Gene therapy in haemophilia: literature review and regional perspectives for Turkey.

Haemophilia is an X-linked lifelong congenital bleeding disorder that is caused by insufficient levels of factor VIII (FVIII; haemophilia A) or factor IX (FIX; haemophilia B) and characterized by spontaneous and trauma-related bleeding episodes. The cornerstone of the treatment, factor replacement, constitutes several difficulties, including frequent injections due to the short half-life of recombinant factors, intravenous administration and the risk of inhibitor development. While extended half-life factors and subcutaneous novel molecules enhanced the quality of life, initial successes with gene therapy offer a significant hope for cure. Although adeno-associated viral (AAV)-based gene therapy is one of the most emerging approaches for treatment of haemophilia, there are still challenges in vector immunogenicity, potency and efficacy, genotoxicity and persistence. As the approval for the first gene therapy product is coming closer, eligibility criteria for patient selection, multidisciplinary approach for optimal delivery and follow-up and development of new pricing policies and reimbursement models should be concerned. Therefore, this review addresses the unmet needs of current haemophilia treatment and explains the rationale and principles of gene therapy. Limitations and challenges are discussed from a global and national perspective and recommendations are provided to adopt the gene therapies faster and more sufficient for the haemophilia patients in developing countries like Turkey.

Whole exome sequencing reveals novel candidate gene variants for MODY.

Maturity-onset diabetes of the young (MODY) is a monogenic subtype of diabetes mellitus. Although 14 genes were associated to different subtypes of MODY, 30-40% of MODY patients have unidentified genetic mutations. In this study, we conducted whole exome sequencing (WES) in four Turkish MODY suspected patients in two families who do not carry any pathological variants in four frequent MODY genes (HNF1A, GCK, HNF1B and HNF4A). Initially, the variants were scanned for the known 14 MODY genes and a gene set related to glucose metabolism. Secondly, the destructive (frameshift, inframe insertion/deletion, initiator codon variant, splice acceptor/ donor variant, stop gained/lost) and missense variants in novel candidate genes shared by two patients of the same family were assessed by different bioinformatic tools. As a result, a total of three rare and novel probably pathogenic heterozygous missense variants (p.His307Gln in c-Myc, p.Asp129Asn in CDK4 and p.Gly107Ser in ARHGDIA) in candidate genes were identified in two families. This study revealed the presence of nonsynonymous alterations in novel candidate genes which were implicated in the insulin secretion. This is the first WES study which reveals novel candidate genes in Turkish MODY patients although functional analyses are required to confirm the pathogenicity of these variants.

Exploring the role of miRNAs in the diagnosis of MODY3

Background/aim: MODY3 associated with HNF1A is the most common form of MODY and is clinically misdiagnosed as type 1 diabetes due to similar clinical symptoms. This study aimed to analyze the role of HNF1A-regulated miRNAs as a biomarker in the diagnosis of MODY3. Materials and methods: MIN6 cells were transfected with the HNF1A cDNA expression vector for overexpression or with siRNA specific to HNF1A to silence its expression. The HNF1A-regulated miRNAs were determined by RNA-Seq of the total RNA extract. Expressions of the candidate miRNAs in blood samples of MODY3, type 1 diabetes, and type 2 diabetes patients and in healthy subjects were compared statistically by Mann-Whitney U tests. Results: This study revealed the presence of 238 known HNF1A-regulated miRNAs in MIN6 cells. miR-129-1-3p, miR-200b-3p, and miR-378a-5p were selected as candidate miRNAs. The expression level of miR-378a-5p significantly decreased in type 2 diabetes and MODY3 patients, while miR-200b-3p expression was significantly decreased only in MODY3 patients. Conclusion: Although further studies with larger numbers of patients are required, this study demonstrated that the expression levels of miR-200b-3p and miR-378a-5p decrease in MODY3 patients and suggests that miR-200b-3p is an especially strong candidate to use clinically for selecting suspected MODY3 patients.

Molecular genetic analysis of the F11 gene in 14 Turkish patients with factor XI deficiency: identification of novel and recurrent mutations and their inheritance within families.

BACKGROUND: Factor XI (FXI) deficiency is an autosomal bleeding disease associated with genetic defects in the F11 gene which cause decreased FXI levels or impaired FXI function. An increasing number of mutations has been reported in the FXI mutation database, most of which affect the serine protease domain of the protein. FXI is a heterogeneous disorder associated with a variable bleeding tendency and a variety of causative F11 gene mutations. The molecular basis of FXI deficiency in 14 patients from ten unrelated families in Turkey was analysed to establish genotype-phenotype correlations and inheritance of the mutations in the patients' families. MATERIAL AND METHODS: Fourteen index cases with a diagnosis of FXI deficiency and family members of these patients were enrolled into the study. The patients' F11 genes were amplified by polymerase chain reaction and subjected to direct DNA sequencing analysis. The findings were analysed statistically using bivariate correlations, Pearson's correlation coefficient and the nonparametric Mann-Whitney test. RESULTS: Direct DNA sequencing analysis of the F11 genes revealed that all of the 14 patients had a F11 gene mutation. Eight different mutations were identified in the apple 1, apple 2 or serine protease domains, except one which was a splice site mutation. Six of the mutations were recurrent. Two of the mutations were novel missense mutations, p.Val522Gly and p.Cys581Arg, within the catalytic domain. The p.Trp519Stop mutation was observed in two families whereas all the other mutations were specific to a single family. DISCUSSION: Identification of mutations confirmed the genetic heterogeneity of FXI deficiency. Most of the patients with mutations did not have any bleeding complications, whereas some had severe bleeding symptoms. Genetic screening for F11 gene mutations is important to decrease the mortality and morbidity rate associated with FXI deficiency, which can be life-threatening if bleeding occurs in tissues with high fibrinolytic activity.

Functional characterisation of the type 1 von Willebrand disease candidate VWF gene variants: p.M771I, p.L881R and p.P1413L.

BACKGROUND: Abnormalities in the biosynthetic pathway or increased clearance of plasma von Willebrand factor (VWF) are likely to contribute to decreased plasma VWF levels in inherited type 1 von Willebrand disease (VWD). Recent studies demonstrated that 65% of type 1 VWD patients have candidate VWF mutations, the majority of which are missense variants. The purpose of this study was to explore the effects of three VWF missense mutations (p.M771I, p.L881R and p.P1413L) located in different functional domains of VWF, reported as candidate mutations in type 1 VWD patients in the course of the MCMDM-1VWD study. MATERIALS AND METHODS: The focus of these studies was on the intracellular biosynthetic processing and localisation of VWF in a heterologous cell system. Molecular dynamic simulation for p.M771I and p.P1413L was also performed to analyse the conformational effects of the changes. RESULTS: As determined by immunofluorescence antibody staining and confocal microscopy of HEK293 cells, the intracellular localisation of recombinant VWF with the p.M771I variation was impaired. Transient transfection studies and phorbol myristate acetate stimulation in COS-7 cells revealed significant intracellular retention. In addition, major loss of VWF multimers was observed for only the p.M771I mutation. Molecular dynamic simulations on p.M771I mutant VWF revealed distinct structural rearrangements including a large deviation in the E' domain, and significant loss of ß-sheet secondary structure. DISCUSSION: The pathogenic effects of candidate VWF gene mutations were explored in this study. In vitro expression studies in heterologous cell systems revealed impaired secretion of VWF and a dominant negative effect on the processing of the wild-type protein for only the p.M771I mutation and none of the mutations affected the regulated secretion.

A common VWF exon 28 haplotype in the Turkish population.

An increasing number of mutations and polymorphisms have been identified in the von Willebrand factor (VWF) gene of patients with von Willebrand disease (VWD). Most of the sequence alterations are within exon 28, duplicated in the VWF pseudogene on chromosome 22. Genetic recombination causing the gene conversion between the VWF gene and its pseudogene is associated with multiple substitutions in the VWF gene and with VWD. In the present study, VWF gene exon 28 was analyzed in 33 patients with VWD by DNA sequencing. A total of 73% of the patients were heterozygous for p.D1472H, p.V1485L, p.1500A, p.1501F, p.L1503P, and p.S1506L single-nucleotide polymorphisms. Family analysis revealed that the gene conversion occurred between the VWF gene and its pseudogene in 3 patients. Case-control association analysis by Haploview 4.2 did not show an association between the haplotype and VWD. In conclusion, a common exon 28 haplotype in the Turkish population, which might have arisen from the gene conversion events in the founder population, was identified.

Higher expression of the novel gene upregulated gene 4 in two acute lymphoblastic leukemia patients with poor prednisolone response.

Elucidation of the molecular mechanisms of leukemogenesis is important for a better understanding of the prognosis of acute lymphoblastic leukemia (ALL). Studies have shown that the expression of upregulated gene 4 (URG4), which promotes cell growth and survival, is increased in different types of carcinomas including hepatocellular carcinoma, gastric cancer and osteosarcoma. Similarly, higher expression of URG4 and cyclin D1 gene might promote proliferation of the blast cells by causing escape from the G1 checkpoint and entry into the S phase. This study reports the high expression level of URG4 in 2 high-risk ALL patients for the first time in the literature. In conclusion, the higher expression of URG4 in our 2 patients suggests that URG4 might be involved in leukemogenesis. Future studies with a large number of high-risk ALL patients and cell culture studies are needed to demonstrate the exact role of URG4 in leukemogenesis.

The molecular genetics of von Willebrand disease.

UNLABELLED: Quantitative and/or qualitative deficiency of von Willebrand factor (vWF) is associated with the most common inherited bleeding disease von Willebrand disease (vWD). vWD is a complex disease with clinical and genetic heterogeneity. Incomplete penetrance and variable expression due to genetic and environmental factors contribute to its complexity. vWD also has a complex molecular pathogenesis. Some vWF gene mutations are associated with the affected vWF biosynthesis and multimerization, whereas others are associated with increased clearance and functional impairment. Moreover, in addition to a particular mutation, type O blood may result in the more severe phenotype. The present review aimed to provide a summary of the current literature on the molecular genetics of vWD. CONFLICT OF INTEREST: None declared.

Molecular characterization of FXI deficiency.

Factor XI (FXI) deficiency is a rare autosomal bleeding disease associated with genetic defects in the FXI gene. It is a heterogeneous disorder with variable tendency in bleeding and variable causative FXI gene mutations. It is characterized as a cross-reacting material-negative (CRM-) FXI deficiency due to decreased FXI levels or cross-reacting material-positive (CRM+) FXI deficiency due to impaired FXI function. Increasing number of mutations has been reported in FXI mutation database, and most of the mutations are affecting serine protease (SP) domain of the protein. Functional characterization for the mutations helps to better understand the molecular basis of FXI deficiency. Prevalence of the disease is higher in certain populations such as Ashkenazi Jews. The purpose of this review is to give an overview of the molecular basis of congenital FXI deficiency.

Cell type-specific regulation of von Willebrand factor expression by the E4BP4 transcriptional repressor.

Mechanisms of tissue-restricted patterns of von Willebrand factor (VWF) expression involve activators and repressors that limit expression to endothelial cells and megakaryocytes. The relative transcriptional activity of the proximal VWF promoter was assessed in VWF-producing and -nonproducing cells, and promoter activity was highest in endothelial cells followed by megakaryocytes. Only basal VWF promoter activity was seen in nonendothelial cells. Here we identify a negative response element located at nucleotides (nts) +96/+105 and demonstrate, using chromatin immunoprecipitation (ChIP) analysis, that in vivo this sequence interacts with the E4BP4 transcriptional repressor. Differences in size and relative abundance of nuclear E4BP4 were observed. In HepG2 cells, low levels of larger forms of E4BP4 are present that directly interact with the negative response element. In VWF-expressing cells, high levels of smaller forms predominate with no evidence of direct DNA binding. However, in endothelial cells, mutation of the VWF E4BP4 binding motif not only restores but also further elevates VWF promoter activity, suggesting that E4BP4 may be part of a coordinated binding complex. These observations implicate this binding motif in repressing both activated and basal levels of VWF transcription by different cell type-specific mechanisms, and support the hypothesis that E4BP4 sequesters negative regulators of transcription, thereby enhancing activated gene expression.

Founder von Willebrand factor haplotype associated with type 1 von Willebrand disease.

To date, no dominant mutation has been identified in a significant proportion of patients with type 1 von Willebrand disease (VWD). In this study, we examined 70 families as part of the Canadian Type 1 VWD Study. The entire VWF gene was sequenced for 1 index case, revealing 2 sequence variations: intron 30 (5312-19A>C) and exon 28 at Tyr1584Cys (4751A>G). The Tyr1584Cys variation was identified in 14.3% (10 of 70) of the families and was in phase with the 5312-19A>C variation in 7 (10.0%) families. Both variants were observed in 2 of 10 UK families with type 1 VWD, but neither variant was found in 200 and 100 healthy, unrelated persons, respectively. Mean von Willebrand factor antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo), and factor VIII coagulant activity (FVIII:C) for the index cases in these families are 0.4 U/mL, 0.36 U/mL, and 0.54 U/mL, respectively, and VWF multimer patterns show no qualitative abnormalities. Aberrant VWF splicing was not observed in these patients, and both alleles of the VWF gene are expressed as RNA. Molecular dynamic simulation was performed on a homology model of the VWF-A2 domain containing the Tyr1584Cys mutation. This showed that no significant structural changes occur as a result of the substitution but that a new solvent-exposed reactive thiol group is apparent. Expression studies revealed that the Tyr1584Cys mutation results in increased intracellular retention of the VWF protein. We demonstrate that all the families with the Tyr1584Cys mutation share a common, evolved VWF haplotype, suggesting that this mutation is ancient. This is the first report of a mutation that segregates in a significant proportion of patients with type 1 VWD.

R506Q (FV Leiden) and R485K Mutations in the Factor V Gene: Incidence in Deep Venous Thrombosis and Hemophilia A Patients.

R506Q (FV Leiden) mutation in exon 10 of the factor V (FV) gene is highly prevalent in European populations and it has been suggested that the coinheritance of FV Leiden mutation may be an important modifier of hemophilia A phenotype. One other substitution R485K in the same exon, with no functional consequences in vitro, is significantly higher in Thailand and has been associated with thrombophilia. In order to see if any correlation exists between R506Q and hemophilia phenotype and between R485K and thrombosis in Turkish patients, DGGE analysis of exon 10 of the FV gene is carried out among deep venous thrombosis (DVT) and hemophilia A patients. Our results indicate that the allelic frequency of the R485K polymorphism is similar to the frequency detected in Europe, and apparently, is not associated with an increased risk of thrombosis in the Turkish population. It is also not possible to show a modifier effect of FV Leiden on hemophilia A phenotype among the limited number of patients included in this study.