The project will Institute of Hematologic Research "Mariano R. Castex" of the National Academy of Medicine
INVESTIGATING:
Mary A. Lazzari. Assistant Director of the Institute of Hematologic Research "Mariano R. Castex. National Academy of Medicine. CONICET. Adriana I.
Woods. CONICET. Leticia
Keller. Biotechnology, National Academy of Medicine.
INTRODUCTION
von Willebrand disease (vWD) is the most common human bleeding disorder (1) (up to 1% in some populations) and is produced by changes in factor von Willebrand (VWF), whose mode of inheritance is autosomal, although acquired cases may arise related to lymphoma and myeloproliferative syndromes. VWD can be divided into three main categories: type 1 and 3 are quantitative deficiencies and type 2 are qualitative deficiencies of vWF molecule. Type 3 is the least common, has very low levels or absent vWF and is inherited in a recessive, in type 2, although the protein may be present, is functionally defective and sub-classified into 4 groups: type 2A, 2M, 2B and 2N (2) with genotypic and phenotypic characteristics different for each subtype.
type 2N vWD (also called variant Normandy) is characterized by have low levels of Factor VIII (FVIII) compared with normal or subnormal levels of vWF and is inherited in a recessive (3). So far, we found that these patients were homozygous or double heterozygous for a series of mutations located between the domains D 'and D3 (4).
stabilization domain D 'requires disulfide bonds and cysteine \u200b\u200b(5), but some authors suggest that other amino acid residues (aa), are also required to maintain the functional conformation of this site. It suggests that the potential induced conformational change Asp116Asn mutation located in domain D3 may explain the loss of binding capacity of vWF to FVIII. It could also affect the formation of disulfide bonds intercadena involved in binding of vWF dimers to form multimers. A subset of type 2N vWD type 2N coheredó mutation in one allele and a mutation of type 1 or type 3 on the other allele. The expression of mutated recombinant vWF confirmed that a single aa substitution produces a decrease in the FVIII binding capacity (6). OBJECTIVES
• Detection of patients with von Willebrand disease type 2N phenotypic and genotypic techniques. Exons 17 to 24 DNA of patients with vWD for the diagnosis of vWD 2N to find known mutations and polymorphisms. Search
new mutations and polymorphisms in exons 25 to 28, and in the domain D3, they would be able to produce conformational changes in the domain D 'which alter the binding of vWF to FVIII.
estimate the prevalence in our variant within the vWD Normandy. Calculate the allele frequencies of different polymorphisms in different exons of the vWF to compare with those recorded in the database of vWF of different ethnic groups.
Using the technique of Conformation-Sensitive Gel Electrophoresis (CSGE) as a method of screening by heteroduplex formation, in order to detect if there was any alteration in a particular exon.
In the case of detecting an altered electrophoretic pattern of exons in the CSGE, sequencing will make them for identification. EXPECTED RESULTS
The results of DNA sequencing of patients will allow us to relate their clinical symptoms with molecular alterations, to determine the prevalence of this variant in our population and find new polymorphisms and mutations.
METHODOLOGY AND WORK PLAN
Selection of samples for phenotypic screening methods:
1. Determination vWF antigen (vWf: Ag) by immunoelectrophoresis according to Laurell technique with anti-vWF polyclonal antibody (DAKO A082) in 1% agarose.
2. Determination of FVIII one stage method, using as a reagent, FVIII-deficient plasma.
3. Ristocetin Cofactor determination. Ristocetin agglutination of platelets in the presence of vWF, through a platelet membrane receptor, glycoprotein Ib, the high molecular weight multimers are required for this interaction while small multimers would be inactive in this regard. This determination is performed by the technique of Macfarlane.
4. Binding method FVIII to vWF. Are chosen patients with decreased levels of FVIII (VN: 0.5-1.5 U / dl) and normal or subnormal vWF: Ag (VN: 0.5-1.5 U / dl) and vWF: RCo (VN: 0.5-1.5 U / dl). The binding capacity of FVIII to vWF was determined using an ELISA previously described (7). The rationale is to incubate the reference samples and patients in polystyrene tubes coated with polyclonal anti-vWF which will join the complex FVIII / vWF samples. Eliminates the endogenous FVIII with CaCl2 and incubated the tubes with purified FVIII commercial. The FVIII bound is determined by the chromogenic substrate technique.
Grouping of patients with link mild or greatly diminished.
Based on the results of the prior art, establishing a ratio between the binding of vWF to FVIII of the patient and their level of vWF: Ag Up to a value of 0.8 is considered a normal link, below it, is considered mild or greatly diminished. DNA extraction
white blood cells.
Blood samples must be collected with EDTA to 2%. It used a commercial kit (Wizard Genomic DNA Purification Kit from Promega). The technique was performed following the manufacturer's instructions.
reaction polymerase chain (PCR).
Once extracted DNA must be the same for PCR amplification. This reaction carried out using 0.2 uL of Taq polymerase 0.5 U / mL, 1 g of DNA, 100 pM of each primer appropriate to the mutation being studied, 200 mmol / L of each deoxynucleotide triphosphate (dNTP) and magnesium chloride.
The following primers have been tested and allow the proper amplification of relevant exons: Exon 17
Primers: forward: 5 'GTG AGT GAG GCG GCA ATA G 3'
reverse: 5 'GAG CGT GGG TCT GAA CGA G 3 '
Exon 18 primers: forward: 5' GAC AAG AGT CCA GAA GGT GTG AGG 3 '.
reverse: 5 'CCT AAG GCC TAC AAA GGG GAA ACT 3'.
Exon 19 primers: forward: 5 'GGG AGA TTT TCA GTG ACT GTC 3'.
reverse: 5' GTG CAC CCT CAC TCC ACC CGC 3'.
Exón 20 Primers: forward: 5' GAT GGG CCC CTC AAA CCC AAG GTG 3'.
reverse: 5' CTC ACC TGC ACC AGA ACG TAC TGG 3'.
Exón 21 Primers: forward: 5' TTC TGG TCT GGT GAG AGC CAG TGG 3'.
reverse: 5' GCT TGG AAA TGT TCC TAT TAA GTG 3'.
Exón 23 Primers: forward: 5' TTC CCC TGA GCC GGA GAG GAT GCT 3'.
reverse: 5' GGA CAT TCC AGG AAG CAA GCT CTA 3'.
Exón 24 Primers: forward: 5'ATA ACC CCA GTT TGA CCA GCT GAC 3'.
reverse: 5'TCA CAC TCT GTG TCC ATA CCT CCA 3'.
Condiciones de la PCR para los exones 18, 19, 20, 21, 22, 23 y 24:
94ºC; 0:30min [94°C;0:30 min/58 ° C, 1:00 min/72 ° C, 1:00 min] x 40 cycles, 72 ° C, 10:00 min.
For exon 17 the temperarure of annealing (TA) is 56 ° C.
For exons 25, 26, 27 and 28 will design specific primers for amplification and test its operation.
DNA sequencing of patient samples.
To isolate the amplified fragment for the exon of interest, purify the PCR product by specific purification columns. After purification, PCR will be a second call sequencing reaction with specific primer (forward or reverse) plus DNA sequencing kit in order to amplify a single strand of DNA. The conditions of the sequencing reaction are: 94 ° C, 3:00 min [94 ° C, 30 ° C seg/50; 15seg/60 ° C, 4:00 min] x 25 cycles. Purified products of the sequencing reaction by precipitation with: absolute ethanol, 3 M sodium acetate, pH 4.8 and DNase free sterile water. The purified product was resuspended in TSR (template supression reagent) and proceed to the sequencing of the samples in the ABI Prism 310 Perkin Elmer.
CSGE (Conformation Sensitive Gel Electrophoresis).
The CSGE is used to detect mutations or polymorphisms based on the property that the change single nucleotide may alter the electrophoretic mobility of the PCR product under mild denaturing conditions (8). Under these conditions the DNA molecules have a stable three dimensional conformation depends on the sequence of nucleotides. Therefore, the single nucleotide change produces a conformational change that can be detected as an increase in the differential migration of homoduplex and heteroduplex in a polyacrylamide gel electrophoresis.
Statistics.
will use the Fisher test or chi square depending on n we obtain and consider a value of <>
significant if
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