RU2458131C1 - Test system for mutation detection in human fumarylacetoacetate hydrolase and alpha-1-antitrypsin genes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is offered is a test system presented by a oligonucleotide kit enabling detecting point mutations in FAH and SERPINA1 genes which induce type 1 tyrosinemia and alpha-1-antitrypsin deficiency respectively. What us described is a method for detection of said point mutations involving two-round multiplex PCR with using the respective kit of specific primers and hybridisation of the prepared PCR products with a biochip containing the test system under the invention.

EFFECT: method enables the short-term accurate genetic analysis.

3 cl, 13 tbl, 19 dwg, 6 ex

Description

Technical field

The invention relates to molecular biology and medicine, and considers the creation and use of a test system for determining the presence of point mutations in the fumaryl acetoacetate hydrolase gene, causing liver damage in newborns.

One of the most frequent metabolic disorders detected during the neonatal period is an increase in the concentration of bilirubin in the blood serum. It is known that the causes of hyperbilirubinemia are: increased bilirubin formation during the breakdown of hemoglobin in the cells of the reticuloendothelial system, decreased conjugation of bilirubin, impaired excretion, or a combination of some of the above factors. The incidence of hyperbilirubinemia is about 55% in full-term infants. In most cases, neonatal hyperbilirubinemia is functional (associated with the immaturity of the enzymatic system) and disappears during the first weeks of life. Nevertheless, an increase in the level of bilirubin may be associated with the pathology of the excretory function of the hepatobiliary system and be malignant, in particular, lead to irreversible damage to the central nervous system.

Over the past decades, understanding of the basics and pathophysiology of many liver diseases has deepened, a number of new nosological forms have been established, the possibilities of effective treatment of many diseases have appeared, in particular, diet therapy (for galactosemia and tyrosinemia) has begun, and liver transplantation has become possible. To date, the number of recognized hereditary liver diseases in newborns is about 20 different forms. The genes of all these diseases are mapped, and in many cases the spectrum of the main pathogenic mutations is determined. For some genes (encoding mainly enzymes), polymorphic variants have also been characterized, represented mainly by single nucleotide substitutions, which can affect the residual activity of the protein. However, the differential diagnosis of most of them is extremely difficult due to the similarity of clinical manifestations. In our country, the number of patients who are quickly and correctly diagnosed is relatively small. Most children die at an early age without adequate treatment, and burdened families do not have information about a hereditary disease.

Since in some cases the causes of malignant hyperbilirubinemia of newborns remain unclear, studying the role of genetic factors in the formation of this pathological condition is an urgent scientific task of great practical importance.

Already in the first month of life, the first clinical signs that indicate a hereditary pathology of the liver and bile ducts and manifest as cholestasis syndrome can be detected. One of the reasons for the violation of the excretory function of the hepatobiliary system in the neonatal period (neonatal cholestasis) is hereditary disease. According to rough estimates, the prevalence of congenital or hereditary liver diseases in children is 1 case per 2500 newborns. It is in such children that severe forms of hepatic pathology develop. A correct and quick diagnosis in this case is very important, because allows you to predict the course of the disease and conduct adequate therapy. However, differential diagnosis of this group of diseases is extremely difficult.

The object of the study is the fumaryl acetoacetate hydrolase and human alpha-1-antitrypsin genes.

The purpose of the work is to develop a system that ensures the diagnosis and genetic analysis of several hereditary diseases from the group of congenital metabolic disorders leading to the development of neonatal hyperbilirubinemia: type 1 tyrosinemia and alpha-1-antitrypsin deficiency.

A test system has been developed, which is a biochip for the detection of mutations and polymorphisms of the fumarylacetoacetate hydrolase and alpha-1-antitrypsin genes on a microchip. In the process, design and optimization of the sequences of oligonucleotide sequences for immobilization on a microchip was carried out. The hybridization conditions on the microchip are selected. As a result of the study, the DNA of children with type 1 tyrosinemia and alpha-1-antitrypsin deficiency carrying nucleotide substitutions in the areas of the studied mutations and polymorphisms of the fumaryl acetoacetate hydrolase and alpha-1-antitrypsin genes was selected. The fragments of these DNA were analyzed by the methods of restriction fragment length polymorphism (RFLP), sequencing, hybridization on a microchip. A comparative analysis of the results obtained by these methods is carried out.

The aim of our work is to develop a system that provides simultaneous diagnosis and genetic analysis of two hereditary diseases from the group of congenital metabolic disorders leading to the development of neonatal hyperbilirubinemia: type 1 tyrosinemia, alpha-1-antitrypsin deficiency. To solve this problem, a test system based on one microchip should be created to detect mutations in the genes of galactose-1-phosphate uridyl transferase; hydrolase fumaryl acetoacetate and in the alpha-1 antitrypsin gene. The developed method provides an accurate genetic analysis and allows you to minimize the time of diagnosis.

Hereditary tyrosinemia type 1.

One of the factors causing severe liver dysfunctions is a violation of tyrosine metabolism caused by mutations in genes encoding enzyme proteins.

Three types of tyrosinemia are known today: tyrosinemia type 1 or hepatorenal tyrosinemia (fumaryl acetoacetate hydrolase deficiency), tyrosinemia type 2 (tyrosine transaminase deficiency), and type 3 tyrosinemia (havkinsuria or 4-hydroxyphenyl pyruvate dioxyne deficiency).

Among these diseases, type 1 tyrosinemia is the most severe, requiring urgent correction of pathology. Mass screening of newborns, conducted in many countries, is aimed at identifying this particular form of tyrosinemia. Timely diagnosis of tyrosinemia allows you to start diet therapy on time and avoid serious consequences for the health of the child, as well as reduce the economic costs associated with medical rehabilitation with late diagnosis.

Tyrosinemia type 1 is described by U.Baber et al. in 1956. The type of inheritance is autosomal recessive. The disease is associated with a deficiency of fumaryl acetoacetate hydrolase, which catalyzes the last stage of tyrosine degradation. Due to the deficiency of the enzyme, tyrosine metabolism is disrupted, its metabolites accumulate in the tissues: fumaryl acetoacetate, maleylacetoacetate, succinylacetone, succinylacetoacetate, which have a toxic effect on liver and proximal renal tubule cells, as a result of which tubular reabsorption processes suffer, first of all. Secondary inhibition of the activity of a number of enzymes takes place: 4-hydroxyphenylpyruvate dioxygenase, porphobilinogen synthase, methionine-adenosyl transferase, delta-aminolevulinic acid dehydratase, gluconeogenesis enzymes, which entails significant biochemical disorders. The antioxidant defense is violated, the total antioxidant activity of the plasma decreases.

The main methods for confirming the diagnosis of tyrosinemia type 1 are biochemical tests based on determining the concentration of tyrosine in the blood and succinyl acetone in the urine. To confirm the diagnosis, the study of the enzyme fumaryl acetoacetate hydrolase in leukocytes or fibroblasts is carried out. Methods have been developed for prenatal diagnosis of the disease by examining succinylacetone in amniotic fluid and the activity of fumarylacetoacetate hydrolase in amniocyte culture or chorionic cells. Difficulties in interpreting metabolic changes are due to the fact that with liver damage of another etiology (both hereditary and non-hereditary), an increase in the level of tyrosine in the blood is possible. Therefore, molecular genetic methods play an important role in the diagnosis of this disease. Mutation analysis in the case of type 1 tyrosinemia may be useful for detecting false-positive results from biochemical tests.

The fumaryl acetoacetate hydroxylase (FAH) gene is mapped to chromosome 15 (locus q23-q25) and consists of 14 exons separated by 13 introns. To date, the gene describes more than 70 mutations, which are mainly represented by missense mutations. Table 1 shows the data for all the mutations of the FAH gene described in the literature.

The most common mutation in patients from Europe, Pakistan, Turkey and the United States is the intron intrusion replacement IVS12 + 5 G> A [Rootwelt H, Høie K, Berger R, Kvittingen EA. Fumarylacetoacetase mutations in tyrosinaemia type I. Hum Mutat. 1996; 7 (3): 239-43; Rootwelt H, Kristensen T, Berger R, Høie K, Kvittingen EA. Tyrosinemia type I-complex splicing defects and a missense mutation in the fumarylacetoacetase gene. Hum Genet 1994 Sep; 94 (3): 235-9]. Mutation IVS 6-1 G> T prevails in Central and Eastern Europe [Bergman AJ, van den Berg IE, Brink W, Poll-The BT, Ploos van Amstel JK, Berger R. Spectrum of mutations in the fumarylacetoacetate hydrolase gene of tyrosinemia type I patients in northwestern Europe and Mediterranean countries. Hum mutat. 1998; 12 (1): 19-26], and in Spain it makes up about 70% of all mutant alleles [Arranz JA, Piñol F, Kozak L, Pérez-Cerdá C, Cormand B, Ugarte M, Riudor E. Splicing mutations, mainly IVS6- 1 (G> T), account for 70% of fumarylacetoacetate hydrolase (FAH) gene alterations, including 7 novel mutations, in a survey of 29 tyrosinemia type I patients. Hum mutat. 2002 Sep; 20 (3): 180-8]. Some populations have their own characteristics of the mutation profile, for example, in the Ashkenazi Jewish population, the Pro261Leu mutation accounts for almost 100% of the mutant alleles [Elpeleg ON, Shaag A, Holme E, Zughayar G, Ronen S, Fisher D, Hurvitz H. Mutation analysis of the FAH gene in Israeli patients with tyrosinemia type I. Hum Mutat. 2002 Jan; 19 (l): 80-l]. In Finland, the Trp262Term mutation accounts for 80% of the mutant alleles, while in patients from other parts of Europe this mutation was not detected [St-Louis M, Leclerc B, Laine J, Salo MK, Holmberg C, Tanguay RM. Identification of a stop mutation in five Finnish patients suffering from hereditary tyrosinemia type I. Hum Mol Genet. 1994 Jan; 3 (l): 69-72].

In order to develop a biochip, we analyzed literature data and mutation frequencies in the FAH gene in patients with type 1 tyrosinemia from the Russian Federation.

The most frequent mutations in the FAH gene in patients with type 1 tyrosinemia from the Russian Federation: Pro342Leu; Argl74Term; IVS 12 + 5 G-> A; IVS6-1 G-> T.

Considering the above, the following mutations in the FAH gene were chosen to create the biochip: Pro342Leu, Argl74Term, IVS 12 + 5 G-> A, IVS6-1 G-> T, IVS7-1 G-> A.

Alpha-1-antitrypsin deficiency.

Alpha-1-antitrypsin deficiency is an autosomal recessive inherited disorder caused by mutations in the SERPINA1 gene. Alpha-1-antitrypsin is an inhibitor of a wide range of serine proteases, playing an important role in protecting tissues from proteolysis. Serine proteases that he inhibits include proteases from the blood coagulation system and fibrinolysis and proteases of the immune response. Violation of the inhibition of these protease groups explains the symptoms of increased bleeding in patients and the frequent death of children from intercurrent diseases and sepsis. Those forms of alpha-1-anitrypsin deficiency that manifest liver disease in the neonatal period lead to an early death. Lighter forms of the disease manifest later. The course of the disease is chronic. It leads to chronic liver failure or less often occurs as asymptomatic hepatosplenomegaly or cholanocarcinoma. In adults, a high incidence of cirrhosis is noted. There is also a high risk of developing emphysema.

The main methods for confirming the diagnosis of alpha-1-antitrypsin deficiency are biochemical tests based on determining the concentration of aflf-1-antitrypsin in the blood plasma. Electrophoretic mobility (isoelectric focusing in a polyacrylamide gel) is also being investigated. In homozygous carriers of mutations, it is changed compared to the control. Mutation analysis in the case of alpha-1-antitrypsin deficiency may be useful for detecting false-positive results from biochemical tests.

The SERPINA1 gene is mapped on chromosome 14 (q32.1 locus) and consists of 5 exons separated by 4 introns. To date, the gene describes more than 40 mutations, which are mainly represented by missense mutations. The most common mutation that causes the development of an early form of the disease is the E342K mutation (Z allele). The second common mutation is the E264V mutation, which in combination with an allele can cause the development of emphysema.

DNA analysis, as a diagnostic-confirming procedure, is based on the determination of mutations using PCR and subsequent restriction analysis.

To create a biochip, frequent mutations in the SERPINA1 gene were selected: E342K, E264V.

The following methods are used to detect point mutations in various genes.

1. The extent of the chain labeled with dideoxyribonucleoside triphosphate (SNP-single nucleotide polymorphism);

2. Allele - specific PCR (Allele specific PCR);

3. The study of fragment polymorphism after restriction (RFLP-restriction fragment length polymorphism);

4. Analysis of conformational polymorphism of single and double stranded DNA (SSCP and DSCP-single- (double) -strand conformation polymorphism);

5. Hybridization on microarrays (Hybridization on microai Tay);

6. Sequencing (sequencing);

7. Dideoxyfingerprinting (ddF-method);

8. PCR heteroduplex analysis (PCR-heteroduplex analysis);

9. The method of imperfect duplex with RNA (RNA mismatch analysis);

10. The method of structurally specific cleavage (Structure-specific endonuclease cleavage);

11. The method of probes (Line Probe assay (LiPA));

12. The PhaB method.

However, all of the above methods have certain disadvantages, which in practice can significantly complicate the mass screening of patients. Thus, chain extension methods and allele-specific PCR (1, 2) require independent reactions to be performed according to the number of mutations studied (i.e., several dozen samples for one patient in the case of the galactose-1-phosphate uridyl transferase gene) and, accordingly, a large number the studied sample.

Methods for studying polymorphism (3, 4), PCR - heteroduplex analysis (8) and the imperfect duplex method with RNA (9) are laborious, take a lot of time, give an indirect conclusion about the type of mutation and require standard standards (for each mutation); In addition, for the imperfect duplex method, increased requirements are imposed on the absence of RNAase. Also, this method is time-consuming and not acceptable for the detection of G-U duplex.

Hybridization on microarrays (5) requires a complex computer calculation of the results and expensive consumables (the microarray itself).

Direct sequencing (6) requires isolation of the gene sequence, has a high cost and requires a sequencing device.

Structurally specific splitting methods (10) and dideoxy fingerprinting (7) require preliminary standardization (selection of conditions), which increases the complexity and the presence of standard standards. In addition, dideoxy fingerprinting is performed with a radiolabeled probe.

The probe method (11) has a high cost and works in the case of a small number of mutations.

The implementation of the PhaB method (12) takes a large amount of time, since it is associated with the replication of phages and subsequent registration of lysis in the culture of M. smegmatis, and it is also very laborious.

These disadvantages are eliminated in the present invention.

The hybridization method on biochips compares favorably with all of the above methods with the ability to determine several mutations present at the same time, low cost, short time to obtain the result. Also, it does not require expensive equipment and highly qualified personnel.

When conducting a patent search, we did not find analogues of the claimed invention. The presented invention has several advantages compared with other methods of medical genetic diagnosis of tyrosinemia type 1.

1. Using the claimed biochip, it is possible to simultaneously detect several different point mutations in the FAH and SERPINA1 gene, which will make it more likely to exclude false-positive results when a patient is diagnosed.

2. The claimed biochip will be more acceptable than the more expensive and time-consuming methods for use in routine medical genetic diagnosis of type 1 tyrosinemia in clinical laboratories.

Thus, our claimed invention will allow us to more easily and accurately determine the presence of mutations in the genes of the RAS and SERPINA1, which will significantly improve the diagnosis of tyrosinemia type I and alpha-1-antitrypsin deficiency.

Disclosure of the invention.

The claimed invention relates to a biochip, which allows to detect the presence of point mutations in a human patient; the use of the specified biochip for the diagnosis of the patient-person, as well as the method of obtaining and using the specified biochip.

A brief description of the figures.

Figure 1. Application of oligonucleotides complementary to various mutations in the FAH gene on a biochip (block 1).

Figure 2. Application of oligonucleotides complementary to various mutations in the SERPINA1 gene on a biochip (block 2).

Figure 3. Detection of the Argl74 mutation (CGA-> TGA). Direct DNA sequencing. Arrows indicate the location of the mutation. A. Fragment of exon 6 of the FAH gene. Norm. B. Fragment of exon 6 of the FAH gene. Mutation Argl74Term in the heterozygous state.

Figure 4. Detection of the mutation IVS6-1 g-> t. RFLP analysis. When an IVS 6-1 G-> T mutation occurs, the restriction site for the restriction endonuclease Bsp 1720 I (GC ^ TNAGC) disappears. Lanes: 1 - molecular weight markers; 2 - fragment of exon 7 of the FAH gene before restriction; 3, 4 - fragment of exon 7 of the FAH gene after treatment with restriction endonuclease Bsp 1720 I, normal; 5 - fragment of exon 7 of the FAH gene after treatment with restriction endonuclease Bsp 1720 I, mutation IVS 6-1 G-> T in a homozygous state; 6 - fragment of exon 7 of the FAH gene after treatment with restriction endonuclease Bsp 1720 I, mutation IVS 6-1 G-> T in the heterozygous state.

Figure 5. Detection of the mutation IVS7-1 g-> a. RFLP analysis. When the IVS7-1 G-> A mutation occurs, a restriction site for the restriction endonuclease Alu I (AG ^ CT) appears. Lanes: 1 - molecular weight markers; 2 - fragment of exon 8 of the FAH gene before restriction; 3 - fragment of exon 8 of the FAH gene after treatment with restriction endonuclease Alu I, normal.

Figure 6. Detection of the mutation IVS 12 + 5 g-> a. Direct DNA sequencing. Arrows indicate the location of the mutation. A. Fragment of intron 6 - exon 7 of the FAH gene. Norm. B. Fragment of intron 6 - exon 7 of the RAS gene, mutation IVS 12 + 5 G-> A in the heterozygous state.

Figure 7. Detection of the Pro342Leu mutation (CCG-> CTG). Direct DNA sequencing. Arrows indicate the location of the mutation. A. exon fragment 12 of the FAH gene. Norm. B. Fragment of exon 12 of the FAH gene, mutation Pro342Leu in a homozygous state.

Figure 8. Detection of mutations E264V (GAA-> GTA) and E342K (GAG-> AAG). Lanes: 1 - molecular weight marker 100bp DNA ladder; 2 - PCR product of exon 5 of the alpha-1-antitrypsin gene before restriction (E342K mutation); 3-PCR product of exon 5 of the alpha-1-antitrypsin gene of the sample with the E342K mutation after restriction with Taq I endonuclease; 4-PCR product of exon 5 of the alpha-1-antitrypsin gene of the control sample (normal) after restriction with Taq I endonuclease; 5-PCR product of exon 3 of the alpha-1-antitrypsin gene before restriction (mutation E264V); 6, 7-PCR products of exon 3 of the alpha-1-antitrypsin gene of control samples (normal) after restriction with Taq I endonuclease

Figure 9. Electrophoregram of multiplex PCR for the first round (FAH gene). Lanes: 1 - molecular weight marker; 2-3 - the first round of PCR: exon fragment 6-7 of the FAH gene (Argl74Term mutations, IVS6-1 gt) - 463 bp + exon 12 fragment of the FAH gene (IVS mutations 12 + 5 ga, Pro342Leu) - 382 bp + exon 8 fragment FAH gene (mutation IVS7-1 ga) - 252 bp

Figure 10. Electrophoregram of multiplex PCR for the second round (FAH gene). Lanes: 1 - molecular weight marker; 2 - second round of PCR: fragment of exon 12 of the FAH gene (mutation IVS 12 + 5 g-a) - 149 bp + fragment of exon 7 of the FAH gene (mutation IVS6-1 g-t) - 129 bp; 3 - second round of PCR: exon 12 fragment of the FAH gene (Pro342Leu mutation) - 140 bp + exon 8 fragment of the FAH gene (IVS37-1 g-a mutation) - 121 bp + exon 6 fragment of the FAH gene (Argl74Term mutation) - 111 bp

Figure 11. Electrophoregram of multiplex PCR for the first round (gene SERPINA1). Lanes: 1 - molecular weight marker; 2-3 - second round of PCR: fragment of exon 3 of the gene SERPINA1 (mutation E264V) - 374 bp + fragment of exon 5 of the gene of SERPINA1 (mutation E342K) - 289 bp

Figure 12. Electrophoregram of multiplex PCR for the second round (gene SERPINA1). Lanes: 1 - molecular weight marker; 2-3 - second round of PCR: fragment of exon 3 of the gene SERPINA1 (mutation E264V) - 139 bp + fragment of exon 5 of the gene of SERPINA1 (mutation E342K) - 92 bp

Figure 13. Hybridization pattern of a DNA sample obtained from a person not carrying a mutation in the FAH gene with a biochip. The location of the points corresponds to figure 1.

Figure 14. Hybridization pattern of a DNA sample obtained from a person carrying the Argl74Term and IVS12 + 5 g-> a mutations in the heterozygous state in the FAH gene with a biochip. The location of the points corresponds to figure 1.

Figure 15. Hybridization pattern of a DNA sample obtained from a person carrying the IVS12 + 5 a-> g mutation in a heterozygous state in the FAH gene with a biochip. The location of the points corresponds to figure 1.

Figure 16. Hybridization pattern of a DNA sample obtained from a person carrying the Argl74Term and IVS6-1 g-> t mutations in the heterozygous state in the FAH gene with a biochip. The location of the points corresponds to figure 1.

Figure 17. Hybridization picture of a DNA sample obtained from a person carrying the Pro342Leu mutation in the homozygous state in the FAH gene with a biochip. The location of the points corresponds to figure 1.

Figure 18. Hybridization picture of a DNA sample obtained from a person not carrying a mutation in the SERPINA1 gene with a biochip. The location of the points corresponds to figure 2.

Figure 19. Hybridization picture of a DNA sample obtained from a person carrying the E342K mutation in a homozygous state in the SERPINA1 gene with a biochip. The location of the points corresponds to figure 2.

The implementation of the invention

The implementation of the invention is illustrated by the following examples.

Example 1. Identification of mutations used to create a biochip.

In order to develop a biochip block-1, we analyzed the literature data and mutation frequencies in the FAH gene in patients with type 1 tyrosinemia from the Russian Federation.

The mutation frequencies of Pro342Leu, Argl74Term, IVS 12 + 5 G-> A, IVS6-1 G-> T are currently 44%, 18%, 18% and 9%, respectively. In the analysis of literature data, mutations and polymorphisms were also selected, which were most often found in other populations.

Considering the above, the following mutations in the FAH gene were chosen to create the biochip: Pro342Leu, Argl74Term, IVS 12 + 5 G-> A, IVS6-1 G-> T, IVS7-1 G-> A.

In order to develop a block-2 biochip, we analyzed literature data and mutation frequencies in the SERPINA1 gene in patients with alpha-1-antitrypsin deficiency from the Russian Federation. The most common mutation that causes the development of an early form of the disease is the E342K mutation (Z allele). The second common mutation is the E264V mutation, which in combination with an allele can cause the development of emphysema.

Considering the above, two mutations in the SERPINA1 gene were chosen to create the biochip: E342K, E264V.

Example 2. Design and optimization of oligonucleotide sequences for immobilization on a microchip.

The design of the oligonucleotide sequences for immobilization on a microchip was carried out taking into account the analysis of the nucleotide sequence of the fumarylacetoacetate hydrolase (FAH) and alpha-1-antitrypsin (SERPINA1) genes in the areas of clinically significant mutations. For this purpose, the Oligo 6 program (USA) was used.

Example 3. Construction of a microchip for the detection of polymorphism in the galactose-1-phosphate uridyl transferase gene.

When choosing polymorphic loci, the analysis of which will be carried out using the biochip according to the invention, they were guided by the following approaches: 1) functionally significant alleles that were found with the greatest frequency were selected in the genes; 2) included only those nucleotide substitutions whose polymorphism has already been studied in a number of populations of Russia.

The nucleotide sequences used to create the biochip according to the invention are shown in tables 1; 2; 3 and 4.

The oligonucleotides shown in Tables 1, 2, 3, 4 were synthesized using a standard phosphoamitide procedure using an automated 394 DNA / RNA Synthesizer (Applied Biosystems, USA). At the 3'-end of the oligonucleotides, there is a free amino group spacer introduced at synthesis using 3'-Amino-Modifier C7 CPG 500 ("Glen Reseach", USA) in order to conduct photoinduced co-polymerization of oligonucleotides and components of a polyacrylamide gel.

In order to increase the reliability of signal determination, each oligonucleotide sample was duplicated. To select the sequence of oligonucleotides that ensure maximum specificity, several types of oligonucleotides complementary to the same mutation or non-mutant sequence were used in the biochip.

The fact of the presence of non-mutant and mutant alleles in the patient’s genome was established by analyzing the fluorescence intensity of the corresponding cells on the chip. The application of oligonucleotides complementary to the normal sequence and various mutations of the FAH gene on the biochip is shown in Figure 1, and complementary to the normal sequence and various mutations of the SERPINA1 gene are shown in Figure 2 (the designation of the oligonucleotides is the same as in tables 1, 2, 3, 4 )

Thus, the designed biochip allows us to determine the presence of mutations in the genes of fumaryl acetoacetate hydrolase (FAH) and alpha-1-antitrypsin (SERPINA1), leading to the onset of the disease.

Example 4. Preparation of DNA samples from healthy donors and patients with type 1 galactosemia for biochip testing.

As a result of the work, DNA samples of children with galactosemia patients were selected for each of the ten studied mutations. These DNA samples are prepared for testing the resulting DNA chip.

All mutations that we selected to create the biochip were confirmed by direct non-radioactive sequencing and / or restriction fragment length polymorphism analysis. As examples in figures 2-8 shows the results of part of the experiments. As can be seen in the figures, mutations are well detected using these methods. Thus, as a result of this work, DNA samples were selected that can be used to test the resulting biochip.

Example 5. Hybridization of the biochip with DNA probes with a known structure and with DNA probes obtained from patients.

As a result of the study, oligonucleotide primers and reaction conditions were selected for the second round of PCR on the sequences of the FAH and SERPINA1 genes obtained in the first round of PCR for further detection of pathogenic significant nucleotide substitutions of the FAH and SERPINA1 genes on the microchip. For the first and second rounds of multiplex PCR of the FAH and SERPINA1 genes, the following oligonucleotide primers were selected (tables 5 and 6, respectively).

Since the exons of the RAS gene are located in a dense cluster, in order to avoid cross-PCR, the second round was carried out as follows: an aliquot from the first round of PCR with a volume of 2 μl followed by multiplex PCR.

1. Fragment of exon 12 of the FAH gene (mutation Pro342Leu) + fragment of exon 8 of the FAH gene (mutation IVS37-1 g-a) + fragment of exon 6 of the FAH gene (mutation Argl74Term).

2. Fragment of exon 12 of the RAS gene (mutation IVS12 + 5 g-a) + fragment of exon 7 of the FAH gene (mutation IVS6-1 g-t).

Electrophoregram multiplex PCR for the RAS gene is presented in figures 9 and 10.

For the SERPINA1 gene, the second round of PCR was performed as follows: an aliquot of the first round of PCR with a volume of 2 μl followed by multiplex PCR: exon 3 fragment of the SERPINA1 gene (mutation E264V) + exon 5 fragment of the SERPINA1 gene (E342K mutation).

Electrophoregram multiplex PCR for the gene SERPINA1 presented in figures 11 and 12.

The reaction mixture (25 μl) at the second PCR stage contained 0.5 pmol of each primer, 2.5 μl of PCR buffer with 2.5 mM MgCl ₂ (Sileks, Russia), 6.25 nM of each of dNTP (Silex ”, Russia), 0.2 nM fluorescently-labeled dUTP-Cy5 and 1 unit. Act. Taq polymerase (Sileks, Russia). For amplification, a programmable DNA amplifier of the DNA Technology company (Russia) was used. After preliminary DNA denaturation (3 minutes at 94 ° C), amplification was performed in the mode: 33 amplification cycles: 94 ° C for 30 sec, 59 ° C for 30 sec, 72 ° C for 1 min. The final synthesis of 72 ° C - 3 minutes.

Microchip hybridization was performed using fluorescence-labeled samples obtained in the second stage of the PCR reaction in 32 μl of a mixture of the following composition: 8 μl of formamide (Serva, USA), 8 μl of 20XSSPE (Promega, USA), 4 μl of amplification and 12 μl of H ₂ O. The hybridization mixture was denatured at 95 ° C (5 min), cooled on ice (3 min), applied to the biochip and left overnight at 37 ° C. The chip was then washed in 1XSSPE for 5 minutes at 20 ° C and dried.

The fluorescent signal from the cells was recorded using a wide-field luminescent microscope equipped with a CCD camera and Imageware software (Biochip-IMB, Russia). We used normalized fluorescence signals Jm = (Im-I ₀ ) / (Bm-I ₀ ), where I _m is the signal intensity per unit area of the internal part of the cell component, B _m is the background signal reflecting the distribution of the microchip illumination, I ₀ is the dark current CCD matrix, am - chip cell number. When analyzing the fluorescence intensity, an average value between two duplicate cells was used. Signals Jm were sorted in ascending order. The reference cell was considered the cell, after which a sharp increase in the normalized signal was observed. It was believed that the cell has a positive signal if its signal exceeded the signal of the comparison cell. Then, cells containing the wild-type nucleotide sequence and the corresponding mutant sequence were compared with each other, revealing a dominant signal that exceeded the threshold value. The threshold signal value was determined statistically by processing more than 20 hybridization patterns. If in the pair of positive signals “wild-type probe - mutant probe” none of the signals dominated, the signals from both cells were considered significant, which corresponded to the heterozygous state of the analyzed locus.

Example 6. Analysis of data obtained by hybridization on a biochip according to the invention.

6.1. The result of hybridization of the PCR reaction products obtained using the genomic DNA of donors not suffering from type 1 tyrosinemia with the biochip according to the invention is shown in figure 13 and table 7.

Table 7 Values of normalized fluorescence signals (Jm) for biochip points. The location of the values in the table corresponds to the location of oligonucleotides in block 1 (see figure 1) Argl74 IVS6 Ivs7 IVS12 Pro342 2.88 3.18 0.19 5.08 0.23 2.58 3.65 0.25 5.64 0.27 0.19 0.31 0.05 1.50 0.06 0.19 0.28 0.05 1.45 0.07 2.18 0.19 2.11 0.15 0.32 0.29

According to the information given in table 7 and figure 13, the level of normalized fluorescence signals (Jm) at the points corresponding to the normal sequence of the FAH gene is several times higher than at the points corresponding to the mutated sequences (in the corresponding pairs of oligonucleotides). Thus, the claimed biochip does not give false positive results in the case of a patient who does not carry these mutations in the FAH gene sequence.

6.2. The result of three different hybridizations of the PCR reaction products obtained using the genomic DNA of donors carrying Argl74Term IVS12 + 5 g-> a mutations with the biochip according to the invention is shown in Figure 14 and Table 8.

According to the information given in table 8 and figure 14, the level of fluorescence signals at the points corresponding to the mutations Argl74Term and IVS12 + 5 g-> a in the FAH gene is the same as at the points corresponding to the sequence that does not contain this substitution in the intron 3. This fact suggests that the mutation is in a heterozygous state. At all other points, the signal level corresponding to normal sequences is several times stronger than the signal corresponding to mutated sequences. Thus, the claimed biochip allows us to establish the presence of mutations Argl74Term and IVS12 + 5 g-> a in the FAH gene with high specificity (even in the heterozygous state). In this case, false positive signals corresponding to other mutations do not occur.

6.3. As a result of hybridization of the PCR reaction products obtained using the genomic DNA of a patient carrying the IVS12 + 5 g-> a mutation in the FAH gene in the heterozygous state, the result is shown in Figure 15 and Table 9.

According to the information given in table 9 and figure 15, the level of fluorescence signals at the points corresponding to the IVS12 + 5 g-> a mutation in the FAH gene is the same as at the points corresponding to sequences that do not contain replacement data. This fact suggests that the mutation is in a heterozygous state. At all other points, the signal level corresponding to normal sequences is several times stronger than the signal corresponding to mutated sequences. Thus, the claimed biochip allows us to establish the presence of the IVS12 + 5 g-> a mutation in the FAH gene with high specificity (even in the heterozygous state). In this case, false positive signals corresponding to other mutations do not occur.

6.4. As a result of hybridization of the PCR reaction products obtained using the genomic DNA of a patient carrying the Argl74Term and IVS6 g-> t mutations in the FAH gene in the heterozygous state, the result is shown in figure 16 and table 10.

According to the information given in table 10 and figure 16, the level of fluorescence signals at points corresponding to the Argl74Term and IVS6 g-> t mutations in the FAH gene is the same as at points corresponding to sequences that do not contain replacement data. This fact suggests that the mutation is in a heterozygous state. At all other points, the signal level corresponding to normal sequences is several times stronger than the signal corresponding to mutated sequences. This fact shows that the claimed biochip allows us to establish the presence of Argl74Term and IVS6 g-> t mutations in the FAH gene with high specificity even in the heterozygous state. In this case, false positive signals corresponding to other mutations do not occur.

6.5. As a result of hybridization of the PCR reaction products obtained using the genomic DNA of a patient carrying the Pro342Leu mutation in the FAH gene in a homozygous state, the result shown in Figure 17 and Table 11 was obtained.

According to the information given in table 11 and figure 17, the level of fluorescence signals at points corresponding to the Pro342Leu mutation in the FAH gene is several tens of times stronger than at points corresponding to sequences that do not contain replacement data. This fact suggests that the mutation is in a homozygous state. At all other points, the signal level corresponding to normal sequences is several times stronger than the signal corresponding to mutated sequences. Thus, the biochip allows us to establish the presence of the Pro342Leu mutation in the FAH gene with high specificity (in the homozygous state). In this case, false positive signals corresponding to other mutations do not occur.

6.6. As a result of hybridization of the PCR reaction products obtained using the genomic DNA of a patient not carrying a mutation in the SERPINA1 gene, the result is shown in figure 18 and table 12.

According to the information given in table 12 and figure 18, the level of normalized fluorescence signals (Jm) at the points corresponding to the normal sequence of the SERPINA1 gene is several times higher than at the points corresponding to the mutated sequences (in the corresponding pairs of oligonucleotides). Thus, the claimed biochip does not give false positive results in the case of a patient who does not carry these mutations in the SERPINA1 gene sequence.

6.7. As a result of hybridization of the PCR reaction products obtained using the genomic DNA of a patient carrying the E342K mutation in the SERPINA1 gene in a homozygous state, the result shown in Figure 19 and Table 13 was obtained.

According to the information given in table 13 and figure 19, the level of fluorescence signals at the points corresponding to the E342K mutation in the SERPINA1 gene is several times stronger than at the points corresponding to sequences that do not contain replacement data. This fact suggests that the mutation is in a homozygous state. At all other points, the signal level corresponding to normal sequences is several times stronger than the signal corresponding to mutated sequences. This fact shows that the claimed biochip allows us to establish the presence of the E342K mutation in the SERPINA1 gene with high specificity (in the homozygous state). In this case, false positive signals corresponding to other mutations do not occur.

Claims (3)

1. The test system, which is a biochip for detecting point mutations in the genes of RAS and SERPINA1, causing human type 1 tyrosinemia and human alpha-1-antitrypsin deficiency, containing oligonucleotides having the sequence Seq ID NO: 1, Seq ID NO: 2 , Seq ID NO: 3, Seq ID NO: 4, Seq ID NO: 5, Seq ID NO: 6, Seq ID NO: 7, Seq ID NO: 8, Seq ID NO: 9, Seq ID NO: 10, Seq ID NO: 11, Seq ID NO: 13, Seq ID NO: 14. Seq ID NO: 15, Seq ID NO: 16, Seq ID NO: 17. 2. The use of the test system according to claim 1 for the detection of point mutations in the FAH and SERPINA1 genes that cause human type 1 tyrosinemia and human alpha-1-antitrypsin deficiency. 3. A method for detecting point mutations in the FAH and SERPINA1 genes that cause human type 1 tyrosinemia and human alpha-1-antitrypsin deficiency, comprising
1) staging a two-round multiplex PCR on the patient’s genomic DNA using primers having the sequences Seq ID NO: 19, Seq ID NO: 20, Seq ID NO: 21, Seq ID NO: 22, Seq ID NO: 23, Seq ID NO: 24, Seq ID NO: 25, Seq ID NO: 26, Seq ID NO: 27, Seq ID NO: 28, ID NO: 29, Seq ID NO: 30, Seq ID NO: 31, Seq ID NO: 32, Seq ID NO: 33, Seq ID NO: 34, Seq ID NO: 35, Seq ID NO: 36, Seq ID NO: 37, Seq ID NO: 38, Seq ID NO: 39, Seq ID NO: 40, Seq ID NO : 41, Seq ID NO: 42 and used according to tables 5 and 6.
2) hybridization of the obtained PCR products with a biochip according to claim 1,
3) analysis of the results of hybridization with the establishment of the presence of a mutation and its homozygous or heterozygous state.

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