RU2402612C2 - Method of screening newborns for monogenic diseases and biochip for said method realisation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: presence or absence of DNA mutations is determined by hybridisation of obtained fragments on specialised oligonucleotide biochip. For this purpose used are amplification of PAH, CFTR, PAX8, GALT genes and obtaining of single-strand fluorescently marked product by method of nick-translation and restriction, prepared is biochip for screening of newborns for diseases, which contains set of immobilised oligonucleotides SEQ ID NO: 1-82. Interpretation of hybridisation results is carried out by comparison of intensity of fluorescent signals, obtained in perfect and imperfect hybridisation.

EFFECT: invention allows to obtain novel accelerated method of mass screening of newborns for presence of predisposition to monogenic diseases.

5 cl, 4 tbl, 4 ex

Description

The invention relates to molecular biology, microbiology and medicine. The invention also relates to the technology of manufacturing biochips that are used in molecular biology for DNA sequencing and mapping, mutation detection, and a number of medical applications.

Accurate diagnosis in combination with a detailed analysis of the type of inheritance of a particular disease is crucial for the formation of risk groups, that is, the selection of families in which the probability of giving birth to sick children is increased. First of all, these are families where there is already or was a child suffering from some kind of monogenic hereditary disease. Phenylketonuria, congenital hypothyroidism, cystic fibrosis and galactosemia are often inherited pathologies in childhood. Cystic fibrosis is one of the most common hereditary diseases caused by a mutation in the transmembrane regulator gene. This disease is characterized by damage to the exocrine glands of vital organs and systems and has a severe course and prognosis especially with late detection.

Timely detection of a predisposition to these diseases and appropriate therapy can dramatically improve the quality of life of children with monogenic diseases.

Currently, there are several dozen methods used to screen newborns for monogenic diseases. For example, the following can be distinguished.

The well-known "Method for the diagnosis of congenital hypothyroidism" using screening control of thyrotropin level (TSH) or the level of total thyroxine (T4) in dry blood spots of newborns by immunofluorescence method using kits "Delfia Neonatal hTSH and T4" (Dedov II, Peterkov V .A., Bezlepkina OB, Alekseeva PM Screening program for early diagnosis and treatment of congenital hypothyroidism in children. Methodical recommendations. M., 1996, - p.14-19). The disadvantages of this method: it is recommended that the control in dynamics after 2-3 weeks to ensure the reliability of screening, as a result, replacement therapy begins no earlier than a month after birth.

The well-known "Method for the diagnosis of cystic fibrosis" (Patent RU No. 2141670, 1999.11.20). A patient's sweat sample is treated with strongly acidic catheonites in acid form in the presence of an acid-base indicator. Pre-strongly acidic cateonites in acid form are washed with distilled water until neutral. Determination of the concentration of sodium chloride is carried out spectrophotometrically, and cystic fibrosis is diagnosed by increasing the concentration of sodium chloride in the sweat of the patient. Using this method is possible starting from only 4 months of a child’s life.

The well-known "Method for the diagnosis of congenital hypothyroidism in newborns" (Patent RU No. 2138807, 1999.09.27). The method is based on crystalloscopy of the blood serum of newborns: a drop of blood is applied to a glass slide, covered with a coverslip and incubated in a thermostat for 2-2.5 hours, the obtained samples are examined on a polarizing microscope; a sample having a characteristic crystalline inclusion of cholesterol reacts to polarizing light in the form of optically active spherulite morphotypes; in healthy individuals, spherulite morphotypes are not detected. The disadvantage of this method is the manual determination of the presence / absence of spherulite morphotypes, in which (especially when conducting screening studies due to the large number of analyzes), the probability of error increases sharply and the cost of the method in man-hours increases. In addition, the method is used only on the 5th day after birth, while every day without replacement therapy for this disease is of great importance for development.

The well-known "Method for the diagnosis of cystic fibrosis" (Patent RU No. 2151188, 2000.06.20). The method consists in the following: genomic DNA is isolated from 5-10 ml of peripheral blood according to the standard method using proteinase K followed by extraction with phenol / chloroform. For amplification of the normal allele, the direct primer 5'-GGCAC-CATTA-AAGAA-AATAT-SATSTT-3 ', ending at the 3'-end with CTT trinucleotide, absent in the mutant allele, is used. For amplification of the deletion allele, the direct primer 5'-GGCAC-CATTA-AAGAA-AATAT-CATTG-G-3 ', without CTT, is used, ending with the subsequent TGG trinucleotide at the 3'-end. As a reverse primer, 5'-CATTC-ACAGT-AGCTT-ACCCA-3 'primer is used to amplify both alleles. The disadvantage of this method is the determination of only one frequent mutation, which is determined in 56% of patients with cystic fibrosis in Russia. The remaining 44% of patients have other mutations and they cannot diagnose cystic fibrosis with this method.

The well-known "Method for determining point nucleotide substitutions in the DNA of mycobacteria, a method for diagnosing the resistance of mycobacteria to rifampicin, a biochip for implementing these methods" (Patent RU.N0 2175015, 2001.10.20). The fragment of the rpoB gene is amplified to obtain a single chain fluorescently labeled product by PCR. A biochip is prepared to determine rifampicin-resistant mycobacteria. The amplified labeled product is hybridized to a biochip. The results of hybridization are recorded and interpreted by comparing the fluorescence intensity of the signals obtained with perfect and imperfect hybridization. The biochip is a microarray with cells in which a set of oligonucleotides is immobilized. The sequences of the latter are given in the description. The upper row of the biochip contains oligonucleotides complementary to wild-type DNA sequences. The corresponding column under each cell contains oligonucleotides that are complementary to mutant DNA sequences that are responsible for replacing one amino acid. Diagnostics of the resistance of mycobacterium tuberculosis to rifampicin is carried out by determining point nucleotide substitutions in the DNA of the pathogen. This method is the closest in essential features to the claimed.

Describes the "Molecular Diagnosis of Galactosemia" (US Patent No. 6420550, 2002.07.16), designed to detect mutations in the GALT gene, using a kit that includes: (a) samples, each of which is from 10 to 25 nucleotides in length, containing complementary sequences to the following sequences: TGGTTGGAT, TGCCGGGTA, TTCCCGCCA, CCAATTATG, CTGGGACCA, and GCCTGTAAG, and (b) amplification primers, each at least 14 nucleotides long, and complementary to the sequence selected from SEQ ID NO: 7, which is the result of amplification, which is the result of amplification SEQ ID NO: 7, which is regions of the GALT gene containing sites mutations. The kit according to the first paragraph, in which the primers include the only kit that amplifies the region of the human GALT gene, including SEQ ID NO: 8. The disadvantage of this method is its limited scope for mass screening of only one disease - galactosemia.

The well-known "Method for the diagnosis of diseases associated with a defect in the transmembrane conductivity regulator for cystic fibrosis" (US Patent No. 7160729, 2007.01.09 Method for detecting diseases that are associated with defects of cystic fibrosis transmembrane conductance regulator (CFTR) protein). The method is based on the destabilization of CFTR-dependent regulation of cell volume and the further determination of the volume of treated cells, while the cells that changed the volume are healthy, and if the cells have not changed the volume, cystic fibrosis is diagnosed

The well-known "Method for genetic screening of congenital metabolic disorders in newborns using tandem mass spectrometry", which consists in restoring the activity of enzymes from a patient’s blood sample and determining the enzymatic activity, concentration of the enzyme or analyte by the amount of fluorescently labeled substrate bound to them. (Application US No. 2005/0048499, TANDEM MASS SPECTROMETRY METHOD FOR THE GENETIC SCREENING OF INBORN ERRORS OF METABOLISM IN NEWBORNS). Disadvantages - the ability to determine genetic abnormalities only by the product of this gene. To implement the method requires very expensive equipment and highly qualified specialists, which also increases the cost of the method.

A common drawback of the above methods: they are impractical to apply for mass screening, because they are laborious and allow you to diagnose only one disease.

A significant achievement of biotechnology in recent years has become biochips. Most accurately, the English name DNA-microarrays describes them, i.e. This is an organized placement of DNA molecules on a special carrier. Unusual devices allow for a short time to identify several thousand allergens, oncogenes, various biologically active substances, and even genetic defects. In a short time, biological microchips have emerged as an independent field of analysis with applications ranging from studying the fundamental problems of molecular biology and molecular evolution to practical applications in medicine, pharmacology, ecology, forensic science, etc.

The technology of biochips replacing entire immunological laboratories makes it possible to increase the productivity of most diagnostic methods by thousands and tens of thousands of times and dramatically reduce the cost of analysis.

Known methods for the manufacture of biochips based on hydrogels, in which the technological cycle consists of the steps of: (1) preparing a substrate, (2) forming a matrix of gel cells on it, (3) applying biological macromolecules to the cells in accordance with a predefined biochip scheme, ( 4) chemical treatment of cells with the aim of immobilization of probe molecules, (5) washing and drying of the obtained biochips.

A known method of preparing a biochip (US Patent No. 5574142, 1996.11.12), according to which a special thin-layer (5 μm) chamber is used with a reaction volume limited on one side by the substrate of the future biochip and on the other a window transparent in the UV region. Biochip cells are formed one after another by cyclically performing the following operations: (1) filling the chamber with a composition with an appropriate probe, (2) polymerizing the composition at the location of the future cell under the influence of UV radiation, focused into a square spot of the required size, (3) washing the chamber before filling it with another solution.

The known "Method for the manufacture of a microchip based on oligonucleotides" (Patent RU No. 2157385, 2000.10.10.), According to which a microchip is made on the basis of oligonucleotides immobilized in an organic gel prepared by copolymerization of unsaturated derivatives of oligonucleotides with unsaturated monomers, moreover, water-salt solutions containing unsaturated monomers, oligonucleotides modified with unsaturated fragments, and a component of a polymerization-inducing polymerization system soluble only in water are applied to eklyannuyu substrate as microdroplets and the copolymerization is carried out by immersing the formed matrix monomers in a water-immiscible organic solvent containing a dissolved catalyst system of the other component.

The invention is known "Composition (K) for immobilization of biological macromolecules in hydrogels by copolymerization, a method for preparing a composition and biochip" (Patent RU No. 2206575, 2003.06.20), which relates to compositions (K) for polymerization immobilization of biological macromolecules in hydrogels during the formation of biochips K = aA + bB + cC + dD + eE, including A - a monomer based on derivatives of acrylic and methacrylic acids; B is a water soluble crosslinking agent; C is a modified biological macromolecule containing an unsaturated group, D is a water-soluble compound as a component of the copolymerization medium; E is water, where a, b, c, d, e is the percentage (X) of each component in the composition (X = m / v100% for solids and X = v / v100% for liquid substances), in which the total content the monomer and the crosslinking agent is in the range of 3-40% (3 (a + b) (40), the ratio of the monomer and the crosslinking agent is in the range 97: 3-60: 40, and the percentage of components C, D and E is in the range (c) from 0.0001% to 10%; (d) from 0% to 90%; (e) from 5% to 95%, the method of their preparation, to modified biologically significant DNA compounds and proteins, a biochip in which silt layer on the substrate is separated by empty gaps into several cells, each cell may or may not contain immobilized macromolecules, and macromolecules immobilized in different cells may differ in nature and properties, and two methods of carrying out a polymerization-chain reaction on a biochip.

However, known compositions for immobilizing biological macromolecules in hydrogels during the formation of biochips, biochips and methods for their manufacture have several disadvantages.

1. The use of only acrylamide as a gel-forming component and N, N'-methylenebisacrylamide as a crosslinking agent in the manufacture of gels greatly limits the spectrum of hydrogels obtained in structure and porosity.

2. The modified oligonucleotides used for the manufacture of biochips contain only the 5'-terminal methacrylamide group bonded to the oligonucleotide molecule with an acid labile phosphoramide bond or the terminal allyl group having low affinity for acrylamide and N, N'-methylene bisacrylamide in the copolymerization reaction.

3. Known biochips are characterized by low reproducibility of the properties of gel cells and heterogeneous distribution in the cell volume of the immobilized compound.

4. A known method of chemical polymerization of the components of the composition in the manufacture of a biochip is carried out in an atmosphere of moist nitrogen, which reduces the efficiency of polymerization.

5. The known method of photoinduced polymerization uses UV radiation with a wavelength of 254 nm, which leads to the destruction of oligonucleotides.

6. Known methods for manufacturing biochips are technically complex and use procedures that do not provide the necessary uniformity and reproducibility of the properties of gel cells and are poorly automated.

The above disadvantages of the methods for the diagnosis of monogenic diseases and biochips for their implementation are eliminated by the claimed invention.

The present invention is the creation of a new accelerated method of mass screening of newborns for a predisposition to monogenic diseases. The basis of the invention is also the task of creating a biochip that ensures the optimal functioning of biologically significant macromolecules immobilized in it.

The problem is solved in a new way to detect a large number of mutations in the DNA of the subject in a single blood test. The claimed method proposes the use of: obtaining single-stranded labeled DNA fragments of the human genome directly from a clinical sample (whole umbilical cord blood). The presence or absence of DNA mutations is determined by hybridization of the obtained fragments on a specialized oligonucleotide biochip. The method also includes methods for recording and interpreting analysis results. Salient features of the method:

(a) isolation of genomic DNA from a clinical sample and amplification of the gene and obtaining a single-stranded fluorescently labeled product; (b) - preparation of a biochip with immobilized nucleotides; (c) - hybridization of the amplified labeled product on a biochip; (d) - registration; and (e) - interpretation of hybridization results by comparing the intensity of fluorescent signals obtained with perfect and imperfect hybridization. What is new in this technique is that at stage (a) amplification of the RAS, CFTR, PAX8, GALT genes and the production of a single-stranded fluorescently labeled product by nick translation and restriction are used, with the restriction enzyme DPN2 applied in stage (a); at the stage (b) a biochip is prepared for screening newborns containing a set of immobilized oligonucleotides SEQ ID 1-82 (Tables 1-4); at the stage (c) carry out the hybridization of the labeled amplified product on the specified biochip; in step (d), the results are recorded at a wavelength of 635 nm and 532 nm for the dyes Su5 and Su3, respectively; at the stage (e) - the interpretation of the results of hybridization is carried out by comparing the intensity of the fluorescent signals obtained with perfect and imperfect hybridization: if in the cell of the first row of such a section the signal intensity is higher than in the other rows and above the threshold level determined by the negative control, the sample is referred to wild type for a given nucleotide (not having a mutation in this position); if the signal intensity in a cell of rows 2, 3 or 4 separately or in pairs (for example, cells of rows 2 and 3) is higher than in the remaining rows and above the threshold level determined by the negative control, the sample is classified as a mutant type for this nucleotide; if the signal intensity in the cell of the first row and one of the cells of the 2nd, 3rd or 4th row is higher than in the remaining cells of the section and above the threshold level determined by the negative control, then the sample is classified as heterozygous.

The size of the amplified single stranded fluorescently labeled fragment is about 60 bp.

The umbilical cord blood of a newborn is used as a clinical sample.

The biochip for screening newborns contains a set of immobilized oligonucleotides SEQ ID 1-82 and 10 non-specific oligonucleotides as a control, the use of which allows the detection of point mutations and deletions in the genes of RAS, CFTR, PAX8, GALT with high specificity, and the order of placement of oligonucleotides provides the most reliable way assess the presence or absence of a mutation and its exact localization. Moreover, the biochip contains a set of oligonucleotides SEQ ID 1-82 and 10 non-specific oligonucleotides as a control, as well as oligonucleotides complementary to oligonucleotides SEQ ID 1-82. And also it contains from 1 to 4 copies of a set of oligonucleotides SEQ ID 1-82.

A new technical result of the invention consists in the fact that the new methods of the invention that we have developed allow us to quickly and accurately analyze the DNA of newborns for a predisposition to the following hereditary diseases: cystic fibrosis, congenital hypothyroidism, galactosemia, phenylketonuria, and only one cord blood sample is sufficient to analyze .

Achieving a new technical result is due to the following provisions.

The use of restriction and nick translation allows to obtain labeled fragments of a small size - about 60 bp - genomic DNA. The resulting single-stranded labeled DNA fragments are capable of entering into a specific hybridization interaction with oligonucleotides immobilized on a biochip. The known arrangement of oligonucleotides on the biochip makes it possible to establish the presence or absence of mutations in the genes responsible for the development of monogenic diseases in a DNA sample. The developed conditions for sample preparation and hybridization provide a high degree of differentiation between those cells of the biochip where hybridization has passed and those where no specific hybridization complexes have arisen. The design of oligonucleotides ensures the absolute specificity of their interaction with the regions of genes responsible for the development of monogenic diseases containing the studied mutation. Registration and processing of results is carried out by a specialized program that allows you to reliably determine the presence of a signal in relation to the background level.

A detailed description of the method and clinical examples of its implementation.

The set of oligonucleotides (biochip cells) shown in SEQ ID I - 82 (Table 1-4) is applied to a glass substrate in the form of droplets with maximum accuracy and reproducibility using robots (e.g., BioOdyssey, Bio-Rad, USA). Each formed cell is a hemisphere with a diameter of 1 μm and a period of about 8 μm and contains an immobilized oligonucleotide probe, complementary to the sequence of the gene region associated with the presence or absence of a predisposition to a monogenic disease.

Oligonucleotides for immobilization on a biochip were synthesized on an automatic synthesizer (type 394 DNA / RNA synthesizer. Applied Biosystems, USA) and contain a spacer with a free amino group 3'-Ammo-Modifier C7 CPG 500 (Glen Research) for subsequent immobilization on a biochip substrate.

Take 1 ml of the cord blood of the newborn, isolate genomic DNA using a commercial DNA Box 500 kit (Protrans).

The resulting genomic DNA is fragmented using restriction enzyme DPN2. After DNA fragmentation, a nick translation reaction was performed using Klenow Fragment (Sigma, 40 u / μl) in the presence of Cy5-dCTP or Cy3-dCTP (Amersham, I mM solution). DNA is then purified using a microcon 30 filter (Amicon / Millipore) and hybridization is carried out on a biochip.

Hybridization of the labeled sample on the biochip is carried out in a buffer of the following composition: 1M GuSCN; 50 mM HEPES, pH 7.5; 5 mM EDTA, pH 7.0. Hybridization is carried out in a commercially-produced hybridization chamber with a volume of 30 μl (Sigma) overnight (16-18 hours) at 65 ° C. Washing is carried out three times in the following buffers.

First wash: 2X SSC, 0.03% SDS, 5 min 70 ° C (for precise separation of the specific from non-specific hybridization signal).

Second wash: 1X SSC, 5 min at room temperature.

Third wash: 0.2X SSC, 5 min at room temperature.

Registration and fixation of the hybridization pattern is carried out using an automatic complex consisting of a Innoscan 700 (Inopsys) type biochip scanner and a computer at a wavelength of 635 nm and 532 nm for Su5 and Su3 dyes, respectively. Processing the intensity of the fluorescent signal is carried out using the program MapixSoftware (Inopsys).

The presence of fluorescence in the cells of the biochip draw conclusions about the presence or absence of the studied polymorphism in a particular gene.

Clinical sample processing.

Isolation of genomic DNA.

Genomic DNA is isolated using a commercially available DNA Box 500 kit (Protrans) according to the manufacturer's recommendations.

Labeling DNA with fluorescent dyes.

The resulting genomic DNA is fragmented using restriction enzyme DPN2. After DNA fragmentation, a nick translation reaction was performed using Klenow Fragment (Sigma, 40 u / μl) in the presence of Cy5-dCTP or Cy3-dCTP (Amersham, I mM solution). DNA is then purified using a microcon 30 filter (Amicon / Millipore) and hybridization is carried out on a biochip.

Hybridization of a labeled sample on a biochip.

Hybridization of the labeled sample on the biochip is carried out in a buffer of the following composition: 1M GuSCN; 50 mM HEPES, pH 7.5; 5 mM EDTA, pH 7.0. Hybridization is carried out in a commercially-produced hybridization chamber with a volume of 30 μl (Sigma) overnight (16-18 hours) at 65 ° C. Washing is carried out three times in the following buffers.

First wash: 2X SSC, 0.03% SDS, 5 min 70 ° C (for precise separation of the specific from non-specific hybridization signal).

Second wash: 1X SSC, 5 min at room temperature.

Third wash: 0.2X SSC, 5 min at room temperature.

Registration and interpretation of hybridization results - stage (d).

Registration and fixation of the hybridization pattern is carried out using an automatic complex consisting of a biochip scanner of the Innoscan 700 type (Inopsys) and a computer at a wavelength of 635 nm and 532 nm for dyes Su5 and Su3, respectively. Processing the intensity of the fluorescent signal is carried out visually or using MapixSoftware (Inopsys).

The interpretation of the results is as follows.

Within each 1-by-4-cell sized area of the biochip, the fluorescence signal intensities are visually or using software. If in the cell of the first row of such a section the signal intensity is higher than in the other rows and above the threshold level determined by the negative control, the sample is assigned to the wild type for this nucleotide (which does not have a mutation in this position). If the signal intensity in the cell of the 2nd, 3rd or 4th row separately or in pairs (for example, cells of the 2nd and 3rd row) is higher than in the remaining rows and above the threshold level determined by the negative control, the sample is to the mutant type for a given nucleotide (the type and location of the mutation is established by comparison with the arrangement of oligonucleotides on the biochip). If the signal intensity in the cell of the first row and one of the cells of the 2nd, 3rd or 4th row is higher than in the remaining cells of the section and above the threshold level determined by the negative control, then the sample is classified as heterozygous (one allele is wild type, second allele - mutant type). As a result, a conclusion is issued on the presence or absence of mutations in the analyzed positions of the corresponding genes.

Oligonucleotide biochip for screening newborns for monogenic diseases.

Oligonucleotides for immobilization on a biochip are synthesized on an automatic synthesizer (type 394 DNA / RNA synthesizer. Applied Biosystems, USA) and contain a spacer with a free amino group 3'-Amino-Modifier C7 CPG 500 (Glen Researh) for subsequent immobilization on a biochip substrate or 5 ' -Amino-Modifier C6 (Glen Researh) for introducing a fluorescent label. The introduction of a fluorescent label Su5 carried out in accordance with the manufacturer's recommendations. (Amersham)

The synthesized set of oligonucleotides (biochip cells) is applied to the glass substrate in the form of drops with maximum accuracy and reproducibility using robots (BioOdyssey, Bio-Rad, USA). Each formed cell is a hemisphere with a diameter of 1 μm and a period of about 8 μm and contains an immobilized oligonucleotide probe, complementary to the sequence of the gene region associated with the presence or absence of a predisposition to a monogenic disease.

The biochip structure.

The biochip is a set of oligonucleotides immobilized on a solid support.

To detect mutations of the SNP type, a two-part oligonucleotide set is used. The first part is a lot of oligonucleotides, each oligonucleotide is exactly complementary to the DNA segment of the gene containing the test position. The second part of the kit consists of oligonucleotides that are exactly complementary to the part of the DNA of the gene containing the test position, with the exception of the nucleotide in the test position, so that 3 oligonucleotides with different nucleotides in the test position fall on one test position. As a result, 4 oligonucleotides, differing in the nucleotide in the studied position, fall to one studied position for one DNA strand. This approach allows us to determine which of the nucleotides A, C, G or T is in the studied position and, accordingly, make a conclusion about the presence or absence of a mutation in this position. The set of biochip oligonucleotides includes oligonucleotides that are complementary to both the forward strand of the gene’s DNA and the reverse. In addition, a set of oligonucleotides can be applied to the biochip in several copies (from 1 to 4 copies). This allows you to increase the sensitivity of the analysis.

To detect deletions, a two-part oligonucleotide set is used. The first part of the set of oligonucleotides is the same as for the detection of mutations of the SNP type. The second part consists of many oligonucleotides that are exactly complementary to the region of the gene’s DNA, except that these oligonucleotides lack nucleotide (s) in the test position. As a result, 2 oligonucleotides are used to study one deletion in one DNA strand. The set of biochip oligonucleotides includes oligonucleotides that are complementary to both the forward strand of the gene’s DNA and the reverse. In addition, a set of oligonucleotides can be applied to the biochip in several copies (from 1 to 4 copies).

Oligonucleotides are synthesized on a DIC automatic synthesizer and applied to the biochip in such a way that oligonucleotides complementary to a portion of the DNA straight chain gene (“direct” oligonucleotides) containing 1 mutation position are arranged in a column one after the other, while the oligonucleotide complementary to the wild type occupies the upper position gene (i.e., not containing a mutation). Oligonucleotides complementary to the region of the DNA reverse chain gene containing the corresponding mutation position are positioned in the same way as the “straight lines”. Oligonucleotides are placed on the biochip randomly, taking into account the above conditions. To increase the sensitivity of the method, the biochip may contain from 2 to several copies of a set of oligonucleotides. In addition, on the biochip there are 10 cells containing oligonucleotides that are nonspecific to the genome under study, which play the role of a negative control.

Statistics and clinical examples

In clinical trials of the proposed method for detecting mutations, 10 newborns with a clinical diagnosis of phenylketonuria, 7 newborns with a clinical diagnosis of cystic fibrosis, 2 newborns with a diagnosis of congenital hypothyroidism, 1 child with a diagnosis of galactosemia and 40 healthy newborns were examined. As a result of testing the proposed method, the following results were obtained:

- among patients with phenylketonuria, 8 newborns were homozygous for the R408W / R408W mutation, 1 was heterozygous with the R408W / R261Q genotype, 1 was heterozygous with the I65T / R408W genotype according to the RAS gene.

- among patients with cystic fibrosis, 6 newborns were DelF508 / DelF508 homozygotes and one turned out to be heterozygous with the GTR542X / DelF508 genotype according to the CFTR gene.

- among patients with congenital hypothyroidism, one child was homozygous for the Q40P / Q40P mutation, and the other was heterozygous with the R31H / X genotype for the PAX8 gene.

- among newborns with a diagnosis of galactosemia, 1 patient was homozygous for the Q188R mutation in the GALT gene.

Clinical example 1.

Patient Sh-va. Phenylketonuria was diagnosed in 2005 as a result of screening studies. The material for the screening study was blanks with the blood of newborns living in the Rostov Region. The level of FA was determined using a “reagent kit for the quantitative determination of FA in dry blood stains using the fluorimetric method - FA-Fluor (Innovative Biotechnologies LLC, Russia) using the Fluoroscan-2 instrument (Labsystems, Finland).

The level of phenylalanine in patient Sh-how at the time of registration (1 month) is 769.8 μmol / L (reference values up to 120.0 μmol / L). At the time of our study (2007), the patient was receiving a diet, and the values of FA were within the physiological norm.

We carried out molecular genetic typing according to the claimed method using a biochip for the 6 most frequent mutations in the RAS gene (FAG) (R408W, P281L, R261Q, R158Q, R252W, I65T).

Using the proposed method, the patient’s genotype Sh-how was established: - R408W / R408W.

Clinical example 2.

Patient A-in. The diagnosis of congenital hypothyroidism was made in 2007 at the age of 1 month as a result of screening studies to determine the level of thyroid-stimulating (TSH) in dry blood spots of newborns by the immunofluorescence method using Delfia Neonatal hTSH kits. In patient A-in, the TSH concentration on the 3rd day after delivery is 124 μU / ml, with a norm of up to 20 μU / ml.

We carried out molecular genetic typing according to the claimed method using the biochip we developed for the 5 most frequent mutations in the PAX8 gene (R31H, Q40P, C57O, L62R, R108X). Using the biochip, the genotype of the patient A -in was determined: - Q40P / Q40P

Clinical example 3.

Patient Ts-ko. The diagnosis of cystic fibrosis was made at the age of 3 years on the basis of the clinical picture - chronic bronchopulmonary process and intestinal syndrome, the presence of cystic fibrosis in siblings and a positive result of a sweat test of sodium chloride concentration.

The concentration of sodium chloride was determined spectrophotometrically. Patient C-co recorded a positive triple sweat test with a sweat chloride content above 60 mm.

We carried out molecular genetic typing according to the claimed method using a biochip for the 5 most frequent mutations in the CFTR gene (delF508, G542X, G551D, N1303K, W1282X).

Using the biochip we developed, the patient’s genotype Ts-ko was established:

- DelF508 / DelF508.

Clinical example 4.

In the newborn K-ova, DNA was isolated from cord blood and analyzed using the proposed method. Genotyping was carried out for the following mutations: in the RAS gene (R408W, P281L, R261Q, R158Q, R252W, I65T), in the CFTR gene (delF508, G542X, G551D, N1303K, W1282X), in the GALT gene (Q188R, K28517, , S135L), in the PAX8 gene (R31H, Q40P, C57O, L62R, R108X) / It was found that the newborn K-s was heterozygous with the R408W / genotype - according to the RAS gene, no other mutations studied were found. When conducting 1 month after the birth of the study in the framework of the screening program, deviations in biochemical parameters were not detected.

The advantages for clinical practice of our proposed diagnostic method and biochip for its implementation are as follows.

The duration of the analysis is 24 hours. The accuracy of the method is 98% and covers 78% of cases of detected monogenic diseases in children.

Hybridization and washing conditions provide a high degree of differentiation between perfect and imperfect hybridization duplexes, and thus allow the procedure to be carried out in an ordinary diagnostic laboratory

The method compares favorably with existing analogues by its simplicity and low cost and can be widely recommended in clinical practice.

Claims (5)

1. A biochip for screening newborns for monogenic diseases, containing a set of immobilized oligonucleotides SEQ ID NO: 1-82, also oligonucleotides complementary to them, and 10 non-specific oligonucleotides as a control, with the number of copies of this set from 1 to 4. 2. A method for screening newborns for monogenic diseases using the biochip according to claim 1, comprising: (a) isolating genomic DNA from a clinical sample and amplifying a gene and obtaining a single chain fluorescently labeled product; (b) preparing a biochip according to claim 1 with immobilized nucleotides; (c) hybridization of the amplified labeled product on a biochip; (d) recording and (e) interpreting the results of hybridization by comparing the intensity of fluorescent signals obtained with perfect and imperfect hybridization, characterized in that stage (a) uses amplification of the RAS, CFTR, PAX8, GULT genes and obtaining a single-chain fluorescently labeled product by nick translation and restriction; in step (b), the biochip according to claim 1 is prepared for screening newborns containing a set of immobilized oligonucleotides SEQ ID NO: 1-82; at the stage (c) carry out the hybridization of the labeled amplified product on the specified biochip; in step (d), the results are recorded at a wavelength of 635 nm and 532 nm for the dyes Su5 and Su3, respectively; at the stage (e), the interpretation of the results of hybridization is carried out by comparing the intensity of the fluorescent signals obtained with perfect and imperfect hybridization: if in the cell of the first row of such a section the signal intensity is higher than in the remaining rows and above the threshold level determined by the negative control, the sample refers to wild type for a given nucleotide (not having a mutation in this position); if the signal intensity in the cell of the 2nd, 3rd or 4th row separately or in pairs (for example, cells of the 2nd and 3rd row) is higher than in the other rows and above the threshold level determined by the negative control, the sample belong to the mutant type for this nucleotide; if the signal intensity in the cell of the first row and one of the cells of the 2nd, 3rd or 4th is higher than in the other cells of the section and above the threshold level determined by the negative control, then the sample is classified as heterozygous. 3. The method according to claim 2, characterized in that the clinical sample includes cord blood of a newborn. 4. The method according to claim 2, characterized in that at the stage (a) the restriction enzyme DPN2 is used. 5. The method according to claim 2, characterized in that the size of the amplified single-stranded fluorescently labeled fragment is about 60 bp