SMN one-step method detection developed based on MALDI-TOF MS and suitable for spinal muscular atrophy molecular diagnosis and neonatal screening

Abstract

According to the invention, MALDI-TOF MS (Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) is adopted to design Assay for detecting copy number quantification and The method comprises the following steps: firstly, by introducing a reference gene and a competitive template, developing CNV Assay (copy number variation analysis) in a small number of samples; and then comparing the data obtained by verification in the clinical queue with the result of the MLPA. Secondly, a pathogenic variation spectrum is established based on SNP (Single Nucleotide Polymorphism), and SMN common mutation sites and sequence variation of the subjects are detected.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis, and in particular, to the application of SMN detection based on MALDI-TOF MS in the molecular diagnosis of spinal muscular atrophy and neonatal screening, and more particularly, to a one-step SMN detection method developed based on MALDI-TOF MS and suitable for the molecular diagnosis of spinal muscular atrophy and neonatal screening. Background Art

[0002] Spinal muscular atrophy (SMA) is a rare autosomal inherited neuromuscular disease, mainly caused by homozygous deletion of the SMN gene (SMN1; OMIM 600354) near the telomeric side of the long arm of chromosome 5. SMN protein deficiency causes degeneration of α motor neurons in the anterior horn of the spinal cord, leading to muscle atrophy and movement disorders, and in severe cases even affects breathing and swallowing functions. SMA has a high disability and mortality rate and is the number one genetic disease "killer" of infants under 2 years old. In the past few years, the launch of three disease-modifying therapeutic drugs has brought hope to SMA patients. Real-world studies have found that gene-targeted therapy has a good response in presymptomatic SMA patients. Therefore, moving the window of therapeutic intervention forward to presymptomatic has the greatest benefit; and conducting SMA newborn screening is the best way to identify presymptomatic patients. However, traditional detection methods cannot effectively cover the full picture of the SMN gene, and are time-consuming and costly, making them unsuitable for large-scale SMA screening. Therefore, a more cost-effective method suitable for large-scale SMA newborn screening and that can also meet clinical diagnosis is needed.

[0003] Specifically, 98% of SMA patients have biallelic SMN1 mutations inherited from both parents, and 2% of patients have a new mutation in one allele. There are two main types of SMA mutation genotypes: 95% are caused by homozygous deletion of biallelic SMN1, i.e. [0+0] type; 5% are caused by compound heterozygous SMN1 mutations, i.e. one allele is deleted and the other allele has a minor pathogenic mutation, i.e. [0+1 d ] type. Patients with both SMN1 alleles having minor pathogenic variants, that is, [1 d +1 d ] genotype, which is very rare. The SMA carrier rate is about 1 / 100 to 1 / 45 in the population, and the carrier rate in the Asian population is about 1 / 48. Among carriers, SMN1 genotypes are mainly divided into 4 types: [1+0] genotype, [2+0] genotype, [1+1 d ] and [2+1 d ] genotypes, with population frequencies of 2.58%, 0.11%, 0.05% and 0.002%, respectively.

[0004] SMN2 is a homologous gene on the centromere side of SMN1, and there are only 5 base differences between the two. Among them, c.840C>T in exon 7 causes 90% of SMN2 mRNA exon 7 to be selectively spliced, and only 10% of SMN2 expresses full-length functional SMN protein. SMN2 copy number is currently recognized as a modifier of SMA. The more SMN2 copies a patient carries, the milder the phenotype. SMN2 copy number is often used as an important reference data in drug experiments that regulate SMN protein as a treatment strategy, and in newborn screening, it is used as an important biological marker for the treatment evaluation of patients before symptoms appear. Although its correlation with phenotype is not completely consistent, the management consensus still regards SMN2 copy number as one of the standard steps for SMA diagnosis. Therefore, for genetic testing of SMA, in addition to SMN1, the copy number of SMN2 is also crucial for the target gene. The detection results of SMN1 copy number variation (CNV) and pathogenic variation are used for disease diagnosis or exclusion diagnosis, and the detection results of SMN2 copy number are used as reference indicators for treatment, clinical management and prognosis evaluation after patient diagnosis.

[0005] There are many clinical technologies that can be used for the diagnosis and screening of SMA. Among them, MLPA can directly detect the SMN1 copy number and clearly distinguish patients, carriers and normal people; it can also detect the SMN2 copy number of patients at the same time, and is the gold standard for diagnosing SMA recommended by the current SMA management consensus. However, MLPA cannot detect small variations in the SMN1 gene and [2+0] genotype carriers. In addition, fluorescent quantitative PCR can also be used to detect SMN copy number, but its specificity is slightly inferior to that of the MLPA method, and SMN2 requires additional probe design for detection. It also cannot be used to detect small variations in the SMN1 gene and [2+0] genotype carriers. In view of this, the application of traditional sequencing methods such as Sanger sequencing and NGS in SMA is also very limited. Some long-read sequencing methods are suitable for the above scenarios, but the high cost and high specimen requirements limit their application scenarios outside of scientific research (such as large-scale newborn screening). Summary of the invention

[0006] The present invention aims to overcome the above-mentioned defects and use MALDI-TOF MS to accurately detect SMN copy number and SMN1 pathogenic sites applicable to the Chinese population. The present invention applies MALDI-TOF MS to samples from China and compares the copy number with the traditional MLPA method. The detection results of the two methods are consistent. In addition, the present invention also identifies [0+1 d ] genotype SMN1 compound heterozygous mutation patients. In addition, the present invention also supplements the potential marker sites of [2+0] genotype SMA carriers, which can assist in identifying silent carriers in combination with clinical manifestations.

[0007] The present invention provides a primer combination, characterized in that it comprises an amplification primer and an extension primer;

[0008] Wherein, the above-mentioned amplification primer is selected from at least one of the amplification primers represented by the sequences of SEQ ID No.1 to SEQ ID No.8;

[0009] The extension primer is at least one selected from the extension primers represented by the sequences of SEQ ID No. 10 to SEQ ID No. 11.

[0010] Furthermore, the present invention also provides the use of the primer combination in preparing a product for detecting the copy number of SMN1 and SMN2.

[0011] Furthermore, the present invention also provides the use of the primer combination in preparing a product for diagnosing SMA in newborns.

[0012] In addition, the present invention also provides a primer combination, characterized in that it includes an amplification primer and an extension primer;

[0013] Wherein, the above-mentioned amplification primer is selected from at least one of the amplification primers represented by the sequences of SEQ ID No.13 to SEQ ID No.30;

[0014] The extension primer is at least one selected from the extension primers represented by the sequences of SEQ ID No.31 to SEQ ID No.39.

[0015] Furthermore, the present invention also provides the use of the primer combination in preparing a product for detecting SMN genotype, characterized in that:

[0016] The above SMN genotypes include minor variations in the SMN1 gene;

[0017] The above-mentioned SMN1 gene minor variations include [2+0] genotype and [0+1 d ]genotype.

[0018] Furthermore, the present invention also provides the use of the primer combination in preparing a product for diagnosing SMA in newborns.

[0019] In addition, the present invention also provides a primer combination, characterized in that it includes an amplification primer and an extension primer;

[0020] Wherein, the above-mentioned amplification primer is selected from at least one of the amplification primers represented by the sequences of SEQ ID No.1-SEQ ID No.8 and SEQ ID No.13-SEQ ID No.30;

[0021] The extension primer is at least one selected from the extension primers represented by the sequences of SEQ ID No.10 to SEQ ID No.11 and SEQ ID No.31 to SEQ ID No.39.

[0022] Furthermore, the present invention also provides the use of the primer combination in preparing a product for detecting the copy number of SMN1, SMN2, and SMN genotype, characterized in that:

[0023] The above SMN genotypes include minor variations in the SMN1 gene;

[0024] The above-mentioned SMN1 gene minor variations include [2+0] genotype and [0+1 d ]genotype.

[0025] Furthermore, the present invention also provides the use of the primer combination in preparing a product for diagnosing SMA in newborns.

[0026] In addition, the present invention also provides a system for detecting SMA, characterized in that it comprises a detection module;

[0027] Wherein, the above detection module uses MALDI-TOF MS to detect / analyze SMN copy number and detect / identify SMN pathogenic sites;

[0028] The above-mentioned SMN pathogenic loci include G.27134t>G and G.27706_27707delat polymorphic loci variants;

[0029] The above detection module also introduces internal reference genes and competition templates;

[0030] During the testing process,

[0031] Select the above-mentioned amplification primers to carry out amplification reaction;

[0032] The extension primers mentioned above are selected to carry out the extension reaction.

[0033] Functions and effects of the present invention:

[0034] In the context of the global promotion of SMA neonatal screening, in order to better promote the screening and diagnosis of SMA, there is an urgent need for a rapid and economical analysis method that can simultaneously quantify the copy number of SMN1 and SMN2 and identify common pathogenic mutations. The method of the present invention should be able to achieve the following effects:

[0035] (1) Identify the copy number of complete SMN1 and SMN2 based on c.840;

[0036] (2) identify common SMN1 pathogenic variants other than the c.840C deletion;

[0037] (3) Assist in identifying [2+0] genotype carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1-1 .Quantitative mechanism of SMN gene. In it, the exogenous oligonucleotide (i.e., competitor template) has the same sequence as the target region to be amplified, except for one base variation. For example, the competitor template targeting SMNE7 (SMN E7_C) contains a G base extension, which is different from the C in SMN1 E7 and the T in SMN2 E7. The experimental process includes 4 steps: 2.5 hours of PCR amplification, 45 minutes of SAP reaction, about 3.5 hours of extension reaction and 1 hour of mass spectrometry analysis. In 384-well plate mode, the whole experimental process takes about 8 hours. SAP, shrimp alkaline phosphatase; SMN, survival motor neuron.

[0039] Figure 1-2 .SMN gene detection flow chart.

[0040] Figure 2 .Schematic diagram of SMN exon 7 (E7) from MALDI-TOF MS. Wherein, C represents E7 of SMN1, T represents E7 of SMN2, and G represents the template competing with SMN E7. MALDI-TOF MS detection of SMN E7 in non-SMA individuals: (A) subjects with both SMN1 and SMN2 E7 (C, T) and (B) after inclusion of competing template (C, G, T); (C) subjects with only SMN1 E7 (0 SMN2 copies, C) and (D) after inclusion of competing template (C, G). SMN E7 MALDI-TOF MS in SMA patients: (E) subjects with only SMN2 E7 (0 SMN1 copies, T) and (F) after inclusion of competing template (G, T). SMA, spinal muscular atrophy; SMN, survival motor neuron.

[0041] Figure 3 Schematic representation of SMN exon 8 (E8) from MALDI-TOF MS. Wherein, G represents SMN1’s E8, A represents SMN2’s E8, and C represents the template competing with SMN’s E8. MALDI-TOF MS detection of SMN E8 in non-SMA individuals: (A) a subject with both SMN1 and SMN2 E8 (G, A) and (B) after inclusion of a competing template (G, C, A); (C) a subject with only SMN1 E8 (0 SMN2 copies, G) and (D) after inclusion of a competing template (G, C). MALDI-TOF MS of SMN E8 in SMA patients: (E) a subject with only SMN2 E8 (0 SMN1 copies, A) and (F) after inclusion of a competing template (C, A). SMA, spinal muscular atrophy; SMN, survival motor neuron.

[0042] Figure 4 .Schematic diagram of internal calibration (IC) of MALDI-TOF MS. RPPH1 was used as the internal calibration for the SMN copy number panel. Where C represents RPPH1 and G represents the template competing with RPPH1. (A) MALDI-TOF MS of RPPH1 in all subjects (C) and (B) after the inclusion of the competing template (G, C).

[0043] Figure 5 Comparison of MLPA and MALDI-TOF MS detection of SMN copy number in 13 DNA samples. The copy number of 13 samples was obtained by two different detection methods. Among them, (A) SMN1 exon 7, (B) SMN2 exon 7, (C) SMN1 exon 8 and (D) SMN2 exon 8. The gray dashed lines represent the cutoff values ​​of MLPA and MALDI-TOF MS, respectively. MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MLPA: multiplex ligation-dependent probe amplification; SMN: survival of motor neuron.

[0044] Figure 6 Comparison of SMN copy number detection by MLPA and MALDI-TOF MS. The copy number of 78 samples was obtained by using two different detection methods: (A) SMN1 exon 7, (B) SMN2 exon 7, (C) SMN1 exon 8, and (D) SMN2 exon 8. The gray dashed lines represent the cutoff values ​​of MLPA and MALDI-TOF MS, respectively. Samples outside the box (red) show ambiguous copy numbers. MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; multiplex ligation-dependent probe amplification (MLPA); SMN, survival motor neuron.

[0045] Figure 7 MALDI-TOF MS technology was used to detect the normal copy number samples, where (A) 20 ng DNA was used to detect the copy number of SMNE7 and e8 in normal samples (2N) (n=38). The copy number variation detection results of different batches of normal samples of SMN1 Exon7 (B), SMN2 Exon7 (C), SMN1 Exon8 (D), and SMN2 Exon8 (E).

[0046] Figure 8 .MALDI-TOF MS was used to identify two normal samples with different DNA copy numbers.

[0047] Fig. 9.Representative Sanger sequencing profile of DNA extracted from an agarose gel of PCR products amplified from pathogenic variants.

[0048] Fig.10 .Schematic diagram of common pathogenic variants of SMN1 detected by MALDI-TOF MS; (A,B) 22dupA is c.22dupA (ps er8lysfs*23) located in exon 1 of the SMN1 gene. (C,D) G400A is C.400g>A (pg lu134lys) in exon 3 of the SMN1 gene. (E,F) G863T is c.863G>T (pg ly279glufs*5) in exon 7 of the SMN1 gene. (G,H) T683A is c.683T>A (pl eu228*), located in exon 5 of the SMN1 gene. (I,J) C689T is c.689C>T (ps er230leu) in exon 5 of the SMN1 gene. (K, L) T835-5G is c.835-5T>G (pg ly279glufs*5) intron 6 of SMN1 gene. The left pictures (A, C, E, G, I, K) are wild type, and the right pictures (B, D, F, H, J, L) are mutant. SMN, survival motor neuron.

[0049] Fig.11 .Genetic analysis of a child with SMA characterized by heterozygous deletion of the SMN1 gene and pathogenic variants, in which (A) MLPA results showed that the copy number of exon 7 / 8 of the SMN1 gene was 1, and the copy number of exon 7 / 8 of the SMN2 gene was 3; (B) MALDI-TOF MS CNV panel analysis of the patient showed that the quantitative results of SMN1 exon 7, SMN2 exon 7, SMN1 exon 8, and SMN2 exon 8 were 0.98±0.14, 2.64±0.16, 0.87±0.03, and 2.79±0.13, respectively (n=3). (C) Schematic diagram of DNA mass spectrometry A815G detection (wild type). (D) Schematic diagram of the patient's pathogenic variant combination (A815G) detected by DNA mass spectrometry. The A815G variant is the c.815A>G (pt yr272cys) variant in exon 6 of the SMN1 gene. (E) Genetic profile of a compound heterozygous SMA patient. MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; multiplex ligation-dependent probe amplification (MLPA); SMA, spinal muscular atrophy; SMN, survival motor neuron.

[0050] Fig.12.DNA mass spectrometry detected the SMN1 gene intron 7 mutation T27134G to G.27134t>G, and the exon 8 mutation 27706-27707delAT to G.27706_27707delat. MALDI-TOF MS detected G.27134t>G and G.27706_27707delat wild type (A, B) and mutant type (C, D) (E, F) One case had G.27134t>G and G.27706_27707delat mutations in three copies of SMN1 gene. DETAILED DESCRIPTION

[0051] The present invention is capable of various modifications and various embodiments, and therefore specific embodiments are illustrated and described in the accompanying drawings. However, this is not intended to limit the present invention to specific implementations, but should be understood to include all modifications, equivalents, and even substitutes that fall within the concept and technical scope of the present invention.

[0052] Example 1. Test method

[0053] 1.1 Sample collection and DNA extraction: All operations were approved by the Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (approval number: n090331). XHEC-C-2023-072-1) and the standards of the Declaration of Helsinki and the International Standard for Good Clinical Practice for Drugs. In 2023-2024, peripheral blood was collected from all study subjects using EDTA tubes. Coriell DNA (ID: NA03813, NA03814, NA12548) was purchased from Coriell Research Institute (https: / / www.coriell.org). Blood samples were collected with the informed consent of parents or legal guardians. Peripheral blood was processed using a commercial magnetic bead extraction kit (Xi'an Tianlong Technology Co., Ltd., China). Subsequently, genomic DNA (gDNA) was extracted using a fully automatic nucleic acid extractor (GeneRotex 96). The concentration and quality of the extracted nucleic acid were determined using a NanoDropTM 2000 spectrophotometer (NanoDrop Technologies Inc., DE, USA). Samples with an A260 / A280 ratio of 1.6 to 2.0 are considered qualified. The concentration was further quantified using the Qubit dsDNA BR Assay Kit on a 2.0 fluorometer (Life Technologies, Grand Island, NY, USA). All DNA samples were stored at -80°C until the start of the experiment.

[0054] 1.2 Panel and primer design: Two different panels (CNV; pathogenic variants) were used for genetic analysis of SMA. For the CNV panel, amplification primers for SMN1 and SMN2 were designed to amplify the exon 7 (E7) and exon 8 (E8) regions, with RPPH1 as an internal reference control. For accurate copy number quantification, competitive templates competing with SMN1 / SMN2 E7 / 8 and RPPH1 were synthesized, and the principle was as described above. Another panel included 7 common Chinese SMN1 pathogenic variants (detection frequency >2) evaluated by the American College of Medical Genetics and Genomics and 2 polymorphic variants associated with the [2+0] genotype. Primers were designed using the Agena Bioscience online software Assay Design Suite (https: / / support.agenabio.com / s / login / ) and synthesized by Sangon Biotech (Shanghai) Co., Ltd. When software design failed, primers were designed manually. All primer sequences are shown in Tables S1-4 below.

[0055] Table S1. Amplification primers for SMN copy number quantification by MALDI-TOF MS

[0056]

[0057] Note: RPPH1 is used as an internal calibrator for the SMN copy number panel. SMN: survival motor neuron.

[0058] Table S2. Extended primers for SMN copy number quantification by MALDI-TOF MS

[0059]

[0060] Note: RPPH1 is used as an internal calibrator for the SMN copy number panel. SMN: survival motor neuron.

[0061] Table S3. Amplification primers for detecting SMN pathogenic variants using MALDI-TOF MS

[0062]

[0063]

[0064] Table S4. Primer extension for detection of SMN pathogenic variants using MALDI-TOF MS

[0065]

[0066] 1.3 MALDI-TOF MS system: Unless otherwise stated, the reagents used in the MALDI-TOF MS system were from Agena Biosciences. The experimental procedures can be roughly divided into PCR amplification of the DNA fragment of interest, shrimp alkaline phosphatase (SAP) reaction (PCR products were treated with SAP to remove excess ddNTPs), single nucleotide extension, and mass spectrometry analysis.

[0067] 1.3.1 PCR amplification: The PCR reagent set includes 0.8μl ddH2O (optional 0.5-1.0μl), 0.5μl buffer (optional 0.1-1.0μl), 0.4μl MgCl2 (optional 0.5-1.0μl), 0.1μl dNTP Mix (optional 0.01-0.2μl), 1μl Primer Mix (optional 0.5-2.0μl), 0.2μl PCR Enzyme (optional 0.1-0.8μl) and 20ng gDNA (2μl) (optional 1-4.0μl), forming a 5μl PCR (optional 2-10μl) system. For the CNV group, in order to maintain a 5 μl PCR system, ddH 2 O was replaced with the same volume of competitive template mix (approximately 62,160 copies, 92,000 copies, and 424,000 copies for exon 7_C, exon 8_C, and RPPH1_C, respectively).

[0068] The reaction program contained the following stages: Stage 1 involved an initial denaturation step at 95°C for 2 minutes. Stage 2 consisted of 45 cycles of denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 60 seconds. Stage 3 involved a final extension step at 72°C for 5 minutes. The reaction was then kept at 4°C for storage.

[0069] 1.3.2SAP reaction: A 2μl SAP system includes 1.53μl ddH2O, 0.17μl SAP buffer and 0.3μl SAP Enzyme.

[0070] The reaction protocol was as follows: incubation at 37°C for 40 minutes, followed by incubation at 85°C for 5 minutes, and then maintained at 4°C.

[0071] 1.3.3 Extension reaction: 2 μl of reagents used for extension reaction include 0.62 μl ddH2O, 0.2 μl BufferPlus, 0.2 μl Termination Mix, 0.04 μl Extend Enzyme and 0.94 μl Extend Primer Mix.

[0072] The reaction protocol consisted of three consecutive stages: (1) a heat denaturation step at 95°C for 30 seconds. (2) The subsequent stages were divided as follows: 1) initial denaturation at 95°C for 5 seconds, 2) a total of 5 cycles, each cycle consisting of 52°C for 5 seconds and then 80°C for 5 seconds, 3) 40 cycles of 1) plus 2). (3) The final stage was incubation at 72°C for 3 minutes, followed by cooling to 4°C.

[0073] 1.3.4 Mass spectrometry analysis: The extension products were desalted and assigned to SpectroCHIP in 384 format. The data were then analyzed using Typer Analyzer (Agena Biosciences, version 4.1). For copy number quantification, the target gene was first normalized according to the peak area of ​​the competitive template, then normalized with a stable diploid internal reference (RPPH1), and finally calibrated using 2N samples (2 copies of SMN1 and SMN2 E7 / 8). The calculation formula is as follows:

[0074]

[0075] 1.4 Multiplex ligation-dependent probe amplification analysis. MLPA analysis of all samples was performed by Shanghai Medici Technology Co., Ltd. (China). SALSA MLPA kit MRC-P060-B2 SMACarrier, Applied Biosystems capillary sequencer, and the publicly available Coffalyser.Net (MRC-Holland, Amsterdam, Netherlands) MLPA analysis software were used for analysis according to the general protocol (https: / / www.mrcholland.com).

[0076] 1.5 Construction of standard mutant plasmids. All mutant plasmids were constructed by Sangon Biotech (Shanghai) Co., Ltd. Briefly, the synthesized target gene was transferred into the pUC57 vector, and plasmid extraction and Sanger sequencing were performed. Plasmid samples with successful mutation were selected for MALDI-TOF MS analysis.

[0077] 1.6 Statistical analysis. In this study, IBM SPSS Statistics (version 29) and GraphPadPrism (version 10) were used to analyze and plot data. All quantitative data were expressed as mean ± standard deviation. P < 0.05 was considered statistically significant.

[0078] This embodiment may be a system for detecting SMA, which includes a detection module;

[0079] The detection module uses MALDI-TOF MS to detect / analyze the SMN copy number and detect / identify the SMN pathogenic sites; the SMN pathogenic sites include G.27134t>G and G.27706_27707delat polymorphic site variations; the detection module introduces the internal reference gene and competitive template;

[0080] During the detection process, the above-mentioned amplification primers are selected to perform an amplification reaction;

[0081] The extension primers mentioned above are selected to carry out the extension reaction.

[0082] The finished product of this embodiment can also be in the form of a detection kit, a detection instrument, etc.

[0083] Example 2. Result analysis

[0084] 2.1 CNV panel design: Based on the genetic attributes of SMA, the capabilities of the MALDI-TOF MS platform were utilized. Figure 1-1 As shown, in this example, a quantitative panel is designed to quantify the copy number of the pathogenic gene SMN1 and the prognostic gene SMN2.

[0085] Generally speaking, for SMN E7 of non-SMA subjects (whether carriers or not), only C peak and T peak or C peak (0 SMN2 copies) can be observed in the mass spectrum ( Figure 2 A&C). When the competing template is introduced, the mass spectrum shows peaks C, G, T, or C and G ( Figure 2 B&D). Since most SMA patients have homozygous deletion of SMN1, only T peak and G,T peak are observed in the mass spectrometry ( Figure 2 E&F). Similarly, SMN E8 and RPPH1 genes showed corresponding peak patterns in MALDI-TOF MS ( Figure 3 , Figure 4 ). As described above in the experimental section, the copy number can be determined after correcting the peak area for the expected region. The development of CNV detection is a two-step process: first, the methodological performance is evaluated using a limited number of samples, followed by validation and application evaluation in clinical cohorts.

[0086] 2.2 SMN copy number quantification results

[0087] The development of CNV tests is a two-step process: first, evaluation of methodological performance using a limited number of samples, followed by validation and application evaluation in clinical cohorts.

[0088] 2.2.1 Methodological performance of CNV detection. In the initial stage, 10 non-SMA patients and 3 Coriell DNA samples (ID: NA03813, SMA patient; NA03814, SMA carrier; NA12548, [2+1] SMN1 genotype) were collected for methodological establishment. For these 13 samples, the SMN copy number detected by MALDI-TOF MS was highly consistent with that by MLPA ( Figure 5 ; Table S5), with a kappa value of 0.935 (P<0.001, Table S6). The detection concentration of these samples was 10 ng / μl per well (i.e., 20 ng gDNA), and the intra-assay precision was 96.15%. MLPA detection found 5 cases of SMN1 and SMN2 E7 / 8 gene duplication. The MALDI-TOF MS quantitative results were 2.00±0.18, 2.03±0.27, 2.03±0.08, and 2.01±0.13, respectively. In addition, the SMN2 gene E7 / 8 of NA03814 was atypical with 5 copies (refer to the MLPA product manual). The quantitative result of SMN2 E7 in the MALDI-TOF MS system was 4.4±0.90, which was within the unclear range ( Figure 5 B).

[0089] Table S5. Comparative analysis of SMN copy number in 13 DNA samples by MALDI-TOF MS and MLPA

[0090]

[0091] *The Medical Research Council of the Netherlands (MRCHolland) used P021-B1 SMA to measure SMN1 and SMN2 copy numbers in samples from the Coriwell Biobank. The SMN copy numbers corresponding to the Coriwell DNA are provided in the kit instructions (P060-B2SMACarrier, P021-B1 SMA). MALDI-TOFMS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; multiplex ligation-dependent probe amplification (MLPA); SMA, spinal muscular atrophy; SMN, survival motor neuron. SMA is 5qSMA caused by pathogenic variants in the SMN1 gene at 5q13.

[0092] Table S6. Kappa consistency evaluation of SMN copy number detection in 13 DNA samples

[0093]

[0094] *The copy number of SMN1 E7 gene was 1.67±0.09 by MALDI-TOFMS and 2.44±0.12 by MLPA (grayscale value). The latter was classified as 3 copies in the kappa consistency test. ΔThe copy number of SMN2E7 was 4.4±0.90 by MALDI-TOFMS and 5 by MLPA. When performing the kappa test, the total number of tests (total) was weighted according to the specific situation. The kappa value was 0.935 (asymptotic standard error 0.045, P<0.001), indicating strong consistency between the two tests. MALDI-TOFMS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MLPA: multiplex ligation-dependent probe amplification; SMN: survival motor neuron.

[0095] 2.2.2 Validation and application evaluation of CNV detection: To validate the performance of the CNV panel, the panel was tested in a clinical cohort of 78 people (Table S7). DNA samples were blindly numbered by personnel not involved in this study. MLADI-TOF MS test results showed that 77 of the 78 samples had an SMN copy number ≤ 3. In addition, the quantitative results showed robust consistency with MLPA ( Figure 6 ; Table S7). Among them, 16 cases had co-deletion of exon 7 and exon 8, and 3 cases had deletion of exon 7 alone. 5 cases were carriers, 38 cases were normal, and the rest of the subjects had different degrees of copy number variation. At 20 ng of gDNA per well, we performed additional replicates on different batches of 2N samples to confirm the batch-to-batch stability of the CNV panel ( Figure 7 In addition, the quantitative results of 20ng gDNA per well were consistent with those of 40ng gDNA ( Figure 8 ).

[0096] 2.3 Detection of intragenic SMN pathogenic variants: Approximately 5% of SMA patients carry a point mutation in one SMN1 allele and a deletion of the other SMN1 allele, or carry biallelic mutations in the SMN1 exons (very rare). Due to the inherent instability of the 5q region, the frequency of de novo mutations is relatively high. Therefore, in addition to quantifying copy number, we also developed a panel of pathogenic variants based on common SMN1 point mutations found in the Chinese population ( Fig. 9 and Fig.10 ).

[0097] The pathogenic variant combination had high sensitivity in the entire cohort, with stable and accurate detection results at the 20ng gDNA level. Notably, one patient in our cohort had a compound heterozygous deletion of SMN1 (specifically, a deletion of one SMN1 gene combined with a pathogenic variant in one allele within the gene). By using an integrated SMN panel containing CNVs and pathogenic variants, we successfully elucidated the pathogenic mechanism of this compound heterozygous deletion ( Fig.11 MALDI-TOFMS accurately identified the c.A815G mutation in this patient ( Fig.11 D), a finding that was confirmed by DNA level sequencing (8 exons within the SMN coding region) and RNA reverse transcription monoclonal sequencing. c.815A>G is located in exon 6 of the only remaining copy of the SMN1 gene. In addition, parental genome sequencing showed that the father carried two SMN1 copies, one of which carried the same c.815A>G mutation observed in the patient, while the mother was identified as an SMA carrier ( Fig.11 E). Apart from this, no other pathogenic variants were found in the cohort.

[0098] 2.4 Detection of SMN1 gene sequence variation: Among SMA carriers, there is a unique [2+0] genotype, which is characterized by the presence of two functional SMN1 gene copies on one chromosome and SMN1 deletion or conversion on the other chromosome. The frequency of the [2+0] genotype in SMA carriers is approximately 4%. Studies have shown that the polymorphic site variations G.27134t>G and G.27706_27707delat have suggestive effects on individuals with the [2+0] genotype. Therefore, we included these two polymorphic sites to capture potential [2+0] carriers of SMA ( Fig.12 AD). After validating the polymorphism combination, we observed G.27134t>G and G.27706_27707delat mutations simultaneously in a subject with three copies of SMN1 ( Fig.12 E&F) (Table S7).

[0099] Table S7. Distribution of polymorphic site variants in the cohort

[0100]

[0101] *Contains three Coriell DNAs (IDs: NA03813, NA03814, NA12548). SMN, survival motor neuron.

[0102] From the above results, it can be found that the product of the present invention can detect the [2+0] genotype during the process of conventional genotype and copy number identification. However, when using the traditional MLPA kit (as shown in the following table), if the [2+0 type] gene is to be detected, an independent MLPA kit is required for determination, which not only increases the probability of missed detection (because it cannot be detected without independent detection), but more importantly, MLPA will increase the screening cost due to its high price.

[0103]

[0104] Primary test

[0105] οSecondary test

[0106] Increased detection of Silent Carriers.

[0107] -Not possible to detect

[0108] ◇MC002: no absolute Copy aside numbers from 0determined.

[0109] √Suitable to detect

[0110] *Exon 1-6deletions are not detected by MC002

[0111] Functions and effects of this embodiment:

[0112] In this study, the SMN CNV assay was first established using MALDI-TOF MS. The initial design was to quantify the SMN copy number by comparing the target gene with the internal reference gene (RPPH1 / TERT) and the 2N calibrator. After repeated adjustments to the primer ratio and reaction procedure, MALDI-TOF MS instrument parameters such as peak area, peak height, and SNR were tried to quantify the copy number. The final results showed that the scheme can accurately identify SMA patients and normal people (2N type calibration samples), and the copy number quantification results of peak area, peak height, and SNR are almost consistent (data not shown). However, the quantitative results of the assay for non-homozygous deletion or non-2N copy number samples (i.e., a small number of SMA carriers or copy number duplication individuals) are poor, and the batch-to-batch differences are large, making it difficult to promote to large-scale application. In order to accurately quantify the copy number, we introduced a competitive template for further correction. Studies have shown that CNVPanel can simultaneously quantify the copy number of exons 7 and 8 of the SMN1 and SMN2 genes. The test results in a small-scale cohort are highly consistent with MLPA, and a small number of samples were reproduced by different experimenters in two laboratories. It should be pointed out that although it is the gold standard for SMN copy number detection, there are differences in the measurement results of MLPA in different laboratories. Therefore, some samples measured in the gray area by MLPA can be compared with the measurement data of MALDI-TOF MS (e.g. NA03814 in this study) to obtain more accurate copy number information. In the future, CNV Panel data of a larger sample population needs to be analyzed to obtain more accurate cut-off values.

[0113] In addition, based on the common SMN1 mutation sites in the Chinese population, we designed a pathogenic mutation panel using MALDI-TOF MS. It is worth mentioning that c.463_464del AA, which was originally included in the mutation panel (gene detection panel can be understood as a collection of genes), is also a common SMN1 mutation site in the Chinese population. However, there is another SNP signal peak near the mass spectrometry peak of this site (most likely rs4915), which interferes with the correct interpretation of its results, so it was eventually discarded by us. Currently, about 90 minor pathogenic variants have been reported, of which nearly 30 have been reported in Chinese patients. The addition of other mutation sites will help screen out more potential [1+1d] type carriers. Interestingly, the combined application of CNV+mutation panel in this study enabled us to accurately capture 1 case of SMN1 [0+1 d] type compound heterozygous mutation. The pathogenic mutation in the SMN1 gene of this patient is c.815A>G, which is inherited from her father. This site has been reported many times in previous studies and is a known pathogenic mutation. This case shows that MALDI-TOF MS has great potential in genetic testing of SMA. In addition, due to the existence of special [2+0] genotype carriers, in order to meet the needs of population screening as much as possible, we introduced two potential genetic markers for [2+0] type carriers. g.27134T>G and g.27706_27707delAT are SMN1 sequence polymorphic sites. Studies have shown that they have a suggestive effect in the [2+0] type silent carrier population. In MALDI-TOF MS, individuals who carry two copies of SMN1 and test positive for g.27134T>G and / or g.27706_27707delAT can be regarded as potential [2+0] genotype silent carriers, and further testing of their parents can determine whether they both carry SMN1 genes on their chromosomes. If the patient has the [2+0] genotype, one parent carries at least 3 copies and the other carries at least 1 copy ([1+0] type carrier).

[0114] In this study, SMA refers to 5q-SMA caused by pathogenic variants of SMN1 located at 5q13. This study describes the feasibility and application prospects of MALDI-TOF MS in SMA diagnosis and neonatal screening.

[0115] Although the above description is centered on the embodiment, it is only an example and does not limit the present invention. It is clear to those skilled in the art that various modifications and applications not illustrated above can be made within the scope of the essential characteristics of the present embodiment. For example, each component specifically shown in the embodiment can be implemented after being modified. Moreover, various differences related to such modifications and applications should be interpreted as being included in the scope of the present invention as defined in the attached claims.

Claims

1. A primer combination, characterized in that: Includes amplification primers and extension primers; Wherein, the amplification primer is selected from at least one of the amplification primers represented by the sequences of SEQ ID No.1 to SEQ ID No.8; The extension primer is selected from at least one of the extension primers represented by the sequences of SEQ ID No.10 to SEQ ID No.

11.

2. Use of the primer combination as claimed in claim 1 in the preparation of a product for detecting the copy number of SMN1 and SMN2.

3. Use of the primer combination as claimed in claim 1 in preparing a product for diagnosing SMA in newborns.

4. A primer combination, characterized in that: Includes amplification primers and extension primers; Wherein, the amplification primer is selected from at least one of the amplification primers represented by the sequences of SEQ ID No.13 to SEQ ID No.30; The extension primer is selected from at least one of the extension primers represented by the sequences of SEQ ID No.31 to SEQ ID No.

39.

5. Use of the primer combination according to claim 4 in preparing a product for detecting SMN genotype, characterized in that: The SMN genotype includes a minor variation in the SMN1 gene; The SMN1 gene minor variation includes [2+0] genotype and [0+1 d ]genotype.

6. Use of the primer combination as claimed in claim 4 in preparing a product for diagnosing SMA in newborns.

7. A primer combination, characterized in that: Includes amplification primers and extension primers; Wherein, the amplification primer is selected from at least one of the amplification primers represented by the sequences of SEQ ID No.1-SEQ ID No.8 and SEQ ID No.13-SEQ ID No.30; The extension primer is at least one selected from the extension primers represented by the sequences of SEQ ID No.10-SEQ ID No.11, and SEQ ID No.31-SEQ ID No.

39.

8. Use of the primer combination according to claim 7 in preparing a product for detecting the copy number of SMN1, SMN2, and SMN genotype, characterized in that: The SMN genotype includes a minor variation in the SMN1 gene; The SMN1 gene minor variation includes [2+0] genotype and [0+1 d ]genotype.

9. Use of the primer combination as claimed in claim 7 in preparing a product for diagnosing SMA in newborns.

10. A system for detecting SMA, characterized in that: Contains detection module; Wherein, the detection module uses MALDI-TOF MS to detect / analyze SMN copy number and detect / identify SMN pathogenic sites; The SMN pathogenic sites include G.27134t>G and G.27706_27707delat polymorphic site variations; the detection module also introduces internal reference genes and competitive templates; During the testing process, Selecting the amplification primers according to claim 1, claim 4, or claim 7 to carry out an amplification reaction; The extension reaction is carried out using the extension primer according to claim 1, claim 4, or claim 7.

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