SMN1 and SMN2 specific LNA q PCR primer and protocol and method therefor

Abstract

The invention relates to a diagnostic kit and method for the diagnosis of SMA. It was characterized by a probe-free, LNA-based assay. The invention enables the differentiation between SMA patients and carriers in a single PCR reaction conducted under uniform conditions. Using LNA-modified primers, differentiation of patients and carriers is achieved with 100% accuracy. In the invention, as a result of the PCR reaction, the SMN2 copy number of affected children is determined with complete accuracy through a cost-effective and rapid protocol. While MLPA, the current gold standard for SMA diagnosis, requires two days and relies on complex analysis software, the proposed method can distinguish SMA patients, carriers, and healthy individuals within approximately 1 hour and minutes. It directly calculates ΔCt values, eliminating the need for sophisticated data analysis tools. As a result, a cheaper, faster and more reliable diagnostic method has been developed offering a practical alternative to probe- or suppressor-based systems.

Description

[0001] SMN1 AND SMN2 SPECIFIC INA q PCR PRIMER AND PROTOCOL AND

[0002] METHOD THEREFOR

[0003] TECHNICAL FIELD

[0004] The invention relates to a diagnostic kit and method for the diagnosis of SMA. it is characterized by a probe-free assay utilizing LNA-modified oligonucleotides.

[0005] BACKGROUND ART

[0006] Spinal Muscular Atrophy (SMA) is a rare neuromuscular disorder that results in the loss of motor neurons and progressive muscle atrophy.

[0007] It is usually diagnosed in infancy or early childhood and is the most common genetic cause of infant mortality if not treated. A milder form of the disease can also occur later in life. The common feature is progressive weakness of voluntary muscles, with the arms, legs and respiratory muscles affected first.

[0008] Associated complications may include poor head control, dysphagia, scoliosis, and joint contractures. The traditional classification is based on the age of onset and severity of symptoms.

[0009] Spinal muscular atrophy is due to an abnormality in the SMN1 gene, which encodes SMN. an essential protein for the survival of motor neurons. The loss of these neurons in the spinal cord disrupts signal transmission between the brain and skeletal muscles. The SMN2 gene is regarded as a disease modifier, since a higher number of SMN2 copies typically correlates with a less severe phenotype. Diagnosis of SMA relies on clinical presentation and is confirmed by genetic tests.

[0010] The document with publication number CN111218506A states that "The invention relates to the field of molecular diagnostics, in particular to a detection kit for copy numbers of the SMN1 and SMN2 genes. The seventh and eighth exons of SMN1 and SMN2 were each simultaneously amplified in separate single-tube PCR reactions using specific primers. Additionally, one or two groups of housekeeping genes are included to monitor efficiency. PCR products are analyzed by capillary electrophoresis, and the copy numbers of the SMN1 and SMN2 genes are determined relative to a normal control with two copies of each gene. A relatively quantitative assessment is then performed to estimate the copy numbers, identifying samples with 0. 1 , 2, 3, or >4 copies. Based on these results, individuals are classified as SMA patients, carriers, or unaffected.

[0011] Starting from DNA samples, resuits can be obtained within 3 hours, the copy number status of SMN1 and SMN2 genes can be determined with only one PCR experiment, the detection technical process is simple and standardization can be easily performed."

[0012] The document with publication number CN116287192A is summarized as follows: "The invention discloses a kit integrating SMN1 and SMN2 copy numbers, small variation and family linkage analysis and the application of the kit. SMN1 and SMN2 and SMN1 pathogenic variation and a molecular genetic marker are amplified simultaneously in a PCR reaction. PCR products are analyzed by capillary electrophoresis. Based on an SMA normal control with two SMN gene copies, the copy numbers of exons 7 and 8 of the SMN1 and SMN2 genes are relatively quantified. SMN genes with 0, 1 , 2, 3, or >4 copies are calculated and used to detect corresponding copy number variations. According to the present invention, it is possible to distinguish between normal SMN gene exon copy numbers, heterozygous deletion mutations, and homozygous deletion mutations. Furthermore, the method enables detection of increased SMN2 transcription and minor pathogenic variations in the SMN1 gene. The invention also provides essential data for family-based analysis and facilitates clarification of the inheritance pattern of the SMN1 gene. Results can be obtained from a DNA sample within 2.5 hours, companion diagnostics covering all modes of genetic variation of SMA can be achieved with only a one PCR experiment, the detection technology is simple in operation, and standardization is easy to achieve." The document with publication number CN113980961A is summarized as follows: "The invention belongs to the field of gene detection and discloses a primer and probe composition for the detection of the SMN1 and SMN2 genes. The primerprobe sequences are shown as SEQUENCE ID NO. 1-8, respectively . The primary Figure 8 shows the comparison of the sequence obtained by bidirectional DNA sequence analysis of the SMN2 PCR products of the BP coded sample with the SMN2 refseq genome sequence using the NCBI Blast application.

[0013] Figure 9 is the amplification curve obtained from Real-Time PCR assay of individuals with homozygous deletion for SMN2 gene and individuals carrying 2 or more SMN2.

[0014] Figure 10 is the SMN1 amplification curves of affected children and healthy controls.

[0015] Figure 11 shows S N2 copy number graph prepared with PCR results of children with SMA.

[0016] Figure 12 shows SMN1 copy number graph obtained from PCR results of carriers with 1 copy of SMN1 gene and healthy controls carrying 2 or more SMN1 genes.

[0017] For ease of understanding, identical reference numerals are used where possible to indicate identical elements in the figures. Figures are not drawn to scale and can be simplified for clarity. It is contemplated that elements and features of an embodiment can be usefully incorporated into other embodiments without the need for further explanation.

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] The preferred alternatives of the method and the appropriate kit embodiments of invention, which are mentioned in this detailed description, are only intended for providing a better understanding of the subject-matter, and should not be construed in any restrictive sense.

[0020] The invention is able to differentiate between patients and carriers with a single PCR reaction performed under the same conditions and can differentiate between patients and carriers with 100% success using LNA primers

[0021] In the invention, the SMN2 copy number of affected children is determined with 100% success using a method that is faster and more cost-effective. The MLPA method, which is the gold standard among the currently used methods, takes 2 days and requires the use of complex analysis software, whereas the alternative method can easily distinguish between SMA patients, SMA carriers and healthy individuals in a PCR reaction that takes 1 hour and 15 minutes and calculates ACt values directly instead of using complex analysis methods and software. As a result, a cheaper, faster and more reliable method has been developed than methods using probes and suppressors. The SMN1 and SMN2 genes are involved in the pathogenesis of recessively inherited SMA disease. SMA is caused by SMN1 gene deletion or mutations.

[0022] Homozygous deletion of SMN1 exon 7 is observed in 95% of patients. SMN1 and SMN2 genes have 99% common DNA sequence.

[0023] Figure 1 shows the base differences in the exon 7-8 region of SMN1 and 2 genes, the 2 genes have only 2 base difference. The fact that there are only 2 base difference in this region, which is critical for SMA , has made it necessary to use relatively more expensive probe systems to distinguish SMN1 and 2 genes.

[0024] This region, which contains the base differences between the two genes, is crucial for SMN protein production. While all SMN proteins synthesized from the SMN1 gene are functional, only 10% of SMN2 gene products are functional.

[0025] In the PCR method, sequence-specific primers are used to target the DNA region whose presence, absence, or quantity is to be determined.

[0026] Base differences between the two sequences allow the design of sequence-specific primers and therefore the determination of which sequence is present and which is absent in the analyzed sample.

[0027] However, due to the high similarity between the SMN1 and SMN2 genes, their specific amplification by PCR has traditionally required the use of probe systems and suppressors. Conventional primers cannot distinguish between such similar regions because they may form missmatch base pairings. The invention demonstrates that a single base difference can be detected using modified LNA primers. LNA primers are chemically modified oligonucleotides that allow more specific binding and help prevent mismatch base pairing when used in PCR reactions.

[0028] However, LNA primers need to be designed and optimized according to the targeted region In our invention, LNA primers were designed to specifically amplify SMN1 and SMN2 genes The position of the primers on SMN1 and SMN2 genes is shown in Figure 2.

[0029] In the DNA sequence representation of the LNA primers that specifically amplify the SMN1 and SMN2 genes, both primers contain LNA-modified bases at their ends.

[0030] Primers containing 5' or dispersed LNA modifications are known to increase their specificity LNA primers bind to the target sequence more strongly than conventional primers and are less likely to make missmatch base pairings than conventional primers. However in case of a base mismatch, LN As cause a change in the 3D structure in the region of the base mismatch, in our invention, we aimed to provide a more specific amplification by ensuring that the 3-dimensional structure change that will occur as a result of incorrect base pairing is at the 3’ end and that DNA polymerase binding is suppressed due to the structure change. The sequences of the primers we designed for this purpose and the properties thereof are given in table 1 .

[0031] Table 1 : Table of primers. Bases indicated with sign are LNA modified bases and all primers are written in the 5 -3' direction. In the invention, SMN1 and SMN2 gene-specific LNA primers were designed. The advantages of primers for the diagnosis of SMA are listed below.

[0032] 1. Since the exon 7-8 region of the SMN1 and SMN2 genes is an A-T rich region, the primer needs to be very long in order to design a primer with a binding temperature of around 60 “C specific to this region, which reduces PCR efficiency. However, by including LNA-modified bases in the sequence, the length of the primers was kept within the range suitable for PCR and the binding temperature was increased.

[0033] 2. By adding LNA-modified bases to the 3' ends of the primers, the structure change that would occur as a result of incorrect base pairing was used as an advantage and as a result, SMN1 primers amplified only the SMN1 sequence and SMN2 primers amplified only the SMN2 sequence.

[0034] 3. The primers for beta actin, which was used as a housekeeping gene, were designed by adding LNA bases to the 3' end of the primers, thus aiming to achieve similar PCR efficiencies.

[0035] In our invention, LNA primers specific to both genes were designed and various temperatures and concentrations were tested in optimization experiments. The annealing temperature was increased as much as possible to maximize specificity and avoid nonspecific binding. Primers were able to bind to their targets from 65 ’C, but nonspecific binding was prevented by increasing the annealing temperature to 61 °C. Although primers could bind to their targets at 55 °C, nonspecific binding was eliminated by increasing the temperature to 61 °C.

[0036] Different primer concentrations were tested and reactions were optimized with the lowest concentrations to minimize cost.

[0037] The standardized PCR results after optimization are shown in Table 2. In ail experiments, 4 pl of DNA was used at a concentration of 5ng / pl.

[0038] Table 2: Table of PCR reaction components and quantities thereof. Water (uL)

[0039] The PCR program used with the determined reaction content is as indicated below in Table 3 on the program screen.

[0040] Table 3: PCR program used in the Light Cycler 480.

[0041] As a result of PCRs performed using the primers listed in Table 1 , the PCR results indicated in Table 2 and the program shown in Table 3, no amplification of S N1 was observed in individuals with homozygous deletion for SMN1. and no amplification was observed in individuals with homozygous deletion fur SMN2, i.e. no SMN2 gene, and no CT value was obtained. The finding suggests that primers and PCR conditions provide specific amplification. However, in order to confirm this finding, the products obtained as a result of PCRs using DNA from individuals carrying both SMN1 and SMN2 genes were analyzed by bidirectional DNA sequencing analysis. Representative results from the DNA sequencing analyses are presented below.

[0042] A sample carrying 3 copies of SMN1 gene and 1 copy of SMN2 gene gave CT for SMN1 and SMN2 genes in Real-time PCR experiments. The chromatograms obtained by bidirectional sequencing of SMN1 gene PCR products with F and R primers that can bind to both SMN1 and S N2 PCR products are shown in Figures 3 and 4. The SMN2 sequence obtained by reading the chromatograms is as follows.

[0043] When the sequence obtained was compared with the relevant region of the SMN2 gene, it was confirmed that they were completely identical and that the sequence obtained as a result of PCR was the SMN2 gene as shown in Figure 8. In Figure 8, in the comparison of the sequence obtained by bidirectional DNA sequence analysis of the SMN2 PCR products of the BP coded sample with the SMN2 refseq genome sequence using the NCBI Blast application, the two sequences are completely identical, the products obtained as a result of SMN2 PCR using the designed SMN2 LNA primers and optimized conditions are belonging to the S N2 gene.

[0044] The findings presented above confirm that the designed SMN1 primers amplify only the SMN1 gene and SMN2 primers amplify only the SMN2 gene. Another evidence for the specificity of the primers is the CT values and amplification curves obtained from PCR reactions.

[0045] Figure 9 shows the PCR amplification curve of individuals with different SMN2 copy numbers The green curve belongs to an individual with a homozygous deletion of the SMN2 gene. 40 cycles of PCR resulted in no amplification in this individual.

[0046] Figure 10 shows the SMN1 amplification curves of affected children and healthy controls. In healthy controls, the SMN1 gene was amplified at cycle 25, whereas in SMA patients, amplification did not occur or CT was obtained after cycle 35. In real time PCR experiments. CT values taken in the last 5 cycles are ignored and samples are considered negative. Therefore, the curves shown in Figures 9 and 10 prove the specificity of the designed primers. As a result of the optimization of the reaction content shown in Table 2 and the optimization of the PCR conditions shown in ‘Table 3, specific product amplification was achieved and gene copy numbers for SMN1 and SMN2 genes were determined using the ACt method. Table 4 shows the results of PCR using DNA samples of children with SMA, i.e. children without SMN1 gene, with homozygous deletion. As expected, ail children are negative for SMN1 . The ACt SMN2 value was calculated by subtracting the mean actin Ct value from the mean SMN2 Ct value. The SMN2 copy number indicated in the table refers to the copy number obtained as a result of LPA analysis.

[0047] Table 3: PCR results of children with SMA. SMN2 copy number refers to the number of copies obtained as a result of MLPA analysis. MLPA analysis is the gold standard method for SMN copy number determination.

Claims

CLAIMSbase of the SMN1 R primer, the marked C base, is converted to I in the SMN2 gene;

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