatives of the two mild patients remained undetermined. By questionnaire survey, only one of the five genetically identified carriers displayed noticeable symptoms of being a carrier. Conclusion: The novel variants c.187_188del and c.1314_1314delA can cause severe hemophilia B. This study did not observe a significant association between symptoms and carrier status in the five carriers. [ABSTRACT FROM AUTHOR]

Hemophilia B (HB) is a hereditary bleeding disorder caused by the genetic variation of the coagulation factor IX (FIX) gene (F9). Several F9 structural abnormalities, including large deletion and/or insertion, have been observed to cause HB development. However, there is limited information available on F9 deep intronic variations. In this study, we report about a novel large deletion/insertion observed in a deep region of F9 intron 1 that causes mRNA splicing abnormalities. The patient was a Japanese male diagnosed with moderate HB (FIX:C = 3.0 IU/dL). The genomic DNA of the patient was isolated from peripheral blood leukocytes. DNA sequences of F9 exons and splice donor/acceptor sites were analyzed via polymerase chain reaction and Sanger sequencing. Variant-affected F9 mRNA aberration and FIX protein production, secretion, and coagulant activity were analyzed by cell-based exon trap and splicing-competent FIX expression vector systems. A 28-bp deletion/476-bp insertion was identified in the F9 intron 1 of a patient with moderate HB. A DNA sequence identical to a part of the inverted HNRNPA1 exon 12 was inserted. Cell-based transcript analysis revealed that this large intronic deletion/insertion disrupted F9 mRNA splicing pattern, resulting in reduction of protein-coding F9 mRNA. A novel deep intronic F9 rearrangement was identified in a Japanese patient with moderate HB. Abnormal F9 mRNA splicing pattern due to this deep intronic structural variation resulted in a reduction of protein-coding F9 mRNA, which probably caused the moderate HB phenotype. • An F9 intronic structural variation was found in a moderate hemophilia B patient. • A part of inverted HNRNPA1 exon 12 was inserted into the F9 deep intron 1. • This F9 deep intronic variation caused aberrant F9 mRNA splicing. • The splicing aberration caused a reduction in the protein-coding F9 mRNA. • The F9 deep intron 1 structural variation resulted in a reduction of FIX production. [ABSTRACT FROM AUTHOR]

Introduction: Hemophilia B is associated with molecular heterogeneity, with more than 1200 unique variants in the F9 gene. We hereby describe the mutational spectrum of severe hemophilia B patients presenting in a tertiary‐care center in India. Method: DNA was extracted from peripheral blood samples of 35 diagnosed severe hemophilia B patients belonging to 32 families, and were subjected to Sanger sequencing. Determination of the effect of novel variants on the protein structure and correlation between genotype and phenotype was attempted using in‐silico tools. Results: Twenty‐seven different mutations were detected in 30 probands, including 20 known and 7 novel variants. Also, we found one suspected case of whole gene deletion. The serine peptidase domain harbored most of the variants (48.1%). Inhibitory antibodies were found in two patients. Conclusions: This study provides a comprehensive mutational spectrum and mutation screening strategy by Sanger sequencing of F9 gene in severe hemophilia B patients, in a resource‐constraint setting. [ABSTRACT FROM AUTHOR]

Background: Hemophilia B (HB) is an X‐linked recessive inherited bleeding disorder caused by mutations in the F9 gene that lead to plasma factor IX deficiency. To identify the causative mutations in HB, a molecular analysis of HB pedigrees in China was performed. Methods: Using next‐generation sequencing (NGS) and an in‐house bioinformatics pipeline, 76 unrelated HB pedigrees were analyzed. The mutations identified were validated by comparison with the results of Sanger sequencing or Multiplex Ligation‐dependent Probe Amplification assays. The pathogenicity of the causative mutations was classified following the American College of Medical Genetics and Genomics guidelines. Results: The mutation detection rate was 94.74% (72/76) using NGS. Of the 76 HB pedigrees analyzed, 59 causative variants were found in 72 pedigrees, with 38 (64.41%) missense mutations, 9 (15.25%) nonsense mutations, 2 (3.39%) splicing mutations, 5 (8.47%) small deletions, 4 (6.78%) large deletions, and 1 intronic mutation (1.69%). Of the 59 different F9 mutations, 10 were novel: c.190T>G, c.199G>T, c.290G>C, c.322T>A, c.350_351insACAATAATTCCTA, c.391+5delG, c.416G>T, c.618_627delAGCTGAAACC, c.863delA, and c.1024_1027delACGA. Of these 10 novel mutations, a mosaic mutation, c.199G>T(p.Glu67Ter), was identified in a sporadic HB pedigree. Using in‐silico analysis, these novel variants were predicted to be disease‐causing. However, no potentially causative mutations were found in the F9 coding sequences of the four remaining HB pedigrees. In addition, two HB pedigrees carrying additional F8/F9 mutations were discovered. Conclusion: The identification of these mutations enriches the spectrum of F9 mutations and provides further insights into the pathogenesis of HB in the Chinese population. [ABSTRACT FROM AUTHOR]

Background: Hemophilia B (HB) is a coagulation disorder with an X‐linked recessive inheritance pattern, caused by plasma FIX deficiency. In Colombia, HB is considered a rare and high‐cost disease, with 362 males reported in 2017. Methods: Here, we characterized 20 HB apparently unrelated families by PCR amplification and Sanger sequencing. Results: Fourteen unique variants were identified: seven missense, three nonsense, one variant in the 3′ UTR region, two large deletions >50 bp, and one intronic substitution that affects splicing c.520+13A>G that was present in 7/20 patients (35%). All these variants have been previously reported in the literature, except for exons 3 and 4, deletions, present in one patient. The genotype‐phenotype association correlates with the reported in the literature, with the exception of one patient. Conclusion: This molecular analysis allowed us to establish the causal variant of HB in 100% of patients, to provide the appropriate genetic counseling to each of the families, and to propose a more cost‐effective carrier analysis. Here, we reported the first variants in Colombian population with Hemophilia B, finding a new variant and one intron recurrent variant present in 35% of patients. [ABSTRACT FROM AUTHOR]

• Mutations in F9 gene may either hinders synthesis and secretion of FIX, or impairs its function. • The novel mutation p.Asp315Tyr happens to affect one critical residue of catalytic triad. • The novel mutation p.Asp315Tyr impairs the proper folding and/or destabilize the overall structure of FIX. [ABSTRACT FROM AUTHOR]

Background: Hemophilia B is an X‐linked recessive disorder caused by mutations in the F9 on Xq27.1. Mainly males are affected but about 20% of female carriers have clotting factor IX activity below 0.40 IU/ml and bleeding problems. Fragile‐X syndrome (FMR1) and FRAXE syndrome (AFF2) are well‐known causes of X‐linked recessive intellectual disability. Simultaneous deletion of both FMR1 and AFF2 in males results in severe intellectual disability. In females the phenotype is more variable. We report a 19‐year‐old female with severe intellectual disability and a long‐standing bleeding history. Methods: A SNP array analysis (Illumina Human Cyto 12‐SNP genotyping array) and sequencing of F9 were performed. Laboratory tests were performed to evaluate the bleeding diathesis. Results: Our patient was diagnosed with mild hemophilia B after finding an 11 Mb deletion of Xq26.3q28 that included the following genes among others IDS,SOX3,FMR1,AFF2, and F9. Conclusion: The case history demonstrates that a severe bleeding tendency suggestive of a hemostasis defect in patients with intellectual disability warrants careful hematological and genetic work‐up even in the absence of a positive family history. This case history demonstrates that a severe bleeding tendency suggestive of a hemostasis defect in patients with intellectual disability warrants careful hematological and genetic work‐up even in the absence of a positive family history. [ABSTRACT FROM AUTHOR]

Introduction Both congenital dysfibrinogenemia and haemophilia B (HB) are rare coagulopathies caused by mutations within the fibrinogen and F9 genes respectively. Aim To investigate the pathogenesis of combined dysfibrinogenemia with HB in a family. Methods Coagulation assays, factor IX ( FIX) activity (one-stage method), fibrinogen activity (Clauss method), antigen (immunoturbidimetry), fibrinogen polymerization and fibrinolysis velocity were measured. The sequences of fibrinogen genes and F9 were amplified by PCR and analysed by sequencing. Results The proband, a 16-year-old boy with HB ( FIX 2 IU dL−1), also had persistently low Clauss fibrinogen level (0.64-0.65 g L−1) with normal antigen level (2.23 g L−1). The mother had a FIX 45 IU dL−1 and similarly discrepant low Clauss fibrinogen (0.79 g L−1) to antigen levels (2.23 g L−1). Thrombin time for both were either slightly prolonged or at boundary value. Genetic analysis of the proband and the mother identified similar mutations in the FGG gene (heterozygous c.1042T>A resulting in p.Phe348Ile or γPhe322Ile in the mature protein) and in the F9 gene (c.1243del p.His415Metfs*11 and c.1245T>A p.His415Gln). The father had no fibrinogen or F9 gene mutations. Plasma fibrinogen polymerization was delayed, but fibrinolysis velocity was normal in the proband and his mother. Conclusion To our knowledge, this is the first report of a family with combined novel dysfibrinogen (Fibrinogen Beijing) and HB with bleeding manifestations. [ABSTRACT FROM AUTHOR]

Background and purpose Recently, we have shown that radiotherapy (RT) combined with the immunocytokine L19-IL2 can induce long-lasting antitumour effects, dependent on ED-B expression and infiltration of cytotoxic T cells. On the other hand, in certain tumours, IL2 treatment can trigger a natural killer cell (NK) immune response. The aim of this study is to investigate the therapeutic effect of our combination therapy in the ED-B positive F9 teratocarcinoma model, lacking MHCI expression and known to be dependent on NK immune responses. Material and methods In syngeneic F9 tumour bearing 129/FvHsd mice tumour growth delay was evaluated after local tumour irradiation (10 Gy) combined with systemic administration of L19-IL2. Immunological responses were investigated using flow cytometry. Results Tumour growth delay of L19-IL2 can be further improved by a single dose of RT administered before immunotherapy, but not during immunotherapy. Furthermore, treatment of L19-IL2 favours a NK response and lacks cytotoxic T cell tumour infiltrating immune cells, which may be explained by the absence of MHCI expression. Conclusion An additive effect can be detected when the NK dependent F9 tumour model is treated with radiotherapy and L19-IL2 and therefore this combination could be useful in the absence of tumoural MHCI expression. [ABSTRACT FROM AUTHOR]

Background: Haemophilia A and B (HA, HB) are the most frequent X-linked bleeding diseases; two-thirds of cases are severe. Methods: We counselled 51 couples for prenatal diagnosis (PD) of haemophilia. In 7/51 (13.7%) cases, the couple decided not to undergo PD because counselling revealed that they were carriers of a mild form of the disease, while we performed 44 PD for severe HA (36 cases) or HB (8 cases). The indication for PD was a haemophilic child (30/44, 68.2%) or an affected family member (12/44, 27.3%); in two cases the non-carrier mother of isolated haemophilic patients requested PD because of the risk of mosaicism. Results: We completed PD in 43/44 cases; in one case, the prenatal sample was contaminated by maternal DNA; however, molecular analysis revealed the female sex of the foetus. We performed PD for 16 of the 36 couples at risk of HA (44.4%) by analysing the intron (IVS)22 inversion; in 1/36 cases (2.8%) the mother had the IVS1 inversion, and in 8/36 (22.2%) the family mutation was identified by sequencing; in 11/36 (30.6%) cases the family mutation was unknown, and PD was performed by linkage (no recombination nor uninformative cases occurred). For HB, in 6/8 (75.0%) cases, PD was performed by DHPLC or by sequencing; in 2/8 cases we tested intragenic markers (again with no cases of recombination or uninformative families). Conclusions: PD in well-equipped laboratories, and multidisciplinary counselling are an aid to planning reproductive and early therapeutic strategies in families with severe haemophilia. [ABSTRACT FROM AUTHOR]

The proteins in the syntaxin family are known to mediate fusion of cytoplasmic vesicles to the target membrane, yet subpopulations of certain syntaxins, including syntaxin4, translocate across the cell membrane in response to external stimuli. Here, we show that extracellularly presented syntaxin4 impacts cell behavior and differentiation in teratocarcinoma F9 cells. While undifferentiated F9 cells extruded a small subpopulation of extracellular syntaxin4 at the lateral cell membrane, the induction of differentiation with all-trans retinoic acid (RA) abolished this localized expression pattern. We found that the cells that were stimulated in a non-directional fashion by extracellular syntaxin4 displayed a flattened shape and retained a substrate-bound morphology even under a long-term, serum-starved cultivation. Such a cellular response was also elicited by a circular peptide composed of the potential functional core of syntaxin4 (AIEPQK; amino acid residues 103∼108) (ST4n1). While the proliferation and metabolism were not affected in these cells, cell-cell interaction became weakened and the expression of vinculin, a regulator of both intercellular and cell-substrate adhesion molecules, was altered. We also found that the expressions of several differentiation markers were up-regulated in cells stimulated with extracellular syntaxin4 and that syntaxin3, another family member, was most prominent. Intriguingly, forced expression of syntaxin3 induced the spread morphology in F9 cells, indicating that syntaxin3 partly mediates the function of extracellular syntaxin4. These results demonstrate the involvement of a non-directional stimulation of extracellular syntaxin4 in the functional and morphological differentiation of F9 cells. [ABSTRACT FROM AUTHOR]

Using dodecyl N-acetylglucosaminide (GlcNAc-C12) as a saccharide primer, we investigated the biosynthetic changes of neolacto-series glycosphingolipids (GSLs) in mouse embryonal carcinoma F9 cells during differentiation induced by retinoic acid plus dibutyryl cyclic AMP (RA/dbcAMP). In the differentiated cells, the glycosylation of GlcNAc-C12 was greatly enhanced. The sugar compositions of glycosylated primers were assigned as Hex-GlcNAc, [Hex]2-GlcNAc, [Hex]2[HexNAc]-GlcNAc, and [NeuAc][Hex]-GlcNAc by liquid chromatography-tandem mass spectrometry. The detection of augmented biosynthesis of endogenous sialylparagloboside indicated that [NeuAc][Hex]-GlcNAc was predicted to be the non-reducing end trisaccharide of sialylparagloboside. The transcription of B3gnt5, B4galt1, Ggta1, Fut4 and St3gal6, encoding glycosyltransferases involved in the neolacto-series glycosphingolipids biosynthesis, was increased, whereas that of Fut9 and St6galI was decreased after RA/dbcAMP treatment. Furthermore, the sialyltransferase activity of ST3GalVI sialylating paragloboside was enhanced with the increase in St3gal6 expression. Since most stage-specific embryonic antigen-1 (SSEA-1) active determinants are carried by glycoproteins in F9 cells, the changes in glycolipid metabolism do not seem to be closely related to loss of cell surface SSEA-1 expression upon F9 differentiation. These results indicate that RA/dbcAMP treatment activates the biosynthesis of neolacto-series GSL and enhances sialylation of paragloboside in F9 cells with down-regulation of Fut9 expression. [ABSTRACT FROM PUBLISHER]

Abstract: We found that the levels of all general transcription factors (GTFs) for RNA polymerase II decreased in F9 cells when the cells were subjected to a differentiation procedure. Different from other GTFs, decrease of TFIIB during the differentiation was suppressed by addition of a proteasome inhibitor, MG132. The half-life of TFIIB in the differentiated cells was remarkably reduced compared with that in the undifferentiated cells. Moreover, it was demonstrated that TFIIB is a poly-ubiquitinated protein. Results of this study suggest that components of the transcription machinery decreased in accordance with cell differentiation and that TFIIB is specifically and rapidly degraded by the ubiquitin–proteasome pathway. [Copyright &y& Elsevier]

Haemophilia B (HB) is a rare X-linked recessive bleeding disorder caused by a mutation in the F9 gene. The aims of this study were to characterize the mutation spectrum of F9 in Korean patients with HB to establish the optimal molecular diagnostic strategy and to find genotype–phenotype correlations. Study subjects consisted of 33 unrelated Korean patients with HB. We performed polymerase chain reaction (PCR) amplification and direct sequencing of all exons and flanking sequences of F9. When large deletion was suspected from PCR failure, exon dosage test using multiplex ligation-dependent probe amplification (MLPA) was performed. We identified disease-causing mutations in 32 out of 33 patients by direct sequencing analyses (mutation detection rate, 97%). A total of 28 unique mutations were detected, including 7 novel ones. Six mutations were recurrent but observed in no more than two patients. In the remaining one patient, exon 1 was not amplified, and MLPA analysis confirmed a large deletion involving exon 1. The genotype–phenotype correlations between the type of mutation and the severity of factor deficiency were not consistent, as has been previously reported. One patient developed inhibitor, and he was the patient with exon 1 deletion. Based on our results from 33 Korean patients with HB, which showed no hotspot for mutations, direct sequencing of all exons with flanking sequences is needed as the first-line test. MLPA can be a feasible platform at clinical laboratories to detect large deletion mutations in suspected cases. [ABSTRACT FROM AUTHOR]

Hemophilia B, an X-linked recessive bleeding disorder, is caused by heterogeneous mutations in the factor IX (F9) gene. Hence, carriers of the disease are usually detected by F9 gene linked RFLP analysis. We aimed to test a set of RFLP markers (DdeI, XmnI, MnlI, TaqI & HhaI), used worldwide for carrier detection, to estimate its heterozygosity in different population groups of India, and identify additional single nucleotide polymorphisms (SNPs) if necessary. A total of 8 population groups encompassing different regions of India, consisting of 107 unrelated normal females without any history of hemophilia B in the family and 13 unrelated obligate carriers were recruited in the study. Regions of F9 gene were amplified by PCR from genomic DNA of the donors followed by restriction enzyme digestion and/or sequencing as appropriate. Combined informativeness for the markers varied between 52–86% among normal females belonging to different geographical locations of India. Haplotype analysis revealed that the most prevalent haplotype lacked the restriction sites for all five RFLP markers. Screening regions of F9 gene that harbor 10 SNPs reported in dbSNP yielded only two SNPs, which increased the overall informativeness in each population group and heterozygosity in the obligate carriers for the disease from 38% to 69%. Our data show that heterozygosity of commonly used RFLP markers is remarkably variable across different regions of India. Thus prudent selection of the markers based on specific population groups including usage of additional markers is recommended for efficient carrier detection. [ABSTRACT FROM AUTHOR]

Bleeding symptoms and clotting activity vary among mutations that alter mRNA splicing of either the factor VIII or factor IX genes. We analyzed splicing mutations in both genes for changes in individual information ( Ri, in bits) involving both donor or acceptor sites. Mutations with low or negative Ri values (<2.4 bits) or significant changes in Ri (Δ Ri ≥ 7 bits) exhibited either reduced protein activity, increased clotting time and bleeding frequency and were predictive of severe disease. Thus, information analysis of splicing mutations may be useful in predicting phenotypes in hemophilia. [ABSTRACT FROM AUTHOR]

Abstract: Combined treatment of teratocarcinoma F9 cells with retinoic acid and dibutyryl-cAMP induces the differentiation into cells with a phenotype resembling parietal endoderm. We show that the levels of cyclin-dependent kinase inhibitor p21/WAF1/Cip1 (p21) protein and mRNA are dramatically elevated at the end of this differentiation, concomitantly with the appearance of p21 in the immunoprecipitated CDK2–cyclin E complex. The induction of differentiation markers could not be achieved by expression of ectopic p21 alone and still required treatment with differentiation agents. Clones of F9 cells transfected with sense or antisense p21 cDNA constructs revealed, upon differentiation, upregulated levels of mRNA for thrombomodulin, a parietal endoderm-specific marker, or increased fraction of cells in sub-G1 phase of the cell cycle, respectively. Consistent with this observation, whereas p21 was strictly nuclear in undifferentiated cells, a large proportion of differentiated cells had p21 localized also in the cytoplasm, a site associated with the antiapoptotic function of p21. Furthermore, p21 activated the thrombomodulin promoter in transient reporter assays and the p21 mutant defective in binding to cyclin E was equally efficient in activation. The promoter activity in differentiated cells was reduced by cotransfection of p21-specific siRNA or antisense cDNA. Coexpression of p21 increased the activity of the GAL-p300(1–1303) fusion protein on the GAL sites-containing TM promoter. This implies that p21 might act through a derepression of the p300 N-terminal-residing repression domain, thereby enhancing the p300 coactivator function. As differentiation of F9 cells into parietal endoderm-like cells requires the cAMP signaling, the results together suggest that the cyclin-dependent kinase inhibitor p21 may promote specifically this pathway in F9 cells. [Copyright &y& Elsevier]

Rcd1, initially identified as a factor essential for the commitment to nitrogen starvation-invoked differentiation in fission yeast, is one of the most conserved proteins found across eukaryotes, and its mammalian homolog is expressed in a variety of differentiating tissues. Here we show that mammalian Rcd1 is a novel transcriptional cofactor and is critically involved in the commitment step in the retinoic acid-induced differentiation of F9 mouse teratocarcinoma cells, at least in part, via forming complexes with retinoic acid receptor and activation transcription factor-2 (ATF-2). In addition, antisense oligonucteotide treatment of embryonic mouse lung explants suggests that Rcd1 also plays a role in retinoic acid-controlled lung development. [ABSTRACT FROM AUTHOR]

We have developed a simple, sensitive and cost-effective direct DNA sequencing procedure for the molecular diagnosis of haemophilia B. All factor IX gene essential regions were amplified under identical thermocycling parameters allowing mutation identification in less than 15 h from blood sample collection. Identical results in terms of accuracy and speed were obtained when using a single hair as the source of DNA. Using this approach, we have found mutations in 10 out of 10 haemophilia B patients, two of which are novel (P287T and S123C). Very suitable for individual studies, this procedure can be easily scaled up for higher throughput. [ABSTRACT FROM AUTHOR]