RU2396354C2 - Method of identifying s65c, h63d, c282y mutations and polymorphism 2 ex + 4t>c in hfe gene - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to molecular biology and can be used in medical practice when diagnosing hereditary hemochromatosis. The method involves carrying out allele- specific polymerase chain reaction (PCR) in the presence of Sybr Green l dye in which mutant and normal alleles are obtained in different reaction vessels, and determination of the end product through analysis of amplicon temperature dissociation curves.

EFFECT: simple method for simultaneous identification of three main mutations (S65C, H63D, C282Y) in the HFE gene associated with development of hereditary hemochromatosis and polymorphism 2 EX +4T→C, used as additional monitoring of the presence of S65C and H63D mutations.

11 dwg, 11 ex

Description

Mutations in the HFE gene (6p21.3) lead to the development of hereditary or classical hemochromatosis, the clinical consequences of which are cirrhosis, diabetes, heart failure and skin pigmentation. One third of deaths in homozygous carriers are caused by hepatocellular carcinoma. The serious consequences of hereditary hemochromatosis can nevertheless be prevented, as the condition of patients can be easily corrected in the early stages of the development of the disease, if mutations can be detected that lead to the development of this disease. Clinically, hereditary hemochromatosis in the early stages is poorly diagnosed.

Hemochromatosis (hereditary or classic) is an autosomal recessive disease (1). The HFE gene is expressed on two alleles, and thus signs of the disease can also develop in heterozygous mutation carriers (1). At the same time, if a person adheres to a special diet that limits the intake of iron in the body, signs of the disease may be absent. Mutations in the HFE gene are a fairly common occurrence. Among hereditary diseases, classical hemochromatosis is considered to be one of the most common diseases. Among the mutations associated with the development of hemochromatosis, the most studied mutation is C282Y. Among the causes of liver cancer, this mutation is in the first place (8.8%), provided that it is homozygous (2). In the group of patients with cirrhosis, homozygous carriers for this mutation occur in 2.6% of cases (3). The prevalence of hemochromatosis in the United States (Utah), according to some data, is defined as 5.6%, with homozygotes being 0.3% and heterozygotes 10.6% (3). According to other sources, the incidence of the disease ranges from 1: 333 to 1: 500 (4.5). The occurrence of mutant alleles in Europe in individual populations is 29.7%, 5.2%, and 3% for H63D, C282Y, and S65C, respectively (6). The mutation 2EX + 4T> C in the splicing site of the second exon is considered by most researchers to be an ordinary polymorphism, but there is no consensus on this. At the same time, it is known that the replacement of the 4th nucleotide T with C in the donor splicing site is in unstable linkage with mutations S65C and H63D.

BACKGROUND

Various approaches to the determination of point mutations in genes are known, including in the HFE gene, in which the melting characteristics of duplexes are used (7-10). The closest analogue of the present invention can be considered the method proposed by Marzilliano N. et al. (8). This method is designed to identify H63D and C282Y mutations by allele-specific amplification of the corresponding gene regions in the presence of the intercalating dye Sybr Green 1 and subsequent registration of the final product by amplicon melting curves. The main disadvantage of the prototype is its lack of resolution in terms of distinguishing between homo and heterozygotes, as well as limiting the analysis to only two mutations, which are known to predispose to the development of the disease.

In the method according to the invention, these disadvantages are eliminated due to a) the inclusion of the third S65C mutation and 2EX + 4T> C polymorphism, which serves as an additional control for the presence of H63D and S65C mutations, and b) separate copies of the mutant and normal alleles for each of the studied mutations. This allows you to increase the resolution of the method and, accordingly, the accuracy of the diagnosis.

The testing procedure is as follows. In one amplifier, PCR was simultaneously performed for eight samples obtained from one DNA sample with allele-specific primers.

Each reaction is performed in a separate test tube or in a separate cell of a 96-well PCR panel, where components of one or another test system are present. A test DNA sample obtained using any known isolation method using any commercial kit (in particular the “GFX Genomic Blood DNA Purificftion Kit” / Amersham /) is added 2 μl to each of the eight reaction mixtures. At the same time, reactions with control DNA are carried out in the same instrument and under the same conditions, in which the presence / absence of mutations was previously confirmed by an independent method.

The test is performed using oligonucleotides having the sequence:

gct-cca-cac-ggc-gac-tct-cat-g-3 '- 63H / primer identifying the normal allele 63 Hys (1)

gct-cca-cac-ggc-gac-tct-cat-c-3 '- 3D

/ primer identifying the mutant allele 63 Asp (2)

acg-tgg-atg-acc-agc-tgt-tcg-tgt-t-3 '- 63F / common primer for test systems (1 and 2)

gct-gtt-cgt-gtt-cta-tga-tca-tga-ga-3 '- 65S / primer identifying the normal allele of 65 Ser (3)

gct-gtt-cgt-gtt-cta-tga-tca-tga-gt-3 '- 65C / primer / identifying the mutant allele 63 Cis (4)

tgc-agc-cac-atc-tgg-ctt-gaa-3 '- 65R / common primer for test systems (3 and 4)

tgg-gga-aga-gca-gag-ata-tac-gtg-3 '- 282C / primer identifying the normal 282 Cis allele (5)

tgg-gga-aga-gca-gag-ata-tac-gta-3 '- 282Y / primer identifying the mutant allele 282 Tyr (6)

ggc-act-cct-ctc-aac-ccc-caa-tag-3 '- 282R / common primer for test systems (5 and 6)

cac-aac-cac-agc-aag-ggt-at-3 '- 2EX + 4FT / primer identifying the normal allele (7)

aca-acc-aca-gca-agg-gta-c-3 '- 2EX + 4FC / primer identifying the mutant allele (8)

gaa-aag-ctc-tga-caa-cct-cag-gaa-3 '-2EX + 4R / common primer for test systems (7 and 8)

A 50 μl reaction mixture consists of:

10x buffer - 5 μl

dATF, dTSTF, dGTF, dTTF - each 5 mM each

specific primers - each 12 pMol

Taq DNA polymerase - 5 units

Sibr Green fluorescent intercalating dye at a final concentration of 1: 10000-1: 100000

DNA - 2 μl

H ₂ O up to 50 μl

35 cycles of the reaction proceed according to the scheme:

95 ° C-30 sec

68 ° C-30 sec

72 ° C-1 min

on any thermocycler with a cell size of 0.2 ml (Cyclotemp 107 / MSTU named after Bauman).

The results are recorded according to the melting curve in an amplifier with an optical module, which allows recording fluorescence with a wavelength of 520 nm. The module heating step is set from 60 ° C to 95 ° C - 1 ° C / min. The point of dissociation of amplicons is in the region of 82-85 ° C. The result of the experiment is taken into account by the presence of a peak corresponding to the dissociation point of the control DNA, the presence / absence of mutations in which is previously confirmed independently. Despite the possibility of calculating the melting temperature for amplicons (11), a comparison of the test results of the test DNA and the control is necessary, since empirical data rarely exactly coincide with the calculated indicators, in addition, they can vary as a result of changes in the reaction conditions, which are difficult to withstand completely from test to test.

If there is a peak corresponding to the PCR product in a test tube with a C282Y mutation test system, the corresponding mutant allele is present in the test DNA. If there is no peak identifying a non-mutant allele, then the C282Y mutation is in the homozygous position. If the mutation is homozygous in a test tube with a test system for the normal allele (C282), the PCR product will be absent and, accordingly, there will be no signal for the dissociation of the PCR product.

At the heterozygous position of the C282Y mutation in the test DNA, the melting peaks of the DNA will be recorded in test tubes with the normal allele and mutant allele test systems.

The absence of a PCR fragment in a test tube with a C282Y mutation test system, while a specific PCR product is in a test tube for a normal allele, determined by the correspondence of the dissociation curve of a control normal DNA fragment, indicates that both alleles of the tested DNA encode cysteine at 282 protein positions ( HFE gene), i.e. no mutation.

The results of testing for mutations H63D, S65C, as well as 2EX + 4T> C polymorphism are evaluated in a similar way.

EXAMPLES OF WORK OF TEST SYSTEMS.

Option No. 1 (figure 1)

- The target DNA contains a C282Y mutation in the heterozygous position. Test systems for normal and mutant alleles produce PCR products, which is recorded by the presence of two dissociation curves 84.7 - 85.1 ° C.

Option No. 2 (figure 2)

- The target DNA contains the C282Y mutation in the homozygous position. The mutant allele test system produces a PCR product, which is detected by the presence of a 84.7 ° C dissociation curve. Whereas the peak of dissociation is not recorded in a test tube with a test system for the normal allele.

Option No. 3 (figure 3)

- The target DNA does not contain the C282Y mutation. The test system for the normal allele produces a PCR product, which is recorded by the presence of a dissociation curve of 85 ° C. Whereas the peak of dissociation is not recorded in vitro with the test system for the mutant allele.

Option No. 4 (figure 4)

- The target DNA contains a H63D mutation in the heterozygous position. Test systems for normal and mutant alleles produce PCR products, which is recorded by the presence of two dissociation curves 84.7 - 85.1 ° C.

Option No. 5 (figure 5)

- The target DNA contains a H63D mutation in the homozygous position. The mutant allele test system produces a PCR product, which is detected by the presence of a 84.8 ° C dissociation curve. Whereas the peak of dissociation is not recorded in a test tube with a test system for the normal allele.

Option No. 6 (Fig.6)

- The target DNA does not contain the H63D mutation. The test system for the normal allele produces a PCR product, which is recorded by the presence of a dissociation curve of 85 ° C. Whereas the peak of dissociation is not recorded in vitro with the test system for the mutant allele.

Option No. 7 (Fig.7)

- The target DNA contains the S65C mutation in the heterozygous position. Test systems for normal and mutant alleles produce PCR products, which is recorded by the presence of two dissociation curves of 83.8 ° C.

Option No. 8 (Fig.8)

- The target DNA does not contain the S65C mutation. The test system for the normal allele produces a PCR product, which is recorded by the presence of a dissociation curve of 83.8 ° C. Whereas the peak of dissociation is not recorded in vitro with the test system for the mutant allele.

(S65C mutation is a rare event, therefore, we were not able to detect this mutation in the homozygous position among the tested DNA samples).

Option No. 9 (Fig.9)

- The target DNA contains a polymorphism 2ex + 4T> C in the heterozygous position. Test systems for the 2x + 4T allele and the 2x + 4C allele produce PCR products, which is recorded by the presence of two dissociation curves 82-82.6 ° C.

Option No. 10 (figure 10)

- The target DNA contains the 2x + 4T allele in the homozygous position. The test system for the 2x + 4T allele produces a PCR product, which is recorded by the presence of a dissociation curve of 82.6 ° C. While the PCR product in a test tube with a test system for the 2x + 4C allele is absent (there is no dissociation curve).

Option No. 11 (11)

- The target DNA contains the 2x + 4C allele in the homozygous position. The test system for the 2x + 4C allele produces a PCR product, which is recorded by the presence of a dissociation curve of 82.6 ° C. While the PCR product in a test tube with a test system for the 2x + 4T allele is absent (there is no dissociation curve).

Information sources

1. Borecki et al. // Am. J. Hum. Genet. 47: 542-550, 1990.

2. Willis et al. // Gut 46: 401-404, 2000.

3. Cartwright et al. // New Eng. J. Mod. 301: 175-179, 1979.

4. Beaumont et al. // New Eng. J. Med. 301: 169-174, 1979.

5. MacSween & Scott et al. //. J. Clin. Path 26: 936-942, 1973.

6. de Juan et al. // Europ. J. Hum. Genet. 9: 961-964, 2001.

7. Smillie D. // J. Clin. Pathol: Mol. Pathol., 51, 232-236, 1998.

8. Marzilliano N. // Haematol, 85, 9, 990-991, 2000.

9. Papp A. et al. // Biotech. 34, 5, 1068-1072, 2003.

10. Germer S. & Higuchi R. // Gen. Res. 9, 72-78, 1999.

11. Lew M. et al. // Clin. Chem., 50, 7, 1156-1164, 2004.

Claims (1)

A method for simultaneously determining the main mutations in the HFE gene associated with hereditary hemochromatosis, involving PCR amplification of the gene regions, which include the sites of the determined mutations, in the presence of an intercalating dye Sybr Green 1 and subsequent identification of the mutations by the dissociation curves of the obtained DNA fragments, characterized in that both three mutations S65C, H63D and C282Y are tested, as well as 2 EX + 4T> C polymorphism, used as an additional control for the presence of S65C and H63D mutations; each of the four determinations is carried out in two samples containing two primers, one of which is the same, and the other provides amplification of either the normal or mutant allele, using the following specific oligonucleotides:
(1) gctccacacggcgactctcatg-3 '(primer identifying the normal 63 allele of His),
(2) gctccacacggcgactctcatc-3 '(primer identifying the mutant allele 63 Asp),
acgtggatgaccagctgttcgtgtt-3 '(primer common to samples (1) and (2));
(3) gctgttcgtgttctatgatcatgaga-3 '(primer identifying the normal 65Ser allele),
(4) gctgttcgtgttctatgatcatgagt-3 '(primer identifying the mutant allele of 65 Cis),
tgcagccacatctggcttgaa-3 '(primer common to samples (3) and (4));
(5) tggggaagagcagagatatacgtg-3 '(primer identifying the normal 282Cis allele),
(6) tggggaagagcagagatatacgta-3 '(primer identifying the mutant allele 282Tur),
ggcactcctctcaacccccacaag-3 '(primer common to samples (5) and (6));
(7) cacaaccacagcaagggtat-3 '(primer identifying the normal allele 2EX + 4T),
(8) acaaccacagcaagggtac-3 '(primer identifying the mutant allele 2EX + 4C),
gaaaagctctgacaacctcaggaa-3 '(primer common to samples (7) and (8)).

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