Recombination between two 14-bp homologous sequences as the mechanism for gene deletion in factor IX Seattle 1.

Factor IXSeattle 1 is a 10-kb intragenic deletion identified in a family that has hemophilia B. By sequencing across the site of the deletion, we discovered at the deletion junction a 13-bp sequence (5' . . . TAGAA-GTTCACTT . . . 3') that was homologous to two 14-bp sequences 10 kb apart in introns D and F of the normal factor IX gene. The presence of these homologous sequences in two different regions of the normal gene allows us to propose that genetic recombination has occurred between the sequences, resulting in the gene deletion. The precise recombination site was able to be localized to one of 5 bp (5' . . . AGTTC . . . 3') in the middle of the homologous sequences. The exact length of the deletion is 10,000 bp.     

An insertion within the factor IX gene: hemophilia BEl Salvador.

A patient with moderate to severe hemophilia B has been found to have a large insertion within his factor IX gene. The site of insertion is located in a DNA segment of approximately 0.8 kb between exon IV and an EcoRI site within intron D. The size of the DNA insertion is approximately 6 kb, and it contains at least two TaqI sites, two EcoRI sites, and one HindIII site. The insert probably originates from outside the FIX gene and does not represent an internal duplication. We propose that this abnormal FIX gene be called FIX El Salvador in recognition of the birthplace of the patient.     

Factor IXMadrid 2: a deletion/insertion in factor IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site.

DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5' end of intron d and the two last coding nucleotides located at the 3' end of exon IV in the normal factor IX gene; this fragment has been replaced by a 47-bp sequence from the normal factor IX gene, although this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends of the deleted DNA fragment.     

Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response

We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10(12) vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of approximately 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression.     

Molecular structure of canine LINE-1 elements in canine transmissible venereal tumor

We determined the 4251-bp sequence of open reading frame 2 (ORF2) of canine LINE-1 retroposon that encodes 1275 amino acids. The truncated LINE-1 inserts associated with transmissible venereal tumor (TVT) of dogs contained the 1378-bp LINE-1 insert (TVT-LINE) flanked by 10-bp direct repeats upstream to c-myc gene. The TVT-LINE elements were composed of 416 bp inverse sequences homologous to the complementary strand of the LINE-1, a 5-bp deletion and 962-bp sequences homologous to the 3' region of the LINE-1.     

Factor IX San Dimas. Substitution of glutamine for Arg-4 in the propeptide leads to incomplete gamma-carboxylation and altered phospholipid binding properties

DNA sequence analysis of the Factor IX gene from a hemophilia B patient (98% Factor IX antigen; less than 0.01 unit/ml clotting activity) has identified a point mutation in exon II. A guanine to adenine transition causes the substitution of a glutamine codon for an arginine codon at -4 in the propeptide of Factor IX. This variant, termed Factor IX San Dimas, circulates in the plasma as proFactor IX with a mutant 18-amino acid propeptide still attached. Like Factor IX Cambridge (Arg-1----Ser), Factor IX San Dimas is unable to express metal-induced epitopes recognized by conformation-specific polyclonal antibodies. Amino acid analysis of the alkaline hydrolysate indicates that purified Factor IX San Dimas contains a reduced number of gamma-carboxyglutamyl residues compared to Factor IX. However, this protein undergoes metal-induced quenching of the intrinsic fluorescence. In addition, Factor IX San Dimas is unable to interact with phospholipid vesicles. The absence of coagulant activity in Factor IX San Dimas can be attributed to impaired calcium-induced conformational changes and loss in the ability to bind phospholipid vesicles in the presence of calcium ions.     

Successful Phenotype Improvement following Gene Therapy for Severe Hemophilia A in Privately Owned Dogs

Severe hemophilia A (HA) is an inherited bleeding disorder characterized by <1% of residual factor VIII (FVIII) clotting activity. The disease affects several mammals including dogs, and, like humans, is associated with high morbidity and mortality. In gene therapy using adeno-associated viral (AAV) vectors, the canine model has been one of the best predictors of the therapeutic dose tested in clinical trials for hemophilia B (factor IX deficiency) and other genetic diseases, such as congenital blindness. Here we report our experience with liver gene therapy with AAV-FVIII in two outbred, privately owned dogs with severe HA that resulted in sustained expression of 1–2% of normal FVIII levels and prevented 90% of expected bleeding episodes. A Thr62Met mutation in the F8 gene was identified in one dog. These data recapitulate the improvement of the disease phenotype in research animals, and in humans, with AAV liver gene therapy for hemophilia B. Our experience is a novel example of the benefits of a relevant preclinical canine model to facilitate both translational studies in humans and improved welfare of privately owned dogs.     

Binding of the Ets factor GA-binding protein to an upstream site in the factor IX promoter is a critical event in transactivation.

Factor IX is an essential vitamin K-dependent serine protease that participates in the intrinsic pathway of coagulation. The protein is expressed exclusively in the liver. The rare Leyden form of hemophilia B (inherited factor IX deficiency) results from point mutations in three proximal promoter elements that decrease factor IX expression. Recovery of expression occurs following puberty, with factor IX protein levels rising into the normal range. We have previously implicated the PAR domain D-site-binding protein (DBP) as well as an upstream element, site 5, as playing important roles in the phenotypic recovery of hemophilia B Leyden. Here we demonstrate that site 5 binds both the CCAAT/enhancer-binding protein (C/EBPalpha) and the ubiquitous Ets factor GA-binding protein (GABPalpha/beta). Transactivation of the factor IX promoter by the PAR proteins DBP and hepatic leukemia factor (HLF) is dependent on the binding of GABPalpha/beta to site 5, and coexpression of these two factors is required for optimal activation of this promoter. The binding of C/EBPalpha to site 5 also augments the activity of GABPalpha/beta. Analysis of the developmental regulation of site 5-binding proteins in rat liver has shown that C/EBPalpha and the GABPbeta subunit increase markedly in the 2 weeks after birth. These observations establish a functional association between the Ets factor GABPalpha/beta and C/EBPalpha and indicate that the two PAR proteins, DBP and HLF, may play complementary roles in factor IX activation. Given the developmental changes exhibited by these proteins, it is likely that they play a role in regulation of the normal factor IX promoter as well as promoters carrying hemophilia B Leyden mutations.     

Mutations in the catalytic domain of human coagulation factor IX: rapid characterization by direct genomic sequencing of DNA fragments displaying an altered melting behavior

Deficiency in coagulation factor IX, a plasma glycoprotein constituent of the clotting cascade, results in hemophilia B, an inherited recessive X-linked bleeding disorder. Some affected individuals, referred to as antigen positive or CRM+, express an inactive factor IX gene product at normal levels and are expected to have natural mutations altering domains of the molecule that are critical for its correct function. The serine protease catalytic domain of activated factor IX, encoded by exons VII and VIII of the gene, is a possible target for such mutations. We designed a strategy allowing rapid analysis of this region through enzymatic amplification of genomic DNA, analysis of the amplification products by denaturing gradient gel electrophoresis, and direct sequencing of the fragments displaying an altered melting behavior. This procedure permitted us to characterize two previously undescribed mutations. Factor IX Angers is a G-to-A substitution generating an Arg in place of a Gly at amino acid 396 of the mature factor IX protein. Factor IX Bordeaux is an A-to-T substitution introducing a nonsense codon in place of the normal codon for Lys at position 411. Moreover, the already described factor IX Vancouver defect was found in three apparently independent families. These results provide further insight into the molecular heterogeneity of hemophilia B. In addition, we demonstrate the usefulness of this rapid screening procedure, which has broad applications in human genetics and can be used as an alternative to RFLP analysis in carrier detection or prenatal diagnosis studies.     

Encapsulated human primary myoblasts deliver functional hFIX in hemophilic mice

BACKGROUND: Hemophilia B is a bleeding disorder caused by defective factor IX (FIX), currently treated by regular infusions of plasma-derived or recombinant FIX. We propose a gene therapy strategy based on the implantation of cells secreting FIX enclosed in alginate microcapsules as a highly desirable alternative treatment. We have reported sustained delivery of human factor IX (hFIX) in immunocompetent mice implanted with encapsulated primary mouse myoblasts engineered to secrete hFIX. As a step towards the treatment of human patients, in this study we report the implantation of encapsulated human primary myoblasts secreting hFIX in hemophilia B mice. METHODS: Human primary myoblasts were transfected with plasmids pKL4M-hFIX, pLNM-betaIXL, pMFG-hFIX, and transduced with retrovirus MFG-hFIX. Two human primary myoblast clones secreting approximately 1 microg hFIX/10(6) cells/day were enclosed in biocompatible alginate microcapsules and implanted intraperitoneally into SCID and hemophilic mice. RESULTS: Circulating hFIX (peak of approximately 120 ng/ml) was detected in hemophilia B mice on day 1 after implantation. Human FIX delivery was transient, however, becoming undetectable on day 14. Concurrently, anti-hFIX antibodies were detected. At the same time, activated partial thromboplastin time (APTT) was reduced from 94 s before treatment to 78-80 s. Tail bleeding time decreased from 15 min to 1.5-7 min after treatment, some mice being normalised. These findings indicate that the delivered hFIX is biologically active. Similarly treated NOD/SCID mice had circulating hFIX levels of 170 ng/ml on day 1 that remained detectable for 1 month, albeit at low levels. Cell viability of microcapsules retrieved on day 60 was below 5%. CONCLUSIONS: Our findings indicate that encapsulated human primary myoblasts secrete functional hFIX. Furthermore, implantation of encapsulated human primary myoblasts can partially correct the phenotype of hemophilia B mice, supporting the feasibility of this gene therapy approach for hemophilia B. However, the long-term viability of the encapsulated human myoblasts must first be improved.     

Nucleotide substitutions at the -6 position in the promoter region of the factor IX gene result in different severity of hemophilia B Leyden: consequences for genetic counseling

Mutations in the promoter region of the factor IX gene result in hemophilia B Leyden, which is characterized by considerable improvement in the disease after puberty. We have found that distinct nucleotide substitutions at the -6 position in the Leyden-specific (LS) region are associated with a different severity of hemophilia B. The proband (aged 2) from one family is a severe hemophiliac with factor IX activity (F.IXC) and antigen (F.IXAg) levels less than 1.0U/dl. F.IXC and F.IXAg levels in two affected uncles are approximately 30% of normal levels. The LS region was targeted for analysis because the phenotypes suggested the inheritance of a factor IX Leyden gene. An abnormal TaqI digestion pattern was found in amplified DNA from the proband, and sequencing showed a G (-6) to C transversion that was linked to the disease in the family. In another family, two brothers (aged 8 and 9) suffer from mild hemophilia with F.IXC ranging from 7 to 10 U/dl and F.IXAg from 3 to 4 U/dl. They are the only documented members of the family with a bleeding tendency. Denaturing gradient gel electrophoresis on amplified fragments from one of the patient's genomic DNA corresponding to the 8 exons and flanking sequences of the factor IX gene suggested a defect only in a segment from the 5 region. This segment showed an altered TaqI digestion pattern, and sequencing demonstrated a G(-6) to A transition that was traced to the patients's mother and a grandmother. The different phenotypes associated with the G (-6) to A purine nucleotide transition compared with a G(-6) to C transversion provide evidence that this area is directly involved in the regulation of the human factor IX gene expression in vivo by binding of regulatory factors. The ability to predict that the conditions of a hemophilia B patient will improve with age has important implications for genetic counseling of the family. Therefore, the LS region should always be included when scanning the factor IX gene for mutations.     

Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.     

Identification of a single nucleotide C-to-T transition and five different deletions in patients with severe hemophilia B.

DNA of 70 unrelated hemophilia B patients, including three inhibitor patients, was analyzed by using various restriction enzymes and was hybridized with both a factor IX cDNA and 3'- and 5'-flanking probes. When the gene was mapped this way, six patients all afflicted with severe hemophilia B were shown to have a deviating hybridization pattern. One inhibitor patient showed a partial deletion of about 9 kb that removes exons a-c. A partial deletion of at least 11 kb that removed exon a and that had a maximum size of 35 kb in the 5'-flanking region could be identified in a patient of unknown status. In another three noninhibitor patients a complete deletion of the factor IX gene and two partial deletions could be observed. The partial deletions are of approximately 8 kb and approximately 1.5 kb, removing exons d and e and exon g, respectively. As detected by oligonucleotide probing, a C-to-T transition at amino acid 338 gave rise to an altered TaqI restriction pattern that could be observed in a sixth patient. The other 64 hemophilia B patients, including two inhibitor patients, showed a hybridization pattern indistinguishable from a normal one.     

Hereditary desmoid disease in a family with a germline Alu I repeat mutation of the APC gene

Two families with autosomal dominantly inherited desmoid tumors have recently been shown to have germline mutations at the 3' end of the APC gene. We subsequently identified an Amish family with autosomal dominantly inherited desmoid tumors. Genetic analysis performed on one family member, a 47-year-old man with multiple desmoid tumors and no colon polyps, revealed a protein truncating mutation in the middle of the APC gene. The truncating mutation is the result of a 337-bp insertion of an Alu I sequence into codon 1526 of the APC gene. The presence of a poly(A) tail at the 3' end of the insertion suggests that the Alu I sequence was inserted by a retrotranspositional event. Germline insertions of Alu I sequences have occasionally been reported to cause other genetic diseases including type I neurofibromatosis, hereditary site-specific breast cancer (BRCA2), and hemophilia B. However, this is the first report of a germline mutation of the APC gene resulting from an Alu I insertion.     

Internal homologies of the Ba fragment from human complement component Factor B, a class III MHC antigen

The amino acid sequence of Ba, a fragment of the complement protein Factor B, has been determined from the sequence of its corresponding cDNA. Ba is composed of 234 amino acids and from the sequence two striking regions of internal homology are apparent which are related to a third less homologous region. Analysis of cloned genomic DNA using an 81-bp cDNA probe containing coding information for part of a leader peptide and nine amino acids at the N terminus of Ba has established the extent of the 5' end of the Factor B gene and shown that the region of the gene encoding Ba is approximately 1.6 kb in length.     

Molecular diagnosis of hemophilia A and B. Report of five families from Costa Rica

Hemophilia A and B are X-chromosome linked bleeding disorders caused by deficiency of the respective coagulation factor VIII and IX. Affected individuals develop a variable phenotype of hemorrhage caused by a broad range of mutations within the Factor VIII or Factor IX gene. Here, were report the results of the molecular diagnosis in a five Costa Rican families affected with Hemophilia. Methods of indirect and direct molecular diagnosis are applied in three Hemophilia A and two Hemophilia B families from Costa Rica as well as preconditions, practicability and facilities of this diagnosis. In two families with Hemophilia A and both families with Hemophilia B the causative mutation could be detected by Southern blotting, polymerase chain reaction or sequence analysis. One Hemophilia A family could only analyzed by linkage analysis using genomic markers.     

Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration

CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications. However, it is still infeasible because homologous recombination (HR) is inefficient, especially for non-dividing cells. To overcome the challenge, we report that a homology-independent targeted integration (HITI) strategy is used for permanent integration of high-specificity-activity Factor IX variant (F9 Padua, R338L) at the albumin (Alb) locus in a novel hemophilia B (HB) rat model. The knock-in efficiency reaches 3.66%, as determined by droplet digital PCR (ddPCR). The clotting time is reduced to a normal level four weeks after treatment, and the circulating factor IX (FIX) level is gradually increased up to 52% of the normal level over nine months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through primer-extension-mediated sequencing (PEM-seq), no significant off-target effect is detected. This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.     

Sequencing, cloning and expression of a β-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium, Caldicellulosiruptor Rt8B.4

Abstract We report here the isolation of a Renibacterium salmoninarum DNA sequence capable of transforming a non-invasive Escherichia coli strain into a microorganism able to enter the fish cell line, CHSE-214. Immunofluorescence and electron microscopy techniques were used to assess the acquired invasive phenotype by HB101 E. coli cells, upon transformation with pPMV-189. This plasmid carries a 2282-bp R. salmoninarum DNA segment. The invasive phenotype is qonserved upon deletion of approximately 1000 bp at the 3' end of the insert. The remaining segment contains an ORF region encoding a putative protein of about 30 kDa.