A primer combination for detecting human SMN1 and SMN2 gene mutations based on NGS method and its application

Abstract

The present invention discloses a primer composition and reagent for detecting human SMN1 and SMN2 gene mutations based on NGS method. The primer composition can specifically amplify all exons and part of introns or UTR adjacent regions of SMN1 and SMN2 genes in a single tube by multiple PCR method, and the constructed library is then used for NGS sequencing, so as to realize the report of the overall copy number of SMN1 and SMN2 genes of patients and the copy number variation of each of the 7th and 8th exons, and also can report the pathogenic point mutations and short deletion / insertion mutations in other regions of the two genes. The present invention can meet the needs of SMA pathogenic gene screening, genetic risk analysis, disease prognosis assessment, treatment guidance, etc. The overall process is simple and efficient, low cost, and has good prospects.

Description

Technical Field

[0001] The present invention relates to the field of molecular screening of genetic diseases, and specifically to a primer composition for detecting human SMN1 and SMN2 gene variations based on an NGS method and an application thereof. Background Art

[0002] Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder and the most common genetic cause of infant death. SMA is characterized by the loss of alpha motor neurons in the spinal cord, resulting in progressive muscle atrophy, weakness, and ultimately paralysis. Other features of SMA include muscle spasticity or tremors, poor weight gain, difficulty sleeping, scoliosis, joint contractures, and congenital heart disease. SMA diagnosis is divided into four groups based on the severity of symptoms and the age of symptom onset, ranging from severe infancy with onset leading to childhood death (Type I) to mild symptoms that do not develop until adolescence or later (Type IV). Although patients with severe SMA can be detected early, many patients with mild SMA are not diagnosed until adulthood.

[0003] SMA is an autosomal recessive genetic disease. According to statistics, its incidence rate is 10 to 20 per 100,000 people, and the carrier rate of the pathogenic gene in the normal population is about 1 / 40~1 / 80. The most common form of SMA is caused by biallelic pathogenic variants of SMN1 on chromosome 5. Most SMA (95%) is caused by homozygous deletion of the SMN1 gene on both chromosomes of the patient, especially the deletion of exons 7 and / or 8 of the gene; a few (5%) are caused by deletion of the SMN1 gene on one chromosome and a functionally disabled mutation of the SMN1 gene on another chromosome. In addition, the SMN2 gene near the SMN1 gene plays an important regulatory role in the severity and progression of SMA. The SMN2 gene sequence is highly homologous to SMN1, with only more than 10 bases different. One base difference leads to a 90% reduction in SMN2 pre-mRNA processing, so it is impossible to produce sufficient protein concentration to compensate for the effects of SMN1 deletion. Even so, an increase in the number of SMN2 copies in some SMA patients can still effectively delay the onset and symptoms of SMA. Another relevant genetic modification site in SMN2 is the c.859G>C variant located in exon 7. Some studies have shown that patients with mutations at this position have milder symptoms even if only two copies of SMN2 are present.

[0004] In recent years, several treatments for SMA have become available. However, the optimal treatment window for SMA progression is narrow in the affected population. Therefore, obtaining reliable SMN1 / 2 gene molecular diagnostic results as early as possible has become crucial for patient care and treatment. However, there are still several challenges in providing molecular diagnosis for SMA patients: First, it is necessary to develop technical means that can efficiently distinguish between the two highly similar genes and analyze SMN1 and SMN2 deletions separately; second, the SMN1 / 2 region is relatively complex and changeable. Not only does the SMN1 and SMN2 gene transform into each other, but in some populations, SMN1 tandem duplications, that is, the "2+1" or "2+2" phenomenon, occur, resulting in an increase in the total copy number of SMN1&2, leading to abnormal results in some traditional detection methods; third, in addition to exons 7 and 8 of SMN1 / 2, mutations in other exons of SMN1 can also cause disease, and it is necessary to detect them, but it is difficult to distinguish whether these mutations occur in SMN1 or SMN2.

[0005] At present, the most common SMA gene detection methods in clinical practice are mainly the following categories: (1) first-generation sequencing (Sanger) technology; (2) restriction fragment length polymorphism (PCR-RFLP) technology; (3) fluorescence quantitative PCR (qPCR) technology; (4) digital PCR (ddPCR) technology; (5) multiple ligation-dependent probe amplification (MLPA) technology; (6) second-generation sequencing (NGS) technology, etc. Sanger and PCR-RFLP technologies are relatively simple, but they can only find homozygous deletions, cannot distinguish between heterozygous deletions and normal people carrying deletions, and cannot analyze point mutations. qPCR and ddPCR technologies have certain subjective influences and are also unable to analyze point mutations. MLPA technology is currently the gold standard for detecting SMA copy number, but it cannot detect unknown point mutations, and the overall operation is relatively cumbersome. In contrast, NGS technology can detect both point mutations and copy number variations of SMN1 / 2, and has a high sequencing throughput, making it suitable for automated screening of a wide range of people. It has great application potential, but it also places high demands on the design capabilities of NGS primer / probe sets for SMA gene testing. Summary of the invention

[0006] Therefore, in order to solve the above-mentioned shortcomings, the present invention provides a primer composition and its application for detecting human SMN1 and SMN2 gene mutations based on NGS method, which is used for molecular detection of human spinal muscular atrophy (SMA) sensitive genes SMN1 and SMN2, and the detection content includes point mutations (SNV) and short insertion / deletions (in_del) in all exon regions, copy number variation (CNV) of SMN1&2 as a whole, and copy number variation of exons 7 and 8 of SMN1 / 2. The detection results can meet the needs of SMA pathogenic gene screening, genetic risk analysis, disease prognosis assessment, treatment guidance, etc.

[0007] The present invention is implemented in this way. The present invention provides a primer composition for SMN1 and SMN2 gene detection, including 48 pairs of primers or a combination of some of the primers, and the forward primer and reverse primer sequences of each pair of primers include a gene-specific amplification region at the 3' end, and a connector matching region connected to the 5' end of the specific amplification region.

[0008] The primer combination of the present invention comprises 48 pairs in total, which are divided into two groups: the first group of primers comprises 22 pairs, which can specifically amplify all exon regions of the SMN1 / SMN2 gene, and part of the introns or UTR regions around the 7th and 8th exons, wherein the sequences of the specific amplification regions at the 3' end of the forward primers are shown as SEQ IDs 1 to 22, respectively, and the sequences of the specific amplification regions at the 3' end of the reverse primers are shown as SEQ IDs 23 to 44, respectively; the second group of primers comprises 26 pairs, which can specifically amplify sequences of other specific regions of the human gene as a reference for the copy number variation of the SMN1 / 2 gene, wherein the sequences of the specific amplification regions at the 3' end of the forward primers are shown as SEQ IDs 45 to 70, respectively, and the sequences of the specific amplification regions at the 3' end of the reverse primers are shown as SEQ IDs 71 to 96, respectively.

[0009] In addition, the 5' end adapter matching region sequence of the forward primer in the above 48 pairs of primers is shown as SEQ ID NO.97, and the 5' end adapter matching region sequence of the reverse primer is shown as SEQ ID NO.98.

[0010] The primer combination can be used to establish a library by a multiplex PCR method, and then the library NGS sequencing method can be used to detect all exon regions and some important introns or UTR adjacent regions of the subject's SMN1 and SMN2 genes, as well as multiple reference sites in the human genome. The types of mutations that can be measured include point mutations, short insertions / deletions, and copy number variations. The results of copy number variations include both the overall copy number amplification / deletion of SMN1&2, as well as the variation of the 7th and 8th exons of the critical SMN1 and SMN2 genes. The overall design of the primer combination allows all amplification of the target fragments and subsequent library construction to be performed in the same tube, avoiding the trouble of separate tubes, saving related materials and time, and the overall cost is low.

[0011] Preferably, the specific amplification region sequences in the forward primer and the reverse primer are 18-25 base sequences among the SEQ IDs 1 to 96.

[0012] Preferably, the linker matching region sequences in the forward primer and the reverse primer are 20-25 base sequences among the SEQ IDs 97 to 98.

[0013] The present invention further protects the use of the above-mentioned SMN1 and SMN2 gene variation detection primer combination in SMA genetic risk analysis and screening of potential subjects that can benefit from treatment.

[0014] The present invention further protects a reagent for detecting SMN1 and SMN2 gene mutations, comprising the above-mentioned primer combination.

[0015] Preferably, at least one of the three pairs of adapters is included, wherein the three pairs of adapters correspond to the three sequencing platforms of Illumina, IonTorrent and MGI respectively, and the adapters contain DNA fragments paired with the sequences of SEQ ID 97-98.

[0016] Preferably, the reagent is suitable for molecular detection of genomic DNA samples of fresh or frozen tissue, whole blood, leukocytes, and saliva.

[0017] The present invention further protects the use of the above reagent in SMN1 and SMN2 gene detection based on the NGS method.

[0018] The present invention has the following advantages: The present invention provides a primer combination and application thereof for detecting human SMN1 and SMN2 gene variations based on an NGS method through improvement, which has the following improvements compared with similar devices or reagents:

[0019] Advantage 1: The primer combination and application thereof for detecting human SMN1 and SMN2 gene variations based on the NGS method described in the present invention, the primer combination adopts a "double coverage" design of two amplicons at important sites, and can effectively detect the copy number variations of the 7th and 8th exons of SMN1 / 2 on the human SMA sensitive genes SMN1 and SMN2, which are closely related to the occurrence and development of the disease.

[0020] Advantage 2: The primer combination and application thereof for detecting human SMN1 and SMN2 gene variation based on the NGS method described in the present invention do not simply default the copy number of SMN1 / 2 to 2, but consider the possibility of overall amplification or deletion of SMN1&2 by comparing the genomic reference sites. Therefore, special cases such as "2+1" and "2+2" can be indicated, providing subjects with an accurate SMN1 and SMN2 gene copy number variation map.

[0021] Advantage 3: The primer combination for detecting human SMN1 and SMN2 gene mutations based on the NGS method and its application described in the present invention can also detect point mutations and short insertions / deletions in all exon regions of the SMN1 / 2 gene. Combined with the above points 1-2, the detection results of the primer combination can meet the needs of SMA pathogenic gene screening, genetic risk analysis, disease prognosis assessment, treatment guidance, etc.

[0022] Advantage 4: The primer combination and application thereof for detecting human SMN1 and SMN2 gene mutations based on the NGS method of the present invention is a single-tube reaction when the reagent constructs the library, and the operation is simple and convenient, and the NGS library construction can be completed within a few hours. The amount of data required for sequencing is low, and the comparison rate, on-target rate and uniformity of the results are good.

[0023] Advantage 5: The primer combination and application thereof for detecting human SMN1 and SMN2 gene mutations based on the NGS method described in the present invention are suitable for the detection of various types of samples such as fresh or frozen tissues, peripheral blood leukocyte DNA, saliva DNA, FFPE, etc. from subjects, and have low requirements for samples. The conventional sample input amount for a single reaction is 20ng, which can be further reduced to 5-10ng through optimization experiments.

[0024] Advantage 6: The primer composition and application thereof for detecting human SMN1 and SMN2 gene variations based on the NGS method described in the present invention are simple in method and low in overall cost, filling the gap in the construction of NGS library by multiplex PCR technology in the field of domestic SMN1 and SMN2 gene detection, and contributing to the transformation of reagents from laboratory to clinical application and in vitro diagnostic reagents, and having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic diagram of the structure of a single primer in a primer composition for detecting human SMN1 and SMN2 gene variations based on the NGS method according to the present invention;

[0026] Figure 2-Figure 16 It is a schematic diagram of the test results of samples SMA-01 to SMA-15 in the embodiments of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below, and the technical solutions in the embodiments of the present invention will be described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The verification method adopted in the multiplex PCR library construction and sequencing process of the present invention is as follows:

[0029] 1. Sample Preparation

[0030] 15 commercial genomic DNA references from different populations were collected. The SMN1 / 2 gene variation of each sample was supported by literature or verified by MLPA.

[0031] 2. Library Construction

[0032] 2.1 First round of PCR

[0033] 2.1.1 Prepare gene-specific PCR reaction solution as shown in the table below and add the corresponding DNA sample to each tube.

[0034]

[0035] 2.1.2 Cover the tube, shake and mix, and then centrifuge quickly.

[0036] 2.1.3 Run the PCR reaction according to the following procedure.

[0037]

[0038] 2.2 Purification of the first round of PCR products

[0039] 2.2.1 After taking each tube out of the PCR instrument, quickly centrifuge it to throw the liquid to the bottom of the tube.

[0040] 2.2.2 Add 1.2 times the volume of purified magnetic beads (AMPure, Beckman, the same below) to each tube, shake to mix and centrifuge quickly. Let stand at room temperature for 5 minutes.

[0041] 2.2.3 Transfer each tube to the magnetic rack and place at room temperature for 5 minutes or until the liquid is clear. Carefully aspirate the supernatant, taking care to avoid touching the magnetic beads.

[0042] 2.2.4 Add 150 μL 70% ethanol to each tube for washing. After incubation for 30 seconds, remove the ethanol and be careful not to touch the magnetic beads. Repeat the washing once.

[0043] 2.2.5 Cover the tube caps, centrifuge each tube quickly, place it on a magnetic rack, and carefully remove the remaining ethanol in the tube.

[0044] 2.2.6 Place at room temperature for 2-3 minutes until the beads are dry. Add 32 μL of nuclease-free water to each tube, cover the tube, shake to mix and centrifuge quickly.

[0045] 2.2.7 Transfer each tube to a magnetic rack and place at room temperature for 5 minutes. Transfer the supernatant to a new tube.

[0046] 2.3 Second round of PCR

[0047] 2.3.1 Prepare the PCR reaction solution with adapters as shown in the table below.

[0048]

[0049]

[0050] 2.3.2 Cover the tube, shake and mix, and then centrifuge quickly.

[0051] 2.3.3 Run the PCR reaction according to the following procedure.

[0052]

[0053] 2.4 Library purification

[0054] 2.4.1 After taking each tube out of the PCR instrument, quickly centrifuge it to discard the liquid to the bottom of the tube.

[0055] 2.4.2 Add 1 volume of purified magnetic beads to each tube, shake to mix and centrifuge quickly. Let stand at room temperature for 5 minutes.

[0056] 2.4.3 Transfer each tube to the magnetic rack and place at room temperature for 5 minutes or until the liquid is clear. Carefully aspirate the supernatant, taking care to avoid touching the magnetic beads.

[0057] 2.4.4 Add 150 μL 70% ethanol to each tube for washing. After incubation for 30 seconds, remove the ethanol and be careful not to touch the magnetic beads. Repeat the washing process.

[0058] 2.4.5 Cover the tube caps, centrifuge each tube quickly, place it on a magnetic rack, and carefully remove the remaining ethanol in the tube.

[0059] 2.4.6 Place at room temperature for 2-3 minutes until the beads are dry. Add 32 μL of nuclease-free water to each tube, cover the tube, shake to mix and centrifuge quickly.

[0060] 2.4.7 Transfer each tube to a magnetic rack and place at room temperature for 5 minutes. Transfer the supernatant to a new tube.

[0061] 2.5 Library quality control

[0062] The library was quantified using Qubit and the fragment size and purity of the library were analyzed using Qseq100.

[0063] 3. Sequencing

[0064] 3.1 Dilute each library to 5 nM according to the concentration, take a small amount of equal volume and mix.

[0065] 3.2 Add an equal volume of 0.2N NaOH for denaturation, and then dilute to 15pM with HT1 solution.

[0066] 3.3 Take 594 μL of 15 pM mixed library and add 6 μL of 12.5 pM PhiX library control.

[0067] 3.4 Add the above 600 μL to the sequencing kit and load it into the Illumina Miseq sequencer for sequencing. The sequencing read length is PE150 and the sequencing depth is not less than 2000x.

[0068] 4. Data Results

[0069] 4.1 Result List

[0070] The test values ​​of 15 human genome reference samples are completely consistent with the reference results, as shown in the following table.

[0071]

[0072]

[0073] *No reference result, the actual measurement is 1 copy

[0074] 4.2 Detailed results are as attached in the instruction manual Figure 2-16 Shown

[0075] The sequences of SEQ ID NO.1 to 98 are as follows:

[0076]

[0077]

[0078]

[0079]

[0080] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer combination for detecting human SMN1 and SMN2 gene variations based on NGS method, characterized in that: The primer combination comprises 48 pairs in total, which are divided into two groups: the first group of primers comprises 22 pairs, which can specifically amplify all exon regions of the SMN1 / SMN2 gene, and some introns or UTR regions around the 7th and 8th exons, wherein the 5' end adapter matching region sequence of the forward primer is shown in SEQ ID NO.97, and the 3' end specific amplification region sequences are shown in SEQ ID 1 to 22, respectively; the 5' end adapter matching region sequence of the reverse primer is shown in SEQ ID NO.98, and the 3' end specific amplification region sequences are shown in SEQ ID 23 to 44, respectively; the second group of primers comprises 26 pairs, which can specifically amplify sequences of other specific regions of the human gene as a reference for the copy number variation of the SMN1 / 2 gene, wherein the 5' end adapter matching region sequence of the forward primer is shown in SEQ ID NO.97, and the 3' end specific amplification region sequences are shown in SEQ ID 45 to 70, respectively; the 5' end adapter matching region sequence of the reverse primer is shown in SEQ ID NO.98, and the 3' end specific amplification region sequences are shown in SEQ ID As shown in 71~96.

2. A reagent for SMN1 and SMN2 gene detection, characterized in that: Comprising the primer composition described in claim 1.

3. The reagent according to claim 2, characterized in that The reagent is suitable for molecular detection of DNA samples from fresh or frozen tissues, whole blood, and saliva.

4. The reagent according to claim 2, characterized in that The reagent is used for the detection of SMN1 and SMN2 genes based on the NGS method.

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