RU2819985C1 - METHOD FOR DIAGNOSING VARIANT SOPH c.5741G>A IN NBAS GENE - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to clinical laboratory diagnostics, and can be used for diagnosis of carriage of mutant gene NBAS with mutation c.5741G>A, leading to development of SOPH-syndrome with autosomal recessive type of inheritance in Yakut population. DNA is recovered. Polymerase chain reaction with fluorescent detection is carried out. Presence of a fluorescence signal on the R6G channel is used to diagnose mutant type c.5741A in the NBAS gene. ROX channel is used to diagnose wild type c.5741G in the NBAS gene. Combined fluorescence of the ROX and R6G channels is used to diagnose the carriage of the mutant gene in a heterozygous state.

EFFECT: method provides simpler and higher accuracy of determining mutation c.5741G>A in 45 exon of the NBAS gene, high sensitivity and specificity at low cost of analysis due to use of a pair of specific primers (SEQ ID NO 1 and 2) and hydrolysis probes (SEQ ID NO 3 and 4).

1 cl, 2 dwg, 1 tbl

Description

The invention relates to the field of medicine and molecular biology and can be used to diagnose a hereditary disease, namely SOPH syndrome (SOPH) in the NBAS gene.

SOPH syndrome, first described in 2010, was considered common only in the Yakut population, however, every year, according to the literature, new cases of SOPH-like conditions are discovered throughout the world. The frequency of heterozygous carriage of a pathogenic mutation was 1300 cases per 100,000 people. conditionally healthy Yakuts, while in the Republican Genetic Register of Hereditary and Congenital Pathology of Yakutia there are more than 93 detected cases of SOPH, which is a frequency of more than 18 patients per 100,000 people. Yakut population (see Zhozhikov L., Sukhomyasova A., Gurinova E. et al. Origins of SOPH syndrome: A study of 93 Yakut patients with review of C-terminal phenotype // Clinical Genetics. 2023. V. 103. No. 6 . pp. 625–635. https://doi.org/10.1111/cge.14319).

In addition, the effect of the founder of this variant was identified in Yakutia, whose age was 804 ± 140 years. Taking into account the date of the study and the sample, these data indicate the beginning of the spread of the G5741 → A mutation in the Yakut population in the first half of the 12th century. (1192nd year ±140 years), which in the history of Yakutia corresponds to the heyday of the Kulun-Atakh archaeological culture. The carriers of this culture were adapted to local conditions and led a nomadic lifestyle in the middle Lena basin (see Fedorova S.A., Khusnutdinova E.K. Genetic Structure and Genetic History of the Sakha (Yakuts) Population // Russ J Genet. 2022. V. 58. No. 12. P. 1409–1426. https://doi.org/10.1134/S1022795422120031).

It is known that SOPH syndrome is associated with the homozygous variant c.5741G>A in the NBAS (neuroblastoma amplified sequence) gene, the locus of which is located on the short arm of the second chromosome. The variant results in the replacement of guanine by adenine in exon 45, which leads to the replacement of arginine by histidine (p. R1914H) in the structure of the NBAS protein. The region where this mutation occurs is localized on that half of the protein that binds the NBAS protein with the RINT1 and ZW10 proteins, which are part of the Syntaxin 18 complex, which, in turn, plays a role in the fusion of cell organelle membranes and transport between the endoplasmic reticulum and the apparatus Golgi. The NBAS gene consists of 52 exons and contains 420 kb. Gene disruptions modulate the expression of 1444 human genes, corresponding to almost 10% of all expressed genes. Some of them are associated with embryonic development and cholesterol biosynthesis (see Zhozhikov LR, Vasilev FF, Maksimova NR The Function of the NBAS Has Been Revealed: What about Its Multisystem Pathologies? // Russ J Genet. 2023. V. 59. No. 4 . P. 317–324. https://doi.org/10.1134/S1022795423040117).

Currently, there are known methods for determining polymorphic variants and mutations, for example, restriction fragment length polymorphism analysis (RFLP), allele-specific PCR, PCR with analysis of high-resolution melting curves (PCR-HRM, High Resolution Melt), direct sequencing, massively parallel sequencing, hybridization on oligonucleotide templates, real-time PCR with fluorescent detection.

At the same time, methods of direct sequencing and massively parallel sequencing are characterized by high cost, complexity and limited use in clinical practice.

The method of analyzing PCR-RFLP restriction fragment length polymorphism takes place in two stages: first, PCR itself is performed and the formation of an amplifier, that is, copies of the DNA section required in the study, then, using a specific restriction enzyme, the desired section responsible for the disease is cut off. After that, the number of bands on the gel is analyzed for the purpose of subsequent diagnostics: the presence of two or more bands (the number depends on the design of the study) indicates a trimmed fragment, while another band, usually the one that is longer, indicates its absence.

According to the PCR method with analysis of melting curves with high resolution (PCR-HRM, High Resolution Melt), a DNA section is first amplified, and then, when the number of copies of the amplification is high enough, the temperature is gradually and slowly increased with luminescence detection. The glow occurs when two DNA strands separate from each other. Diagnosis is based on the temperature at which this separation occurs. If the glow occurs at a relatively low temperature, this indicates weak coupling between the chains. Conversely, when bonds are broken at high temperature, it indicates strong bonding, which shows complementary bonding.

The disadvantages of the known method are low specificity and the need for certain mutations such as large deletions and missense variants, which limits diagnostic capabilities, especially in the case of hereditary diseases, such as SOPH syndrome, in which PCR-HRM does not cause a significant shift in the annealing temperature two strands of DNA.

Real-time PCR using fluorescent probes is based on the same principle as conventional PCR, but with real-time fluorescence detection during the amplification process. Luminescence occurs when the probe molecules, consisting of a fluorophore and a quencher, connected to an oligonucleotide, dissociate. Each fluorophore has its own wavelength, which ensures diagnostic specificity: one fluorophore corresponds to one probe, and their attachment to the matrix depends on complementarity with it. This method was chosen by the co-authors for experimental work.

The well-known method involves the development of primers, that is, oligonucleotides of a certain sequence corresponding to the desired region where the mutation is located, as well as the development of probes for detecting two alleles - diseased and healthy. For each of these alleles, a probe is selected, and for each probe a quencher is selected, after which the required temperature regime for such a set of test systems is determined, that is, the temperature at which the DNA chains will dissociate and anneal again, giving luminescence and amplification. In addition, DNA samples are selected for control tests, including DNA from diseased carriers and healthy people.

When analyzing the state of the art, inventions were also identified using PCR techniques with fluorescent detection for diagnosis of carriage (determination of genotype) of other hereditary and multifactorial diseases, for example, see RU No. 2304170 (cl. C12Q1/68, published 08/10/2007), RU No. 2534817 (cl. C12N15/11, published 12/10/2014), RU No. 2505608 (cl. C12Q1/68, G01N33/50, published 01/27/2014), RU No. 2556808 (cl. C12Q1/68, published 20.07 .2015), RU No. 2518301 (cl. C12Q1/68, published 06/10/2014), RU No. 2675324 (cl. G01N33/50, C12Q1/68, published 12/18/2018), RU No. 2739943 (cl. C12Q1/ 68, published 12/30/2020), RU No. 2445368 (cl. C12Q1/68, published 03/20/2012), RU No. 2631824 (cl. C12Q1/68, published 09.26.2017), RU No. 2451086 (cl. C12Q1) /68, published 05.20.2012). At the same time, no direct analogues to the method of diagnosing SOPH using the PCR method have been identified from the state of the art.

The objective of the claimed invention is to develop an optimal method for determining a person’s genotype based on the c.5741G>A mutation in exon 45 of the NBAS gene, responsible for the development of SOPH syndrome.

The technical result of the invention is to simplify the method and increase the accuracy of determining the c.5741G>A mutation in exon 45 of the NBAS gene, ensuring high sensitivity and specificity at low cost of the study, as well as increasing the availability of such studies, since the claimed solution can be carried out on standard well-known equipment.

To solve this problem, a method for diagnosing carriage of the mutant NBAS gene with the c.5741G>A mutation, leading to the development of SOPH syndrome with an autosomal recessive type of inheritance in the Yakut population, includes DNA isolation and polymerase chain reaction with fluorescent detection using a pair of primers AATGCAGGGTTCTTTGGTCTCA ( SEQ ID NO 1), TGAGCTTCGTCTTCTGAGTTTCTT (SEQ ID NO 2) and hydrolysis probes ROX-CTGTCTGTGGAAGCCCGT-BHQ2 (SEQ ID NO 3), R6G-GCTGTCTGTGGAAGCCCATA-BHQ1 (SEQ ID NO 4), where the presence of a fluorescence signal through the R6G channel is used to diagnose a mutant type c.5741A in the NBAS gene, according to the ROX channel - wild type c.5741G in the NBAS gene, according to the joint luminescence of the ROX and R6G channels - carriage of the mutant gene in a heterozygous state.

A comparative analysis of the features of the claimed solution with the features of analogues indicates that the claimed solution meets the “novelty” criterion.

The set of features of the invention provides a solution to the stated technical problem, namely, obtaining a molecular genetic method for diagnosing the hereditary disease SOPH syndrome, including the selection of specific original oligonucleotide sequences (primers), allele-specific hydrolysis probes and optimization of conditions for real-time PCR with fluorescent detection .

The claimed technical solution is illustrated in the drawing, where in FIG. Figure 1 shows the results of amplification on a 3% agarose gel, with the length of the amplification fragments being 140 nucleotide pairs (bp) (M – pUC19/MspI marker; 1, 2 – SOPH patients, homozygotes for the c.5741G>A mutation in the gene NBAS; 3, 4 – parents of patients with SOPH, heterozygous carriers of the c.5741G>A mutation in the gene NBAS; 5 – conditionally healthy individual, carrier of the wild type allele (allele c.5741G in the gene NBAS); 0 – deionized water); in fig. 2 is an example of interpretation of the results obtained using the developed primers and probes for real-time PCR, in the form of a graph of the distribution of genotypes at the locus under study (samples with fluorescence by the R6G probe of more than 800 RFU and fluorescence by the ROX probe of less than 200 RFU - patients with SOPH, homozygotes for the c.5741G>A mutation in the gene NBAS; samples with approximately the same luminescence for both probes are heterozygous carriers of the c.5741G>A mutation in the gene NBAS; samples with fluorescence using the R6G probe of less than 200 RFU and fluorescence of more than 600 RFU using the ROX probe are conditionally healthy individuals, carriers of the wild type allele (allele c.5741G in the gene NBAS); sample without glow – deionized water).

In the claimed technical solution, which includes DNA extraction from peripheral blood lymphocytes, amplification is carried out in a mixture of Taq polymerase, which has 5'-exonuclease activity, and two pairs of oligonucleotide sequences (primers and hydrolysis probes) labeled with various fluorescent dyes. The fluorescent dyes ROX and R6G are attached to nucleotide C at the 5' ends of the hydrolysis probes. The fluorescence quenchers BHQ2 and BHQ1 are attached to the 3'-terminal nucleotide T. In the experimental work, the nucleotide sequences of the hydrolysis probes were selected so that they hybridized with the internal region of the amplification product in the region where the target mutation c.5741G>A in the NBAS gene is located ( see table). In this case, the probe with the fluorescent dye/quencher pair ROX/BHQ2 was complementary to the wild type (allele c.5741G), and R6G/BHQ1 was complementary to the mutant (allele c.5741A). If the probe does not hybridize during the amplification process, then a resonance energy transfer occurs from the excited fluorophore at the 5' end of the probe to the quencher located at its 3' end, as a result of which there will be no luminescence. If the probe is completely complementary to the DNA section, it will hybridize to form a duplex. This duplex will then be cleaved by Taq polymerase, as a result of which the fluorophore and quencher molecules will move away from each other. As a result, an increase in the level of fluorescence of the dye will be observed. Thus, measuring the fluorescence intensity at wavelengths corresponding to the R6G and ROX fluorophores will allow us to determine the genotype of the sample under study.

The claimed method is carried out as follows.

Genomic DNA was isolated from biological material. It is recommended to use venous peripheral blood as a material for research. DNA was isolated from biological material using the standard phenol-chloroform extraction method or using commercial reagent kits. The concentration of the isolated DNA samples was measured by spectrophotometry. Then the DNA samples under study were diluted with deionized water to a concentration of 15 ng/μl.

Before amplification, the PCR mixture, primers, probes and DNA samples were completely thawed, thoroughly mixed by vortex, followed by centrifugation.

The amplification reaction was carried out on a programmable thermal cycler with an optical fluorescence detection system in a volume of 10 μl of the reaction mixture of the following composition: 1 μl of a mixture of forward and reverse primers (10 pmol/μl of each primer), 0.5 μl of each probe (10 pmol/μl), 5 µl 2.5x PCR buffer (KCl, Tris-HCl pH8.8, 6.25 mM MgCl ₂ , Taq DNA polymerase 5 units/µl, 2.5 mM dNTP), 2 µl deionized water, 1 µl genomic DNA concentration 15 ng/µl. In fig. Figure 1 shows the results of amplification in an agarose gel.

The real-time PCR reaction was performed under the following conditions: initial denaturation at 95°C for 5 minutes, followed by 40 amplification cycles, each consisting of a denaturation step at 95°C for 15 seconds and a combined annealing-extension step at 60°C for within 50 seconds.

After PCR, the results were analyzed using thermal cycler software with the ability to perform real-time PCR (“allelic discrimination”). If there was a fluorescence signal through the R6G channel, the sample was diagnosed as containing the c.5741G>A mutation in the NBAS gene. A sample positive for the ROX fluorophore was interpreted as normal (wild type). In the presence of two fluorescence signals R6G and ROX, the result was interpreted as a heterozygous state/carrier of the c.5741G>A mutation in the NBAS gene (see Fig. 2).

The developed method for diagnosing a hereditary disease was tested by testing genomic DNA samples from 10 SOPH patients, 10 relatives of SOPH patients and 10 apparently healthy individuals living in the Republic of Sakha (Yakutia). Molecular genetic studies of carriage of the c.5741G>A mutation in the gene NBAS These samples were studied four times. Earlier diagnosis of SOPH patients and their relatives (carriers of the c.5741G>A mutation in the gene NBAS) was carried out using the direct Sanger sequencing method. A molecular study using the developed method showed that all SOPH patients were homozygous for the c.5741G>A mutation in the gene NBAS, and their relatives (parents) were heterozygous carriers of this pathogenic variant. All controls were carriers of the wild type allele (allele c.5741G in the gene NBAS).

Thus, the claimed method for diagnosing SOPH syndrome makes it possible to make an accurate diagnosis, conduct prenatal DNA diagnostics and prospective medical and genetic counseling, for example, for those getting married. The technical solution is characterized by ease of implementation, accuracy and accessibility for implementation in a molecular genetic laboratory.

The list of oligonucleotides was prepared according to Standard ST.26 using WIPO Sequence software “Recommended Standard for the Presentation of Lists of Nucleotide and Amino Acid Sequences Using XML (Extensible Markup Language)”.

Table

Oligonucleotide primers and probes for detection of the c.5741G>A mutation in the NBAS gene

Primer/Probe Sequence (5’-3’) Primer (direct) AATGCAGGGTTCTTTGGTCTCA Primer (reverse) TGAGCTTCGTCTTCTGAGTTTCTT Probe (wild type) ROX-CTGTCTGTGGAAGCCCGT-BHQ2 Probe (mutant type) R6G-GCTGTCTGTGGAAGCCCATA-BHQ1

Claims (1)

A method for diagnosing carriage of the mutant NBAS gene with the c.5741G>A mutation, leading to the development of SOPH syndrome with an autosomal recessive mode of inheritance in the Yakut population, including DNA isolation and polymerase chain reaction with fluorescent detection using a pair of primers AATGCAGGGTTCTTTGGTCTCA (SEQ ID NO 1 ), TGAGCTTCGTCTTCTGAGTTTCTT (SEQ ID NO 2) and hydrolysis probes ROX-CTGTCTGTGGAAGCCCGT-BHQ2 (SEQ ID NO 3), R6G-GCTGTCTGTGGAAGCCCATA-BHQ1 (SEQ ID NO 4), where the presence of a fluorescence signal through the R6G channel is used to diagnose the c.5741A mutant type in the NBAS gene, in the ROX channel – wild type c.5741G in the NBAS gene, in the joint luminescence of the ROX and R6G channels – carriage of the mutant gene in a heterozygous state.