RU2524433C1 - Set of sequences of oligonucleotides for diagnostics of germinal mutations in gene ret, associated with genetic predisposition to cancer of thyroid - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to molecular biology and oncology and may be used for diagnostics of terminal mutations in gene RET, associated with genetic predisposition to cancer of thyroid (CT). The set of sequences of oligonucleotides has the following nucleotide composition: 5'-CATGGCCACTCCCAGTGC-3', 5'-CACAGGGACTCCTCAGCAC-3', 5'-CACCCCCACCCACAG-3',5'-GAGATGGGTGGCTTGTG-3', 5'-GGCTAGTGCTGTCAGGCC-3', 5'-CTAAGCACCCTAGACGCG-3', 5'-CAGGGATAGGGCCTGG-3', 5'-CCCCAAGAGAGCAACACC-3', TACGAGCCGTGCCT, GATGGGCTGCGCAG, CACGTGCTGGGCCT, TAGAAGCTGTACAT, CACGAGAAGTGGAG, TACGAGCTGAGCTG, GGAAATGGATGGGATTTG, CCATATGGACGGCAATTC.

EFFECT: invention enables to carry out efficient diagnostics of germinal mutations in gene RET, associated with genetic predisposition to cancer of thyroid, based on the frequency of mutations among patients in the Russian population.

4 tbl, 2 ex

Description

The invention relates to medicine, namely molecular biology and oncology.

Known nucleotide sequence for the detection of mutations of exon 11 of the RET gene (US Patent No. 6171791 B1).

Disadvantages: the small length of the studied DNA (only 11 exons from 1810 to 1845 positions), the lack of an accurate determination of the risks of thyroid cancer (thyroid cancer), the complexity of the study and the high cost of research (sequencing).

A known invention is dedicated to the detection of RET / PTC oncogenes in case of papillary thyroid cancer (Ukrainian Patent No. 29847 U).

Disadvantages: the complexity of the process (RNA isolation using the reverse transcription and amplification method), the study of somatic mutations, rather than germline mutations, which does not allow assessing the risk of developing the disease, studying only papillary cancer.

The objective of the invention is the creation of an optimal set of oligonucleotide sequences for the analysis of germline mutations in the RET gene associated with a hereditary predisposition to thyroid cancer, based on mutation frequencies among patients in the Russian population.

The problem is solved by creating an optimal set of oligonucleotide sequences for the detection of terminal germline mutations C634 in exon 11 and M918T in exon 19 of the RET gene using multiplex "nested" polymerase chain reaction (PCR) and allele-specific hybridization with a set of immobilized differentiating oligonucleotides.

The technical result of the claimed invention is the creation of an optimal set of oligonucleotide sequences for the diagnosis of germline mutations in the RET gene associated with a hereditary predisposition to thyroid cancer based on mutation frequencies among patients in the Russian population.

The technical result is achieved by selecting the optimal set of oligonucleotide sequences for multiplex "nested" PCR and hybridization, which are used to diagnose germline mutations in the RET gene associated with a hereditary predisposition to thyroid cancer in patients in the Russian population with the following nucleotide composition:

5'-CATGGCCACTCCCAGTGC-3 '

5'-CACAGGGACTCCTCAGCAC-3 '

5'-CACCCCCACCCACACAG-3 '

5'-GAGATGGGTGGCTTGTG-3 '

5'-GGCTAGTGCTGTCAGGCC-3 '

5'-CTAAGCACCCTAGACGCG-3 '

5'-CAGGGATAGGGCCTGG-3 '

5'-UDPAAGAGAGCAACACC-3 '

TACGAGCCGTGCCT

GATGGGCTGCGCAG

CACGTGCTGGGCCT

TAGAAGCTGTACAT

CACGAGAAGTGGAG

TACGAGCTGAGCTG

GGAAATGGATGGGATTTG

CCATATGGACGGCAATTC

Selection of oligonucleotide sequences includes: synthesis of oligonucleotides of SEQ ID NO: 9-16, strictly complementary to the target DNA sequence and corresponding to the temperature conditions of hybridization; amplification of RET gene fragments using multiplex “nested” PCR in stage I, in which the DNA sample is used as a matrix, a set of oligonucleotides with the sequences shown in SEQ ID NO: 1-8, with the formation of a biotype-labeled PCR product in stage II PCR; the presence of a set of immobilized oligonucleotides with the sequences shown in SEQ ID NO: 9-16; hybridization of the labeled PCR product with a set of immobilized oligonucleotides.

To conduct molecular genetic studies, the biological material was genomic DNA isolated from peripheral blood lymphocytes.

Amplification oligonucleotides and oligonucleotide probes were synthesized using phosphodiester and hydrophosphoryl methods. The synthesis of oligonucleotides was carried out using an automatic DNA / RNA synthesizer.

Oligonucleotides for hybridization of alleles of the RET gene associated with a hereditary predisposition to thyroid cancer were selected in such a way as to identify all the point mutations selected for analysis

To determine the point mutations of the RET gene, 8 highly specific differentiating oligonucleotide probes were selected and synthesized, the structure of which ensured binding only to fully complementary target DNAs.

PCR was performed using thermostable Taq polymerase in the appropriate buffer on a Dyad instrument. PCR mixture volume of 25 Phase I l included: 1 × PCR buffer (Tris-HCl 67 mM, pH 8,6, (NH _{4) 2} SO ₄ 166 mM, Triton X-100 0,01%), MgCl ₂ 1.5 mM, 0.2 mM each of dNTP, Taq polymerase 2.5 U, 0.2 μM oligonucleotides.

Amplification included: denaturation of double-stranded DNA at t = 94 ° C for 3 min 30 sec, then 35 cycles of the polymerase chain reaction: at t = 94 ° C for 30 sec, at t = 60 ° C for 20 sec, at t = 72 ° C for 10 sec, then elongation at t = 72 ° C for 3 min. The mixture of stage II PCR contained 0.2 μM forward primer, 2 μM reverse biotype-labeled primer. As a matrix, 2 μl of the PCR stage I product was added to the mixture and amplification was performed, including: denaturation at t = 94 ° C for 3 min 30 sec, then 35 PCR cycles: at t = 94 ° C for 30 sec, at t = 61 ° C for 20 sec, at t = 72 ° C for 10 sec, elongation at t = 72 ° C for 3 min A single-stranded amplicon capable of hybridization with allele-specific DNA probes was obtained. Amplification of each DNA sample was carried out in a separate tube. The oligonucleotides used for immobilization carry at the 5'- or 3'-end an active group that provides immobilization.

A set of differentiating oligonucleotides was applied to the surface of a membrane containing active groups.

In the multiplex "nested" reaction of stage I PCR, oligonucleotides SEQ ID NO: 1, 2, 5, 6 were used.

In the multiplex "nested" reaction of the second stage of PCR, oligonucleotides SEQ ID NO: 3, 4, 7, 8 were used.

Table 1 presents the sequence of oligonucleotides for the multiplex "nested" reaction of I and II stages of PCR.

Table 1 DNA locus Specific sequence number Title 5'-3 'sequence Length, nucleotide pairs Oligonucleotide Annealing Temperature S634 one 634_F1 5'-CATGGCCACTCCCAGTGC-3 ' eighteen 63 2 634_R1 5'-CACAGGGACTCCTCAGCAC-3 ' eighteen 63 3 634_F2 5'-CACCCCCACCCACACAG-3 ' eighteen 62 four 634_R2 5'-GAGATGGGTGGCTTGTG-3 ' eighteen 63 M918T 5 918_F1 5'-GGCTAGTGCTGTCAGGCC-3 ' eighteen 62 6 918_R1 5'-CTAAGCACCCTAGACGCG-3 ' eighteen 62 7 918_F2 5'-CAGGGATAGGGCCTGG-3 ' 16 60 8 918_R2 5'-UDPAAGAGAGCAACACC-3 ' eighteen 60

Table 2 presents the sequence of oligonucleotide probes used for hybridization.

table 2 Specific sequence number Mutation Probe name Sequence 5'-3 ' 9 C634WT c634_wt TACGAGCCGTGCCT 10 C634R c634_r GATGGGCTGCGCAG eleven C634g c634_g CACGTGCTGGGCCT 12 C634Y c634_y TAGAAGCTGTACAT 13 C634W c634_w CACGAGAAGTGGAG fourteen C634S c634_s TACGAGCTGAGCTG fifteen M918WT m918_wt GGAAATGGATGGGATTTG 16 M918T m918_t CCATATGGACGGCAATTC

Table 3 presents the oligonucleotides of SEQ ID NO for "nested" PCR: 1-4 for the C634 DNA locus and 4-8 for the M918T DNA locus.

Table 3 S634 one 634F1 5'-CATGGCCACTCCCAGTGC-3 ' 2 634R1 5'-CACAGGGACTCCTCAGCAC-3 ' 3 634F2 5'-CACCCCCACCCACACAG-3 ' four 634R2 5'-GAGATGGGTGGCTTGTG-3 ' M918T 5 918F1 5'-GGCTAGTGCTGTCAGGCC-3 ' 6 918R1 5'-CTAAGCACCCTAGACGCG-3 ' 7 918F2 5'-CAGGGATAGGGCCTGG-3 ' 8 918R2 5'-UDPAAGAGAGCAACACC-3 '

Table 4 presents the sequence of immobilized oligonucleotides: 9-16 for hybridization.

Table 4 9 TACGAGCCGTGCCT 10 GATGGGCTGCGCAG eleven CACGTGCTGGGCCT 12 TAGAAGCTGTACAT 13 CACGAGAAGTGGAG fourteen TACGAGCTGAGCTG fifteen GGAAATGGATGGGATTTG 16 CCATATGGACGGCAATTC

A hybridization mixture with a total volume of 500 μl included formamide 100 μl, 20 × SSPE (sodium chloride, sodium phosphate, EDTA) 100 μl and amplification 20 μl. The prepared hybridization mixture was denatured at t = 95 ° C for 5. min, cooled in ice for 2 min, then the membrane with immobilized oligonucleotides was incubated in the hybridization mixture for 10-12 hours. After completion of the incubation, washing from the unreacted hybridization mixture in 1 × SSPE buffer at room temperature for 15 min.

Then, nonspecific binding sites were blocked; for this, the membrane was incubated in blocking buffer, which included BSA (bovine serum albumin). Washed the SSC membrane (saline-sodium citrate) with buffer (0.15 M NaCl) twice. After this, incubation was carried out with a conjugate diluted in buffer (streptavidin-alkaline phosphatase). Unbound conjugate was removed from the SSC membrane with buffer. The membrane was incubated with buffer (Tris-HCl 0.1 M pH 7.6, MgCl ₂ 10 mM, ZnCl ₂ 10 mM, Triton X-100 0.1%) for 2 min. After this, the membrane was incubated in a staining buffer containing substrates HCT and BHIF (5-bromo-4-chloro-3-indolylphosphate and nitrosine tetrazolium). To terminate the reaction, the membrane was washed with distilled water.

The alkaline phosphatase enzyme converted a colorless substrate to the final reaction product of a blue-lilac color. A staining reaction was observed at the binding sites of the probe and the complementary sequence.

The presence of one of the mutant alleles (C634WT, C634R, C634G, C634Y, C634W, C634S, M918WT, M918T) in the studied sample was determined taking into account the location of oligonucleotide probes on the membrane. The presence of mutations in the patient’s genotype confirms the genetic diagnosis of a hereditary predisposition to thyroid cancer or determines a high risk of its development during life.

The invention is illustrated by examples 1,2:

Example 1

Patient R., 9 years old

Preliminary diagnosis: suspected syndrome of multiple endocrine neoplasia type 2B (MEN 2B).

Family history: oncological diseases are not burdened.

Study: determination of mutations in the RET proto-oncogene associated with the development of multiple endocrine neoplasia 2B syndrome (MEN 2B).

Study material: DNA isolated from peripheral blood lymphocytes.

Result: a germinal missense mutation of the M918T river was detected (s.2753T> C) in the heterozygous state in the 918 codon 16 of exon of the RET proto-oncogen associated with the MEN2B syndrome. The diagnosed mutation is registered in the international MEN2 Database ARUP [arup.utah.edu].

The final diagnosis: syndrome of multiple endocrine neoplasias type 2B (with m MEN 2B). High risk of bilateral pheochromocytoma, multiple neuromas of the gastrointestinal tract.

Given the results of molecular genetic studies, the patient belongs to the highest risk group (level D) of the development of prognostically unfavorable (aggressive) forms of medullary thyroid cancer, with a 50% risk of developing adrenal pheochromocytoma.

The type of inheritance is autosomal dominant.

Recommended by:

1. Considering the complete penetrance of the RET proto-oncogen, it is advisable for the patient to undergo surgical treatment in the amount of total thyroidectomy with lymphodisection;

2. dynamic observation:

- observation by an oncologist, endocrinologist, geneticist;

- control of the level of calcitonin basal and stimulated in dynamics + ultrasound of the neck;

- control of total and ionized calcium in dynamics;

- control of metanephrine of blood plasma and daily urine + ultrasound of the retroperitoneal space for the timely diagnosis of pheochromocytoma in dynamics;

3. DNA diagnostics for the presence of mutations in the RET gene of relatives of the 1-11 degree of relationship.

Example 2

Patient P., 13 years old

Diagnosis: suspected syndrome of multiple endocrine neoplasia type 2A (MEN 2A).

Family history: burdened with medullary thyroid cancer.

Research: determination of mutations in the RET proto-oncogene.

Study material: DNA isolated from peripheral blood lymphocytes.

Result: a germline missense mutation of p.C634Y was revealed in the heterozygous state in the 634 codon of the RET proto-oncogen. The diagnosed mutation is registered in the international MEN2 Database ARUP [amp.utah.edu] as a clinically significant option.

Conclusion: hereditary RET-associated medullary thyroid cancer as part of the MEN2A syndrome.

Given the results of molecular genetic studies, the patient belongs to the high-risk group (level D) of the development of prognostically unfavorable (aggressive) forms of medullary thyroid cancer with a 50% risk of developing adrenal pheochromocytoma.

Recommended:

1. Considering the complete penetrance of the RET proto-oncogen, it is advisable for the patient to undergo surgical treatment in the amount of total thyroidectomy with lymphodisection;

2. dynamic observation:

- determination of the level of basal and pentagastrin-stimulated calcitonin;

- control of metanephrine of blood plasma and daily urine + ultrasound of the retroperitoneal space for the timely diagnosis of pheochromocytoma in dynamics;

3. DNA diagnostics for the presence of mutations in the RET gene of relatives of the I-II degree of kinship.

Claims (1)

A set of oligonucleotide sequences for the diagnosis of germline mutations in the RET gene associated with a hereditary predisposition to thyroid cancer, having the following nucleotide composition:
5'-CATGGCCACTCCCAGTGC-3 '
5'-CACAGGGACTCCTCAGCAC-3 '
5'-CACCCCCACCCACACAG-3 '
5'-GAGATGGGTGGCTTGTG-3 '
5'-GGCTAGTGCTGTCAGGCC-3 '
5'-CTAAGCACCCTAGACGCG-3 '
5'-CAGGGATAGGGCCTGG-3 '
5'-UDPAAGAGAGCAACACC-3 '
TACGAGCCGTGCCT
GATGGGCTGCGCAG
CACGTGCTGGGCCT
TAGAAGCTGTACAT
CACGAGAAGTGGAG
TACGAGCTGAGCTG
GGAAATGGATGGGATTTG
CCATATGGACGGCAATTC.