WO2018117986A1

WO2018117986A1 - A method for detecting a copy number of smn1 gene

Info

Publication number: WO2018117986A1
Application number: PCT/TH2016/000105
Authority: WO
Inventors: Objoon TRACHOO; Panthan BHAKBHOOM
Original assignee: Leader Medical Genetics And Genomics Co Ltd
Current assignee: Leader Medical Genetics And Genomics Co Ltd
Priority date: 2016-12-23
Filing date: 2016-12-23
Publication date: 2018-06-28
Anticipated expiration: 2019-06-23

Abstract

A method for detecting a copy number of SMN1 includes extracting genomic DNA from a biological sample of a human subject, contacting a primer of an SMN1 exon7 to the biological sample and a PCR mixture to obtain a sample mixture, performing a PCR reaction to the sample mixture for collecting a testing result, and analyzing the copy number of SMN1 of the testing result by comparing with genomic DNA controls which have nl-n4 copy number of SMN1 gene. Additionally, the disclosed embodiments may involve a kit for detecting a copy number of SMN1 which substantially comprises SMN1 primers, albumin primers, a PCR mixture and frozen DNA controls.

Description

A METHOD FOR DETECTING A COPY NUMBER OF SMNl GENE Field of Technology

The present disclosure relates to a method for detecting SMNl gene. More particularly, the present disclosure relates to a method for detecting a copy number of SMNl gene in normal phenotype population as premarital screening. In addition, the present disclosure relates to a kit for detecting a copy number of SMNl gene by using the disclosed method.

Background

Spinal muscular atrophy (SMA) is a severe autosomal recessive disease having an overall incidence of 1 in 6,000 to 10,000 live births and a carrier frequency of 1 in 40 to 50 (Tae-Mi Lee et al., J Korean Med Sci 2004; 19: 870-3). An expression of SMA is related to two genes, namely SMNl and SMN2. The former is directly used for diagnosis SMA disease, while the latter is associated with the onset age, risk of death and survival of SMA patients. Approximately 94% of SMA affected patients are associated with the homologous deletion of SMNl gene in exon7, while for the rest of 6%, it has been reported for the cause of mutations.

By having only five-base pair difference in 3' region, a false positive result from SMN2 always occurs for SMNl detection. The number of SMNl gene is detected for assessment the genetic status, for example, a number copy of 0, 1, and 2 are interpreted to an affected subject, a carrier, and a normal subject, respectively. The false negative from SMN2 therefore causes an incorrect assessment of target copy number. It is desirable to expressly determine the copy number of SMNl exon7 for providing genetic or premarital counseling to normal population for prevention a severe autosomal recessive disease as such.

Thus, there exists a need for a method for detecting a copy number of SMNl gene to eliminate contamination from pseudo-SMNl gene for achieving accurate results.

Summary of the Invention

An object of the present disclosure is aimed at a method for detecting a copy number of SMNl . The quantitative polymerase chain reaction (qPCR) is performed by using known designed SMN 1 primer and inventively comparing with a copy number control ( 1 n-n) under certain PGR protocols for removal a false negative from SMN2 and increase an accuracy of SMN1 detection. Further, the PCR result is quantifiably inteφreted by comparative cycle threshold (AACT) and relative normalized expression to the results of DNA controls. Thus, the number of SMN1 gene is precisely detected, in absence of misinterpretation of SMN2, to identify the SMN1 genetic status.

A further object of the present disclosure is to provide a kit for detecting a copy number of SMN1 for facilitating medical staffs to identify SMN1 genetic status. The kit includes SMN1 primers, albumin primers, PCR mixture, and frozen DNA controls of I , 2n, 3n, and 4n.

Brief Description of the Drawings

Embodiments of the present disclosure are described hereinafter with reference to the figures, in which:

FIGs. 1A-1D show a range of annealing temperatures for detecting the copy number of SMN1.

FIG. 2 shows a detectable gap between a true and a false positive curves at the annealing temperature of 60°C.

FIG. 3 A is a comparison of relative normalized expression between a subject having in copy comparing with ln-4n of DNA controls.

FIG. 3B is a melt curve of the subject (In).

FIG. 4 is a comparison of relative normalized expression between a subject having 2n copy comparing with l -4n of DNA controls.

FIG. 4B is a melt curve of the subject (2n).

Detailed Description

Hereinafter, the invention shall be described according to representative or preferred embodiments of the present invention and by referring to the accompanying description and drawings. However, it is to be understood the description and drawings corresponding to such embodiments are for purpose of clarity and to aid understanding, and it is envisioned that individuals having ordinary skill in the relevant art may devise various modifications without departing from the scope of the invention as defined by the appended claims. Aspects of a method for detecting a copy number of SMNl

In accordance with various embodiments, the present disclosure relates to a method for detecting a copy number of SMNl. Substantially, the method comprises the steps of extracting a genomic DNA from a biological sample of a subject; contacting an SMNl primer to the biological sample and a PCR mixture to obtain a sample mixture; performing a PCR reaction to the sample mixture in specific conditions for collecting a testing result; and analyzing the copy number of SMNl of the testing result by comparing with genomic DNA controls.

In accordance with many embodiments, a biological sample of an interested subject is mainly collected from blood, or salivary secretion for DNA extracting. For example, the peripheral blood is drawn at least 3-5 cc. to process for extracting a genomic DNA which is required 25 ng per a testing well. The DNA extraction process is operated by the QIAamp DNA mini kit (Qiagen, Germany) and is measured by a spectrophotometer sold under the trademark NanoDrop™ 2000 (Thermo Fisher Scientific, DE). Also, the real-time PCR detection system sold under the trademark CFX96 Touch™ (Bio-Rad Laboratories, Inc, USA) is used for PCR reaction.

In accordance with several embodiments, the SMNl primer has been designed to have a sequence of in exon 7 at position 6 and intron 7 at position +2: forward primer: 5'- CCTTTTATTTTCCTTACAGGGTTTC-3'; reverse primer: 5'-

GATTGTTTTACATTAACCTTTCAACTTTT-3' (Lee et al. 2004). The primers for the reference gene, exon 12 of human serum albumin are: forward primer; 5'- AGCTATCCGTGGTCCTGAAC-3 ' ; reverse primer: 5'-

TTCTC AGAAAGTGTGC AT ATATCTG-3 ' (Lee et al. 2004). Further, the designed primer as such has been contacted to the extracting DNAs of the subject and a PCR mixture for obtaining a sample mixture. In accordance with a number of embodiments, qPCR has been performed to the sample mixture under specific conditions. For example, supermix sold under the trademark SsoFast™ EvaGreen of Bio-Rad Laboratories Inc, USA, is used for PGR mixture. For the positive control of the disclosed method, each set of three wells containing 10 μL of supermix reagent, 25 ng of In genomic DNA, and 475 nM of SMN exon7 Primer together with another set of three wells by containing 10 iL of the same PCR reagent, 25 ng of In genomic DNA, and 150 nM of albumin primers for PCR control reaction, whereas the negative control PCR is performed by using DNA control of 2n-4n. The PCR reaction is operated in triplicates in the condition of initial denaturation step of 95 °C for 10 minutes, follow by 35 cycles of denaturation at 95°C for 15 seconds and 60°C for 60 seconds. The biological sample after extracting to 25 ng of genomic DNA is tested with the same environments as such to detect the copy number of SMN1. With respect to annealing temperature, at the range of 60-62°C, there is an absence of misinterpretation from SMN2 interference. As shown in FIGs. 1 A- 1D, copy number of On from an affected SMN1 patient, has been tested to verify the specific range of the annealing temperature which is able to remove the SMN2 misinterpretation. FIGs. I A- IB illustrate that the annealing temperature which is less than 60°C may introduce the false positive of SMN1 resulting in a misinterpretation of the copy number of SMN1 , whereas FIGs. 1C-1 D illustrate that at the specific range of annealing temperature, 60-62°C, there is an absence of SMN I curve. More specifically, FIG. 2 illustrates the false positive curve of SMN1 (SMN2 CT=38.98, ALB CT=24.76) which is presented after the true positive curve of SMNl fSMNl CT=24.65, ALB CT=24.77) at least 14 cycles. The wider detectable gap between the SMN1 and the false positive curves as such is advantageous to make the correctness of SMN 1 interpretation. As a result, the copy number of SMN1 is accurately interpreted to determine the genetic status of an interested subject.

In accordance with most embodiments, by using a positive control (n=l) and negative control (n > 2) for comparison with the PCR results of the subject and the controls, copy number In is indicated that the subject is an SMA carrier, whereas 2n or more of the copy number is responsible for non-carrier status. The testing result from qPCR reaction has been further evaluated on the basis of comparative cycle threshold (AACT) and relative normalized expression to determine the copy number of the subject.

For example, the CT values as shown in Table 1 is computed by the following equation (Thomas D Schmittgen, Nature Protocol, 2008) for obtaining the comparative cycle threshold (AACT) for further determining the copy number of the subject. Examples hereafter are demonstrated the normalized expression method. The values from the calculation are relatively compared in each batch of PCR.

For a copy number ln-4n, each average CT, from three values (CTl, CT2, and CT3), of the SMN1 gene in object (ln-4n) and normal (2n) samples is shown in Table 1.

Table 1 : The average CT of SMN1 gene

For a copy number ln-4n, each average CT, from three values (CTl, CT2, and CT3), of albumin (ALB) in object (ln-4n) and normal (2n) samples is shown in Table 2.

Table 2: The average CT of albumin control In 2n 3n 4n Sample (2n) average CT(SMNl) 25.47 24.85 24.12 24.30 24.91 average CT(Albumin) 24.03 24.44 24.40 24.89 24.55

Table 3: The average CT of the SMN 1 gene, albumin, and samp le (2n).

MCT

[(CT gene of interest - CT internal control) sample A - (CT gene of interest - CT internal control) sample B)]

By applying the average CT values from Table 3 to the equation, each fold change due to treatment is demonstrated as follows:

For the copy number In, fold change due to treatment _ 2"delta delta CT

_ ₇-[(25.47-24.03)-(24.85-24.44)]

= 0.48971

For copy number 2n. fold change due to treatment = 2 ^■delta delta CT

_ 2-[(24.91-24.55)-(24.85-24.44)]

1.035265

For copy number 3n. fold change due to treatment = 2 -delta delta CT _ 2-[(24.12-24.40)-(24.85-24.44)J

0.69

= 2^'

= 1.613284

For copy number 4n, fold change due to treatment = 2 ^•delta delta CT

= 2 -[(24.30-24.89)-(24.85-24.44)|

= 2'

= 2

For the normal control (2nX fold change due to treatment = 2 -delta delta CT

_>-[(24.85-24.44)H24.85-24.44)]

-o

= 2

Table 4: The quantitative value of AACT and its standard deviation (SD) comparing to subject FIG. 3A and FIG .4A demonstrate relative normalized expression a subject with an SMN carrier status. To analyze the subject result, the normalized expression is relatively comparable between the quantifiable value of the subject and the ln-4n controls. In case that the calculated subject value is closed to which copy number, it has been classified to that number. In FIG. 3A and FIG. 4A, the subject values are relevant to In, and 2n, resulting in the interpretatipn of a carrier, and a non-carrier, respectively. Further, the negative controls, 3n and 4n, present more additional advantages not only on demonstrating a non-carrier status, but also on counselling the genetic status of the descendants. For example, an offspring of 4n subject is determined for a non-carrier status only for detection copy number at least 3n, not 2n.

Additionally, the melt curve is conducted for the evidence of one amplicon. FIG. 3B and FIG. 4B show that at a starting temperature of 65°C with an increase of 0.5°C interval in each cycle until reaching 95°C, only one peak is demonstrated to verify the exist of one amplicon. In accordance with many embodiments, a method for manufacturing an application for detecting a copy number of SMN1 is started from preparing an SMN1 exon7 primer which is used for contacting with genomic DNA extracted from a biological sample of an interested subject and a PCR mixture in certain environment of PCR reaction. The sequence of the SMN1 exon7 primer may include forward primer: 5'- CCTTTTATTTTCCTTAC AGGGTTTC-3 ' ; reverse primer: 5'-

GATTGTTTTACATTAACCTTTC AACTTTT-3 ' . Further, DNA controls, which have a copy number of In, 2n, 3n, and 4n, are frozen and used for relatively evaluation of normalized expression with the subject's result. Aspects of a Kit for Detecting a Copy Number of SMN1

The present disclosure further involves a kit for detecting a copy number of SMN1. Specifically, the kit comprises a test module for DNA extracting of a subject, the S3VTN1 primers, the albumin primers, PCR mixture, and the frozen DNA controls. Additionally, a user instruction which describes the specific amount of all composition and the steps of performing PCR reaction may be included. In accordance with a number of embodiments, the sequence of SMN 1 exon7 primers is forward primer: 5 ' -CCTTTTATTTTCCTTACAGGGTTTC-3 ' ; reverse primer: 5'- GATTGTTTTACATTAACCTTTCAACTTTT-3 ' (Lee et al. 2004) at a concentration of 475 nM for detection the SMNl copy number of the subject. The designed primer has been reacted with the subject's DNA extracted from the biological sample. The amount of extracted DNA is required approximately 25 ng per a testing well.

In accordance with various embodiments, albumin primers, forward primer: 5'- AGCTATCCGTGGTCCTGAAC-3'; reverse primer: 5'-

TTCTC AGAAAGTGTGC ATATATCTG-3 ' (Lee et al. 2004), have been prepared for reassure a performance of PGR reaction. The amount of the albumin primers is required approximately 1 0 nM per a testing well.

In accordance with most embodiments, there is a specific PCR mixture compatible to the disclosed method. The PCR reagent of supermix sold under the trademark SsoFast™ EvaGreen® (Bio-Rad Laboratories, Inc, USA), is prepared for use in the amount of 10 in each well.

In accordance with embodiments, the frozen DNA controls, as positive and negative DNA controls, are used to validate the accuracy of the copy number of the subject. A copy number of In is the positive control for interpreting a carrier of SMNl, while copies number of 2n-4n are the negative controls for interpreting a non-carrier of SMN 1 .

The PCR reaction is described to perform in the specific conditions. For example, 475 nM of the SMNl exon7 primers, 150nM of the albumin primers, and 10

of the PCR mixture are run in triplicate with the initial denaturation step of 95°C for 10 minutes, followed by 35 cycles of denaturation at 95 °C for 15 seconds and 60°C for 60 seconds. However, the annealing temperature may vary from 60 to 62°C because at this specific range, the false positive curve of SMNl is occurred at least 14 cycles after the SMNl curve. The detectable gap of at least 14 cycles is able to clearly distinguish the true and the false positive curves. REPRESENTATIVE EXAMPLES EXAMPLE ONE

Experiments have been conducted to prove an interference of SMN2 by having certain PCR conditions. The new qPCR protocol of the present disclosure has been developed to influence the specificity and sensitivity of the disclosed method by reducing the possibility of the non- SMN1 primer binding occurring. Having played an important role of PCR reaction, the annealing step is optimized to allow the specific binding site by lowering the temperature after denaturation step.

For the following experiments, which have the same protocol as the present disclosure regardless the disclosed annealing temperature, the testing samples has no SMNl gene. However, an experiment has an annealing temperature of 58°C, as illustrated in FIG. 1A having the albumin curve (ALB Cq=24.35) to confirm the performance of the PCR reaction, and the subsequent curve of SMNl exon 7 (SMNl Cq=36.82) which reflects a false amplification from SMN2 gene, nearly homologous to SMNl .

Another experiment having an annealing temperature of 59°C, as shown in FIG. IB, remains a presence of a false positive from SMN2 as well.

Having an attempt to reduce the misinterpretation as such, inventors have created a range of annealing temperatures capable of a correction of SMNl copy number detection. The experiments, as shown in FIG. 1C and FIG. ID, have been conducted for gaining a higher specificity and sensitivity of the detection method. The albumin curves remain existing to ensure the PCR reaction. In absence of SMNl curve from the On sample, the range of the annealing temperature at 60-62°C is verified the actual copy number of SMNl gene. As a result, the present disclosure is able to achieve an accurate number of SMNl gene by effectively removal a false positive problem.

Results

FIG. 1A and FIG. I B show that in the condition of the annealing temperatures of 58 and 59°C, the SMNl curve remains detectable, whereas FIG. IC and FIG. I D surprisingly demonstrate the accuracy of the experimental data without a false detection of SMNl at the annealing temperatures of 60-62°C. EXAMPLE TWO

Experiments have been conducted to demonstrate a positive result interpretation of the present disclosure. A subject sample has been tested for detecting a copy number of SMN1 exon7. By following the zero false positive protocol, the result is compared with the control (ln-4n) and purely verify to one amplicon, SMN1, by means of a melt peak analysis.

The expression of a testing sample is relatively normalized to the positive and negative controls of present disclosure. Having one copy number of SMN1 gene, the positive control (In) indicates a carrier status of SMA disease. Contrary to the negative controls (n > 2), the normal genetic status has been confirmed.

The quantitative value (AACT) of the subject is relatively compared to the normal expression of ln-4n. In case that the value of the sample is in the boundary of In copy number, a positive control, the subject is classified to have only one SMN1 gene as shown in FIG. 3 A.

Table 5: The quantitative value of AACT and its standard deviation (SD) comparing, to I n subject.

After PCR process, the melt point curve analysis has been additionally performed to support that the disclosed method has one amplicon of SMN1 , as shown in FIG. 3B having only one peak at approximately 76°C. The quantitative result from the present disclosure is correctly reflected the genetic status of the In subject.

EXAMPLE THREE

Experiments have been conducted to demonstrate another negative result interpretation of the present disclosure. A subject is performed under specific PCR conditions according to the present disclosure to detect the copy number of SMN1 gene. FIG. 4A shows the two copies of SMNl of the testing subject. The bar chart of the sample is close to the range of 2n and the one melt peak of FIG. 4B is verified the existence of one amplicon of SMNl .

Table 6: The quantitative value of ΔΔΟΤ and its standard deviation (SD) comparing to 2n subject.

Claims

1. A method for detecting a copy number of an SMN1 gene comprising:

extracting genomic DNA from a biological sample of a subject;

contacting a primer of an SMN1 exon7 to the biological sample and a PC mixture to obtain a sample mixture;

performing a PCR reaction to the sample mixture for collecting a testing result; and analyzing the copy number of SMN I of the testing result by comparing with genomic

DNA controls.

2. The method of claim 1, wherein the biological sample is at least one of blood and salivary secretion.

3. The method of claim I, wherein a concentration of the SMN1 exon7 is 475 nM.

4. The method of claim I, wherein an annealing temperature of the PCR reaction is in the range of 60-62°C for achieving a detectable gap between a true positive curve and a false positive curve of the SMN1 gene.

5. The method of claim 4, wherein the detectable gap is at least 14 cycles.

6. The method of one of claims 1 and 4, wherein the PCR reaction is performed under an initial denaturation step of 95 °C for 10 min, followed by 35 cycles of a denaturation at 95°C for 15 s and 60°C for 60 s.

7. The method of claim 1, wherein the genomic DNA controls are a positive DNA control and a negative DNA control.

8. The method of one of claims 1 and 7, wherein the positive DNA control is In of the SMN gene.

9. The method of one of claims 1, 7, and 8, wherein the negative DNA controls are > 2n of the SMN1 gene, preferably 2n-4n.

10. A kit for detecting a copy number of SMN1 gene comprising:

SMN1 exon7 primers;

albumin primers;

a PCR mixture; and

frozen DNA controls,

wherein the copy number of SMN1 gene is detectable via a PCR reaction.

1 1. The kit of claim 10, wherein a sequence of the SM 1 exon7 primers is forward primer: 5 ' -CCTTTT ATTTTCCTT AC AGGGTTTC-3 ' ; reverse primer: 5'- GATTGTTTT AC ATTAACCTTTC AACTTTT-3 ' .

12. The kit of one of claims 10 and 11, wherein a concentration of the SMN1 exon7 primers is 475 nM.

13. The kit of claim 10, wherein a sequence of the albumin primers is forward primer: 5'- AGCTATCCGTGGTCCTGAAC-3 ' ; reverse primer: 5'- TTCTC AGAAAGTGTGCATATATCTG-3 ' .

14. The kit of one of claims 10 and 13, wherein a concentration of the albumin primers isl50 nM.

15. The kit of claim 10, wherein the frozen DNA controls comprises a negative control and a positive control.

16. The kit of one of claims 10 and 15, wherein the negative control is In of the SMN1 gene.

17. The kit of one of claims 10, 15, and 16, wherein the positive control is > 2n of the SMN1 gene, preferably 2n-4n.

18. The kit of claim 10, wherein an annealing temperature of the PCR reaction is in the range of 60-62°C for achieving a detectable gap between a true positive curve and a false positive curve of the SMN1 gene.

19. The kit of claim 18, wherein the detectable gap is at least 14 cycles.

20. The kit of one of claims 10,18, and 19 wherein the PCR reaction is substantially performed under an initial denaturation step of 95°C for 10 min, followed by 35 cycles of a denaturation at 95°C for 15 s and 60°C for 60 s.