Study of Hemophilia A Caused by Defect in the FVIII Gene

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Study of Hemophilia A Cause by Defect of FVIII Gene and Detection of FVIII Antibodies in Plasma

Keywords: Hemophilia A, Factor VIII, ELISA, Immunostaining, Antibody.


Graphics

  1. Figure of Immunostaining of both patient and normal people’s liver cells.

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  1. Figure of treatment to different patient within same symptom.

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  1. General information of Hemophilia A

Hemophilia A is an X-linked recessive bleeding disorder due to a deficiency in the activity of coagulation factor VIII (FVIII). The disorder is clinically heterogeneous with variable PASI, depending on the plasma levels from coagulation factor VIII (1). Intracranial hemorrhage can occur and lead to drastic complications. Bleeding from tongue or lip lacerations is often incessant (2). Early reports of hemophilia families beginning with a newspaper account in 1792 and described with a bizarre X-linked bleeding disorder (3,4). Most heterozygous female carriers of hemophilia A have concentrations of clotting factor VIII (5). In addition, intron 22 of the human F8 gene is hypomethylated and methylated on the inactive X. Japanese showed 45% heterozygosity and Asian Indians showed 13%; polymorphism was not found in American blacks or Caucasians (5). For further study, DNA diagnosis can be helpful in early care of hemophilia (6). In 2008, Lavery introduced other strategies for preimplantation genetic diagnosis of hemophilia (7). In 1995, a high transduction efficiency and a high rate of factor VIII production was demonstrated as gene therapy (8). For recent research, 382 different mutations in the F8 gene were identified (9). However, in 2009, mismatched factor VIII replacement therapy was found might be a risk factor for the development of anti-factor VIII alloantibodies (10). In the future study, one of the subject should be find other more reliable and effective genetic therapy to cure this disease.

  1. Genetic information of coagulation factor VIII Gene

Hemophilia A is an X-linked recessive bleeding disorder caused by a deficiency in the activity of coagulation factor VIII (11). This is a large plasma glycoprotein that functions in the blood coagulation cascade as a cofactor for the factor IXa -dependent activation of factor X. Factor VIII is tightly associated in the blood with von Willebrand factor, which serves as a protective carrier protein for factor VIII (12). The purpose of study this gene is because approximately 10 to 20% of patients with severe hemophilia A develop antibodies, known as inhibitors, to factor VIII following treatment with exogenous factor VIII. Most of these patients have nonsense mutations or deletions in the F8 gene (13).

The F8 gene contains 26 exons and spans 186 kb (14). It is a complex of a large inert carrier protein and a non-covalently bound small fragment which contains the procoagulant active site (15). The F8 gene is located on the long arm of the X chromosome at position 28. It also contains 3 copies of F8A and its adjacent regions, 1 in intron 22 and 2 telomeric and upstream to the F8 gene transcription start site (16). The F8 gene is expressed in human liver, spleen, lymph nodes, and a variety of other tissues, but not in bone marrow, peripheral blood lymphocytes, or endothelial cells (17).

The important studies of F8 gene is basically on clinical. In a study of 147 sporadic cases of severe hemophilia A, 126 patients (85.7%) were identified as the causative defect in the F8 gene (18). Mutations in the F8 gene lead to the production of an abnormal version of coagulation factor VIII or reduce the amount of this protein (19). In a study of 83 patients with hemophilia A, 2 different point mutations were identified, one in exon 18 and one in exon 22, that recurred independently in unrelated families. Each mutation produced a nonsense codon by a change of CG to TG. Some mutations, such as the large inversion described above, almost completely eliminate the activity of coagulation factor VIII and result in severe hemophilia (20).

  1. cDNA information:
  1. Homo sapiens coagulation factor VIII, procoagulant component (F8), transcript variant 1, mRNA (ACCESSION# NM_000132).
  2. Species of the gene: Homo sapiens (human).
  3. Size of cDNA: 9048 pb. Size of ORF: 7056 bp.
  4. Predicted protein size: 2351 residues.
  5. Homology information:

There are 7 genes identified as putative homologs of one another during the construction of HomoloGene. The F8 gene is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, and zebrafish. 78 organisms have orthologs with human gene F8.

  1. Up Primer (Tm=72): 5’ GAATTCatgcaaatagagctctccacctgc 3’

Down Primer (Tm=72): 5’ GAATTCcagaacctccatcctcagggcaa 3’

  1. Sequence Reference:

172 atgcaaata

181 gagctctcca cctgcttctt tctgtgcctt ttgcgattct gctttagtgc caccagaaga

241 tactacctgg gtgcagtgga actgtcatgg gactatatgc aaagtgatct cggtgagctg

301 cctgtggacg caagatttcc tcctagagtg ccaaaatctt ttccattcaa cacctcagtc

361 gtgtacaaaa agactctgtt tgtagaattc acggatcacc ttttcaacat cgctaagcca

421 aggccaccct ggatgggtct gctaggtcct accatccagg ctgaggttta tgatacagtg

481 gtcattacac ttaagaacat ggcttcccat cctgtcagtc ttcatgctgt tggtgtatcc

541 tactggaaag cttctgaggg agctgaatat gatgatcaga ccagtcaaag ggagaaagaa

601 gatgataaag tcttccctgg tggaagccat acatatgtct ggcaggtcct gaaagagaat

661 ggtccaatgg cctctgaccc actgtgcctt acctactcat atctttctca tgtggacctg

721 gtaaaagact tgaattcagg cctcattgga gccctactag tatgtagaga agggagtctg

6721 gagttgatgg gctgtgattt aaatagttgc agcatgccat tgggaatgga gagtaaagca

6781 atatcagatg cacagattac tgcttcatcc tactttacca atatgtttgc cacctggtct

6841 ccttcaaaag ctcgacttca cctccaaggg aggagtaatg cctggagacc tcaggtgaat

6901 aatccaaaag agtggctgca agtggacttc cagaagacaa tgaaagtcac aggagtaact

6961 actcagggag taaaatctct gcttaccagc atgtatgtga aggagttcct catctccagc

7021 agtcaagatg gccatcagtg gactctcttt tttcagaatg gcaaagtaaa ggtttttcag

7081 ggaaatcaag actccttcac acctgtggtg aactctctag acccaccgtt actgactcgc

7141 taccttcgaa ttcaccccca gagttgggtg caccagattg ccctgaggat ggaggttctg

  1. FVIII gene cloning
    1. Process of cell line:

Use schwann cells of healthy people for preparing RNA extraction.

  1. mRNA purification:

Lysis Buffer was used to break cells and harvest total RNA. Dynabeads® mRNA DIRECT™ Kit can be used to get mRNAs. Then, these mRNAs were stored at -80. (RNAnase remover needs to be used.)

  1. RT-PCR and verification:

Two designed primers, mRNA template, dNTPs, RNase free water, and other enzyme are used for perform RT-PCR to get cDNA of Factor VIII gene by QIAGEN® One Step RT-PCR Kit. Set up the RT-PCR cycle according to Tm calculated. After that, cDNA products need to be purified by using MinElute column Kit, and stored in -4. In addition, little part of PCR products need to be used for verification. The size of DNA products can be screened on gel with standard ladder, check if match the size of PCR product.

  1. Plasmid construction:

PcDNA3.1(-) was chosen as the vector to express FVIII gene. PcDNA3.1(-) has promoter and Ampicillin and Neomycin marker that can be expressed in mammalian cell. Cut cDNA and the vector by the restriction enzyme EcoRI. After that, cDNA and the vector were linked with T4 ligase overnight at room temperature.

  1. Transformation:

This plasmid with the target gene was transformed into E.coli and cultured on the LB plate contains Ampicillin.

  1. Verification:

After cultured in E.coli, plasmid was extracted from E.coli with mini prep. Lysis solutions, cold ethanol and centrifugation was needed at mini prep. Some extracted plasmids were digested with EcoRI and go for southern blot. Two bands should be shown on the gel and check the size of small bands if fit the cDNA. The rest target gene was sent to sequence to test whether has a mutation or insert correctly in plasmid. After verification, the plasmid extractions were stored in -20 for stored.

  1. Gene expression
    1. Transfection:

Schwann cells of Hemophilia A patients is used as the targeted cells. These cells were pre-cultured in 6-well plates to about 60% confluent. The hybird plasmids were transfected into the patients’ cells using lipofectamine 2000 from Invitrogen and Opti-MEM from GIBCO. In transfection process, the plasmids contains FVIII gene was delivered into targeted cells. The none-gene control (NGC) experiment should be performed as no transfected patients’ cells.

  1. Gene expression validation:

Western blot was used to validate the protein expression. After 48 hours of transfection, Western blot was used to both positive control and NGC.(By using WesternDot™ 625 Goat Anti-Rabbit Western Blot Kit.) As the predicted result, the successfully transfected cells produce the FVIII protein. In the contrary, the NGC would not make this protein but defected FVIII proteins. The correct size protein band could be seen to verify cDNA expression.

  1. Protein localization:

Immunostaining was used to detect the localization and distribution of protein. After transfection, block cells with Blocking Buffer for 60min. Incubated overnight at 4 after adding primary antibody to cells. In addition, a fluorochrome-conjugated secondary antibody was added for incubation at room temperature for 2 hours. Another color of fluorescence labeled antibodies of beta-actin were also loaded into the reaction. Observing the stained cell culture under fluorescent microscope and checking the distribution of protein.

Proposal of study

  1. Experimental suggestion:

Gene therapy and FVIII protein supplement is the main hopeful treatment to Hemophilia A. However, the gene therapy has not been successfully performed in human because of many problem (21). So, it is important to know whether a specific Hemophilia A patient is due to cause by non-synthesis protein or FVIII inhibitor. Moreover, the one of reliable treatment is supplement of FVIII protein. But after long-term treat with FVIII gene, most patient shows a relapse even give more FVIII protein (22). Most symptom is due to antibody work as an inhibitor against FVIII protein but not all. In this case, a detection of this antibody is important to tell whether the patients need an immune tolerance induction therapy.

  1. Experiment design:
    1. Immunostaining:

Immunostaining was used to detect the localization and distribution of FVIII protein in patient liver cell. Both patient liver cells and normal liver cells were incubated after adding primary antibody. In addition, a fluorochrome-conjugated secondary antibody was added for incubation. ER-Tracker™ and BODIPY® FL C5-Ceramide were used to stain ER and Golgi. Observing the stained cell culture under fluorescent microscope and checking the distribution of protein.

  1. ELISA:

ELISA is used to detect the antibody in Hemophilia A patient that have used FVIII protein supplement for several years. The plasma samples of both patient and normal people were collect and diluted with Coating Buffer. A ELISA plate was used by adding the dilute solution of antibody. Blocking Buffer was then added after incubation, and continue incubate overnight. After that, HRP-conjugated Factor-VIII Recombinant was added to each well of the plate. As the detection, TMB Reagent and Stop Buffer to finish the reaction and read at 450nm in plate reader.

  1. Predicted result:

For immunostaining, there are three possible: â‘ Patient’s sample have more FVIII protein on ER than Golgi compare to normal people. This may shows a defect of FVIII gene cause a mis-expression and mis-fold FVIII gene that leads to lack of FVIII protein. â‘¡The FVIII protein have the same amount at ER and Golgi. This may shows the FVIII gene can be express properly but inhibit by other protein in patients’ body. â‘¢There’s no protein on ER OR Golgi in patients’ cell but in cytosol. This may shows the defect of FVIII gene may lead a lacking of ER signal that cannot secret the FVIII protein.

For ELISA, normally the absorbance of patients’ cell should be larger than normal people. If there’s no significant difference that may due to other reason and these patient should take a gene therapy instead of immune tolerance induction therapy.

  1. Budget

mTERIAL

Price

company

Lipofectamine® 2000 Transfection Reagent

$ 528

Invitrogen

Opti-MEM® I Reduced Serum Medium

$18.3

GIBCO

Dynabeads RNA DIRECT Kit

$554

One-Step RT-PCR Master Mix Reagents Kit

$149

QIAGEN

Veriti® 96-Well Fast Thermal Cycler

$7990

Invitrogen

pcDNA™3.1 (-)

$504.51

Invitrogen

Plasmid Miniprep Kit

$75.63

Invitrogen

StandardDNASequencing

$5.00

Bio Basic

WesternDot® 625 Goat Anti-Rabbit Western Blot Kit

$337

Thermo Fisher Scientific Inc.

CFX96 Touch™ Real-Time PCR Detection System

About $17000

BIO-BAD

Transfection Buffer

$100

Histogene® LCM Immunofluorescence Staining Kit

$870.00

Invitrogen

Human Schwann cell line

About $1000

ER-Tracker™

$290/100

Thermo Fisher Scientific Inc.

BODIPY® FL C5-Ceramide

$246/250

Thermo Fisher Scientific Inc.

Coagulation Factor-VIII Human Recombinant

$1200/250IU

PROSPEC

Other lab martials

$800

Total

$31668

REFERENCE

  1. Mannucci, P. M., Tuddenham, E. G. D. The hemophilias--from royal genes to gene therapy. New Eng. J. Med. 344: 1773-1779, 2001. Note: Erratum: New Eng. J. Med. 345: 384 only, 2001.
  2. Antonarakis, S. E., Kazazian, H. H., Tuddenham, G. D. Molecular etiology of factor VIII deficiency in hemophilia A. Hum. Mutat. 5: 1-22, 1995.
  3. McKusick, V. A. Hemophilia in early New England: a follow-up of four kindreds in which hemophilia occurred in pre-Revolutionary period. J. Hist. Med. 17: 342-365, 1962.
  4. Ratnoff, O. D., Lewis, J. H. Heckathorn's disease: variable functional deficiency of antihemophilic factor (factor VIII). Blood 46: 161-173, 1975.
  5. Inaba, H., Fujimaki, M., Kazazian, H. H., Jr., Antonarakis, S. E. MspI polymorphic site in intron 22 of the factor VIII gene in the Japanese population. Hum. Genet. 84: 214-215, 1990.
  6. Baty, B. J., Drayna, D., Leonard, C. O., White, R. Prenatal diagnosis of factor VIII deficiency to help with the management of pregnancy and delivery. (Letter) Lancet 327: 207 only, 1986. Note: Originally Volume I.
  7. Lavery, S. Preimplantation genetic diagnosis of haemophilia. Brit. J. Haemat. 144: 303-307, 2008.
  8. Dwarki, V. J., Belloni, P., Nijjar, T., Smith, J., Couto, L., Rabier, M., Clift, S., Berns, A., Cohen, L. K. Gene therapy for hemophilia A: production of therapeutic levels of human factor VIII in vivo in mice. Proc. Nat. Acad. Sci. 92: 1023-1027, 1995.
  9. Santacroce, R., Acquila, M., Belvini, D., Castaldo, G., Garagiola, I., Giacomelli, S. H., Lombardi, A. M., Minuti, B., Riccardi, F., Salviato, R., Tagliabue, L., Grandone, E., Margaglione, M., the AICE-Genetics Study Group. Identification of 217 unreported mutations in the F8 gene in a group of 1,410 unselected Italian patients with hemophilia A. J. Hum. Genet. 53: 275-284, 2008.
  10. Viel, K. R., Ameri, A., Abshire, T. C., Iyer, R. V., Watts, R. G., Lutcher, C., Channell, C., Cole, S. A., Fernstrom, K. M., Nakaya, S., Kasper, C. K., Thompson, A. R., Almasy, L., Howard, T. E. Inhibitors of factor VIII in black patients with hemophilia. New Eng. J. Med. 360: 1618-1627, 2009. Note: Erratum: New Eng. J. Med. 361: 544 only, 2009.
  11. Mannucci, P. M., Tuddenham, E. G. D. The hemophilias--from royal genes to gene therapy. New Eng. J. Med. 344: 1773-1779, 2001. Note: Erratum: New Eng. J. Med. 345: 384 only, 2001.
  12. Toole, J. J., Knopf, J. L., Wozney, J. M., Sultzman, L. A., Buecker, J. L., Pittman, D. D., Kaufman, R. J., Brown, E., Shoemaker, C., Orr, E. C., Amphlett, G. W., Foster, W. B., Coe, M. L., Knutson, G. J., Fass, D. N., Hewick, R. M. Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature 312: 342-347, 1984.
  13. Antonarakis, S. E., Kazazian, H. H., Tuddenham, G. D. Molecular etiology of factor VIII deficiency in hemophilia A. Hum. Mutat. 5: 1-22, 1995.
  14. Gitschier, J., Wood, W. I., Goralka, T. M., Wion, K. L., Chen, E. Y., Eaton, D. H., Vehar, G. A., Capon, D. J., Lawn, R. M. Characterization of the human factor VIII gene. Nature 312: 326-330, 1984.
  15. Zacharski, L. R., Bowie, E. J. W., Titus, J. L., Owen, C. A., Jr. Synthesis of antihemophilic factor (factor VIII) by leukocytes: preliminary report. Mayo Clin. Proc. 43: 617-619, 1968.
  16. Freije, D., Schlessinger, D. A 1.6-Mb contig of yeast artificial chromosomes around the human factor VIII gene reveals three regions homologous to probes for the DXS115 locus and two for the DXYS64 locus. Am. J. Hum. Genet. 51: 66-80, 1992.
  17. Wion, K. L., Kelly, D., Summerfield, J. A., Tuddenham, E. G., Lawn, R. M. Distribution of factor VIII mRNA and antigen in human liver and other tissues. Nature 317: 726-729, 1985.
  18. Antonarakis, S. E., Kazazian, H. H., Tuddenham, G. D. Molecular etiology of factor VIII deficiency in hemophilia A. Hum. Mutat. 5: 1-22, 1995.
  19. Gitschier, J., Wood, W. I., Tuddenham, E. G. D., Shuman, M. A., Goralka, T. M., Chen, E. Y., Lawn, R. M. Detection and sequence of mutations in the factor VIII gene of haemophiliacs. Nature 315: 427-430, 1985.
  20. Youssoufian, H., Kazazian, H. H., Jr., Phillips, D. G., Aronis, S., Tsiftis, G., Brown, V. A., Antonarakis, S. E. Recurrent mutations in haemophilia A give evidence for CpG mutation hotspots. Nature 324: 380-382, 1986.
  21. Pipe, W. (2004) Coagulation factors with improved properties for hemophilia gene therapy. Seminars in Thrombosis and Hemostasis 30, 227-237.
  22. Van der Bom JG, Mauser-Bunschoten EP, Fisher K, etal. Age at first treatment and immune tolerance to factor VIII in severe hemophilia [J]. Thromb Haemost, 2003, 89(3): 475-479.

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