RU2551985C2 - Set of oligonucleotide probes, dna-microchip, method of its obtaining, set for molecular-genetic research of human and their application - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of biochemistry, in particular to set of oligonucleotide probes for diagnostics of human population, living on RF territory, for hereditary monogenic diseases, by detection of mutations and/or polymorphisms. Also claimed are method of obtaining DNA-microchip, set for research of population of people, as well as method of detection of associated with monogenic diseases mutations and/or polymorphisms, in which claimed set of oligonucleotide probes is applied.

EFFECT: invention makes it possible to increase self-descriptiveness, accuracy and reduce research term.

4 cl, 3 tbl, 3 ex

Description

Technical field

The invention relates to biotechnology, namely to molecular genetic studies of humans for the diagnosis of hereditary diseases, predisposition to multifactorial diseases with a genetic component, and a personal genetically determined reaction to pharmaceutical preparations.

About half of all cases of disease inheritance occur when the mother and father do not suffer from hereditary diseases, but are hidden carriers of damaged genes. If a child inherits from each parent such a damaged gene, this will lead to the development of the disease. The risk of giving birth to a sick child in parent parents is 25%, which is characterized as high from the medical genetic point of view. Guided by information on their own genetic characteristics obtained through molecular genetic studies, as well as using modern reproductive medicine capabilities, future parents will be able to avoid the risks of conceiving a child with a severe or fatal hereditary disease and give birth to a healthy child.

Description of the prior art

A known method of screening newborns for such hereditary diseases as cystic fibrosis, congenital hypothyroidism, galactosemia, phenylketonuria in a single blood test. The presence or absence of DNA mutations is determined by hybridization of the obtained fragments on a specialized oligonucleotide biochip. For this, amplification of PAH, CFTR, PAX8, GALT genes and the production of a single chain fluorescently labeled product by nick translation and restriction are used, a biochip is prepared for screening newborns for diseases containing a set of immobilized specific oligonucleotides. Interpretation of the results of hybridization is carried out by comparing the intensity of the fluorescent signals obtained with perfect and imperfect hybridization. Despite the fact that such a study allows you to get a new accelerated method of mass screening of newborns for a predisposition to monogenic diseases, it has limited information content.

The principle of selection of polymorphisms is described, described in EP 2463384. However, the authors studied and selected polymorphisms that determine the occurrence of sudden cardiac death without taking into account their territorial prevalence.

Description of the invention

The objective of this development is to create a method for studying human DNA for the presence of a large number of mutations and / or polymorphisms using a biochip.

Another objective is the development and application of a unique set of oligonucleotide probes that allow the determination of mutations and / or polymorphisms included in the diagnosticum (biochip), which can be used to diagnose the most common mutations and polymorphisms associated with the most common for populations of people (ethnic groups) that inhabit a certain geographical area, hereditary and multifactorial diseases with a hereditary component.

The main distinguishing feature of the microchip is the first stage of its development - the determination of mutations and / or polymorphisms that can be detected using it. At this stage, they proceed from the genetic characteristics of the populations inhabiting a certain territory: the diagnosticum (microchip) includes only the most frequent mutations and / or polymorphisms associated with the most frequent hereditary and multifactorial diseases with a hereditary component in this territory. Thus, the effectiveness and informativeness of diagnostics in a specific territory with the help of such a diagnosticum increases compared to direct analogues, due to the method of selection of detected mutations, and not due to an increase in their number.

The composition of mutations detected using a microchip is optimized and strictly specific for ethnic groups inhabiting a given territory for a long time. Thus, the effectiveness and information content of genetic diagnosis of hereditary diseases using this kit is much higher than using existing analogues.

By the frequency of occurrence of a mutation or polymorphism is meant its prevalence in a given population or in a given ethnic group based on the number of cases of its homozygous carriage per 1000 people. At the same time, global frequency means its average frequency among the studied populations of the planet. Data on global frequencies of mutations and / or polymorphisms and frequencies in a given population are obtained from the scientific literature.

In one aspect, the invention relates to a set of oligonucleotide probes capable of detecting a complex (set) of mutations and / or polymorphisms for molecular genetic research (screening) of a person, wherein:

(a) at least 10% (10% to 95%) of the total number of mutations and / or polymorphisms that can be determined must have a frequency of occurrence among ethnic groups living in the selected territory for a long time, or in the selected population people, exceeding the average global incidence by at least two times (the ratio of the frequency of occurrence in a population living in a selected territory, or in a selected population to the global average frequency is> 2);

(b) at least 5% (5% to 90%) of mutations and / or polymorphisms should not occur in ethnic groups living in the selected area for a long time, or in the selected population of people other than the selected one;

(c) no more than 10% (from 0% to 10%) of mutations and / or polymorphisms having a world average frequency of occurrence more than two times higher than the frequency of occurrence in ethnic groups living in the selected territory for a long time, or in the selected human populations (the ratio of the global average frequency to the frequency of occurrence in a population living in a selected territory, or in a selected population> 2);

(d) not more than 5% (from 0% to 5%) of mutations and / or polymorphisms that are not found in ethnic groups living in the selected territory for a long time, or in the selected population;

(e) the remaining content (from 0-85%) of mutations and / or polymorphisms occurring in ethnic groups living in a selected territory or in a selected population may be equal to the global average or may not exceed more than twice (ratio the frequency of occurrence in the population living in the selected territory, or in the selected population to the global average frequency is ≤2).

During the research it was found that it is the selection of the studied mutations that is associated with the effectiveness and informativeness of diagnostics in a particular territory, which increase due to the fact that the most common and specific mutations specific to this territory are taken for diagnosis.

In another aspect, the invention relates to a method for producing a DNA microarray.

DNA microarrays are prepared by printing oligonucleotide probes containing DNA regions that allow the detection of selected mutations and / or polymorphisms on a glass plate coated with aminosilane (see WO 2004073988).

A distinctive feature of our developed method is the stage of preliminary selection of mutations according to the algorithm established by us. The method allows to obtain a unique microchip for molecular genetic diagnostics of a person belonging to an ethnic group living in a certain geographical area for a long time, and to increase the accuracy and information content of the study.

A method of obtaining a DNA microarray for molecular genetic research of a person living in a selected territory or in a selected population, includes:

I) selection of mutations and / or polymorphisms, including:

(a) selection of at least 10% (10% to 95%) of the total number of mutations and / or polymorphisms that can be determined having a frequency of occurrence among ethnic groups living in the selected territory for a long time, or in the selected population people, exceeding the average global incidence by at least two times (the ratio of the frequency of occurrence in a population living in a selected territory, or in a selected population to the global average frequency is> 2);

(b) the selection of at least 5% (from 5% to 90%) of mutations and / or polymorphisms should not occur in ethnic groups living in the selected territory for a long time, or in the selected population of people other than the selected one;

(c) the selection of not more than 10% (from 0% to 10%) of mutations and / or polymorphisms having a world average frequency of occurrence more than twice the frequency of occurrence in ethnic groups living in the selected territory for a long time, or a selected population of people (the ratio of the world average frequency to the frequency of occurrence in a population living in a selected territory, or in a selected population> 2);

(d) Selection of not more than 5% (from 0% to 5%) of mutations and / or polymorphisms not found in ethnic groups living in the selected territory for a long time, or in the selected population;

(e) the selection of the remaining mutations and / or polymorphisms (from 0-85%) found in ethnic groups living in a selected territory, or in a selected population, can be equal to the global average or cannot exceed it more than twice (ratio the frequency of occurrence in a population living in a selected territory, or in a selected population, to the global average frequency is ≤2);

II) obtaining oligonucleotide probes containing DNA regions, allowing the detection of selected mutations and / or polymorphisms;

III) applying oligonucleotide probes to a glass plate coated with aminosilane or another nucleic acid adhesive, one of the sides of the plate being processed in such a way that the laser beam propagates in it due to the effect of total internal reflection.

Preferably, the probes are selected so as to accurately detect a mutation or polymorphism, indicated both on the sense and on the antisense DNA strand.

To detect mutations and polymorphisms, probes are selected according to the following criteria:

- lack of internal secondary structure;

- the ability to participate in the specific elongation of gene fragments containing regions that subsequently identify mutations and polymorphic variants of genes.

This microchip device can be used as part of a kit for molecular genetic diagnostics based on the elongation of primers on DNA microarrays.

The developed DNA microchip as part of the kit allows one-time detection of carriage of mutations and / or polymorphisms associated with hereditary diseases, and is used for molecular genetic diagnosis of hereditary diseases; predisposition to multifactorial diseases with a genetic component and a personal genetically determined reaction to pharmaceuticals.

The kit for molecular genetic research of a person living in a selected territory or in a selected population includes:

- A set of enzymes and reagents for preparing a matrix of the reaction of elongation of primers on a microchip (Eng. Arrayed Primer Extention - APEX), consisting of DNA polymerase, magnesium solution, solutions of disoxynucleotides, uracil-N-glycosylase, alkaline phosphatase of shrimp.

- Primers for PCR reaction multiplex

- Set for cleaning PCR products

- APEX reaction kit: DNA polymerase, magnesium solution, fluorescently labeled dideoxynucleotide solutions for chain elongation

- DNA microchip adapted for molecular genetic research of people living in a certain geographical area.

The use of kits based on APEX technology is described in US 20070134691.

The method described in EP 1088214 was chosen as a method for analyzing a DNA microarray, which allows to increase the accuracy, simplify the method of genetic diagnostics and make it economically viable.

The method of molecular genetic research of a person belonging to an ethnic group living in a selected area for a long time, or in a selected population, involves the use of a new kit for diagnostics and recording of analysis results by comparing the intensity of fluorescent signals when irradiating a DNA microchip with lasers of different wavelengths .

The implementation of the invention

The implementation of the claimed group of inventions is shown by the example of the development of tools for diagnosis and its implementation for a population of people living in the territory of the Russian Federation, however this principle can be applied to any territory or state.

The list and sequence of probes containing sites that further identify the mutations and polymorphic gene variants shown in Table 1 are presented in Table 2.

As mutations that belong to category (a) of the selection algorithm, we can cite the following:

Gene Nucleotide substitution Disease CFTR c. 489 + 1G> T Cystic fibrosis Pah c.1222C> T Phenylketonuria BTD c.98_104delGCGGCTGinsTCC Biotinidase Deficiency

As mutations belonging to category (b) of the selection algorithm, the following can be cited:

Gene Nucleotide substitution Disease TCIRG1 c.807 + 5G> A Autosomal recessive osteopetrosis Vhl c.598A> C Autosomal recessive erythrocytosis Gjb1 c.259C> G Charcot-Marie-Tooth disease, type 1A

As mutations belonging to category (c) of the selection algorithm, the following can be cited:

Gene Nucleotide substitution Disease IKBKAP c. IVS20 + 6T> C Familial disautonomy DHCR7 c.278C> T SLOS Syndrome Hexa c.805 + 1G> A Tay-Sachs disease

As mutations belonging to category (d) of the selection algorithm, the following can be cited:

Gene Nucleotide substitution Disease SLC26A4 c.2168A> G Pendred's syndrome SLC26A4 IVS7AS, A-G, -2 Pendred's syndrome NPHS1 c.3325C> T Finnish nephrotic syndrome

As mutations belonging to category (e) of the selection algorithm, the following can be cited:

Gene Nucleotide substitution Disease Mefv c.2080A> T Recurrent disease MTND1 c.3460G> A Leber optic atrophy Gjb2 c.35delG Sensorineural hearing loss

The microchip differs in the nucleotide sequence of the probes mounted on the DNA microchip for the passage of the primer elongation reaction on the chip (Eng. Arrayed Primer Extention - APEX). During the reaction, amplified and fragmented parts of the genome containing the analyzed mutations are denatured and applied to the DNA microarray.

The claimed DNA microchip is used as part of the kit (see table 3) for the diagnosis of hereditary diseases, carried out in several stages:

1) Obtaining human genomic DNA directly from a clinical sample (whole peripheral or umbilical cord blood, or erythrocyte mass or other biological sample).

2) Amplification of DNA sections containing mutations and polymorphisms using specifically selected primers.

3) Purification of PCR products.

4) Fragmentation of PCR products.

5) Hybridization of the obtained single-stranded fragments on a DNA microarray followed by the completion of probes on one nucleotide specifically labeled with a fluorescent marker at the mutation site. In this case, samples of the analyzed DNA are used as a matrix.

6) Registration of the analysis results by comparing the intensity of the fluorescence signals, upon irradiation of the DNA microchip with lasers of different wavelengths.

PCR and DNA fragmentation

Polymerase chain reaction (PCR) is used to amplify (increase the copy number) sections of genomic DNA carrying target mutations or single nucleotide polymorphisms (SNPs).

Part of dTTP is replaced in the PCR mix by dUTP, which allows further fragmentation using Uracil N-Glycosylase (UNG). PCR products are combined, concentrated and purified from non-integrated dNTPs using shrimp alkaline phosphatase (SAP). After treatment with SAP and UNG, the samples are heated to inactivate the enzymes and cleave the DNA at the site of insertion of uracil. Fragmentation of long PCR products provides better hybridization with complementary oligonucleotides immobilized on glass.

Conducting an APEX Reaction

Immediately before the chip primer elongation reaction (Arrayed Primer Extention - APEX), fragmented PCR products are denatured and transferred in the reaction mixture to the developed chip (an array of oligonucleotides on glass). The reaction mixture contains a buffer, single-stranded DNA, thermostable DNA polymerase, and four different, individually fluorophore-labeled terminator nucleotides.

Matrix-independent DNA polymerase reaction is carried out at a gradually increasing temperature in order to minimize the formation of undesirable secondary structures by oligonucleotides, in addition, to ensure effective hybridization with the target DNA fragments and to maintain a high level of polymerase activity. After incubation, the free and non-covalent bonded material is washed, which ensures the best signal-to-noise ratio.

Detection

Treated slides carrying an array of oligonucleotides that underwent an APEX reaction are scanned in a Genorama® QuattroImager ™ microarray scanner (see EP 1088214). Four lasers (alone) are used to excite different dyes. Four well spectrally separated dyes are excited by total internal reflection of the laser beam in the thickness of the glass slide, acting as a light guide. The light emitted by the fluorophores in response to excitation is recorded using a CCD camera.

Since four different dyes are used for each reaction, four different images of the emitted light are taken for each microchip. Each image corresponds to its own dye, respectively, reflects the pattern of embedding on the chip of one of the four terminator nucleotides.

Analysis of images and information.

Scanning is accompanied by analysis using Genorama® Genotyping Software ™ (“laser disk for genotyping”) to translate information about the fluorescence pattern into a nucleotide sequence. First, the signal intensities of the corresponding terminator nucleotide dyes are normalized. The signal intensities at each location of the oligonucleotides in all four images are compared, and the most intense signal is interpreted as the corresponding nucleotide. Depending on the embedding of which nucleotide occurred at the point of mutation, a conclusion is made about the status of its carriage in a particular patient.

Thus, a unique set of mutations and polymorphisms can be genotyped using the developed microchip.

High specificity (98%) and sensitivity (96%) were ensured when this microchip was used as part of the kit for genetic diagnosis using the elongation reaction of primers on DNA microarrays.

This microchip allows detection of mutations and polymorphisms, as well as associated hereditary monogenic diseases and predispositions to multifactorial diseases, including cystic fibrosis in one study, shown in Table 1. The DNA microchip is focused on the Russian population and is intended to diagnose the most frequent mutations associated with hereditary diseases, and the most common polymorphisms associated with a predisposition to multifactorial diseases and spread to territis Rhee Russian Federation.

Clinical examples

Clinical example 1.

Patient R-va. The material for the screening study was a sample of the erythrocyte mass of cord blood.

We conducted molecular genetic typing using the inventive DNA microarray for 216 mutations.

Using the proposed DNA microarray in a patient with P-howl, a mutation associated with galactosemia in the homozygous form of C.-119 del / del in the GALT gene was detected.

Clinical example 2.

Patient K-va. The material for the screening study was a peripheral blood sample.

We conducted molecular genetic typing using the inventive DNA microarray for 216 mutations.

Using the proposed DNA microarray, a mutation in the mitochondrial genome associated with hereditary optic neuropathy of Leber's optic nerve was revealed in a patient K. m. 1527A in the MTCYB gene.

Clinical example 3.

Patient C-yang. The material for the screening study was a sample of the erythrocyte mass of cord blood.

We conducted molecular genetic typing using the inventive DNA microarray for 216 mutations.

Using the proposed DNA microarray in patient C-yang, a mutation associated with a periodic disease in homozygous form c.442C / C in the MEFV gene was detected.

DNA microchip can be used for molecular genetic diagnostics in hospitals, clinical diagnostic laboratories and research institutes. These studies will allow healthy people to obtain information about their genetic characteristics to maintain their own health, as well as to prevent the birth of children with severe hereditary pathology in their family.

Table 1 Gene Mutation (trivial name) Nucleotide substitution (according to HGVS nomenclature) Amino acid substitution (according to the HGVS nomenclature) Disease CFTR 394deltt c.262_263deltt p.Leu88Ilefs * 22 Cystic fibrosis CFTR 621 + 1G-> T c. 489 + 1G> T Cystic fibrosis CFTR R334w c.1000C> T p.Arg334Trp Cystic fibrosis CFTR G551d c. 1652G> A p.Gly551Asp Cystic fibrosis CFTR 2184dela c.2052dela R. Lys684Asnf * 38 Cystic fibrosis CFTR 2183AA-> G c.2051_2052delaai nsg p.Lys684Serfs * 38 Cystic fibrosis CFTR 2184insa c.2052_2053insa p.Gln685Thrfs * 4 Cystic fibrosis CFTR S1196X c.3587C> G p.Ser1196 * Cystic fibrosis CFTR 3849 + 10kbc-> T c. 3717 + 12191C> T Cystic fibrosis CFTR N1303K c.3909C> G p.Asn1303Lys Cystic fibrosis CFTR I1005R c. 3014T> G p.Ile1005Arg Cystic fibrosis Pah c.1222C> T p.Arg408Trp Phenylketonuria Pah c.842C> T p.Pro281Leu Phenylketonuria Pah IVS12 + 1g-> a c.1315 + 1G> A Phenylketonuria Pah c.473G> A p.Arg158Gln Phenylketonuria Pah c.754C> T p.Arg252Trp Phenylketonuria Pah c.1045T> C p.Ser349Pro Phenylketonuria Pah c.1242A> G p.Tyr414Cys Phenylketonuria

Pah IVS10nt546 IVS10-11G> A Phenylketonuria Pah IVS4 + 5G> T Phenylketonuria Pah c.143T> C p.Leu48Ser Phenylketonuria Pah c. 1206C> T p.Ala403Val Phenylketonuria Pah c.929C> T p.Arg243 * Phenylketonuria Pah c.781C> T p.Arg261 * Phenylketonuria Pts C.95A> G C.94A> G p.Ser32Gly Type III phenylketonuria Pts C.216T> A C.216T> A p.Asn72Lys Type III phenylketonuria Pts c.318C> T C.317C> T p.Thr106Met Type III phenylketonuria Galt c.584T> C c.584T> C p.Leu195Pro Galactosemia Galt c.384A> G c.855G> T p.Lys285Asn Galactosemia Galt -119_-116delgtca c.-119_-116delgtca Galactosemia Galt IVS2-2A> G c.253-2A> G Galactosemia FKRP c.341C> G p.Ala114Gly Lumbo-limb muscular dystrophy type 2I Capn3 C.550dela p.Thr184fs * Lumbo-limb muscular dystrophy type 2A Capn3 c.598-612del15 p.Phe200_Leu204del Lumbo-limb muscular dystrophy type 2A SGCG c.87 dupt p.Gly30Trpfs * 30 Waist-limb muscular dystrophy type 2C

SGCG c.525delt p.Phe175Leufs * 20 Lumbo-limb muscular dystrophy type 2C SGCG c.848G> A p.Cys283Tyr Lumbo-limb muscular dystrophy type 2C SGCB c.377_384dupca gtagga c.377_384dup8 p.Gly129Glnfs * 2 Lumbo-limb muscular dystrophy type 2E SGCA c.229C> T p.Arg77Cys Lumbo-limb muscular dystrophy type 2D SGCA c.850C> T p.Arg284Cys Lumbo-limb muscular dystrophy type 2D Mfn2 C.280C> T p.Arg94Trp Charcot-Marie-Tooth Neuropathy 2A LMNA c.398G> T p.Arg133Leu Charcot-Marie-Tooth neuropathy 2 B1 LMNA c.892C> T p.Arg298Cys Charcot-Marie-Tooth neuropathy 2 B1 LMNA c.l411C> T p.Arg471Cys Charcot-Marie-Tooth neuropathy 2 B1 LMNA c.1579C> T p.Arg527Cys Charcot-Marie-Tooth neuropathy 2 B1

Gjb1 c.259C> G p.Pro87Ala Charcot-Marie-Tooth Neuropathy IX Gjb1 c.425G> A p.Arg142Gln Charcot-Marie-Tooth Neuropathy IX Gjb1 c.67C> T c.64C> T p.Arg22 * Charcot-Marie-Tooth Neuropathy IX Gjb1 c.277A> G p.Met93Val Charcot-Marie-Tooth Neuropathy IX Gdap1 C.715C> T p.Leu239Phe SH3TC2 c.3325C> T p.Arg1109 * Charcot-Marie-Tooth Neuropathy 2H FIG4 c.122T> C p.Ile41Thr Charcot-Marie-Tooth 4J Neuropathy FGD4 c.893T> G p.Met298Arg Charcot-Marie-Tooth Neuropathy 4H FGD4 c.893T> C p.Met298Thr Charcot-Marie-Tooth Neuropathy 4H IKBKAP c. IVS20 + 6T> C c.2204 + 6T> C Familial disautonomy IKBKAP c.2087G> C p.Arg696Pro Familial disautonomy Bscl2 Asn88ser c.455A> G p.Asn152Ser Spastic paraplegia of Silver TOR1A (DYT1) IVS4 + 5g-> t c. 748 + 5G> T DOPA-independent torsion dystonia

PNKD c.66C> T c.20C> T p.Ala7Val Paroxysmal nonkinesiogenic dyskinesia PNKD c.72C> T c.26C> T p.Ala9Val Paroxysmal nonkinesiogenic dyskinesia CLCN1 Gly190Ser c.568_569GG> TC p.Gly190Ser Myotonia Thomsen CLCN1 Ala493Glu c. 1478C> A p.Ala493Glu Myotonia Thomsen CLCN1 c.2680C> T p.Arg894 * Myotonia Thomsen CLCN1 c. 180 + 3A> T Myotonia Thomsen CLCN1 c.1261C> T p.Arg421Cys Myotonia Thomsen CLCN1 C1649c> T p.Thr550Met Myotonia Thomsen Pex7 c.875A> T p.Leu292 * Point chondrodysplasia SCN4A c.3938C> T p.Thr1313Met Hyperkalemic periodic paralysis SCN4A c.2111C> T p.Tyr704Met Hyperkalemic periodic paralysis SCN4A c.2023C> G p.Arg675Gly Normokaliemic periodic paralysis SCN4A c.2006G> A p.Arg669His Hypokalemic periodic paralysis

Nf1 c.1070T> C p.Leu357Pro Type I Neurofibromatosis Nf1 c.2970delaat c.2970del3 p.Met991del Type I Neurofibromatosis ATP7B c.3207C> A p.H1069Q Wilson-Konovalov disease ATP7B c.2906G> A p.R969Q Wilson-Konovalov disease ATP7B c.2299insc c.2304insc p.m769fs Wilson-Konovalov disease ATP7B c.3532dela p.t1178fs * Wilson-Konovalov disease TCIRG1 c.807 + 5G> A Autosomal recessive osteopetrosis Vhl c.598C> T Arg200Trp Autosomal repressive erythrocytosis LAMA3 c.151insg c.152dupg Laringo-oniho-kutanaya syndrome NPHS1 Arg1109X c.3205C> T p.Arg1069X Finnish nephrotic syndrome Mefv c.2080A> T p.Met694Leu Recurrent disease Mefv c. 1437C> G p.Phe479Leu Recurrent disease Mefv c.2282G> A p.Arg761His Recurrent disease

DHCR7 c.278C> T p.Thr93Met SLOS Syndrome DHCR7 c.453G> A p.Trp151 * SLOS Syndrome DHCR7 c.452G> A p.Trp151 * SLOS Syndrome DHCR7 c.976G> T p.Val326Leu SLOS Syndrome Acadm c.985A> G p.Glu304Lys MCAD deficiency GBA IVS2 + 1G> A c. 115 + 1G> A Gaucher disease GAA IVS1AS-13T> G c.-32-13T> G Pompe disease GAA c.271G> A p.Asp91Asn Pompe disease IDUA c.296C> T c.208C> T p.Gln70 * Gurler's disease Hexa Ex11, 4bp INS c.1278instatc p.Tyr427fs Tay-Sachs disease Hexa IVS12 + 1G> C c.1421 + 1G> C Tay-Sachs disease Hexa IVS9 + 1G> A p.1073 + 1G> A Tay-Sachs disease Hexa c.1444G> A p.Gly482Lys Tay-Sachs disease SERPINA1 E342K, (Z mut) c.1096G> A p.Glu366Lys Alpha-1-aminotrypsin deficiency SERPINA1 c. 792 A> T (S mut) c.863A> T p.Glu288Val Alpha-1-aminotrypsin deficiency SERPINA1 p.Met358Arg c. 1145T> G p.Met382Arg Alpha-1-aminotrypsin deficiency Ryr1 c. 1840C> T p.Arg614Cys Malignant hyperthermia Ryr1 c.1021G> A p.Gly341Arg Malignant hyperthermia Ryr1 c.6502G> A p.Val2168Met Malignant hyperthermia CACNA1 c.3333G> A p.Arg1086His Malignant

S hyperthermia ASPA c.854A> C p.Glu285Ala Canavan Disease SMPD1 p.Arg496Leu c.1487C> T p.Arg498Leu Niman-Peak Disease AB SMPD1 Leu302Pro c.911T> C p.Leu304Pro Niman-Peak Disease AB SMPD1 c.1267C> T p.His423Tyr Niman-Peak Disease AB LCT c.-13910C> T c.1917 + 326C> T Lactose intolerance Polg c.2243G> C p.Trp748Ser Mitochondrial Depletion Syndrome and DNA Polg c.752C> T p.Thr251Ile Mitochondrial Depletion Syndrome and DNA Surf1 c.845-846 delct p.Ser282fs Leia's Syndrome Surf1 326insAT delTCTGCCAGCC c.311-321del10ins2 p.Leu105 * Leia's Syndrome MTTL1 c.3252A> G c.3252A> G MELAS Syndrome MTND5 c.13730A c.13730A Leber optic atrophy MTND1 c.3460G> A c.3460G> A Leber optic atrophy MTND4 c.11778G> A c.11778G> A Leber optic atrophy MTND4L c.10663T> C c.10663T> C Leber optic atrophy

MTND6 c.14459G> A c.14459G> A Leber optic atrophy MTCO3 c.9438G> A c.9438G> A p.Gly78Ser Leber optic atrophy MTCO3 c.9804G> A c.9804G> A p.Ala200Thr Leber optic atrophy ABCA4 (ABCR) c.2588G> C p.Gly863Ala Type 1 Stargard disease ABCA4 (ABCR) c.3113C> T p.Ala1038Val Type 1 Stargard disease ABCA4 (ABCR) c.5882G> A p.Gly1961Glu Type 1 Stargard disease OPA1 c.1334G> A p.Arg445His Atrophy of the optic nerve with deafness OPA1 Tyr582Cys c. 1756A> G Tp.yr619cys Atrophy of the optic nerve with deafness COCH c.208C> T p.Pro51Ser Autosomal dominant deafness Gjb2 35delg c.31_35delg p.Gly12fs Hereditary hearing loss Gjb2 IVS1 + 1G> A (-3201G> A) c.-23 + 1G> A Hereditary hearing loss Gjb2 312_325del14 c.313_326del14 p.Arg104fs Hereditary hearing loss SLC26A4 c.1246A> C p.Thr416Pro Pendred's syndrome

SLC26A4 c.2168A> G p.His723Arg Pendred's syndrome SLC26A4 IVS7AS, A-G, -2 c.919-2A> G Pendred's syndrome SLC26A4 c.1151A> G p.Glu384Gly Pendred's syndrome ADA c.631C> T p.Arg211Cys Adenosine deaminase deficiency HBB c.25_26delaa p.Lys9fs * Beta thalassemia HBB IVS-II-1 (G-> A) c. 315 + 1G> A Beta thalassemia HBB IVS-I-110 (G-> A) c. 93-21G> A Beta thalassemia HBB IVS-I-6 (T-> C) c. 92 + 6T> C Beta thalassemia HBB Codon 44 (-C) c.135delc p.Ser45fs * Beta thalassemia HBB IVS-II-745 (C-> G) c. 316-106C> G Beta thalassemia HBB Codons 8/9 (+ G) c.27_28insg Beta thalassemia HBB IVS-I-1 (G-> A) p. 92 + 1G> A Beta thalassemia ABCD1 c.1411_12insa p.Gln472fs * 555 Adrenoleukodystrophy ABCD1 Arg617His c.1520G> A p.Arg617His Adrenoleukodystrophy ABCD1 c. 1553G> A p.Arg518Gln Adrenoleukodystrophy ABCD1 c. 1666G> A p.Arg554His Adrenoleukodystrophy ABCD1 Arg660Trp c.19780T p.Arg660Trp Adrenoleukodystrophy

Hmbs c.499C> T p.Arg167Trp Acute intermittent porphyria Hmbs c.518G> A p.Arg173Gln Acute intermittent porphyria Hmbs c.331G> A p.Gly111Arg Acute intermittent porphyria KCNQ1 IVS1 + 1G> A 386 + 1G> A LQT syndrome PRNP c.598G> A p.Glu200Lys Hereditary prion diseases SLC26A2 c.835C> T p.Arg279Trp Cramping dysplasia Flg 4-BP DEL, 2282CAGT c.2282 del4 p.Val762fs * Ichthyosis vulgar PKHD1 p.107C> T p.Thr36Met Autosomal recessive polycystic kidney disease LMNA c.1445G> A p.Arg482Gln Family Partial Lipodystrophy, Dannigan Type LMNA c.1445G> T p.Arg482Leu Family Partial Lipodystrophy, Dannigan Type LMNA c. 1751G> A p.Arg584His Family Partial Lipodystrophy, Dannigan Type AGT c.803T> C p.Met268Thr Predisposition to late gestosis Fgb c.-467G> A c.-463G> A Stroke predisposition

Fgb c.-156C> T Stroke susceptible F2 c. * 97G> A G.25313G> A Predisposition to thrombophilia F5 c.1601G> A p.Arg534Gln Predisposition to thrombophilia Mthfr c.665C> T p.Ala222Val Predisposition to thrombophilia Mtr c.2756A> G p.Asp919Gly Predisposition to thrombophilia CALCR Pro447leu c. 1442T> C p.Leu481Pro Predisposition to osteoporosis Gstp1 c.313A> G p.Ile105Val Regulation of xenobiotic detoxification NAT-2 c.341T> C p.Ile114Thr Regulation of xenobiotic detoxification NAT-2 c.481C> T p.Leu161Leu Regulation of xenobiotic detoxification NAT-2 c.590G> A p.Arg197Gln Regulation of xenobiotic detoxification CYP2C9 c.817dela p.Lys273fs * Regulation of Warfarin Metabolism CYP4F2 c. 1297G> A p.Val433Met Regulation of Warfarin Metabolism

Ggcx c. 2084 + 45G> C Regulation of Warfarin Metabolism NOD2 c.2722G> C p.Gly908Arg Crohn's disease NOD2 c.3019_3020insc p.Leu1007fs * Crohn's disease DLG5 c.419G> A p.Arg140Gln Crohn's disease ALDH2 c.1510G> A p.Glu504Lys Regulation of alcohol metabolism ADH1B c.143A> G p.His48Arg Regulation of alcohol metabolism OPRM1 c.118A> G p.Asn40Asp Regulation of opiate metabolism ANKK1 c.2137G> A p.Glu713Lys Regulation of opiate metabolism Hfe c.187C> G p.His63Asp Hemochromatosis Hfe C.845G> A p.Cys282Tyr Hemochromatosis CSTB (STFB) Unferricht-Lundborg disease FGFR2 c.1025G> A p.Cys342Tyr Cruson Syndrome FGFR2 c.758C> G p.Pro253Arg Pfeiffer Syndrome BTD c.98_104delGCGGCTGINSTCC c.98_104delGCGGCTGinsTCC p.Cys33fs * Biotinidase Deficiency BTD C.16120T p.Arg538Cys Biotinidase Deficiency

BTD c.511G> A p.Ala171Thr Biotinidase Deficiency PPT1 c.364A> T p.Arg122Trp Type 1 neuronal ceroid lipofuscinosis Dysf c.855 + 1delg Lumbo-limb muscular dystrophy type 2B Dysf c. 937 + 1G> A Lumbo-limb muscular dystrophy type 2B Dysf c.1566C> G p.Tyr522 * Lumbo-limb muscular dystrophy type 2B Dysf c.3373delg p.Glu1125Lysfs * 9 Lumbo-limb muscular dystrophy type 2B Dysf c.4872_4876delins cccc p.Glu1624Aspfs * 9 Lumbo-limb muscular dystrophy type 2B Dysf c.5979dupa p.Glu1994Argfs * 3 Lumbo-limb muscular dystrophy type 2B Dysf c.6124C> T p.Arg2042Cys Lumbo-limb muscular dystrophy type 2B Dysf c.200_201delinsat p.Val67Asp Lumbo-limb muscular dystrophy type 2B

CFTR c.276G> C p.Glu92Asp Lumbo-limb muscular dystrophy type 2B CSTB (STFB) c.del313-314 c.del313-314 Unferricht-Lundborg disease CCR5 Delta_32 Delta_32 Regulation of HIV entry into the cell CCR5 p.303T> A p.303T> A p.Cys101 * Regulation of HIV entry into the cell CCR5 c.-22459A> G c.-22459A> G Regulation of HIV entry into the cell SRY1 (sy14) del SY14 Male infertility AZFC Sy254 del SY254 Male infertility AZFA Sy84 del SY84 Male infertility AZFB Sy1237 del SY1237 Male infertility AZFB SY1235 del SY1235 Male infertility AZFB SY1302 del SY1302 Male infertility AZFA SY1316 del SY1316 Male infertility AZFC Sy1196 del SY1196 Male infertility AZFC Sy1191 del SY1191 Male infertility

AZF SRY1-6 del SY1-6 Male infertility AZFC Sy1125 del SY1125 Male infertility AZFC Sy1206 del SY1206 Male infertility

table 2 The sequence of probes attached to the DNA microarray. Gene Nucleotide substitution (according to HGVS nomenclature) APEX reaction probe APEX antisense probe CFTR c.262_263delTT ATGTTTTTTCTGGAGATTTATGTTCTATGGAATCTTT TGAATGTACAAATGAGATCCTTACCCCTAAATATAAA CFTR c. 489 + 1G> T CAGATGAGAATAGCTATGTTTAGTTTTGATTTATAAGAAG ATGGGGCCTGTGCAAGGAAGTATTA CFTR c.1000C> T CCAAGTTTGCAGAGAAAGACAATATAGTTCTT AATGAGATGGTGGTGAATATTTTCC CFTR c. 1652G> A GAAGGTGGAATCACACTGAGTGGAG TGCTAAAGAAATTCTTGCTCGTTGA CFTR c.2052delA CTGTCTCCTGGACAGAAACAAAAAAA CAAACTCTCCAGTCTGTTTAGAAGATTG CFTR c.2051_2052delA AinsG GCTCCTGTCTCCTGGACAGAAACAAAAAA CAAACTCTCCAGTCTGTTTAAAAGATTG CFTR c.2052_2053insA TGTCTCCTGGACAGAAACAAAAAAAA CTCTCCAGTCTGTTTAAAAGATTGTTTTTTTT CFTR c.35870G CAACTCTCGAAAGTTATGATTATTGAGAATT CCAGATGTCATCTTTCTTCACGTGT CFTR c. 3717 + 12191C> T TCCATCTGTTGCAGTATTAAAATGG CATTTCCTTTCAGGGTGTCTTACTC CFTR c.3909C> G GAAAGTATTTATTCTTTCTGGAACATTTAGAAAAAA CACTCCACTGTTCATAGGGATCCAA CFTR c. 3014T> G TTGTTATTAATTGTGATTGGAGCTA GGGTTCTAAAACTGCGACAACTGCT Pah c.1222C> T TAGGAACTTTGCTGCCACAATACCT GTCGTAGCGAACTGAGAAGGGCC

Pah c.842C> T GATTTCTTGGGTGTCCTGGCCTTCC GTCTGATGTACTGTGTGCTGTGGAAGACT Pah c.1315 + 1G> A GCTTAAGATTTTGGCTGATTCCATTAACA GAGTGGCCTCGTCAGGTGTAAATTACTTA Pah c.473G> A AAAGATCCTGTGTACCGTGCAAGAC GTAGGCAATGTCAGCAAACTGCTTC Pah c.754C> T TGGCTGGCCTGCTTTCCTCT CACGCCACCCAAGAAATCCC Pah c.1045T> C GGATATGGTGCTGGGCTCCTG GGTCATACCTGTAATTCACCAAAGGATG Pah c.1242A> G TCGGCCCTTCTCAGTTCGCT AATCCTTTGGGTGTATGGGTCG Pah IVS10-11G> A TGATCCTGATTTAACAGTGATAATAACTTTTCACTT TTCTCTGATAAGCAGTACTGTAGGCCC Pah IVS4 + 5G> T GCAAGACGGAAGCAGTTTGCTGGTGA ATCTCATCCTACGGGCCATGGA Pah c.143T> C CTCACTCAAAGAAGAAGTTGGTGCAT CTCAAATAAGCGCAATACTTTGGCC Pah c. 1206C> T CATGAAAAGTACTGTGGCTACCATGAA AGGGCCGAGGTATTGTGGCA Pah c.929C> T CCCAGCTTGCACTGGTTTCCGCCTC GAGGAAAGCAGGCCAGCTACAGGTC Pah c.781C> T TTCTTGGGTGTCCTGGCCTTC TGATGTACTGTGTGCAGTGGAAGACTC Pts p. 94A> G TGACTTTTTTTTTTTTTTTTTGGTCAGTAAATTTCTA CCAAACAGTTTCAAGTTTTCTTCATCAC Pts c.216T> A CCTGCTACGGGAATGGTTATGAA ACGTCCATATATTTTTTGAGATCAGCCAG

Pts c.317C> T TTTTTTTTGTTTTTGTTTTTTTTTTCTTATAGCA TCCCACATATAAACAGCTACATTTTCAGTC Galt c.584T> C ACAGGTATGGGCCAGCAGTTTCC CCTCACACTGGGCAATATCTGGC Galt c.855G> T CCATCATGAAGAAGCTCTTGACCAA GAAAGGACGTCTCAAAGAGGTTGTCATA Galt c.-119_-116delGTCA CAGGGCAGCCCAGTCAGTCA TGACCAGCCGCCAGCACG Galt c.253-2A> G GTGCTTCTAGCCTATCCTTGTCGGT GTGCTATCGTACTGGGGATTCACC FKRP c.341C> G TGAACCGGCCAGCCGCAG TAGGTCTCCGGGCGCGAG TGGACCGGCAAGCCGCAG Capn3 c.550delA GTGGTTATAGATGACTGCCTGCCA GGTGAAAACCAGTTGATTGTTGTACGT Capn3 c.598-612del15 CAAGTCCAACCACCGCAATGAG GCTTACTTAGCATAAGCCTTCTCCAGC SGCG c.87 dupT TTTTGTTTAGGGCCTGAAGGGGCCTTTTT CAAGGGGTGTCTCCACTGAATGTTCAAAA SGCG c.525delT GAGAATCAGTATGTCTACAAAATTGGCATTTAT AGACAGCGCTTTCTCCAGCCA SGCG c.848G> A CCGGTGTGAGCACCACGT CAGATGTGGTTGTGCTCCTGG SGCB c.377_384dup8 ATCCACCCTGTTTATAAAAGCACAGTAGGA AATTTTCATTTCGCCTTCCTCCTACTGT SGCA c.229C> T GACCTGCCCCGGTGGCTC GGCTGCGGTGGGTGTAGC SGCA c.850C> T ACCCACTGAGGCCCCAGAC CCAGAGCATCCACCAAGAAGTCAC Mfn2 c.280C> T AGAAGCATCAGTGAGGTGCTGGCTC AAACCCACTTTCATGTGCCTCC

LMNA c.398G> T TGACCTGATAGCTGCTCAGGCTC AGACCCTCCAGGTCCTTCAGC LMNA c.892C> T CTGCAGCAATCGCGCATC GCCAGAGAGGCTGTCGATGC LMNA c.1411C> T CATGGGGAATTGGCAGATCAAG CAAGGGATCATCTCCATTCTGGC LMNA c.1579C> T GGGCTGCGGGAAAAGCCTG CAGTGGAGTTGATGAGAGCCGTAC Gjb1 c.259C> G TGCAGCTCATCCTAGTTTCCACC CATGGCGACGAGGAGAGCTG Gjb1 c.425G> A CTATGTCATCAGCGTGGTGTTCC ATGAAGACGTCCTCAAACAACAGC Gjb1 c.64C> T CGGCATTCTACTGACATTGGC GATGAAGATGACCGAAAGCCATACTC Gjb1 c.277A> G CCAGCTCTCCTCGTGGCC GCTGGTGAGCCACGTGCA Gdap1 c.715C> T GAAGAGGGCGAGCAACCTTGG AGGGTGAAGGATTCACCGCAGA SH3TC2 c.3325C> T ACAGGCATCATGCAGTCGAGTACTAC GCAAACTGCACAGCTTGCACTTACTC FIG4 c.122T> C GCAGAAACGAAATATCGTGTGTTGAAGA TGACCAAATCTTTTGGTTCTGTTCTATCA FGD4 c.893T> G TCAGAAATTGGCACCATTCCTTAAGA GCATTATCAAATCCTTTCACATATTCTCCATAC FGD4 c.893T> C TCAGAAATTGGCACCATTCCTTAAGA GCATTATCAAATCCTTTCACATATTCTCCATAC IKBKAP c.2204 + 6T> C ATTCGGAAGTGGTTGGACAAGTAAG AACTAGTCGCAAACAGTACAATGGC IKBKAP c.2087G> C CTGCGGAAAGTGGAGAGGGGTTCAC GTCCTGGGGCACAACAGTGACAATC

Bscl2 c.455A> G TCTGCTCCTTCCCTGTTGCCA GTCCACCCTTAGTCAGCGAGACA TOR1A (DYT1) c. 748 + 5G> T GTGTCGGTTTTTGAATAACAAGAACAGTGA CAGGCGGTGGATGGCCCTA PNKD c.20C> T CATGGTGGCGGTGGTAGCTG CGGCCCTTCAGCGCCGTA PNKD c.26C> T CGGCGTTGGTAGCTGCTACGG GCCCCCCGGACCTTCAGC CLCN1 c.568_569GG> TC CACATAATCTTTCAACGCTTTTAGGCCTT GATGTATTGTCTTCATTTCGGGGATT CLCN1 c. 1478C> A CTTCCTTTTATCTTCCCTCTAGGAGCTG CATGATTTCTCCTACCAGCCTTCCAAAT CLCN1 c.2680C> T AGCTTCCGGAACACGACTTCAACT GCAGGTGCCCCGGTACTCTTTC CLCN1 c. 180 + 3A> T AACGTCCAGCCCACACAGGT CTCCCCTCTCCCCTTAGAGCACTT CLCN1 c.1261C> T CTTCCTCTCCCAGTTGATGCCC GTCAAACAAAGTACTGATGGCTTCGC CLCN1 c1649C> T CACAGCTGTGATTTGCATCGAATTAA CAGCATGTGAGCAATCTGACCC Pex7 c.875A> T TGGAGCATCATACAGAGTTTACTTGTGGTT TGAGTGGGGCTCTGAATACTGAAGTCT SCN4A c.3938C> T TACTTAGGGGGGGAAAGACATCTTTATGA TCATGTCGTTATAGTATTTCTTCTGTTCCTCC SCN4A c.2111C> T GGGGCGCTGGGTAACCTGA AACACGATGATAGCCAGCACCAGC SCN4A c.2023C> G CCTCCCTGCTTGGCAGCTG CGACTTGACCAGCTTGAAGACCC SCN4A c.2006G> A CCAACATACAGGGACTGTCTGTGCTAC GGTTTTGTGTACCAGACGGAAGGAG

Nf1 c.1070T> C TTTTCTGTTGGGGTTTTTATAGAACCTGC CCTCTTGAGAATGGCTTACTTGGATTAAAA Nf1 c.2970del3 CATCTAGGGCAAGCTAGCATTGAAAC AGTAGAATGCTTACCTGACCAGATTTAACATC ATP7B c.3207C> A GACTGCGGAGGCCAGCAGTGAACA TGGTGACTGCCACGACCAAGGG ATP7B c.2906G> A CACATCTCCCAGACAGAGGTGATCATCC CGTGATGGACGTCTGGAAAGCAAAC ATP7B c.2304insC ACATTCTTCGACACGCCCCCC AATGAACACAAAGAGCATGGGGGGG ATP7B c.3532delA GACAGACCACGAGATGAAAGGACAG GTCAATAGCCACAAGGATGGCTG LAMA3 c.152dupG GTCAAAGTCAACTGCAAGCGAGTTATG GTTACCTGGCTGGGTCTAAACTCCAC NPHS1 c.3205C> T TCATCCTGGAAGGTTGGAAGAGGAC GGCTCTCCTCATATTCGTTCCTGACTC Mefv c.2080A> T GAATGGCTACTGGGTGGTGATAATG GACGCCTGGTACTCATTTTCCTTCA Mefv c. 1437C> G ACTTCCTGGAGCAGCAAGAGCATTT CCACGTCCTCCAGTGAGGCCACAAA Mefv c.2282G> A CAACCTATCTTCAGCCCTGGGACAC AGGAGCTGTGTTCTTCCCTCCATCA DHCR7 c.278C> T ATCTGGGCCAAGACTCCACCTATAA GGTATAGATCTGGGCGGCTTTCCTC DHCR7 c.453G> A ATTAGATCAATGGCCTGCAAGCCTG AAACCAGAGCAGGTGCGTGAGGAG DHCR7 c.452G> A TATCAGATCAACGGCCTGCAAGCCT AACCAGAGCAGGTGCGTGAGGAGC DHCR7 c.976G> T GCCCCCTAGGGTCTGTACTTG CAGCTGCACGGGGTGGTACA

Acadm c.985A> G ATTTATGCTGGCTGAAATGGCAATG CTCTCTGGTAACTCATTCTAGCTAGTTCAACTT GBA c. 115 + 1G> A CTTCAGGCAGTGTCGTGGGCATCAG CCTCCCCACTGCCTTGACTCACTCA GAA c.-32-13T> G CTCCCTGCTGAGCCCGCTT TCCTACAGGCCTGCGGGAGAAG GAA c.271G> A CCCCCCCAACAGCCGCTTC GCCTTGTCAGGGGCGCAAT IDUA c.208C> T TGACCAGTACTTCCTCAGCTGGGAC CACATAGGCGAGGTTGAGCTGCT Hexa c.1278insTATC GCCCCCTGGTACCTGAACCGTATATC TCCTTCCAGTCAGGGCCATAGGATA Hexa c.1421 + 1G> C ACACAAACCTGGTCCCAAGGCTCTG CCACCTCCCCCCCGAAAACCCTTA Hexa p.1073 + 1G> A AGCTGGAGTCCTTCTACATCCAGAC GACCCCACCCACCCTCCTTCCTTCCTCA Hexa c.1444G> A CCAGAGCAGGTGCTGTTGCC GTCTACTTGTTGCTCCACAGCCTTT SERPINA1 c.1096G> A TGCATAAGGCTGTGCTGACCATCGAC 132AGCAGCTTCAGTCCCTTTCT SERPINA1 c.863A> T TGATGAGGGGAAACTACAGCACCTGG TTGGTGATGATATCGTGGGTGAGTTCATTT SERPINA1 c. 1145T> G GCCATGTTTTTAGAGGCCATACCCA GAACTTGACCTCGGGGGGGATAGAC Ryr1 c.18400T CAGTGTGTGTGTAATGGTGTGGCTGTA AGTTCTCAGTAATAAGATCTTGGTTGGAGC Ryr1 c.1021G> A TGGGCCCCCCTGAGATCAAGTAC CTGCACCAAGCACAGTGACTCCC Ryr1 c.6502G> A CAAATCCGCTCGCTGCTCATC TCCTGGGGGCCCATCTGCA

CACNA1S c.3333G> A ACCTGTCCTCTTGTATGTGTCACAGC CTTTCAGGGCATACTGTACACATTGG ASPA c.854A> C ACCGTGTACCCCGTGTTTGTGAATG TTCTTTCTTTTCGTAATATGCGGCC SMPD1 c.1487C> T CAGCCCCACATCCTTGCAAGTTACC GGAGTAGTTTCCATCTATTTGGTACACA SMPD1 c.911T> C GGCCCTGACCACCGTCACAGCAC CACTGGCCCCAGGAACTTCCTCACA SMPD1 c.1267C> T TGATTACGATCCTTAATTCTCCCTACTAGGTG CCTGGGGGAATGTGGCCAATTATAT LCT c.1917 + 326C> T TGCGCTGGCAATACAGATAAGATAATGTAG GAGGAGAGTTCCTTTGAGGCCAGGG Polg c.2243G> C GACGTGGACATCCCTGGCTGCT ACCTTGTGAGGCAGCTTGAAAAAC Polg c.752C> T CCTCATCCCCCTGGAGGTCCCTA GGGTGGGGCTGCTTGCACCA Surf1 c.845-846 delCT TTGCTTTCAACCCCTAGGTATGGACTCT AAACCACAGGTAGGATGTAGCTGCAGAG Surf1 c.311-321del10ins2 GAGTTCTGGCTGAGCCTGTCCC GGGGCAGCCATGCACTCACTC MTTL1 c.3252A> G TTTGTTAAGATGGCAGAGCCCGGTA ACCTGTGACTGTAAAGTTTTAAGTTTTTATGCGA MTND5 c.13730A CAGCCGGAAGCCTATTCGCAG CGGGGGAAATGTTGTTAGTAATGAGAAAT MTND1 c.3460G> A GGGCTACTACAACCCTTCGCTGAC GGGGCTCTTTGGTGAAGAGTTTTTATGG MTND4 c.11778G> A AAACTCAAACTACCAACGCACTCACAGTC TTTGAAGTCCTTGAGAGAGGATTATGATG

MTND4L c.10663T> C TAGCCAATATTGTGCCTATTGCCATACTAG TGTTTCGCAGGCGGCAAAG MTND6 c.14459G> A ATGCCTCAGCATACTCCTCAATAGCCATC GGAATGATGGTTGTCTTTGGATATACTACAG MTCO3 c.9438G> A ACCACACACCACCTGTCCAAAAAA TAATAAATAGGATTATCCCGTATCGAAGGC MTCO3 c.9804G> A GCATCTCCGGCTCAACATTTTTTTGTA TGAAGTCCGTGGAAGCCTGTGG ABCA4 (ABCR) c.2588G> C CGTTCGACTTTCTCTGTTTATTTGGTCTCTATTTTTAG CCAAGGAAGTGGGGTTCCATAGTCT ABCA4 (ABCR) c.3113C> T CCCAGCTGAAAGGAAAGTCCCAGGAGGAGG CAACATGGCTTCCATCTCAAGCTGG ABCA4 (ABCR) c.5882G> A CCAGCCCAGCAGTGGAGAGGCTGTGTGTCG AGTACCCACCTCTCCAGGGCGAACT OPA1 c.1334G> A TCAAATGGATCTGTGGATGCTGAAC ATTTGCCTGACCAAGTCTGTAACAATACTG OPA1 c. 1756A> G TTTTAACCTTGAAACTGAATGGAAGAATAACT TCAAGTTACCGCAGGCGAGGA TCIRG1 c.807 + 5G> A AGCTAGGAGCTGCAGGAGGTGG TCCGGAAGGCCGGGGGCA Vhl c.598C> T CCAAATGTGCAGAAAGACCTGGAG AATGCGCTCCTGTGTCAGCC SOS c.208C> T GGAAAGAGAAAACAGATGTCCTCTGC CCTCAAGAGGGAAGCCCCCCTG Gjb2 c.31_35delG GGCACGCTGCAGACGATCCTGGGGG GTGGAaTGTTTGTTCACACCCCCCC Gjb2 c.-23 + 1G> A CCGCCaCGCTTCCTCCCGACGCAG GCAGTCCGGGGCCGGCGGGCTCA

Gjb2 c.313_326del14 CTACCGGAGACATGAGAAGAAGAGG GATGTCCTTAAATTCACTCTTTATCTCCCC SLC26A4 c.1246A> C CCGCACaGCCGTCCAGGAGAGC GTTCCTACCTGTGTCTTTCCTCCAG SLC26A4 c.2168A> G GAAAGGACACATTCTTTTTGACGGTCC CACTTGGTTCTGTAGATAGAGTATAGCATCA SLC26A4 c.919-2A> G GAGTTTTTAACATCTTTTGTTTTATTTC TATGAAATGGCAGTAGCAATTATCGTC SLC26A4 c.1151A> G CGTTGTCATCCAGTCTCTTCCTTAGG TGCTGATCCCAAAGGCAATGAAT ADA c.631C> T AGGCTTTGAAGAGCGGCATTCAC CCTCCCCGGCGTGGACAGTAC HBB c.25_26delAA TGGTGCATCTGAATCCTGAGGAG CCCACAGGGCAGTAACGGCAGAC HBB c. 315 + 1G> A GCACGTGGATCTTGAGAACTTCAGG AAACATCAAGGGTCCCATAGACTCA HBB c. 93-21G> A GCACTGACTCTCTCTGCCTATT GCAGCCTAAGGGTGGGAAAATAGAC HBB c. 92 + 6T> C GGTGGTGAGGCACTGGGCAGGTTGG CTTAAACCTGTCTTGTAACCTTGAT HBB c.135delC CTTGGACCCAGAGGTTCTTTGAGTC ATCAGGAGTGGACAGATCCCCAAAG HBB c. 316-106C> G TTCATATTGCTAATAGCAGCTACAATCCAG TCCCAACCATAAAATAAAAGCAGAATGGTA HBB c.27_28insG ACCATGGTGCACCTGACTCCTGAGGAGAAG ACCTTGCCCCACAGGGCAGTAACGGCAGAC HBB p. 92 + 1G> A AAGTTGGTGGTGAGGCCCTGGGCAG ACCTGTCTTGTAACCTTGATACCAA

ABCD1 c.1411_12insA GGCCAGGTGGTGGATGTGGAA TGTTCTCGCAGATGATCCCCTGTT ABCD1 c.1520G> A AAGCAGAGAATCGGCATGGCCC GGAGTGCTCACCTGTGGTAGAACATG ABCD1 c. 1553G> A TGCAGCAAGAGCTCCCTGTTCC GCCAGAGCCCACCCAGGATC ABCD1 c. 1666G> A CCTACATGTCTGTGGGCTCCCTGC GAGTCCGGGTAGATCACCTGGTCA ABCD1 c.1978C> T GCCCTGCTCTCCATCACCCAC GCACCTACCACAGGGAGGGCC Hmbs c.499C> T CTGTGGTCTTTAGCAACTCTCCACAG GCCGTGTGTTGAGGTTTCCCC Hmbs c.518G> A CAGCGGAGAAACCTCAACACCC CTGCTCGTCCAGTTTCCGAAGC Hmbs c.331G> A TGCTTCCTCCTGGCTTCACCATC GCAAGACTCTTACTTGCAGATGGCTC KCNQ1 386 + 1G> A TGCTTCGTTTACCACTTCGCCGT TCGCCGGTGGCGATACTCA PRNP c.598G> A CACCAAGGGGGAGAGACTTCACC CGCTCCATCATCTTAACGTCGGTCT SLC26A2 c.835C> T CAAATATCTTCTTGGGCTCAACCTTCCT ATGAGTGAGCCCACACCATTAGTCC Flg c.2282 del4 GGGACATTCAGAAGACTCAGACACACAGT AGCCTGTCTATGGCCTGACACTG PKHD1 p.107C> T AAGAAGGTAGCCTTGCAGGGGGGAA CCTACCATCAAAAATGACTGTGATCCAC LMNA c.1445G> A GAATGTAGATGATCCCTTGCTGACTTACC TCAGGGTGAACTTTGGTGGGAAC LMNA c.1445G> T GAATGTAGATGATCCCTTGCTGACTTACC TCAGGGTGAACTTTGGTGGGAAC LMNA c. 1751G> A GCTGAGTACAACCTGCGCTCGC AGGTCCCGCACAGCACGGTG

AGT c.803T> C TGGAAGACTGGCTGCTCCCTGA GGTGCTGTCCACACTGGCTCCC Fgb c.-463G> A GATAAACACATGATGATATAACATTACTATTGATTTTAAT ACAATGACATAATTCTATTTCAAAAGGGGC Fgb c.-156C> T AGACCAACAAAGAATAATAGTTGTATGACAAGTAAATAAG TCATTTAAGCAACATCTTCCCAGCAAA F2 g.25313G> A TATGGTTCCCAATAAAAGTGACTCTCAGC GAATAGCACTGGGAGCATTGAGGCT F5 c.1601G> A TGTAAGAGCAGATCCCCGGACAGGC TTACTTCAAGGACAAAAACACCTGTATTCCT Mthfr c.665C> T TTGAAGGAGAAGGTGTCTGCGGGAG CAAAGAAAAGCTGAGTGATGATGAAATCG Mtr c.2756A> G AAATCATGGAAGAATATGAAGATATTAGACAGG CTACCACTTACCTTGAGAGACTCATAATGG CALCR c. 1442T> C CCAATTTACATCTGCCATCAGGAGC TTGGTTGTTGGCTGGTTCATTCCTC Gstp1 c.313A> G GGAGGACCTCCGCTGCAAATAC TCACATAGTTGGTGTAGATGAGGGAGA NAT-2 c.341T> C TCACCTTCTCCTGCAGGTGACCA CGACAATGTAATTCCTGCCGTCA NAT-2 c.481C> T TGCTTGACAGAAGAGAGAGGAATCTGGTAC ACTGCTCTCTCCTGATTTGGTCCA NAT-2 c.590G> A CACCAAAAAATATACTTATTTACGCTTGAACCTC AGGTATGTATTCATAGACTCAAAATCTTCAATTGTT CYP2C9 c.817delA GATTGCTTCCTGATGAAAATGGAGA GCGGTCACATAACTAAGCTTTTGTTTACATTTTACCT

CYP4F2 c. 1297G> A CGGAACCCATCACAACCCAGCT ACCTCAGGGTCCGGCCACA Ggcx c. 2084 + 45G> C TTGTCATTGACATCATATGTTGGCAA CTCTCCCCAGGGGAAAGTTACCAAG NOD2 c.2722G> C GACTCTTTTGGCCTTTTCAGATTCTGG CCCTCGTCACCCACTCTGTTGC NOD2 c.3019_3020insC AAGCCCTCCTGCAGGCCC GATGGTGTCATTGCTTTCAAGGG DLG5 c.419G> A CACCACCCCTCCTCACTGACC GGTTCTCCACCTTCTCATTCACTTGC ALDH2 c.1510G> A GAGTACGGGCTGCTGGCATACACT AGGTCCCACACTCACAGTTTTCACTT ADH1B c.143A> G TCTGTAGATGGTGGCTGTAGGAATCTGTC TGCCACTAACCACGTGGTCATCTGTG OPRM1 c.118A> G GGGTCAACTTGTCCCACTTAGATGGC GATCGCATGGGTCGGACAGGT ANKK1 c.2137G> A ACAGCCATCCTCAAAGTGCTGGTC GTCCAGCTGGGCGCCTGCCT Hfe c.187C> G TGACCAGCTGTTCGTGTTCTATGAT CGGGGCTCCACACGGCGACTCTCAT Hfe c.845G> A CCTGGGGAAGAGCAGAGATATACGT ATCTAGGCCTGGGTGCTCCACCTGG CSTB (STFB) GCGACGAGGACTTCGTACACCTG GTTTTCATGATGGAGAGATTGGAACACTC FGFR2 c.1025G> A TTTGAGGACGCTGGGGAATATACGT TATCCCAATAGAATTACCCGCCAAG FGFR2 c.758C> G CCTCTCTCCACCAGAGAGATCGC CTTGGAGGATGGGCCGGTGA BTD c.98_104delGCGGCTGinsTCC AGCCAAAAGTAAGCTTGCTCTTTTTCCTCT CTCCCAGGGCAACCACGTAA

BTD c.1612C> T GTGACGGCGGCTCTCTATGGG CACACTTTTCCTAGTCCCTCTCATACAAGC BTD c.511G> A CATCAGGGGAGATATGTTCTTGGTG CAAGGCTCCTTTGTCTCAAGATTGG PPT1 c.364A> T AATGGCATCTCTGAGCCACTGCCC CGGAGCTAAAACTCTCTTTTATTCCGTAGG Dysf c.855 + 1delG TGTTTGATGAGCCCATCTTTATCACG CGGAGAACACTTTGACTGCTGAGACATAC Dysf c. 937 + 1G> A TGTGCACCATTTACAGAGAGCCCC TACGGCCAAAGTGGAGAGAACTCA Dysf c.1566C> G TCCCCACTTTTGGGCCCTGCTA CTCTGGGACTGCCATAGAGGTTGAT Dysf c.3373delG CGGCGTGATGGATGACAAGAGT AAGGTGGAGACGGACATGGAATCTT Dysf c.4872_4876delinsCCCC AACTACATCCCCTGCACGCTGGA CCAAGATGCCCCAATTTACTTTCCAAATA Dysf c.5979dupA TGGAAATGACCTTGGAGATTGTAGCA CCGCTCCTCATGCTCACTCTCT Dysf c.6124C> T CATGAAGTTCATCCTGTGGCGG AGGATGATGGCCCACCGGAAAC Dysf c.200_201delinsAT TGGACCAGGGCTCTGAGCTTCATG CCCATCTTCTCATGGTCTTTGACCAC CFTR c.276G> C ATTTCTCTGTTTTTCCCCTTTTTGTAGGA GTAAGAGAGGCTGTACTGCTTTGGTGAC CSTB (STFB) c.del313-314 GACCTATTTCTGATCCTGACTTTGGACA TTTGTCAGTCTTCTGGCTGAAGGGC CCR5 Delta_32 TACACCTTCAGCTCTCATTTTCCATACA CCAGCCCCAAGATGACTATCTTTAATGT

CCR5 p.303T> A CCCTGTGGGACTTTGGAAATACAATGTG AAGCCTATAAAATAGAGCCCTGTCAAGAGTTG CCR5 c.-22459A> G ATACGGGGAGAGTGGAGAAAAAGGG ATCCTGGACTTCAGATTAACCCTGTGT SRY1 (sY14) DEL AAGATGGCTCTAGAGAATCCCAGAATG CCAGCTGCTTGCTGATCTCTGAGTTTC AZFc DEL TTTGACCCATAGGTACTAAAAATATCTTTGACA CCCGAATGACCAGCAGCCCT AZF a DEL CTTAATCTGCACGAAACATGGGCTG ACACTAAAGAAAGGGTCCCACTGAATCT AZFb DEL CTGGTGAAGCTAGTGAAGAAAGTTTATTTTCAG TTCCGTTGTTAGAGGCATATTTAAATTTTTAGAG AZFb DEL CAGTGAAACTGCAGCTGTCAAATGA TCCCACCTCGTGTCTCTTATCAATTC AZFb DEL CCAAATATCAGATCTGTGAGCCTTCCTAA GGCTCTGCCTGTAGCATTTTCCTCTG AZF a DEL AAAATTATCTGGGCATCAGCATGTGG TTCATTTGGGCTAAAAACTAGTCCTAATGC AZFc DEL TTCGCCTTCCGACGAGGACGATACT ACCTCTTAAGCTAGGAGCTCTAACCAATTAC AZFc DEL ACTTTTTACCATTTTTATACACTTTTATATCACAAATC TACCGTTATAACAGGTAATTAAAACTGCTAAACAT AZF DEL TCGGCTTCAGTAAGCATTTTCCACT AGAGATCAGCAAGCAGCTGGGATAC AZFc DEL TTCTCTGTCTTACGGTGGAAGACAGAAC GGAATTAAAATTAGACTTTTGTCTACACTACACTG

AZFc DEL CTTGGTTCCCCCGCTTGCTGT TTTGACCAAATTTTTCTAACCAACGTAGAG

Table 3 Diagnostic kit. No. Name one APEX Matrix Preparation Kit: 1.1. Hot Start DNA Polymerase (10 U / μl) 1.2. 10X Buffer for PCR reaction with (NH ₄ ) ₂ SO ₄ 1.3. 25 mM MgCl ₂ 1.4. 2.5 mM dNTP mixture (20% dUTP) 1.5. Uracil-N-Glycosylase (UNG) (1 unit / μl) 1.6. 10x UNG Reaction Buffer 1.7. Shrimp alkaline phosphatase (sAP) 1 u / μl 2 APEX Reaction Kit 2.1. APEX Reaction Mixture 2.2. Antifade 3 Set for cleaning PCR products 3.1. Cleaning columns and collection tubes 3.2. Binding buffer 3.3. Wash buffer 3.4. Elution buffer four DNA chips for the diagnosis of hereditary diseases, including cystic fibrosis 5 Cover slip “Lifter Slips” 6 Alcanox Powder 7 Cover glass 8 Primers for the reaction of the NDP multiplex

Claims (4)

1. A set of oligonucleotide probes containing DNA regions shown in Table 2 for the diagnosis of a population of people living in the Russian Federation for hereditary monogenic diseases by detecting mutations and / or polymorphisms selected in such a way that:
(a) at least 10% of mutations and / or polymorphisms have a frequency of occurrence in the selected ethnic group and / or population of people that exceeds the global incidence by at least two times;
(b) at least 5% of mutations and / or polymorphisms do not occur in ethnic groups and / or populations of people other than the one selected;
(c) no more than 10% of mutations and / or polymorphisms have a frequency of occurrence that is more than twice the frequency of occurrence in a selected ethnic group and / or population;
(d) no more than 5% of mutations and / or polymorphisms do not occur in the selected ethnic group and / or human population;
(e) the remaining mutations and / or polymorphisms have a frequency of occurrence in the selected ethnic group and / or human population equal to the global frequency of occurrence or not exceeding it more than twice. 2. A method of obtaining a DNA microarray for the diagnosis of a population of people living in the territory of the Russian Federation for hereditary monogenic diseases by detecting mutations and / or polymorphisms, including:
I) obtaining oligonucleotide probes containing DNA regions shown in table 2, allowing the detection of mutations and / or polymorphisms from a list including:
(a) at least 10% mutations and / or polymorphisms with a frequency of occurrence in a selected ethnic group and / or population of people that exceeds the global incidence by at least two times;
(b) at least 5% mutations and / or polymorphisms not found in ethnic groups and / or populations of people other than the one selected;
(c) not more than 10% of mutations and / or polymorphisms with a frequency of occurrence more than twice the frequency of occurrence in a selected ethnic group and / or population;
(d) no more than 5% mutations and / or polymorphisms not found in the selected ethnic group and / or human population;
(e) other mutations and / or polymorphisms with a frequency of occurrence in the selected ethnic group and / or population of people equal to the global frequency of occurrence or not exceeding it more than twice;
II) applying oligonucleotide probes to a glass plate coated with a nucleic acid adhesive, with one side of the plate being processed in such a way that the laser beam propagates in it due to the effect of total internal reflection. 3. A set for the study of the population of people living in the territory of the Russian Federation on hereditary monogenic diseases by detecting mutations and / or polymorphisms, including:
a) a kit for preparing a primer elongation reaction matrix on a DNA microarray
(APEX);
b) primers for the PCR reaction are multiplexed;
c) a kit for cleaning PCR products;
d) an APEX reaction kit according to claim 1;
e) DNA microarray obtained by the method according to claim 2. 4. A method for detecting mutations and / or polymorphisms associated with hereditary monogenic diseases in a population of people living in the territory of the Russian Federation, comprising:
1) obtaining human genomic DNA, PCR,
2) purification and fragmentation of PCR products,
3) conducting the APEX reaction on the DNA microchip, characterized in paragraph 2,
4) detection of mutations and / or polymorphisms by comparing the intensity of the fluorescence signals, when the DNA microchip is irradiated with lasers of different wavelengths.