A method for diagnosing a predisposition to hemochromatosis

 

(57) Abstract:

The invention relates to biotechnology, molecular biology and medicine. The method consists in the detection of polymorphic variants of the gene HLA-H (Cys282Tyr and his63asp alleles) method length polymorphism fragments using primers flanking the sites of the gene, where appropriate localized mutations with the introduction in the case of mutations his63asp alleles in one of the primers restriction site nucleases Ksp221 for a clear differentiation of Homo - and heterozygous genotypes. The method consists in the amplification of DNA segments of the gene HLA-H, contains the location of a possible finding of mutations Cys282Tyr and his63asp alleles, primers 5'CTACC-CCCAG-AACAT-CACCA-3' and 5'CTCCA-ATGAC-TAGGG-TGCCA-3' in the case of the Cys282Tyr mutation and primers 5'-CCCTC-TCCAC-ATACC-CTTGC-TG-3 - and 5'-AAGCT-TTGGG-CTACG-TGGAT-GATCA-G-3' in the case of mutations his63asp alleles with subsequent hydrolysis of the first of the above amplification the Rsal restriction enzyme, and the second is a restriction enzyme Ksp221, while analysis of the hydrolysis products of amplification carried out in polyacrylamide gel. The method is simple and accurate diagnosis and enables the screening of large arrays of DNA samples for face recognition prone to hemochromatosis. Diagnosis of this disease to organic lesions of 2 ill.

The invention relates to molecular biology and medicine and can be used to identify polymorphic variants (mutations) of a gene HLA-H (Cys282Tyr and his63asp alleles) associated with the predisposition of a person to hemochromatosis.

Diagnosis of mutations to the clinical manifestations of hemochromatosis can prevent the development of disease and as a consequence of cirrhosis and liver cancer. The greatest probability of developing hemochromatosis holders of the Cys282Tyr mutation in the homozygous state and mixed heterozygotes for mutations Cys282Tyr and his63asp alleles. The frequency of these variants of the gene HLA-H in different populations Caucasoids is 3-10% for the Cys282Tyr and 8-23% for his63asp alleles that demonstrates the need for mass screening of the population at risk for liver disease.

Known methods of diagnosing a predisposition to hemochromatosis are to amplify regions of the gene that contains the location of a possible finding of mutations, the hydrolysis of amplified DNA by nuclease, restriction site which is combined with location-specific mutations, and subsequent electrophoresis hydrolyzed DNA. In other words, the detection of polymorphic variants includes the analysis of the polymorphism of the lengths restrictio possible finding of mutations Cys282Tyr and his63asp alleles.

Known methods are different sets of primer pairs to two amplificare parts of the gene HLA-H, used in the analysis restrictase and conditions of the electrophoresis. From the selection of primers, restricts and conditions of PCR and electrophoresis depends on the unambiguous interpretation of the data RFLP analysis.

Known, for example, a method for the diagnosis of hemochromatosis using for hydrolysis of amplification of restricted SnaBI and BclI and subsequent agarose gel electrophoresis (J. Hepatol., 1997 27(5): 773-779). When using this method it is impossible to clearly distinguish between heterozygotes and homozygotes for the Cys282Tyr mutation, as well as heterozygotes and carriers of the normal allele by mutation his63asp alleles in the case of incomplete hydrolysis of amplification restrictase. This can lead to the incorrect identification of the genotype of the patient and consequently to incorrect diagnosis. Therefore, when using this method requires additional verification of doubtful cases (repeated tests).

Closest to the claimed method is a method for the diagnosis of hemochromatosis by hydrolysis of amplification two restrictable: RsaI and SnaBI for the Cys282Tyr mutation, BclI and MboI - for mutations his63asp alleles prototype (Tissue Antigens 199 the practical object of the invention is the simplification of the method and improving the accuracy of diagnosis.

The goal of the project is achieved by the proposed method lies in the following.

To identify the Cys282Tyr mutation spend amplification of the corresponding DNA with a pair of primers: 5'-CTACC-CCCAG-AACAT-CACCA-3' and 5'CTCCA-ATGAC-TAGGG-TGCCA-3', complementary to the DNA sequences analyzed gene in positions 6586-6567 and 6941-6960 mode 95oC - 3 min, then 35 cycles: 94oC - 1 min, 50oC - 1 min, 72oC - 1,1 minutes Amplified DNA size 394 N. p. hydrolyzing the restriction enzyme RsaI. The most common allele of the gene HLA-H due to the presence of a single RsaI restriction site cleaved into two fragments of length 126 268 N. p. Rare allele with the Cys282Tyr mutation has an additional restriction site and is cleaved so three fragments: 29, 126, 239 N. p. Significant differences in the lengths of restriction fragments make it possible to differentiate all the alleles of the gene HLA-H on this section.

To identify mutations his63asp alleles amplification of the corresponding fragment is carried out with a pair of primers: 5'-CCCTC-TCCAC-ATACC-CTTGC-TG-3' and 5'-AAGCT-TTGGG-CTACG-TGGAT-GATCA-G-3', complementary areas 4929-4907 and 4717-4743 gene HLA-H with an unpaired nucleotide at position 4740 mode 95oC - 3 min, then 35 cycles: 94 the positions 4740 to provide an additional restriction site Ksp221. Next, the amplified DNA size 212 N. p. hydrolyzing the restriction enzyme Ksp221. The most common allele of the gene HLA-H contains two restriction site and is cleaved into fragments of length 20, 21 and 171 N. p. Allele carrying the mutation his63asp alleles, has only one restriction site due to artificial replacement at position 4740 in primer and is cleaved into fragments of 20 and 192 N. p. that allows us to identify the electrophoretic. The presence during electrophoresis bands of DNA larger speaks of incomplete cleavage fragment by restriction enzyme.

Determining significant difference between the proposed method in comparison with the prototype is the following:

- to identify the Cys282Tyr mutation spend amplification of the corresponding DNA using primers 5'-CTACC-CCCAG-AACAT-CACCA-3' and 5'-CTCCA-ATGAC-TAGGG-TGCCA-3' followed by hydrolysis of amplified DNA with the restriction enzyme RsaI, and to identify mutations his63asp alleles carry out the amplification of the corresponding DNA using primers 5'-CCCTC-TCCAC-ATACC-CTTGC-TG-3' and 5'-AAGCT-TTGGG-CTACG-TGGAT - GATCA-G-3' followed by hydrolysis of amplified DNA with the restriction enzyme Ksp221 that allows you to simplify the way and at the same time improves diagnostic accuracy due to precise differentiation of allelic States of the gene HLA-H;

Rhino separation of DNA fragments formed after the restriction.

When this method of identifying the Cys282Tyr mutation after treatment with restriction enzyme in the case of homozygotes for the normal allele results in two DNA fragment length 126 268 N. p., in the case of homozygotes for the mutation - three fragment length 29, 126, 239 N. p., and in the case of heterozygotes four fragment length 29, 126, 239, 268 N. p. In the case of incomplete cleavage of the initial DNA fragment by restriction enzyme arise fragments of greater length.

When detecting mutations his63asp alleles after treatment with restriction enzyme in the case of homozygotes for the normal allele occur three DNA fragments of length 20, 21 and 171 N. p., in the case of homozygotes for the mutation - two fragments of length 20 and 192 N. p. , and in the case of heterozygotes for this mutation - four fragments of length 20, 21, 171 and 192 N. p. In the case of incomplete cleavage of the initial DNA fragment by restriction enzyme arise fragments of greater length.

Thus, a greater likelihood of suffering from hemochromatosis patients have, in the analysis of DNA and the Cys282Tyr mutation in the picture electrophoresis present only strip length 29 (possibly invisible), 126, 239 N. p. (clearly discern regardless of the result of the analysis on the his63asp alleles mutation, and patients, the analysis of DNA and the mutation Cys28 DNA mutation his63asp alleles - strips 20, 21 (possibly invisible), 171 and 192 N. p. (clearly distinguished).

The invention is illustrated by the following examples of specific performance.

To identify the Cys282Tyr mutation of the gene HLA-H performed PCR with 1 µg of genomic DNA in mode 95oC - 3 min, then 35 cycles: 94oC - 1 min, 50oC - 1 min, 72oC - 1,1 min in buffer containing 0,067 M Tris-HCl, 1.8 mm MgCl2-mercaptoethanol, 0.01% tween-20, in the presence of 0.2 mm of deoxynucleosides and 1 unit of Taq polymerase using a pair of primers 5'-CTACC-CCCAG-AACAT-CACCA-3' and 5'CTCCA-ATGAC-TAGGG-TGCCA-3' at a concentration of 0.6 μm. Amplificatoare DNA fragments by length 394 N. p. precipitated with an equal volume of a solution of polyethylene glycol 6000, 2.5 M NaCl, collected by centrifugation, washed with 75% ethanol, dissolved in water and hydrolyzing the restriction enzyme RsaI. The length of the products of the restriction appreciate after electrophoresis in 4% polyacrylamide gel followed by color bromide by ethidium and photography in the ultraviolet. Fragments of length 29 N. p. in consideration not accept because of the small size. As a result, in the case of homozygotes for the normal allele were clearly visible bands of DNA of length 126 268 N. p. (Fig. 1, b), in the case of homozygotes for the mutation Cys282 length served as the completeness of the cleavage fragment by restriction enzyme.

To identify mutations his63asp alleles using 1 µg of genomic DNA. The reaction mixture containing 0,067 M Tris-HCl, 1.8 mm MgCl2, 10 mm-mercaptoethanol, 0.01% tween-20, 0.2 mm deoxynucleotides, 0.6 μm primers 5'-CCCTC-TCCAC-ATACC-CTTGC-TG-3' and 5'-AAGCT-TTGGG-CTACG-TGGAT-GATCA-G-3', were heated at 95oC for 3 min, in the process of this solution add 1 unit of Taq polymerase, and then immediately perform PCR at 94oC - 1 min, 55oC - 1 min, 72oC - 1 min for 35 cycles. The PCR product length 212 N. p. precipitated with an equal volume of a solution of polyethylene glycol 6000, 2.5 M NaCl, collected by centrifugation, washed with 75% ethanol, dissolved in water and treated with restriction enzyme Ksp221. The length of the products of the restriction appreciate after electrophoresis in 4% polyacrylamide gel followed by color bromide by ethidium and photography in the ultraviolet. Fragments of length 20 and 21 N. p. in consideration not accept because of the small size. As a result of this homozygous for the normal allele clearly identified along the strip length 171 N. p. (Fig.2, a), homozygous for the mutation his63asp alleles - band 192 N. p. (Fig.2). Heterozygotes were characterized by the presence of two bands - 171 and 192 N. p. (Fig.2, b). The absence of DNA bands of greater length is CONSOB diagnostics were examined in 32 healthy people and 5 patients with clinical signs of hemochromatosis. As a result, among patients with hemochromatosis was discovered one carrier of the Cys282Tyr mutation in the homozygous state and two mixed heterozygotes for mutations Cys282Tyr and his63asp alleles among healthy a single media such genotypes have been identified.

The use of the proposed method will allow for a comparison with prototype:

- improves diagnostic accuracy due to the unambiguous differentiation of different allelic States of the gene HLA-H,

- to simplify the way through the use of a smaller number of enzymes, and, therefore, fewer stages in the analysis.

A method for diagnosing a predisposition to hemochromatosis by electrophoretic analysis of the lengths of restriction fragments of amplification products of the two sites of the DNA of the gene HLA-H, contains the location of a possible finding of mutations Cys282Tyr and his63asp alleles, wherein in the amplification plot containing the possible location of the Cys282Tyr mutation, using primers with 5'-CTACC-CCCAG-AACAT-CACCA-3' and 5'CTCCA-ATGAC-TAGGG-TGCCA-3', and if amplification section, containing the possible location of mutations his63asp alleles-primer - 5'- CCCTC-TCCAC-ATACC-CTTGC-TG-3' and 5'-AAGCT-TTGGG-CTACG-TGGAT-GATCA-G-3' introduced in one of the restriction site Rsp221, with poseduoilis hydrolysis products of amplification carried out in polyacrylamide gel.

 

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