WO2020244482A1 - 以smnp作为对照检测smn基因拷贝数的方法 - Google Patents

以smnp作为对照检测smn基因拷贝数的方法 Download PDF

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WO2020244482A1
WO2020244482A1 PCT/CN2020/093694 CN2020093694W WO2020244482A1 WO 2020244482 A1 WO2020244482 A1 WO 2020244482A1 CN 2020093694 W CN2020093694 W CN 2020093694W WO 2020244482 A1 WO2020244482 A1 WO 2020244482A1
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primer
smn1
smn2
smnp
sequence
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张晔
王怡慧
张奇
孟妍
朱海燕
刘玉瑛
陈初光
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北京阅微基因技术有限公司
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Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method and a detection kit for detecting the copy number of Survival of Motor Neuron (SMN) genes (Survival of Motor Neuron, SMN) in vitro.
  • SSN Motor Neuron
  • SMA Spinal muscular atrophy
  • SMA Spinal muscular atrophy
  • the carrier rate and incidence of SMA are basically the same among all races.
  • the carrier frequency is 1/40 to 1/60, and the incidence in newborns is about 4 to 10/100,000.
  • SMA is a neuromuscular disorder characterized by the degeneration of motor neuron cells in the anterior horn of the spinal cord, which can cause symmetrical and progressive muscle weakness and muscle atrophy in the limbs and trunk (Neurotherapeutics. 2008; 5:499-506) ). According to the patient’s age of onset, exercise capacity, and life span, the International SMA Federation divides typical SMA into three types: Type I, Type II, and Type III (Neuromuscular Disorders. 1991; 1(2): 81; Neuromuscular disorders. 1992; 2(5-6):423-428).
  • SMA type I onset 6 months ago, also known as Werdnig-Hoffmann disease, infantile SMA, etc. This type accounts for about 50% of all SMA patients. Children with this type have severe progressive muscle weakness and weakened muscle tone, and generally cannot sit alone. If there is no treatment, the survival period will not exceed 2 years.
  • SMA type II onset at 6 to 18 months, is also called juvenile SMA, intermediate SMA, etc. Some can sit and some can stand, but most can't walk, and often suffer from respiratory dysfunction. The general life expectancy is more than two years, and some can survive to adolescence or even longer.
  • SMA type III onset after 18 months, is also called Kugelberg-Welander disease, mild SMA, etc. It has strong clinical heterogeneity and usually develops, but the proximal muscles of infancy usually have problems. Most patients can Walking, only slightly weak. Adolescence may recur and the life expectancy is normal.
  • SMA type 1 congenital type, has severe joint contractures, facial paralysis and respiratory failure, and often has spontaneous fractures and is extremely weak. The disease started before birth and died within one month after birth.
  • SMA type IV adult type, usually onset after 35 years of age, mainly manifested by slow and gradual proximal weakness and muscle atrophy of the upper and lower limbs, and can walk in adulthood.
  • the most important pathogenic gene of SMA is located in the 5q13.2 region of chromosome 5. This region as a whole presents a huge inverted repeat structure, which also causes non-allelic homologous recombination to occur in this region, causing abnormalities such as gene deletion or duplication.
  • SMA survival motor neuron
  • the SMN gene is 20kb in full length and contains 9 exons (1, 2a, 2b, 3-8).
  • the SMN1 and SMN2 gene sequences are highly identical in all gene regions including the promoter. At present, it is found that SMN1 and SMN2 genes have only 5 base differences (rs1454173648, INS6-45, G/A; rs4916, Exon 7+6, 840th base, C/T; rs212214, INS7+100A/G; rs1244569826, INS7+215, A/G; rs1323191655, Exon8+245G/A), they are distributed in the region between intron 6 and exon 8.
  • the SMN gene transcription product is about 1.7 kb in length and encodes a 294 amino acid SMN protein, which is involved in the formation of a multi-protein complex related to RNA processing.
  • SMN protein is commonly expressed in human tissues, and the anterior horn motor neurons of the spinal cord have a higher demand for it. If the SMN protein expression level is too low, it will cause neuron death and cause muscle atrophy.
  • SMN1 can express stable and fully functional SMN protein. Although SMN2 and SMN1 gene sequences are very similar, most of the transcripts transcribed by SMN2 in vivo are not spliced correctly, and only about 10% of mRNAs are spliced correctly and translated into SMN protein with normal activity. Most of the transcripts lack the seventh exon and are called ⁇ 7SMN2. These proteins lack normal SMN protein function and will be quickly degraded. Therefore, SMN2 cannot fully compensate for the lack of SMN protein caused by SMN1 gene deletion or mutation, but its copy number will affect the disease severity of SMN1 deletion SMA patients.
  • the reason for the different splicing of SMN1 and SMN2 is that the two genes have a different base on exon 7.
  • the sixth position (Exon 7+6) of exon 7 is the 840 nucleotide in the coding region.
  • the middle is C and the SMN2 is T (840C>T).
  • This base difference is believed to affect the structure and function of the splicing enhancer in this region, and ultimately cause the difference in RNA splicing mode. Therefore, the base at this position, 840C>T, is a key base that affects whether a normal SMN protein can be produced, and it is also a key base that distinguishes the functional difference between SMN1 and SMN2.
  • SMN1 and SMN2 Due to the high sequence of SMN1 and SMN2, and their inverted repetitive structure on the chromosome, non-allelic homologous recombination may occur between the two genes. This recombination is an important reason for the deletion or duplication of the SMN gene, as well as the conversion of SMN1 and SMN2 genes.
  • SMNP survival of motor neuron 1, telomeric pseudogene
  • SMNP is located in the 9p21.3 region, with a total length of about 1643bp.
  • the mRNA homology between SMNP and SMN1 is more than 80%, and all 9 exons and the last intron have corresponding homologous fragments. It is speculated that SMNP is a pseudogene formed by reverse transcription of SMN RNA. There are no other genes within 1MB upstream and downstream of SMNP.
  • SMNP small cell genome
  • SMN1 copy number can effectively screen SMA carriers.
  • "2+0" means that the individual has 2 copies of SMN1, but the two copies are on one chromosome and there is no SMN1 copy on the other chromosome.
  • This genotype is also a carrier of SMA, and it is possible to pass chromosomes without SMN1 to the offspring, resulting in SMA patients.
  • SNP sites are linked to "2+0" in Jews, and have a certain correlation with "2+0” in some other races (Human Mutation; 2000, 15: 228).
  • the SMN1 and SMN2 genes are highly homologous, with only 5 bases different.
  • the detection needs to have good specificity, so that the detection signal of SMN1 is not affected by SMN2.
  • This method uses multiple sets of specific probes to hybridize with SMN1 exon 7 and other related positions as well as a large number of control sites, and connect and amplify. By comparing the amount of SMN1 exon 7 product with each control site, the SMN1 copy number can be quantitatively determined. Since there is no more accurate method for SMN1 copy number detection for a long time, the MLPA method is relatively the most widely used method in scientific research and clinical detection. However, this method is complicated in operation, high in cost, high in requirements for testing samples, and cumbersome data analysis. Moreover, each test requires multiple control samples to be tested at the same time, and the test results are corrected based on the results of the control samples. Improper selection of the control samples or abnormal results can also lead to detection errors.
  • qPCR has excellent quantification ability on a larger scale, but it can distinguish between 1 copy or 2 copies of a gene. Theoretically, the difference between the CT value of 1 copy and the CT value of 2 copies is only 1. It is necessary to ensure that samples with a CT difference of 1 are effectively distinguished, which requires high detection stability and repeatability.
  • qPCR uses relative quantification, and other control gene signals and SMN1 signals need to be used to calculate ⁇ CT. Ensuring that the primers and probes corresponding to SMN1 and the control gene maintain the same amplification efficiency is a prerequisite for effective detection.
  • qPCR is a feasible method, but it has higher requirements for actual quality and control of testing conditions.
  • ddPCR In addition to complex operation and high cost, ddPCR also requires the detection of internal reference genes, similar to qPCR, which requires high detection stability and repeatability.
  • This method uses a pair of common primers to amplify the relevant regions of SMN1 and SMN2 exon 7 and determine the melting curve of the product. Due to the individual base differences in the SMN1 and SMN2 sequences, the melting curve peaks of the two homozygous double-strands and hybrid double-strands are different, which will cause the two products to show a specific pattern on the melting curves.
  • the usual method is to amplify or capture the relevant regions of SMN1 and SMN2 exon 7, build a library and sequence, and determine the copy number ratio of SMN1 and SMN2 according to the ratio of 840C/T. But only this result cannot determine the final value of the copy number, and other methods need to be used to obtain the total copy number of SMN1 and SMN2.
  • the calculation of the total copy number can be determined according to a specific algorithm based on the number of reads of the sequencing results of a large number of other genes detected at the same time and the total number of reads of the SMN gene.
  • the above method can effectively detect the copy number of SMN1, but the operation is complicated, the cost is high, and the result calculation is complicated, and also requires a large number of internal reference gene detection, which requires high control of reaction conditions.
  • An object of the present invention is to provide a method for accurately detecting SMN1 and/or SMN2 gene copy number using SMNP as a control site in view of the existing problems and deficiencies of the current SMN1 and/or SMN2 gene copy number quantitative detection method.
  • the inventors used the endogenous homologous pseudogene SMNP as the reference gene, and after extensive analysis and experiments, they designed primers that can simultaneously amplify SMN1 and SMNP, or simultaneously amplify SMN2 and SMNP, or simultaneously amplify SMN1, SMN2 and SMNP.
  • primers that can simultaneously amplify SMN1 and SMNP, or simultaneously amplify SMN2 and SMNP, or simultaneously amplify SMN1, SMN2 and SMNP.
  • the target site and the control site can be amplified and detected simultaneously. In this way, the difference between different primers caused by various changes in conditions is avoided.
  • the effect on the target site and the control site is the same, that is, the amplification test results are tolerant to various condition changes and unfavorable factors, which improves quantification Capability and detection stability.
  • the present invention provides the following technical solutions.
  • the inventors compared the homology between SMN1 and SMNP sequences, and the results are shown in Figure 1. According to the results, among the 5 bases where SMN1 and SMN2 are inconsistent, the sequence near INS6-45, Exon7+6 and the upstream sequence have poor homology with SMNP sequence; the sequence near Exon8+245 also has homology with SMNP sequence. Poor; the sequence near INS7+100 and INS7+215 and the upstream sequence have good homology with the SMNP sequence.
  • the first choice for detection is the Exon7+6 (840C>T) site located on the 7th exon, because this site is a functional site that causes the different splicing modes of SMN1 and SMN2.
  • the sequences around and upstream of this site have poor homology with SMNP, and it is difficult to design primers to realize the co-amplification of SMN1 or SMN2 and SMNP. Therefore, the inventors designed detection primers based on regions with good sequence homology, especially those near INS7+100 and INS7+215.
  • SMN1 and SMN2 are A, while SMN2 is G.
  • the primers at this site can simultaneously amplify SMN1 and SMNP, but not SMN2; similarly, in INS7+ At position 215, the bases of SMN2 and SMNP are G, and SMN1 is A.
  • the primers at this position can simultaneously amplify SMN2 and SMNP, but not SMN1.
  • the copy number of SMN1 or SMN2 can be determined by comparing the relative amounts of different products.
  • the detection results reflect the copy number of INS7+100 and INS7+215, not the copy number of Exon7+6. Although these sites are very close, there is no guarantee that they will be closely linked. In fact, there will be a certain ratio of conversion between SMN1 and SMN2. If a switch happens between the detection site and Exon7+6 site, the detection result will be biased. In order to avoid the deviation of the detection result caused by the conversion, the inventor provides a method for detecting whether there is a conversion, and the first detection result can be corrected according to the conversion type. While performing conversion detection, primers can also be added to detect more relevant sites, such as other control sites and SMA-related pathogenic sites and "2+0" related sites.
  • the technical scheme provided by the present invention uses a pair of primers to simultaneously amplify the sequence of SMN1/2 containing Exon7+6 sites, and determine the copy number ratio of the two.
  • the sites that can satisfy the simultaneous amplification of SMN1, SMN2 and SMNP with the same primers are used as control sites to determine the total copy number of SMN1 and SMN2.
  • the amplified detection results can tolerate various condition changes and various interference factors, and the number of product reads corresponding to each gene can be increased. Accurate reaction template copy number ratio.
  • the present invention provides a method for detecting the copy number of motor neuron survival genes SMN1 and/or SMN2 in a target genome, wherein the method can amplify the SMN1 and/or SMN2 gene and the target region of the SMNP gene in the genome with a specific primer combination , And then use the SMNP amplified product as a reference to determine the copy number of SMN1 and/or SMN2 genes by comparing the relative amount of the amplified product.
  • the method provided by the present invention includes the following steps:
  • step 2) In the presence of the primer combination, using the genomic DNA of step 1) as a template to amplify the SMN1 and/or SMN2 gene and the target region of the SMNP gene targeted by the primer combination;
  • the specific primer combination 1 for SMN1 gene can amplify SMN1 and SMNP genes in the genome but does not amplify the target region of SMN2 gene, and the detection result can distinguish SMN1 amplification products and SMNP amplification products;
  • the specific primer combination 2 for SMN2 gene can amplify SMN2 and SMNP genes in the genome but does not amplify the target region of SMN1 gene, and the detection result can distinguish SMN2 amplification products and SMNP amplification products; and/or
  • the specific primer combination 3 for SMN1 and SMN2 genes can amplify target regions of SMN1, SMN2 and SMNP genes in the genome, and the detection result can distinguish SMN1 amplification products, SMN2 amplification products and SMNP amplification products.
  • step 3 the length and amount of the amplified product are detected, and wherein
  • SMN1 and SMNP have different lengths of amplification products; for primer combination 2, SMN2 and SMNP have different lengths of amplification products; and/or for primer combination 3, SMN1, SMN2, and SMNP have different lengths of amplification products.
  • the amplified product is detected by a method selected from the following: electrophoresis, fluorescence quantification and mass spectrometry, such as capillary electrophoresis.
  • the method of the present invention further includes detecting gene conversion between SMN1 and SMN2 genes. For example, detecting the transition between INS7+100 and Exon7+6; and/or detecting the transition between INS7+215 and Exon7+6.
  • the first primer of the primer set 1 is located in the first consensus sequence region of the SMN1 and SMNP genes, and the first primer sequence is identical or complementary to at least a part of the first consensus sequence
  • the consensus sequence is SEQ ID NO: 1 (ATGAGAATTCTAGTAGGGATGTAG)
  • the first primer sequence is preferably SEQ ID NO: 7 (GAGAATTCTAGTAGGGATG).
  • the second primer sequence of the primer set 1 is located in the second consensus sequence region of SMN1 and SMNP genes, but the sequence of the SMN2 gene in the corresponding region is inconsistent with the second consensus sequence, and the second The primer sequence is complementary or identical to at least a part of the second consensus sequence.
  • the second consensus sequence is SEQ ID NO: 2 (ATGTTAAAAAGTTGAAAGGTTAATGTAAAACA), and the second primer sequence is preferably SEQ ID NO: 6 ( ATGTTAAAAAGTTGAAAG).
  • the third primer sequence of the primer combination 2 is located in the third consensus sequence region of SMN2 and SMNP genes, but the sequence of SMN1 gene in the corresponding region is not consistent with the third consensus sequence, and the third primer The sequence is complementary or identical to at least a part of the third consensus sequence.
  • the third consensus sequence is SEQ ID NO: 3 (ACTGGTTGGTTGTGTGGAA), and the third primer sequence is preferably SEQ ID NO: 8 (TGGTTGGTTGTGTG).
  • the fourth primer sequence of the primer set 2 is located in the fourth consensus sequence region of the SMN2 and SMNP genes, and the fourth primer sequence is identical or complementary to at least a part of the fourth consensus sequence
  • the consensus sequence is SEQ ID NO: 4 (GATCTGTCTGATCGTTTCTTTAGTGGTGTCATTTA) or SEQ ID NO: 5 (AATGAGGCCAGTTATCTTCTATAAC).
  • the fourth primer sequence is preferably SEQ ID NO: 9 (GATCGTTTCTTTAGTGGTGTCAT).
  • At least one primer in the primer combination is modified or substituted with a modified base to replace a normal base.
  • the modification is selected from fluorescent group modification, phosphorylation modification, phosphorothioate modification, Locked nucleic acid modification and peptide nucleic acid modification; the primer sequence in the primer combination replaces, adds or deletes one or more nucleotides compared with the complementary sequence of the corresponding region on the template, while retaining its ability to initiate an amplification reaction.
  • the amplification in the method of the present invention is carried out by polymerase chain reaction (PCR), and the PCR amplification is carried out in one or more reaction systems.
  • PCR polymerase chain reaction
  • the present invention also provides a method for diagnosing the risk or severity of spinal muscular atrophy (SMA) in a subject or its offspring, which comprises detecting the motor neuron survival gene SMN1 and/or in the genome of the subject Copy number of SMN2.
  • SMA spinal muscular atrophy
  • the invention also provides a kit for detecting SMN1 and/or SMN2 gene copy number.
  • Figure 2A shows the results of DNA samples from SMA patients
  • Figure 2B shows the results of DNA samples from SMA carriers
  • Figure 2C shows the results of DNA samples from normal humans.
  • FIG. 3A is a graph of the detection result of a normal sample without conversion
  • FIG. 3B is a graph of the detection result of a sample with conversion.
  • Figure 5 Scatter plot of peak area ratios obtained from 2802 samples.
  • SMN1 and SMN2 were determined by ARMS (Amplification Refractory Mutation System) PCR.
  • the primers used include:
  • SMN1-F 5’AT(+G)TTAAAAAGTTGAAAG 3’ (SEQ ID NO: 6);
  • SMN1-R 5’FAM-GAGAATTCTAGTAGGGATG 3’ (SEQ ID NO: 7);
  • SMN2-F 5’TG(+G)TTGGTTGTGTG 3’ (SEQ ID NO: 8);
  • SMN2-R 5'FAM-GATCGTTTCTTTA(+G)TGGTGTCAT 3'(SEQ ID NO: 9).
  • (+G) means that the G base at this position is modified with LNA (Locked Nucleic Acid), which is used to enhance the primer binding ability and indirectly improve the specificity of the primer.
  • LNA Locked Nucleic Acid
  • SMN1-F is completely consistent with SMN1 and SMNP sequence, but not consistent with SMN2 sequence
  • SMN2-F is completely consistent with SMN2 and SMNP sequence, but not consistent with SMN1 sequence
  • SMN1-R and SMN2-R are completely consistent with SMN1, SMN2 and SMNP sequence Consistent.
  • the combination of SMN1-F and SMN1-R can specifically amplify SMN1 and SMNP, but not SMN2.
  • the SMN1 and SMNP products amplified by the two primers are 103bp and 100bp, respectively.
  • the combination of SMN2-F and SMN2-R can specifically amplify SMN1 and SMNP, but not SMN2.
  • the SMN2 and SMNP products amplified by the two primers are 293bp and 283bp, respectively.
  • the two pairs of primers can be used alone or together. Each product can be identified by detecting the size of the product.
  • the PCR amplification system also contains the following components: DNA polymerase (2G Robust, KAPA Biosystems); UDPase enzyme; amplification buffer.
  • Each amplification system includes: 5 ⁇ l of 4 primer mixture, 10 ⁇ l amplification buffer, 1 ⁇ l DNA polymerase and UDPase enzyme, 1 ⁇ l DNA of the sample to be tested, and 20 ⁇ l with sterile water. ;
  • the reaction conditions are: 50°C, 5 minutes; 95°C, 5 minutes; 30 cycles of 94°C, 30 seconds, 58°C, 30 seconds, 72°C, 30 seconds; 72°C, 10 minutes;
  • GeneMapper software including Panel, Bin, corresponding Analysis Method, internal standard files, input sample source data (.fsa file), and select the previously imported file in the relevant parameter selection column, analyze data.
  • Figure 2A shows the results of DNA samples from SMA patients
  • Figure 2B shows the results of DNA samples from SMA carriers
  • Figure 2C shows the results of normal human DNA samples.
  • the corresponding template can be determined according to the fragment size.
  • the expected sizes of SMN1 and SMNP products are 103bp and 100bp, respectively.
  • the peak area ratio of the two products reflects the ratio of the two product amounts, that is, the ratio of the corresponding initial template amount, that is, the ratio of SMN1 and SMNP copy numbers; similarly, for the two products that detect SMN2 copy numbers, SMN2 and SMNP products are expected
  • the sizes are 293bp and 283bp, respectively.
  • the ratio of the peak area of the two products reflects the ratio of the amount of the two products, that is, the ratio of the corresponding initial template amount, that is, the ratio of SMN2 and SMNP copy numbers.
  • the peak area ratios of SMN1 to SMNP and SMN2 to SMNP of the three samples were 0.00, 1.07; 0.49, 1.05; 1.03, 0.95, respectively.
  • the copy numbers of SMN1 and SMN2 in the three samples tested are: 0, 2; 1, 2; 2, 2, respectively. This result is completely consistent with the MLPA result. It shows that the method for detecting SMN1 and SMN2 gene copy numbers with SMNP as a control provided by the present invention is accurate and intuitive, does not rely on other control sites, and does not require complicated correction algorithms.
  • SMN1 and SMN2 Due to the high sequence of SMN1 and SMN2, and their inverted repetitive structure on the chromosome, non-allelic homologous recombination may occur between the two genes. This recombination is an important reason for the deletion or duplication of the SMN gene, as well as the conversion of the SMN1 and SMN2 genes. This conversion may cause the Exon 7+6 position on exon 7 to be different from other bases. The base is not chained.
  • the primers can be designed and another PCR reaction can be performed to detect whether there is a conversion. If there is a conversion, the original detection result can be corrected according to the type of conversion.
  • the following first set of primers is used to detect whether the conversion between INS7+100 and Exon7+6 occurs, including 4 primers:
  • SMN1+6 5'CATTCCTTTA G TTTCCTTACAGGGT A TC 3'(SEQ ID NO: 10);
  • SMN2+6 5'CCTT A ATTTTCCTTACAGGG A TTT 3'(SEQ ID NO: 11);
  • SMN1+100 5'HEX-TTACATTAACCTTTCAACT A TTTA 3'(SEQ ID NO: 12);
  • SMN2+100 5'HEX-ACATTAACCTTTCAAC A TTCTA 3'(SEQ ID NO: 13).
  • the second set of primers is used to detect whether the conversion between INS7+215 and Exon7+6 occurs, including 4 primers:
  • SMN1+6 5'CATTCCTTTA G TTTCCTTACAGGGT A TC 3'(SEQ ID NO: 14);
  • SMN2+6 5'CCTT A ATTTTCCTTACAGGG A TTT 3'(SEQ ID NO: 15);
  • SMN1+215 5'HEX-GTGAAAGTATGTTTCTTCCA G AT 3'(SEQ ID NO: 16);
  • SEQ ID NO: 10 has the same sequence as SEQ ID NO: 14
  • SEQ ID NO: 11 has the same sequence as SEQ ID NO: 15.
  • the two sets of primers can be amplified separately or simultaneously in the same PCR reaction.
  • SMN1+6 can specifically bind to the template whose Exon7+6 position is the C base, that is, the corresponding sequence of SMN1.
  • SMN2+6 can specifically bind to the template whose Exon7+6 position is the T base, that is, the corresponding sequence of SMN2.
  • SMN1+100 and SMN2+100 can specifically bind to the corresponding sequences of SMN1 and SMN2 at INS7+100, respectively
  • SMN1+215 and SMN2+215 can specifically bind to the corresponding sequences of SMN1 and SMN2 at INS7+215, respectively.
  • the first set of primers has different sizes for different types of template amplification products, and can be used to determine whether and what conversion occurs between INS7+100 and Exon7+6.
  • the normal SMN1 gene is C in Exon7+6, and INS7+100 is A.
  • the primers SMN1+6 and SMN1+100 can correspond to two points respectively to realize PCR amplification.
  • the size of the amplified product is 197bp; the normal SMN2 gene is in Exon7. +6 is T, INS7+100 is G, primers SMN2+6 and SMN2+100 can correspond to two points respectively, and the size of the amplified product is 191bp; if conversion occurs, Exon7+6 is T, INS7+100 is For A, the primers SMN2+6 and SMN1+100 can correspond to two points respectively, and the size of the amplified product is 193bp.
  • the SMN1 copy number calculated based on the base copy of INS7+100 will be higher than the copy number calculated based on Exon7+6.
  • Exon7+6 of the SMN gene that undergoes conversion is T, which is the same as SMN2, and it will show similar functions to SMN2 in vivo; if conversion occurs, Exon7+6 is C and INS7+100.
  • the primers SMN1+6 and SMN2+100 can correspond to two points respectively, and the size of the amplified product is 195bp.
  • the SMN1 copy number calculated by the base copy of INS7+100 will be lower than the copy number calculated by Exon7+6.
  • the converted SMN gene Exon7+6 is referred to as C, which is the same as SMN1 and exhibits similar functions to SMN1 in vivo.
  • the second set of primers can be used to determine whether and what conversion occurs between INS7+215 and Exon7+6.
  • Normal SMN1 gene, normal SMN2 gene, the size of the amplified products of type I conversion and type II conversion between the two points were 315bp, 309bp, 311bp, and 313bp.
  • the PCR amplification system also contains the following components: DNA polymerase (2G Robust, KAPA Biosystems); UDPase enzyme; amplification buffer.
  • the tested samples are human peripheral blood samples, and the copy numbers of SMN1 and SMN2 genes in each sample have been tested by the MLPA method.
  • Each amplification system includes: 5 ⁇ l of a total of 6 primer mixtures, 10 ⁇ l amplification buffer, 1 ⁇ l DNA polymerase and UDPase enzyme, 1 ⁇ l blood sample to be tested, supplemented with sterile water 20 ⁇ l;
  • the reaction conditions are: 50°C, 5 minutes; 95°C, 5 minutes; 30 cycles of 94°C, 30 seconds, 58°C, 30 seconds, 72°C, 30 seconds; 72°C, 10 minutes;
  • GeneMapper software including Panel, Bin, corresponding Analysis Method, internal standard files, input sample source data (.fsa file), and select the previously imported file in the relevant parameter selection column, analyze data.
  • the result obtained by the method in Example 1 is actually the copy number of SMN1 at INS7+100 and the copy number of SMN2 at INS7+215. And what needs to be detected is the copy number of Exon7+6 position. In most samples, the SMN gene did not switch between INS7+100 and Exon7+6, and between INS7+215 and Exon7+6. The detected result is the copy number of SMN1 and SMN2 at Exon7+6. . However, if conversion occurs, the copy number obtained by the method of Scheme 1 is inconsistent with the copy number of Exon7+6 locus and needs to be corrected.
  • the sample corresponds to the sample.
  • the result of the detection of the sample by the method in Embodiment 1 is 1 copy of the SMN1 gene and 2 copies of the SMN2 gene.
  • the detection result of the detection system of this embodiment shows that the sample has a type II conversion at the corresponding position.
  • the copy number of SMN1 gene in this sample is 2 (or more), and the copy number of SMN2 gene is 1 (the result of the detection conversion system has SMN2 products, so it will not be less than 1).
  • the copy number of SMN1 in this sample is 1, and it will be misjudged as an SMA carrier. After correction, the SMN copy number of this sample is 2 (or more), which is normal.
  • the copy number of exon 7 of the SMN1 gene in this sample is 2, and the copy number of exon 8 of this sample is 1.
  • the copy number of exon 7 of SMN2 gene is 1, and the copy number of exon 8 is 2. This result is consistent with our revised result.
  • the SMN gene of the sample has indeed undergone conversion, so the copy numbers of exons 7 and 8 are inconsistent.
  • the SMN gene copy number is detected through two PCR amplification detection reactions. One test is used to detect the copy number of SMN1 and SMN2, and the other test is used to detect whether there is a conversion between SMN1 and SMN2.
  • other sites have been added to the system, including SMA-related pathogenic SNPs, "2+0" related sites, and other control sites.
  • the primers of the first system include:
  • the primers of the second system include:
  • the first system is used to detect SMN1 and SMN2 copy numbers.
  • three pairs of primers were also set to amplify the sex chromosome locus Amel and two STR sites D5S818 and TH01. In addition to their routine control functions, they can also monitor whether samples are contaminated and prevent sample confusion.
  • the second system is used to detect whether a conversion occurs between SMN1 and SMN2.
  • three control sites (Amel, D5S818 and TH01) corresponding primers are also set. It also sets up primers for amplifying the full length of exons 1, 2a, 2b, 3, 4, 5, 6, and 8 of the SMN gene, which can detect whether there are mutations of exon length changes (such as Exon1 22insA mutation) , Exon8g.27706-27707 del AT mutation).
  • Some relatively high incidence of pathogenic SNP sites and ARMS primers corresponding to "2+0" related sites are also set, which can be used with primers for amplifying exons to achieve the detection of target pathogenic SNPs.
  • pathogenic SNP sites are based on the relatively high incidence of pathogenic sites included in the OMIM database and the relatively high incidence of pathogenic sites reported in the literature (BMC Medical Genetics.2012.13:86).
  • All SMA-related sites detected include: SMN1 gene 22insA, 683T>A, 400G>A, 689C>T, 830A>G, 835-1G>A, 863G>T, 5C>G, 305G>A, 815A>G , 821C>T, 785G>T, 399_402 del AGAG, g.27134T>G, g.27706-27707 del AT.
  • the PCR amplification system also contains the following components: DNA polymerase (2G Robust, KAPA Biosystems); UDPase enzyme; amplification buffer.
  • Each sample is amplified with two sets of primers in two reactions.
  • the specific detection steps are the same as in Example 2.
  • Figure 4A shows the test result of the first system of one of the samples. Comparing the peak areas of the corresponding products, it can be seen that the copy number of SMN1 is 2 and the copy number of SMN2 is 1.
  • Figure 4B shows the test result of the second system of the same sample. There is no conversion for this sample. Therefore, the copy number result obtained by the first system is an accurate result and does not need to be corrected.
  • the carrier frequency is 1.36%, which is basically the same as reported in the literature.
  • the abscissa is the ratio of SMN1 and SMNP product peaks amplified simultaneously with SMN1-F and SMN1-R primers; the ordinate is the ratio of SMN2 to SMNP product peaks amplified simultaneously with SMN2-F and SMN2-R primers.
  • the data points corresponding to each sample are concentrated in several regions, and the boundaries between the regions are clear. Especially the points near 0.5 on the abscissa are clearly distinguished from other data points with larger abscissas. This shows that the test results have excellent performance in distinguishing 1 copy of SMN1 gene (SMA carriers) and 2 or more copies (normal people). Choosing SMNP as a control is the fundamental reason for the excellent quantitative and distinguishing ability of the test.
  • the technical solution provided by the present invention can be combined with other detection methods, such as NGS detection.
  • a pair of primers were used to simultaneously amplify the sequence of SMN1/2 containing Exon7+6 sites, and determine the copy number ratio of the two.
  • the sites that can satisfy the simultaneous amplification of SMN1, SMN2 and SMNP with the same primers are used as control sites to determine the total copy number of SMN1 and SMN2.
  • the amplified detection results can tolerate various condition changes and various interference factors, and the number of product reads corresponding to each gene can be increased. Accurate reaction template copy number ratio.
  • the target region includes SMN1, SMN2 and SMNP highly homologous regions, SMN1, SMN2 base regions with differences, SMN1/2 and some other gene exons and The area within a certain range of its upstream and downstream.
  • the primer sequence information is as follows:
  • the above primers cover all exons and part of the upstream and downstream sequences of SMN1/2.
  • primers There are 4 pairs of more special primers in the primers, SMA-E3-F/R, SMA-I7-F/R, SMA-I7-F1/SMA-E8-R, SMA-E8_1-F/R, they can use the same The primers, with the same binding capacity, simultaneously amplify SMNP at the SMN1 and SMN2 sites and the control site.
  • Kit Use KAPA's Hyper Prep Kit to construct a library for the target region products amplified by the first step primers.
  • kit instructions to fill in and add A, linker connection, magnetic bead purification, library amplification, and library purification to complete the library construction of the target region of the sample.
  • the Illumina high-throughput sequencing platform is used for library sequencing, and NextSeq 500 System, Mid Output Flow Cell is used for PE150 sequencing.
  • routine data analysis including:
  • NGSQCToolkit Version 2.3.3 Use data processing software to perform quality control on sequencing data (reads), and remove reads whose sequencing is below the quality requirements (CutOffReadLen 80, CutOffQualScore 20);
  • Sequencing depth statistics using perl scripts to count the number of reads at specific locations.
  • the SMA-I7-F/R amplification product contains the INS7+100 site.
  • SMN1 and SMN2 products can be distinguished according to the base of this position; SMA-I7-F1/SMA-E8-R amplification
  • the product contains the INS7+215 site.
  • the SMN1 and SMN2 products can be distinguished based on the base of this position; the SMA-E8_1-F/R amplification product contains Exon8+245 site, which distinguishes SMNP based on sequence On the basis, the SMN1 and SMN2 products can be distinguished according to the base of this position; SMA-E3-F/R does not contain the difference between SMN1 and SMN2, it is impossible to distinguish between the two, only SMNP can be distinguished according to the sequence; SMA-E7-F/R The amplified product contains Exon7+6 site, and SMN1 and SMN2 products can be distinguished based on the base of this position.
  • Exon7+6 locus that really affects the function of SMN gene.
  • the copy number detected by other sites such as INS7+100 is generally consistent with the copy number results of Exon7+6 sites, but if the SMN1 and SMN2 genes are converted (such as sample 4), the measurement results will be biased.
  • the total copy number of SMN1 and SMN2 can be obtained from the sequencing results of the three sets of primers SMA-I7-F/R, SMA-I7-F1/SMA-E8-R, and SMA-E8_1-F/R. From the sequencing results of SMA-E7-F/R primers at Exon7+6 sites, the ratio of SMN1 and SMN2 copy numbers can be obtained. Combining the total copy number of SMN1 and SMN2 and the copy number ratio, the exact copy number of SMN1 and SMN2 at Exon7+6 site can be obtained.
  • the total copy numbers of SMN1 and SMN2 were the same at 4 sites where SMNP was amplified simultaneously.
  • the total number of copies of the 4 samples are: 2, 4, 4, and 3.
  • the copy number ratios of SMN1 and SMN2 of the 4 samples were 0:N, 1:3, 1:1, 2:1.

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Abstract

本发明提供一种检测目标基因组中运动神经元存活基因SMN1和/或SMN2拷贝数的方法，包括：以特异性引物组合能够扩增基因组中SMN1和/或SMN2基因以及SMNP基因的目标区域，然后以SMNP扩增产物为参照，通过比较扩增产物相对量确定SMN1和/或SMN2基因的拷贝数。本发明还提供了检测SMN1和/或SMN2拷贝数的试剂盒。

Description

以SMNP作为对照检测SMN基因拷贝数的方法
本申请要求于2019年06月06日提交中国专利局、申请号为201910492012.3的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，特别涉及一种体外检测运动神经元存活基因(Survival of Motor Neuron，SMN)拷贝数的方法及检测试剂盒。
背景技术
脊髓性肌萎缩症，Spinal muscular atrophy(SMA)，是一种较为常见的常染色体隐性遗传病。SMA的携带率和发病率在各人种中基本相同，携带者频率1/40～1/60，新生儿中发病率约4～10/100,000。
SMA是一种神经肌肉紊乱疾病，以脊髓前角运动神经元细胞退行性变为特征，会导致肢体和躯干出现对称性、进行性的肌无力和肌萎缩(Neurotherapeutics.2008；5:499-506)。根据患者的发病年龄、运动能力和寿命等，国际SMA联合会将典型SMA分为I型、II型和III型等3个型别(Neuromuscular Disorders.1991；1(2):81；Neuromuscular disorders.1992；2(5-6):423-428)。
SMA I型，6个月前发病，也被称为Werdnig-Hoffmann病、婴儿期SMA等。该型占所有SMA患者人数的约50％。该型患儿有严重的进行性的肌无力与肌张力减弱，一般不能独坐，如果没有治疗一般生存期不会超过2年。
SMA II型，6至18个月发病，也被称为青少年型SMA、中间型SMA等。有一些能坐，部分能站，但是大多不能行走，常出现呼吸功能障碍。一般寿命超过两岁，部分可存活至青春期甚至更长。
SMA III型，18个月后发病，也被称为Kugelberg-Welander病、轻型SMA等，有很强的临床异质性，通常能发育，但是婴儿期近端肌肉通常会出问题，多数患者能行走，只是有轻微无力。青春期病情有可能出现反复，寿命多正常。
在典型的SMA I、II和III型之外还有0型和IV型。
SMA 0型，先天型，有严重的关节挛缩、面瘫和呼吸衰竭，常出现自发性 骨折和极其瘦弱。出生前就开始发病，生后一个月内死亡。
SMA IV型，成人型，一般于35岁以后发病，主要表现为缓慢逐渐发生的上下肢近端无力和肌肉萎缩，成年期都能够行走。
SMA最主要的致病基因定位于五号染色体5q13.2区域。该区域整体呈现为一个巨大的反向重复结构，这也导致该区域内容易发生非等位基因间同源重组，造成基因缺失或重复等异常。
在该区域上的两个高度同源的基因被命名为运动神经元存活基因(survival motor neuron,SMN)。研究发现，靠近端粒的SMN1是SMA的致病性基因。如果两个SMN1基因拷贝全部缺失或带有致病性突变则会导致SMA；靠近中心体的SMN2不是SMA致病性基因，但其拷贝数与SMA临床表现严重程度相关(Cell 80:155-165,1995)。
SMN基因全长20kb，含有9个外显子(1、2a、2b、3-8)。SMN1和SMN2基因序列在包括启动子在内的全部基因区域高度一致。目前发现SMN1和SMN2基因只有5个碱基的区别(rs1454173648，INS6-45，G/A；rs4916，Exon 7+6，第840碱基，C/T；rs212214，INS7+100A/G；rs1244569826，INS7+215，A/G；rs1323191655，Exon8+245G/A)，它们分布在内含子6到外显子8之间的区域内。SMN1和SMN2基因所有外显子序列仅存在两个碱基不同，其一是外显子7中的一个不同碱基，为同义突变；其二是外显子8中的一个不同碱基，位于终止密码子后，对蛋白编码无影响。因此，SMN1和SMN2所编码的氨基酸序列是完全一致的。
SMN基因转录产物长约1.7kb，编码294个氨基酸的SMN蛋白，参与构成与RNA加工有关的多蛋白复合体。SMN蛋白在人体组织中普遍表达，脊髓前角运动神经元对其需求较高，若SMN蛋白表达水平过低，会使神经元死亡，导致肌肉的萎缩。
SMN1可以表达稳定、完整的功能的SMN蛋白。虽然SMN2与SMN1基因序列极为相近，但SMN2在体内转录出的大部分转录产物没有正确剪接，仅有约10％的mRNA拼接正确并翻译出具有正常活性SMN蛋白。大部分转录本缺失了第七外显子，被称作Δ7SMN2，这些蛋白缺乏正常SMN蛋白功能且会被很快降解。因此SMN2的不能完全代偿SMN1基因缺失或突变带来的SMN蛋白的不足，但其拷贝数量会影响SMN1缺失SMA患者的疾病严重程度。
造成SMN1和SMN2剪接不同的原因，在于两基因在7号外显子上的一个碱基不同，外显子7的第六位(Exon 7+6)即编码区的840位核苷酸，在SMN1 中为C而在SMN2中为T(840C>T)。该碱基差异被认为影响了该区域剪切增强子的结构和功能，并最终造成RNA剪接方式的不同。所以该位置的碱基，840C>T，是影响能否产生正常SMN蛋白的关键碱基，也是区分SMN1和SMN2功能差异的关键碱基。
由于SMN1和SMN2序列高度一致，及它们在染色体上反向重复的结构，两基因之间有可能发生非等位基因间同源重组。这种重组是造成SMN基因发生缺失或重复的重要原因，也会造成SMN1与SMN2基因发生转换(conversion)。
人类基因组中，与SMN1和SMN2外显子区域同源性最高的是SMNP(survival of motor neuron 1,telomeric pseudogene)。SMNP位于9p21.3区域，全长约1643bp。SMNP与SMN1的mRNA同源性超过80％，全部9个外显子和最后一个内含子都有对应同源片段。推测SMNP是SMN RNA逆转录形成的假基因。SMNP上下游1MB范围内无其他基因。SMNP所在区域没有类似SMN1和SMN2所在基因组区域的反向重复结构，所以不会发生非等位基因间同源重组及引起拷贝数变化。目前SMNP基因没有明确的功能报道，也不与SMA疾病相关。
95～98％的SMA患者存在SMN1第7外显子和/或第8外显子的纯合缺失(Hum.Genet.12:1015-1023,2004)，另外约5％的患者存在SMN1杂合缺失且仅存的SMN1拷贝上存在致病性突变或其他未知致病原因(Am J.Hum.Genet.64:1340-1356,1999)。因此，定量检测SMN1基因第7外显子拷贝数是SMA基因筛查和产前分子诊断的主要策略。
对SMN1拷贝数的检测，可有效对SMA携带者进行筛查。但SMA携带者中的“2+0”型，目前还没有有效的检测方法。“2+0”指，个体带有2个拷贝的SMN1，但两个拷贝位于一条染色体上，另一条染色体上没有SMN1拷贝。这种基因型也是SMA携带者，有可能将不含SMN1的染色体传给子代，产生SMA患者。有文献报道了一些SNP位点在犹太人中与“2+0”连锁，且在其他一些人种中有与“2+0”有一定相关性(Human Mutation；2000,15:228)。
对于SMN1拷贝数检测的特殊性和困难在于：
1.要能有效区分0拷贝(患者)，1拷贝(携带者)和2个及以上拷贝(正常人)。如果只能区分有和无，则只能用于患者确诊，不能用于携带者筛查。为了携带者筛查，尤其需要准确、稳定的定量检测能力，有够有效区分1和2拷贝。
2.SMN1和SMN2基因高度同源，两者仅有5个碱基不同。检测需要有良好的特异性，使SMN1检测信号不受SMN2影响。
当前已有的，对SMN1拷贝数检测的方法主要有：
1.MLPA(Multiplex Ligation-dependent probe amplification)
该方法使用多组特异性探针与SMN1外显子7和其他相关位置以及大量对照位点位置杂交，并连接扩增。通过比较SMN1外显子7产物量与各个对照位点产物量，可定量确定SMN1拷贝数。由于长期以来对SMN1拷贝数检测没有更为准确的方法，MLPA方法是科研和临床检测上相对应用最广泛的方法。但该方法操作复杂，成本高，对待检测样本要求高，数据分析繁琐。而且每次检测要求同时检测多个对照样本，根据对照样本结果对检测结果进行修正，对照样本选取不当或结果异常也会导致检测错误。
2.qPCR
qPCR在较大尺度具备优秀的定量能力，但在区分基因的1个拷贝或2个拷贝上定量能力一般。理论上，1拷贝的CT值和2拷贝的CT值差异只有1，要保证有效区分CT差异为1的样本，对检测稳定性和重复性要求很高。另外qPCR采用的是相对定量，需要用其他对照基因信号与SMN1信号共同计算ΔCT。保证SMN1和对照基因所对应的引物和探针保持同样的扩增效率是检测有效的前提，如果检测条件发生变化(包括扩增条件、体系成分和体积、样本浓度和纯度等)，不同引物和探针保持的扩增效率会产生差异，进而影响检测结果。总之，qPCR是一个可行的方法，但对实际质量、检测条件控制等要求较高。
3.ddPCR(Droplet Digital PCR)
该方法定量能力较好，可有效对SMN1拷贝数进行检查。除了操作复杂、成本高外，ddPCR也需要对内参基因进行检测，类似于qPCR，对检测稳定性和重复性要求很高。
4.高分辨熔解曲线HRM
该方法以一对共同的引物扩增SMN1和SMN2外显子7相关区域，对产物测定熔解曲线。由于SMN1和SMN2序列上个别碱基差异，两种纯合双链和杂合双链的熔解曲线峰值不同，会导致两种产物在熔解曲线上表现出特定的样式。该方法的问题是，能定量确定SMN1和SMN2的拷贝数比例，但不能最终确定数值。如无法区分SMN1:SMN2＝1:2或者2:4，而且对SMN2拷贝数为0的样本也无法检测。为了解决这些问题，还需要引入其他检测，如确定SMN1 和SMN 2总拷贝数等。由于SMN1和SMN2拷贝数组合较多，区分不同的熔解曲线样式也较为不便及易发生错误。
5.NGS(Next Generation Sequencing)
通常的方法是扩增或捕获SMN1和SMN2外显子7相关区域，建库测序，根据840C/T比例确定SMN1和SMN2的拷贝数比例。但只有这个结果无法确定拷贝数最终数值，还需要通过其它方法获得SMN1和SMN 2的总拷贝数。总拷贝数的计算可以根据同时检测的大量其他基因的测序结果reads数与SMN基因总reads数，按照特定算法进行确定。上述方法可以有效检测SMN1拷贝数，但操作复杂、成本较高、结果计算复杂，且同样需要大量内参基因检测，对反应条件控制要求较高。
此外还有Sanger测序、单链构象多态性分析(PCR-SSCP)、变性高效液相色谱分析(DHPLC)等方法，这些方法都存在定量能力低、稳定性差、操作繁琐、有可能需要其他检测修正结果等问题，不适于大规模临床检测使用。因此，迫切需要一种快速准确定量SMN1和/或SMN2基因拷贝数的检测方法。
发明内容
本发明的一个目的是针对目前SMN1和/或SMN2基因拷贝数定量检测方法存在的问题与不足，提供一种以SMNP作为对照位点准确检测SMN1和/或SMN2基因拷贝数的方法。
发明人以内源同源假基因SMNP作为参照基因，经过大量分析和试验，设计出能够同时扩增SMN1和SMNP，或同时扩增SMN2和SMNP，或同时扩增SMN1、SMN2和SMNP的引物，实现了以同样的引物，以同等的结合能力，对目的位点和对照位点同时扩增检测。这样就避免的不同引物受各种条件变化所引起的差异。无论反应条件、体系成分浓度、抑制剂等因素如何影响扩增效率，对目的位点和对照位点的影响是一致的，即，扩增检测结果耐受各种条件变化和不利因素，提升定量能力和检测稳定性。
为了实现SMN拷贝数的检测，首先需要区分SMN1和SMN2基因，两者在整个基因区域内只有5个碱基不同，只能通过这5个碱基体来区分。为实现这一目的，本发明提供如下技术方案。
发明人比较了SMN1与SMNP序列的同源性，结果如图1所示。根据结果可知，SMN1和SMN2不一致的5个碱基中，INS6-45、Exon7+6位点附近序列及上游序列与SMNP序列同源性差；Exon8+245位点附近序列与SMNP序列 同源性也较差；INS7+100和INS7+215附近序列及上游序列与SMNP序列同源性较好。
5个差异位点中，检测目标首选的是位于第7外显子上的Exon7+6(840C>T)位点，因为该位点是导致SMN1和SMN2剪切方式不同的功能性位点。但是该位点周边和上游的序列与SMNP同源性较差，难以设计引物实现对SMN1或SMN2与SMNP的共同扩增。因此，发明人以序列同源性较好的区域，尤其是INS7+100和INS7+215位点附近序列，设计检测引物，
既能同时扩增SMNP，也能区分SMN1和SMN2。具体地，在INS7+100位点，SMN1和SMNP的碱基是A，而SMN2为G，该位点的引物可以实现同时扩增SMN1和SMNP，但不扩增SMN2；类似地，在INS7+215位点，SMN2和SMNP的碱基是G，而SMN1为A，该位点的引物可以实现同时扩增SMN2和SMNP，但不扩增SMN1。通过比较不同产物的相对量即可确定SMN1或SMN2的拷贝数。
但是，对INS7+100和INS7+215位点的检测，其检测结果体现的是INS7+100和INS7+215位点的拷贝数，而不是Exon7+6位点的拷贝数。虽然这些位点距离很近，但并不能保证它们一定紧密连锁。事实上，SMN1和SMN2之间会有一定比例的转换(conversion)。如果恰好在检测位点和Exon7+6位点间发生转换，则检测结果会有偏差。为了避免转换引起的检测结果偏差，发明人提供了一个检测是否存在转换的方法，可以根据转换类型对第一个检测结果进行修正。在进行转换检测的同时，还可以增加引物检测更多的相关位点，如其它对照位点和SMA相关的致病性位点以及“2+0”相关位点等。
本发明提供的技术方案使用一对引物同时扩增SMN1/2包含Exon7+6位点的序列，确定两者拷贝数比例。同时，以能够满足以相同的引物同时扩增SMN1、SMN2和SMNP的位点作为对照位点，用以确定SMN1和SMN2的总拷贝数。由于是以同样的引物，以同等的结合能力，对三个基因相应区域同时扩增检测，扩增检测结果可以耐受各种条件变化和各种干扰因素，各基因对应产物reads数能更为准确的反应模板拷贝数比例。而且这样只用少量位点即可有效确定SMN1和SMN2的总拷贝数，不用使用大量对照位点，降低了体系复杂度、降低了成本，而且不必使用较为复杂的算法对结果进行修正。
本发明提供一种检测目标基因组中运动神经元存活基因SMN1和/或SMN2拷贝数的方法，其中所述方法以特异性引物组合能够扩增基因组中SMN1和/或SMN2基因以及SMNP基因的目标区域，然后以SMNP扩增产物为参照，通过比较扩增产物相对量确定SMN1和/或SMN2基因的拷贝数。
本发明提供的方法包括如下步骤：
1)提供含有目标基因组DNA的样品；
2)在所述引物组合存在下，以步骤1)所述基因组DNA为模板，扩增引物组合所针对的SMN1和/或SMN2基因以及SMNP基因的目标区域；和
3)检测扩增产物，以SMNP扩增产物为参照，确定SMN1和/或SMN2基因在目标基因组中的拷贝数；
其中，针对SMN1基因的所述特异性引物组合1能够扩增基因组中SMN1和SMNP基因但不扩增SMN2基因的目标区域，并且检测结果能够区分SMN1扩增产物和SMNP扩增产物；
针对SMN2基因的所述特异性引物组合2能够扩增基因组中SMN2和SMNP基因但不扩增SMN1基因的目标区域，并且检测结果能够区分SMN2扩增产物和SMNP扩增产物；和/或
针对SMN1和SMN2基因的所述特异性引物组合3能够扩增基因组中SMN1、SMN2和SMNP基因的目标区域，并且检测结果能够区分SMN1扩增产物、SMN2扩增产物和SMNP扩增产物。
在一个实施方案中，在步骤3)中检测扩增产物的长度和量，并且其中
对于引物组合1，SMN1和SMNP的扩增产物长度不同；对于引物组合2，SMN2和SMNP的扩增产物长度不同；和/或对于引物组合3，SMN1、SMN2和SMNP的扩增产物长度不同。
其中，在步骤3)中通过选自以下的方法检测扩增产物：电泳、荧光定量和质谱，例如毛细管电泳。
在一个实施方案中，本发明的方法还包括检测SMN1和SMN2基因之间的基因转换。例如检测INS7+100位点和Exon7+6位点之间的转换；和/或检测INS7+215位点和Exon7+6位点之间的转换。
在一个实施方案中，所述引物组合1的第一引物位于SMN1和SMNP基因的第一共有序列区域，并且该第一引物序列与所述第一共有序列的至少一部分是一致的或互补的，例如所述共有序列是SEQ ID NO:1(ATGAGAATTCTAGTAGGGATGTAG)，所述第一引物序列优选为SEQ ID NO:7(GAGAATTCTAGTAGGGATG)。
在一个实施方案中，其中所述引物组合1的第二引物序列位于SMN1和SMNP基因的第二共有序列区域，但SMN2基因在对应区域的序列与该第二共 有序列不一致，并且所述第二引物序列与所述第二共有序列的至少一部分是互补的或一致的，例如所述第二共有序列是SEQ ID NO:2(ATGTTAAAAAGTTGAAAGGTTAATGTAAAACA)，所述第二引物序列优选为SEQ ID NO:6(ATGTTAAAAAGTTGAAAG)。
在一个实施方案中，所述引物组合2的第三引物序列位于SMN2和SMNP基因的第三共有序列区域，但SMN1基因在对应区域的序列与该第三共有序列不一致，并且所述第三引物序列与所述第三共有序列的至少一部分是互补的或一致的，例如所述第三共有序列是SEQ ID NO:3(ACTGGTTGGTTGTGTGGAA)，所述第三引物序列优选为SEQ ID NO:8(TGGTTGGTTGTGTG)。
在一个实施方案中，所述引物组合2的第四引物序列位于SMN2和SMNP基因的第四共有序列区域，并且该第四引物序列与所述第四共有序列的至少一部分是一致的或互补的，例如所述共有序列是SEQ ID NO:4(GATCTGTCTGATCGTTTCTTTAGTGGTGTCATTTA)或SEQ ID NO:5(AATGAGGCCAGTTATCTTCTATAAC)所述第四引物序列优选为SEQ ID NO:9(GATCGTTTCTTTAGTGGTGTCAT)。
本发明提供的方法中，所述引物组合中的至少一条引物添加有修饰或以修饰碱基取代正常碱基，例如所述修饰选自荧光基团修饰、磷酸化修饰、硫代磷酸化修饰、锁核酸修饰和肽核酸修饰；所述引物组合中的引物序列与模板上对应区域的互补序列相比替换、增加或删除一个或更多个核苷酸，同时保留其引发扩增反应的能力。
本发明方法所述扩增通过聚合酶链式反应(PCR)进行，所述PCR扩增在1或更多个反应体系中进行。
本发明还提供一种诊断受试者或其后代中脊髓性肌萎缩症(SMA)发生风险或严重程度的方法，其包括检测所述受试者的基因组中运动神经元存活基因SMN1和/或SMN2的拷贝数。
本发明还提供一种检测SMN1和/或SMN2基因拷贝数的试剂盒。
附图说明
图1，SMN1与SMNP的序列同源性比较。“*”表示SMN1与SMNP序列一致的碱基，方框表示SMN1与SMN2不一致的5个碱基，下划线表示SMN1外显子7和外显子8对应序列。
图2，以SMNP为对照，通过ARMS PCR确定SMN1和SMN2拷贝数检测结果。图2A为SMA患者DNA样本检测的结果，图2B为SMA携带者DNA样本检测的结果，图2C为正常人DNA样本检测的结果。
图3，SMN1和SMN2之间是否发生转换的检测结果。图3A为没有发生转换的正常样本的检测结果图，图3B为发生了转换的样本的检测结果图。
图4，通过两个扩增检测反应对SMN基因拷贝数及SMA相关位点进行检测的结果。
图5，2802例样本检测得到的峰面积比值散点图。
具体实施方式
实施例1
以SMNP为对照，通过ARMS(Amplification Refractory Mutation System)PCR确定SMN1和SMN2拷贝数。
所使用的引物包括：
SMN1-F：5’AT(+G)TTAAAAAGTTGAAAG 3’(SEQ ID NO:6)；
SMN1-R：5’FAM-GAGAATTCTAGTAGGGATG 3’(SEQ ID NO:7)；
SMN2-F：5’TG(+G)TTGGTTGTGTG 3’(SEQ ID NO:8)；
SMN2-R：5’FAM-GATCGTTTCTTTA(+G)TGGTGTCAT 3’(SEQ ID NO:9)。
“(+G)”表示该位置G碱基带有LNA(Locked Nucleic Acid)修饰，用于增强引物结合能力，并间接提升引物特异性。
其中SMN1-F与SMN1和SMNP序列完全一致，但与SMN2序列不一致；SMN2-F与SMN2和SMNP序列完全一致，但与SMN1序列不一致；SMN1-R和SMN2-R与SMN1、SMN2和SMNP序列全一致。
SMN1-F与SMN1-R组合，可以实现特异性地扩增SMN1和SMNP，但不会扩增SMN2，两条引物扩增得到的SMN1与SMNP产物大小分别为103bp和100bp。SMN2-F与SMN2-R组合，可以实现特异性地扩增SMN1和SMNP，但不会扩增SMN2，两条引物扩增得到的SMN2与SMNP产物大小分别为293bp和283bp。两对引物可以单独使用也可一起使用。各产物可以通过检测产物大小进行识别。
除引物外，PCR扩增体系中还包含以下组分：DNA聚合酶(2G Robust，KAPA Biosystems)；UDPase酶；扩增缓冲液。
对3个样本进行检测，包括一例SMA患者(SMN1拷贝数为0)，一例携带者(SMN1拷贝数为1)，和一例正常人(SMN1拷贝数为2)，各样本SMN1和SMN2基因拷贝数已经过MLPA方法测定。
具体检测步骤如下：
1)收集外周血样本，提取基因组DNA；
2)配制PCR扩增反应体系，每个扩增体系包括：4条引物的混合液5μl，扩增缓冲液10μl，DNA聚合酶及UDPase酶1μl，待检样本DNA 1μl，以无菌水补足20μl；
3)PCR扩增，反应条件为：50℃，5分钟；95℃，5分钟；30个循环的94℃，30秒、58℃，30秒、72℃，30秒；72℃，10分钟；
4)将扩增产物进行毛细管电泳；
5)数据分析，向GeneMapper软件中导入相关文件，包括Panel、Bin、对应的Analysis Method、内标文件，输入样品源数据(.fsa文件)，在相关参数选择栏中选定之前导入的文件，分析数据。
毛细管电泳结果见图2。其中图2A为SMA患者DNA样本检测的结果，图2B为SMA携带者DNA样本检测的结果，图2C为正常人DNA样本检测的结果。
如图2所示，在预期大小范围内有若干产物峰，可以根据其片段大小确定其对应的模板。对于检测SMN1拷贝数的两个产物，SMN1和SMNP产物预期大小分别为103bp和100bp。两产物峰峰面积比例反映两者产物量比例，也就是对应起始模板量的比例，即SMN1和SMNP拷贝数的比例；类似地，对于检测SMN2拷贝数的两个产物，SMN2和SMNP产物预期大小分别为293bp和283bp bp。两产物峰峰面积比例反应两者产物量比例，也就是对应起始模板量的比例，即SMN2和SMNP拷贝数的比例。
三个样本的SMN1与SMNP峰面积比及SMN2与SMNP峰面积比分别为：0.00、1.07；0.49、1.05；1.03、0.95。已知基因组中SMNP拷贝数为2，由此可以得出所测三个样本中SMN1和SMN2拷贝数分别为：0、2；1、2；2、2。该结果与MLPA结果完全一致。表明本发明提供的以SMNP为对照检测SMN1和SMN2基因拷贝数的方法准确、直观，不依赖其他对照位点、不需要复杂的 校正算法。
实施例2
检测SMN1和SMN2之间是否发生转换
由于SMN1和SMN2序列高度一致，及它们在染色体上反向重复的结构，两基因之间有可能发生非等位基因间同源重组。这种重组是造成SMN基因发生缺失或重复的重要原因，也会造成SMN1和SMN2基因发生转换(conversion)，这种转换有可能会造成外显子7上的Exon 7+6位置与其他差异碱基不连锁。可通过设计引物，进行另一个PCR反应来检测是否存在转换，如果存在转换，可以根据转换类型对原先的检测结果进行修正。
下面第一组引物用于检测INS7+100和Exon7+6之间是否发生转换，包括4条引物：
SMN1+6：5’CATTCCTTTA GTTTCCTTACAGGGT ATC 3’(SEQ ID NO:10)；
SMN2+6：5’CCTT AATTTTCCTTACAGGG ATTT 3’(SEQ ID NO:11)；
SMN1+100：5’HEX-TTACATTAACCTTTCAACT ATTTA 3’(SEQ ID NO:12)；
SMN2+100：5’HEX-ACATTAACCTTTCAAC ATTCTA 3’(SEQ ID NO:13)。
第二组引物用于检测INS7+215和Exon7+6之间是否发生转换，包括4条引物：
SMN1+6：5’CATTCCTTTA GTTTCCTTACAGGGT ATC 3’(SEQ ID NO:14)；
SMN2+6：5’CCTT AATTTTCCTTACAGGG ATTT 3’(SEQ ID NO:15)；
SMN1+215：5’HEX-GTGAAAGTATGTTTCTTCCA GAT 3’(SEQ ID NO:16)；
SMN2+215：5’HEX-GAAAGTATGTTTCTTCC TCAC 3’(SEQ ID NO:17)。其中，SEQ ID NO:10与SEQ ID NO:14的序列一致，SEQ ID NO:11与SEQ ID NO:15的序列一致。
以下划线标注的是人为设计的与基因组序列不一致的碱基，用于增加引物特异性及均衡扩增效率。
两组引物可以分别完成扩增，也可在同一个PCR反应中同时完成扩增。
其中，SMN1+6可特异性结合Exon7+6位为C碱基的模板，即SMN1对应序列。SMN2+6可特异性结合Exon7+6位为T碱基的模板，即SMN2对应序列。类似地，SMN1+100和SMN2+100可分别特异性结合SMN1和SMN2在INS7+100位置对应序列，SMN1+215和SMN2+215可分别特异性结合SMN1和SMN2在INS7+215位置对应序列。
第一组引物对不同类型的模板扩增产物大小不同，可以用于确定INS7+100和Exon7+6之间是否发生转换及发生何种转换。
正常SMN1基因在Exon7+6位为C，INS7+100位为A，引物SMN1+6和SMN1+100可分别对应两位点，实现PCR扩增，扩增产物大小为197bp；正常SMN2基因在Exon7+6位为T，INS7+100位为G，引物SMN2+6和SMN2+100可分别对应两位点，扩增产物大小为191bp；如果发生转换，Exon7+6位为T，INS7+100位为A，引物SMN2+6和SMN1+100可分别对应两位点，扩增产物大小为193bp。此时以INS7+100位置碱基拷贝计算SMN1拷贝数，会比以Exon7+6位点计算的拷贝数高。我们称此为I型转换，发生转换的SMN基因Exon7+6位为T，与SMN2分型相同，体内会表现出类似SMN2的功能；如果发生转换，Exon7+6位为C，INS7+100位为G，引物SMN1+6和SMN2+100可分别对应两位点，扩增产物大小为195bp。此时以INS7+100位置碱基拷贝计算SMN1拷贝数，会比以Exon7+6位点计算的拷贝数低。我们称此为II型转换，发生转换的SMN基因Exon7+6位为C，与SMN1分型相同，体内会表现出类似SMN1的功能。
类似地，第二组引物可以用于确定INS7+215和Exon7+6之间是否发生转换及发生何种转换。正常SMN1基因、正常SMN2基因、两位点间发生I型转换、II型转换的扩增产物大小依次为315bp、309bp、311bp、313bp。
除引物外，PCR扩增体系中还包含以下组分：DNA聚合酶(2G Robust，KAPA Biosystems)；UDPase酶；扩增缓冲液。
所检测的样本为人外周血样本，各样本SMN1和SMN2基因拷贝数已经通过MLPA方法检测。
具体检测步骤如下：
1)收集外周血样本；
2)配制PCR扩增反应体系，每个扩增体系包括：共6条引物的混合液5μl，扩增缓冲液10μl，DNA聚合酶及UDPase酶1μl，待检血液样本1μl，以无菌水 补足20μl；
3)PCR扩增，反应条件为：50℃，5分钟；95℃，5分钟；30个循环的94℃，30秒、58℃，30秒、72℃，30秒；72℃，10分钟；
4)将扩增产物进行毛细管电泳；
5)数据分析，向GeneMapper软件中导入相关文件，包括Panel、Bin、对应的Analysis Method、内标文件，输入样品源数据(.fsa文件)，在相关参数选择栏中选定之前导入的文件，分析数据。
电泳结果见图3。
其中图3A所示检测结果中，在检测INS7+100和Exon7+6之间是否发生转换的区域(panel“CONV I”)，只有SMN1和SMN2对应的产物，没有转换对应的产物；在检测INS7+215和Exon7+6之间是否发生转换的区域(panel“CONV II”)，也是只有SMN1和SMN2对应的产物，没有转换对应的产物。此结果表明该样本不存在转换。
其中图3B所示检测结果中，在检测INS7+100和Exon7+6之间是否发生转换的区域，除了SMN1和SMN2对应的产物，还有II型转换对应产物；在检测INS7+215和Exon7+6之间是否发生转换的区域，除了SMN1和SMN2对应的产物，也有II型转换对应产物。此结果表明该样本存在转换，发生在INS7+100和Exon7+6之间，是II型转换。
实施例1中方法所检测得到的结果，实际上是SMN1在INS7+100位置的拷贝数，及SMN2在INS7+215位置上的拷贝数。而需要检测的是Exon7+6位置的拷贝数。在大多数样本中，SMN基因在INS7+100与Exon7+6位点间、INS7+215与Exon7+6位点间没有发生转换，所检测的结果就是SMN1和SMN2在Exon7+6位置的拷贝数。但如果发生了转换，方案1方法得到的拷贝数就与Exon7+6位点拷贝数不一致，需要进行修正。
如本实施例中图B对应样本，该样本以实施例1中方法检测得到的结果为，SMN1基因1拷贝，SMN2基因2拷贝。本实施例检测体系检测结果表明，该样本在相应位置有II型转换。也就是说，存在Exon7+6位为C(SMN1分型)，INS7+100位为T(SMN2分型)，以及Exon7+6位为C(SMN1分型)，INS7+215位为T(SMN2分型)。此时以INS7+100位置碱基拷贝计算SMN1拷贝数，会比以Exon7+6位点计算的拷贝数低，以INS7+215位置碱基拷贝计算SMN2拷贝数，会比以Exon7+6位点计算的拷贝数高。
结合转换结果进行修正，该样本SMN1基因拷贝数为2(或更多)，SMN2 基因拷贝数为1(检测转换体系结果中有SMN2产物，所以不会比1更少)。
如果不进行修正，该样本SMN1拷贝数为1，会被误判为SMA携带者。经过修正，该样本SMN拷贝数为2(或更多)，是正常人。
经MLPA方法验证，此样本SMN1基因第7外显子拷贝数为2，第8外显子拷贝数为1。SMN2基因第7外显子拷贝数为1，第8外显子拷贝数为2。此结果与我们修正后的结果一致。而且从MLPA结果也可看出该样本SMN基因确实发生了转换，所以才会造成第7、8外显子拷贝数不一致。
实施例3
同时检测SMN1和SMN2基因拷贝数和各种SMA相关位点
如上所述，通过两个PCR扩增检测反应完成对SMN基因拷贝数进行检测。其中一个检测用于检测SMN1和SMN2拷贝数，另一个检测用于检测SMN1和SMN2之间是否发生转换。此外在体系中还增加了其他位点，包括SMA相关的致病性SNP，“2+0”相关位点，其它对照位点等。
第一体系的引物包括：
Figure PCTCN2020093694-appb-000001
Figure PCTCN2020093694-appb-000002
第二体系的引物包括：
Figure PCTCN2020093694-appb-000003
Figure PCTCN2020093694-appb-000004
Figure PCTCN2020093694-appb-000005
第一体系用于检测SMN1和SMN2拷贝数。除去实施例1中使用的4条用于确定拷贝数的引物外，还设置了三对引物，分别扩增性染色体基因座Amel，以及两个STR位点D5S818和TH01。它们除了常规对照作用外，还可以监控样本是否发生污染及防止样本混淆。
第二体系用于检测SMN1和SMN2之间是否发生转换。除去实施例2中用于检测INS7+100和Exon7+6之间是否发生转换的4条引物外，也设置了三个对照位点(Amel、D5S818和TH01)对应引物。还设置了用于扩增SMN基因第1、2a、2b、3、4、5、6、8外显子全长的引物，可以检测是否存在外显子长度变化类型的突变(如Exon1 22insA突变、Exon8g.27706-27707 del AT突变)。还设置了一些相对高发的致病性SNP位点和“2+0”相关位点所对应的ARMS引物，可以和扩增外显子的引物一起实现对目的致病性SNP的检测。
致病性SNP位点选择依据是，OMIM数据库中收录的相对高发的致病性位点，及文献报道的相对高发的致病性位点(BMC Medical Genetics.2012.13:86)。“2+0”相关位点g.27134T>G和Exon8g.27706-27707 del AT，源于参考 文献(Human Mutation(2000)15:228)。
所有检测的SMA相关位点包括：SMN1基因22insA、683T>A、400G>A、689C>T、830A>G、835-1G>A、863G>T、5C>G、305G>A、815A>G、821C>T、785G>T、399_402 del AGAG、g.27134T>G、g.27706-27707 del AT。
除引物外，PCR扩增体系中还包含以下组分：DNA聚合酶(2G Robust，KAPA Biosystems)；UDPase酶；扩增缓冲液。
所检测的样本共2802例，为新生儿外周血样本。
每个样本用两个反应分别以两组引物进行扩增。具体检测步骤同实施例2。
图4A为其中一个样本第一体系检测结果。比较相应产物峰峰面积可知，其SMN1拷贝数为2，SMN2拷贝数为1。
图4B为同一样本第二体系检测结果。该样本不存在转换。故第一体系得到的拷贝数结果就是准确结果，无需修正。特殊地，我们发现在5C>G对应位置有产物峰(箭头所示)，提示该样本存在5C>G致病性突变。其他对照位点正常，也没有其他致病性突变目的峰。
2802例样本中，检出II型转换4例，未检出I型转换。
经过修正，2082例样本中，检出携带者38例，其余为正常人。携带者频率为1.36％，与文献报道基本一致。
2082例样本中，检出致病性突变1例。即图4所示样本检出5C>G突变。
2082例样本中，未能通过两个“2+0”相关位点检测出任何“2+0”样本。推测犹太人中“2+0”相关位点对应的单倍型，在中国人群中不存在或频率极低。
对所有携带者样本、转换样本、致病性突变样本，以及随机选取的50例正常样本，进行了MLPA或测序验证，所有结果全部一致。
以2802例样本第一体系检测得到的峰面积比值绘制散点图，如图5所示。
其中横坐标为，以SMN1-F和SMN1-R引物同时扩增的SMN1与SMNP产物峰的比值；纵坐标为以SMN2-F和SMN2-R引物同时扩增的SMN2与SMNP产物峰的比值。
如图可见，各样本对应的数据点集中分布于几个区域，且各区域间分界明确。尤其是横坐标0.5附近的点，与横坐标更大的其他数据点区分非常明确。这说明检测结果对与区分SMN1基因1拷贝(SMA携带者)和2个或以上拷贝(正常人)，性能优异。选择SMNP作为对照，是检测展现优秀的定量能力 和区分能力的根本原因。
实施例4
基于NGS平台检测SMN1和SMN2拷贝数
本发明提供的技术方案可以与其它检测方法结合应用，如NGS检测。使用一对引物同时扩增SMN1/2包含Exon7+6位点的序列，确定两者拷贝数比例。同时，以能够满足以相同的引物同时扩增SMN1、SMN2和SMNP的位点作为对照位点，用以确定SMN1和SMN2的总拷贝数。由于是以同样的引物，以同等的结合能力，对三个基因相应区域同时扩增检测，扩增检测结果可以耐受各种条件变化和各种干扰因素，各基因对应产物reads数能更为准确的反应模板拷贝数比例。而且这样只用少量位点即可有效确定SMN1和SMN2的总拷贝数，不用使用大量对照位点，降低了体系复杂度、降低了成本，而且不必使用较为复杂的算法对结果进行修正。
1.样本目标区域PCR扩增
设计引物对目标区域进行PCR扩增，得到目标区域产物，目标区域包括SMN1、SMN2及SMNP高度同源区域，SMN1、SMN2有差异的碱基区域，SMN1/2以及其它一些基因的外显子及其上下游一定范围内的区域。
引物序列信息如下表：
Figure PCTCN2020093694-appb-000006
Figure PCTCN2020093694-appb-000007
以上引物覆盖了SMN1/2所有外显子及部分上下游序列。
引物中较为特殊的引物有4对，SMA-E3-F/R、SMA-I7-F/R、SMA-I7-F1/SMA-E8-R、SMA-E8_1-F/R，它们可以以同样的引物，以同等的结合能力，对SMN1和SMN2位点以及对照位点SMNP同时扩增。
1.样本目标区域文库构建
使用KAPA公司的Hyper Prep Kit，对通过第一步引物扩增得到的目标区域产物进行文库构建。按照试剂盒说明书描述步骤进行补平加A、接头连接、磁珠纯化、文库扩增以及文库纯化，完成对样本目标区域的文库构建。
2.测序
采用Illumina高通量测序平台进行文库测序，使用NextSeq 500 System，Mid Output Flow Cell进行PE150测序。
3.结果分析
首先进行常规数据分析，包括：
使用数据处理软件(NGSQCToolkit Version 2.3.3)对测序数据(reads)进 行质控，去除测序低于质量要求(CutOffReadLen 80，CutOffQualScore 20)的reads；
使用比对软件(BWA Version 0.7.15-r1140)将测序reads比对至参考基因组(hg19)；
测序深度(depth)统计，使用perl脚本统计特定位置reads数。
主要分析以下5对引物的reads数：能同时扩增SMN1、SMN2和SMNP的四对引物(SMA-E3-F/R、SMA-I7-F/R、SMA-I7-F1/SMA-E8-R、SMA-E8_1-F/R)以及扩增外显子7的SMA-I7-F/R。这些引物扩增产物的reads对确定SMN1和SMN2拷贝数提供直接信息。其他所有位点检测结果与致病性突变有关，与确定SMN1和SMN2拷贝数无关。
SMA-I7-F/R扩增产物包含INS7+100位点，在根据序列区分SMNP基础上，可根据该位置碱基区分SMN1和SMN2产物；SMA-I7-F1/SMA-E8-R扩增产物包含INS7+215位点，在根据序列区分SMNP基础上，可根据该位置碱基区分SMN1和SMN2产物；SMA-E8_1-F/R扩增产物包含Exon8+245位点，在根据序列区分SMNP基础上，可根据该位置碱基区分SMN1和SMN2产物；SMA-E3-F/R不包含SMN1和SMN2差异位点，无法区分两者，只能根据序列区分SMNP；SMA-E7-F/R扩增产物包含Exon7+6位点，可根据该位置碱基区分SMN1和SMN2产物。
检测的4例样本在这5个位点的reads数统计如下表：
Figure PCTCN2020093694-appb-000008
Figure PCTCN2020093694-appb-000009
真正影响SMN基因功能的是Exon7+6位点。通过INS7+100等其他位点检测得到的拷贝数在一般情况下与Exon7+6位点拷贝数结果一致，但如果SMN1和SMN2基因发生了转换(如样本4)，测定结果会有偏差。
由SMA-I7-F/R、SMA-I7-F1/SMA-E8-R、SMA-E8_1-F/R这三组引物的测序结果可得到SMN1和SMN2总拷贝数。由Exon7+6位点的SMA-E7-F/R引物测序结果可以得到SMN1和SMN2拷贝数的比例。结合SMN1和SMN2拷贝数总数和拷贝数比例，即可得到SMN1和SMN2在Exon7+6位点的准确拷贝数。
根据4个样本的reads数，计算得到相应reads数比例，结果如下表所示：
Figure PCTCN2020093694-appb-000010
Figure PCTCN2020093694-appb-000011
所检测4个样本，在4个同时扩增SMNP的位点，得到的SMN1和SMN2总拷贝数都一致。4个样本总拷贝数依次为：2、4、4、3。
根据SMA-E7-F/R引物测序结果，得到4个样本的SMN1与SMN2拷贝数比例依次为：0:N、1:3、1:1、2:1。
综合SMN1和SMN2总拷贝数和拷贝数比例，可以简单地得到，4给样本SMN1拷贝数和SMN2拷贝数依次为：0/2、1/3、2/2、2/1。
以上结果与MLPA结果完全一致。
由于使用了同时扩增SMNP的位点，使得SMN1和SMN2总拷贝数检测更准确且简单，不必再使用额外的对照位点及复杂的算法修正。

Claims (28)

  1. 一种检测目标基因组中运动神经元存活基因SMN1和/或SMN2拷贝数的方法，其中所述方法以特异性引物组合能够扩增基因组中SMN1和/或SMN2基因以及SMNP基因的目标区域，然后以SMNP扩增产物为参照，通过比较扩增产物相对量确定SMN1和/或SMN2基因的拷贝数。
  2. 权利要求1的方法，其包括
    1)提供含有目标基因组DNA的样品；
    2)在所述引物组合存在下，以步骤1)所述基因组DNA为模板，扩增引物组合所针对的SMN1和/或SMN2基因以及SMNP基因的目标区域；和
    3)检测扩增产物，以SMNP扩增产物为参照，确定SMN1和/或SMN2基因在目标基因组中的拷贝数；
    其中，针对SMN1基因的所述特异性引物组合1能够扩增基因组中SMN1和SMNP基因但不扩增SMN2基因的目标区域，并且检测结果能够区分SMN1扩增产物和SMNP扩增产物；
    针对SMN2基因的所述特异性引物组合2能够扩增基因组中SMN2和SMNP基因但不扩增SMN1基因的目标区域，并且检测结果能够区分SMN2扩增产物和SMNP扩增产物；和/或
    针对SMN1和SMN2基因的所述特异性引物组合3能够扩增基因组中SMN1、SMN2和SMNP基因的目标区域，并且检测结果能够区分SMN1扩增产物、SMN2扩增产物和SMNP扩增产物。
  3. 权利要求2的方法，其中在步骤3)中检测扩增产物的长度和量，并且其中
    对于引物组合1，SMN1和SMNP的扩增产物长度不同；
    对于引物组合2，SMN2和SMNP的扩增产物长度不同；和/或
    对于引物组合3，SMN1、SMN2和SMNP的扩增产物长度不同。
  4. 权利要求2或3的方法，其中在步骤3)中通过选自以下的方法检测扩 增产物：电泳、荧光定量和质谱，例如毛细管电泳。
  5. 权利要求2的方法，其中在步骤3)中检测扩增产物的序列和量。
  6. 权利要求1至5任一项的方法，还包括检测SMN1和SMN2基因之间的基因转换。
  7. 权利要求2至6任一项的方法，其中所述引物组合1的第一引物位于SMN1和SMNP基因的第一共有序列区域，并且该第一引物序列与所述第一共有序列的至少一部分是一致的或互补的，例如所述共有序列是SEQ ID NO:1(ATGAGAATTCTAGTAGGGATGTAG)。
  8. 权利要求2至7的方法，其中所述引物组合1的第二引物序列位于SMN1和SMNP基因的第二共有序列区域，但SMN2基因在对应区域的序列与该第二共有序列不一致，并且所述第二引物序列与所述第二共有序列的至少一部分是互补的或一致的，例如所述第二共有序列是SEQ ID NO:2(ATGTTAAAAAGTTGAAAGGTTAATGTAAAACA)。
  9. 权利要求2至8任一项的方法，其中所述引物组合2的第三引物序列位于SMN2和SMNP基因的第三共有序列区域，但SMN1基因在对应区域的序列与该第三共有序列不一致，并且所述第三引物序列与所述第三共有序列的至少一部分是互补的或一致的，例如所述第三共有序列是SEQ ID NO:3(ACTGGTTGGTTGTGTGGAA)。
  10. 权利要求2至9任一项的方法，其中所述引物组合2的第四引物序列位于SMN2和SMNP基因的第四共有序列区域，并且该第四引物序列与所述第四共有序列的至少一部分是一致的或互补的，例如所述共有序列是SEQ ID NO:4(GATCTGTCTGATCGTTTCTTTAGTGGTGTCATTTA)或SEQ ID NO:5(AATGAGGCCAGTTATCTTCTATAAC)。
  11. 权利要求2至10任一项的方法，其中所述引物组合1的第一引物和第二引物分别包含SEQ ID NO:6和SEQ ID NO:7所示序列或由其组成；所述引物组合2的第三引物和第四引物分别包含SEQ ID NO:8和SEQ ID NO:9所示序列或由其组成。
  12. 权利要求6至11任一项的方法，其中所述引物组合进一步包括引物组合4和/或引物组合5，用于检测SMN1和SMN2之间的基因转换；
    例如所述引物组合4能够检测INS7+100位点和Exon7+6位点之间的转换；所述引物组合5能够检测INS7+215位点和Exon7+6位点之间的转换。
  13. 权利要求12的方法，其中所述引物组合4包含至少4条引物，所述4条引物分别包含SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示序列或由其组成；所述引物组合5包含至少4条引物，所述4条引物分别包含SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示序列或由其组成。
  14. 权利要求1至13任一项的方法，其中所述引物组合中的至少一条引物添加有修饰或以修饰碱基取代正常碱基，例如所述修饰选自荧光基团修饰、磷酸化修饰、硫代磷酸化修饰、锁核酸修饰和肽核酸修饰。
  15. 权利要求1至14任一项的方法，其中所述引物组合中的引物序列与模板上对应区域的互补序列相比替换、增加或删除一个或更多个核苷酸，同时保留其引发扩增反应的能力。
  16. 权利要求1至15任一项的方法，其中所述扩增通过聚合酶链式反应(PCR)进行。
  17. 权利要求16的方法，其中所述PCR扩增在1或更多个反应体系中进行，例如1、2、3、4或5个反应体系，每个所述PCR反应体系各自的引物选自引物组合1、引物组合2、引物组合3、引物组合4和引物组合5，或它们的 组合。
  18. 一种诊断受试者或其后代中脊髓性肌萎缩症(SMA)发生风险或严重程度的方法，其包括
    使用权利要求1至17任一项所述方法检测所述受试者的基因组中运动神经元存活基因SMN1和/或SMN2的拷贝数。
  19. 一种检测SMN1和/或SMN2基因拷贝数的试剂盒，其包含
    针对SMN1基因的引物组合1，其能够扩增基因组中SMN1和SMNP基因但不扩增SMN2基因的目标区域，并且检测结果能够区分SMN1扩增产物和SMNP扩增产物；
    针对SMN2基因的引物组合2，其能够扩增基因组中SMN2和SMNP基因但不扩增SMN1基因的目标区域，并且检测结果能够区分SMN2扩增产物和SMNP扩增产物；和/或
    针对SMN1和SMN2基因的引物组合3，其能够扩增基因组中SMN1、SMN2和SMNP基因的目标区域，并且检测结果能够区分SMN1扩增产物、SMN2扩增产物和SMNP扩增产物。
  20. 权利要求19的试剂盒，其中所述引物组合1包含第一引物和第二引物，
    所述第一引物位于SMN1和SMNP基因的第一共有序列区域，并且该第一引物序列与所述第一共有序列的至少一部分是一致的或互补的，例如所述共有序列是SEQ ID NO:1(ATGAGAATTCTAGTAGGGATGTAG)，以及
    所述第二引物序列位于SMN1和SMNP基因的第二共有序列区域，但SMN2基因在对应区域的序列与该第二共有序列不一致，并且所述第二引物序列与所述第二共有序列的至少一部分是互补的或一致的，例如所述第二共有序列是SEQ ID NO:2(ATGTTAAAAAGTTGAAAGGTTAATGTAAAACA)。
  21. 权利要求19或20的方法，其中所述引物组合2包含第三引物和第四引物，
    所述第三引物序列位于SMN2和SMNP基因的第三共有序列区域，但 SMN1基因在对应区域的序列与该第三共有序列不一致，并且所述第三引物序列与所述第三共有序列的至少一部分是互补的或一致的，例如所述第三共有序列是SEQ ID NO:3(ACTGGTTGGTTGTGTGGAA)，以及
    所述第四引物序列位于SMN2和SMNP基因的第四共有序列区域，并且该第四引物序列与所述第四共有序列的至少一部分是一致的或互补的，例如所述共有序列是SEQ ID NO:4(GATCTGTCTGATCGTTTCTTTAGTGGTGTCATTTA)或SEQ ID NO:5(AATGAGGCCAGTTATCTTCTATAAC)。
  22. 权利要求19至21任一项的试剂盒，其中所述第一引物和第二引物分别包含SEQ ID NO:6和SEQ ID NO:7所示序列或由其组成；所述第三引物和第四引物分别包含SEQ ID NO:8和SEQ ID NO:9所示序列或由其组成。
  23. 权利要求19至22任一项的试剂盒，进一步包括引物组合4和/或引物组合5，用于检测SMN1和SMN2之间的基因转换；
    例如所述引物组合4能够检测INS7+100位点和Exon7+6位点之间的转换；所述引物组合5能够检测INS7+215位点和Exon7+6位点之间的转换。
  24. 权利要求23的试剂盒，其中所述引物组合4包含至少4条引物，所述4条引物分别包含SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示序列或由其组成；所述引物组合5包含至少4条引物，所述4条引物分别包含SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示序列或由其组成。
  25. 权利要求19至24任一项的试剂盒，进一步包括引物组合6，所述引物组合6能够检测脊髓性肌萎缩症相关的Amel基因及两个STR位点D5S818和TH01，所述引物组合6对应的核苷酸序列如下表
    Figure PCTCN2020093694-appb-100001
    Figure PCTCN2020093694-appb-100002
  26. 权利要求19至25任一项的试剂盒，其中至少一条引物添加有修饰或以修饰碱基取代正常碱基，例如所述修饰选自荧光基团修饰、磷酸化修饰、硫代磷酸化修饰、锁核酸修饰和肽核酸修饰。
  27. 权利要求19至26任一项的试剂盒，其中至少一条引物的序列与模板上对应区域的互补序列相比替换、增加或删除一个或更多个核苷酸，同时保留其引发扩增反应的能力。
  28. 权利要求19至27任一项的试剂盒，其还包含使用说明。
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