GB2435928A

GB2435928A - Spinal muscular atrophy screening

Info

Publication number: GB2435928A
Application number: GB0604674A
Authority: GB
Inventors: Yi-Ning Su
Original assignee: BIONET CORP
Current assignee: BIONET CORP
Priority date: 2006-03-08
Filing date: 2006-03-08
Publication date: 2007-09-12
Also published as: GB0604674D0

Abstract

A method of determining susceptibility of to spinal muscular atrophy (SMA) comprises analysing the survival motor neuron (SMN) locus to determine the copy number of the SMN gene, and the presence and copy number of a mutated SMN gene. The method is able to determine the relative copy numbers of the SNM1 and SNM2 genes. Preferably, the method uses DNA denaturing high performance liquid chromatography (DHPLC). Primers and kits for carrying out the method are provided.

Description

1 2435928

METHOD OF DETERMINING SUSCEPTIBILITY TO SPINAL

MUSCULAR ATROPHY (SMA) AND THE RELATED PRIMER PAIRS

1. Field of the Invention

The present invention relates to a gene detection method, particularly to a method for detection of survival motor neuron (SMN) genes and spinal muscular atrophy (SMA) carriers.

2. Description of Related Art

Spinal muscular atrophy results from mutation of the survival motor neuron gene on human chromosome 5q13, and the mutation causes the degeneration of spinal cord anterior horn cells that are also called motor neurons, and results in muscular atrophy symptom. The muscular atrophy symptoms are initially manifested as muscular atrophy of the palms and fmgers, and then progressively move into the shoulder, neck, tongue and respiratory system. A patient dies from the inability to swallow and respiratory failure. In Taiwan, the disease occurs approximately 1 in 10,000 live births and has a carrier frequency of 1 to 3%.

When one of the alleles is mutant, the other allele may produce sufficient proteins with normal function to express a normal physical condition.

The foregoing condition is called autosornal recessive disorder. SMA is an autosomal recessive disorder. The carrier expresses a normal phenotype but the genotype is heterozygote. When parental genotypes are both heterozygote, the genotypes of the offspring may be a homozygote and express SMA symptom symptom.

The allele of chromosome 5 in a normal human is two highly homologous SMN genes including an SMN1 gene that is near telomeric genes and an SIvIN2 gene that is near centrornic genes. Only a few people have an SMN1 gene without having an SM1'2 gene. The SMNl and SMN2 genes have the following differences. (1) They have 5 base pairs (bp) different at the 3-end.

(2) Both Slv[Nl and SMTh42 genes will translate a protein.

However, the mRNA transcribed from an SMIN2 gene often loses exon 7, and the protein translated from the above mRNA would be unstable. If deletion or transvertion between SMN 1 and SMN2 genes and leads to a loss of an SMN 1 gene in a person, the person will display the SMA symptom. The expression of the SMN2 gene is related to the intensity of the symptom. There are three types of SMA symptom. SMA type I displays most acute SMA symptom and both SMN1 genes on the alleles of SMA type I patients are lost.

About 95% SMA patients are SMA type I patients and have SMN1 gene with deletion at exon 7 and exon 8. SMA type II and SMA type III are different in the mutation or deletion of the SMN 1 gene. Therefore, the copy numbers of SMN1 gene and SMN2 gene will affect the level of SMA disease.

The most well known method to clinically detect SMA symptom is PCR-RFLP (polymerase chain reaction restriction enzyme fragment length polymorphism). The fragment amplified by the PCR includes SMN1 and SMN2 genes, but only the SMN2 gene fragment can be recognized and cut by a specific restriction enzyme. The SMN 1 gene fragment cannot be recognized and cut by the specific restriction enzyme. Therefore, after treating with the specific restriction enzyme, the amplified gene fragments are separated by electrophoresis The method for detecting SMA is time-consuming.

Another method to detect the SMA symptom includes automatically sequencing the SMA nucleotide sequence by an auto-sequencer and fmdmg the nucleotides difference between the SMN I gene and the SMN2 gene. The method depends on the auto-sequencer for detecting the difference between two genes. However, the cost of the apparatus and materials is very high, and the apparatus must also be operated by a highly trained and skilled technician.

Therefore, the method cannot be at many medical facilities.

The medical cost of SMA symptom is very expensive to the patient, the patient's family and ultimately to the whole society. The conventional detecting method only confirms the existence of the SMA symptom but do not identify SMA carriers. Therefore, a method is still required that can be rapidly, correctly and inexpensively to find a mutant gene of SMA symptom, to give inherit information, or to detect a carrier The current invention is particularly useful for detecting the SMA symptom.

An aspect of the present invention is related to a method of determining the susceptibility of an individual to spinal muscular atrophy (S MA), comprising obtaining a DNA sample from an individual to be assessed, analyzing the nucleotides present at the locus of a survival motor neuron (S MN) gene, identifying the copy number of the survival motor neuron gene and, if a survival motor neuron gene mutation is present, identifying the copy number of the mutated gene, and determining the susceptibility of the individual to SMA, wherein the presence of the survival motor neuron gene mutation and the copy number of the mutated survival motor neuron gene are indicative of a greater likelihood of susceptibility to SMA in the individual as compared with an individual without the mutation.

Preferably, the individual to be assessed is an individual at risk for development of SMA.

Preferably, the SMN gene is SMN1.

Preferably, the method further comprising amplifying the nucleotides present at the locus of a SMN gene before analyzing the nucleotides.

Preferably, the nucleotides are amplified by a polymerase chain reaction (PCR).

Preferably, the nucleotides are amplified using a first primer comprising the sequence of SEQ ID NO: 1 and a second primer comprising the sequence of SEQ ID NO: 2.

Preferably, the mutated SMN gene is SMN2 Preferably, the copy numbers of the SMN 1 gene and the SMN2 gene are identified by DHPLC.

Preferably, the method further comprises determining the ratio of SIvIN1 to SMN2.

Preferably, the presence of SMN2 and the copy number of SMN2 are indicative of a greater likelihood of susceptibility to SMA in the individual as compared with an individual without the mutation.

An aspect of the present invention is related to a primer set consisting essentially of a first primer of the sequence of SEQ ID NO: 1 and a second primer of the sequence of SEQ ID NO: 2.

An aspect of the present invention is related to a kit of determining the susceptibility of an individual to spinal muscular atrophy (SMA), which comprises an above primer set.

Preferably, the kit further comprises a means for DHPLC.

Other objectives, advantages and novel features of the invention will become more apparent from the following detailed description.

The present invention relates to a method for determining susceptibility to motor neuron disease. The method is carried out by a DNA denaturing high performance liquid chromatography (DHPLC) apparatus to detect mutant genes relating to spinal muscular atrophy (SMA) symptom. The DNA DHPLC apparatus was invented by Professor Peter Oefher at Stanford University in the U.S.A. and can automatically detect and confirm a mutation, even a single nucleotide mutation. The mechanism for detecting the small mutation is to denature a polyinerase chain reaction (PCR) product for opening a DNA helix, to separate a mispaired strand and a normal strand, to determine their retention times by a column and to treat the mispaired and normal strands with UV light to obtain a result.

The method for determining susceptibility to motor neuron disease in accordance with the present invention comprises providing a gene nucleotide, amplifying the gene nucleotide with a primer pair to obtain a PCR product and analyzing the PCR product by a DNA denaturing high performance liquid chromatography (DHPLC) apparatus. The primer pair used to amplify the gene nucleotide is designed according to a SMA symptom relevant gene fragment, such as a survival motor neuron (SMN) gene. To successful amplify the target gene, the method comprises using a forward primer, SEQ ID NO: 1, and a reverse primer, SEQ ID NO: 2, in the primer pair, or any primer pairs that may amplify an SMN gene with a PCR reaction.

Preferably, the method further comprises contrasting the result of the PCR product analysis from the DNA DHPLC apparatus with a standard graph of an SMN gene. Because the DNA DHPLC apparatus distinguishes even a few different nucleotides between an SMNI gene and an SMN2 gene, the standard graph can be made recording the different retention times of the SMN 1 gene and the SMN2 gene in the column.

The method specifically amplifies a gene containing an SMN gene fragment, and then successfully distinguishes an extremely few differences between an SMThl gene and an SMN2 gene. Whether the SMN1 gene and the SMN2 gene exist or not and the ratio of the SMN1 gene and the Slv[N2 gene are the major factors in determining a person's health. Therefore, the method is not only for detecting SMA patients, but also for identifying SMA carriers.

The method detects a one point mutation on the nucleotide to distinguish between an SMA patient and an SMA carrier. Advantageously, if the results show a single peak at a specific nucleotide location, the sample is a homozygote sample. If the results show two peaks at the specific nucleotide location and has different retention times, the sample is a heterozygote sample.

The method provides a rapid, inexpensive and reliable way to detect an SMA carrier and an SMA patient. Also, the method reduces the cost for required apparatus and materials.

The present invention also provides a kit for determining susceptibility to motor neuron disease (MND) comprising primer SEQ ID NO: 1 and SEQ ID NO: 2.

The following definitions are provided to preclude any ambiguity in the explanation of the invention.

A "sample" is any sample comprising a biological material containing nucleic acids. In practice, the "sample" used with the method may be whole blood, serum, urine, saliva, cerebrospinal fluid, semen, tears, thi-oat swab, rectal swab, feces and the like.

The "amplification method" refers to a polymerase chain reaction (PCR), a ligase chain reaction (LCR) or strand displacement amplification (SDA). If PCR is selected, the replication composition would include, for example, nucleotide triphosphates, two primers with appropriate sequences, DNA or RNA polymerase and proteins.

A "primer" refers to an oligonucleotide (synthetic or occurring naturally) capable of acting as a point of initiation of nucleic acid synthesis or replication along a complementary strand when placed under conditions in which synthesis of a complementary stand is catalyzed by a polymerase.

An "allele" refers generally to one copy of a naturally occurring gene or a particular chromosome region in a diploid subject. A diploid subject has two sets of chromosomes and two copies of a particular gene and thus two haplotypes of any region of the chromosome and two alleles of any polymorphic site within the gene or chromosome region.

This invention is illustrated further by the following non-limiting examples. All of the literature and publications recited in the context of the present disclosure are incorporated herein by reference.

Examples

Example 1: Amplification of an SMN 1 gene and an SMN2 gene A human blood sample was extracted by Puregene kits (Gentra Systems) to obtain genornic DNA. The genomic DNA was amplified by polymerase chain reaction (PCR) for detection.

The SMN 1 gene and the SMN2 gene differ in 5 base pairs. The product amplified by the PCR reaction will comprise SMN 1 and SMN2 sequences, but the PCR reaction cannot distinguish the difference between the SMN 1 sequence and the SMN2 sequence. Because a person who has an SIv[N1 gene with exon 7 is healthy, existence of exon 7 will be the key factor in detecting the SMA symptom.

The primer pair was designed according to a fragment near the SMN exon 7 on the DNA sequence. A forward primer SEQ ID NO: 1 and a reverse primer SEQ ID NO: 2 were used for amplifying the SMN fragment.

DNA amplification by PCR was canied out in a reaction mixture containing bOng genomic DNA, 100iM dNTPs, 0.5 unit polymerase (AmpliTaq GoldTM, PE Apilied Biosystems), lox buffer 11(10mM Tris-HC1, pH=8.3, 50mM KCI) and 2.SLl, 2nilVI MgC12 (GeneAmp).

DNA amplification was caiiied out on a MBS thennocycler (ThermoHybaid) and the samples were denatured at 95 C for 10 minutes. Then the denatured samples were treated with 35 thermal cycles and elongated for 10 minutes at 72 C. Each thermal cycle included denaturation for 30 seconds at 94 C, primer annealing for 45 seconds at 53 C and elongation for 45 seconds at 72 C.

Example 2: Distinguishing of the SMN1 gene products and the SIVIN2 gene products by the DNA DHPLC apparatus The software played on the DNA DHPLC apparatus is Transgenomic Wave nucleotide fragment analysis system (Transgenomic Inc.). The system used a DNASep column (Transgenomic Inc.) mounted on an automatic DHPLC apparatus, the DNASep colunm contains styrene divinylbenzene particles which has a diameter of 2mm and is non-poriness. Because a DNA molecule contains different bases and the bases have different sfructures, different DNA molecules has different retention time when passing through a DHPLC column. After isolating by the DHPLC column, the DNA molecules were analyzing by an UV meter from absorbance at 260nm. The agents used for mobile phase in the DHPLC apparatus all are DHPLC grade agents, wherein eluent A containing 0.1 M TEAA (TransgenornicTM) and 500 Lii acetonitrile (9017-03 J. T. Baker), and eluent B containing 0.1 M TEAA which containing 25% acetoniti-ile.

20i1 of amplified DNA fragments were directly added to the DHPLC apparatus, heated at 95 C for 5 minutes and slowly cooled from 95 C to 25 C in 70 minutes. Simultaneously, eluent B was injected into the DHPLC apparatus at an initial rate of 0.9 mi/mm, which was gradually increased by 2% every 4.5 minutes. The entire injection required about 10 minutes.

Example 3: Preparation of the SMTh4 1 gene and the SMN2 gene sequence standard graph Sequence standard graphs of the SMN1 gene and the SMN2 gene were prepared for contrast with the sample' sequence graphs.

Firstly, Sjil of PCR products respectively of the SIVIN I genes and the SIvIN2 genes were mixed respectively with 5j.il of pGEM-Teasy Vector to obtain two 1 Ol mixtures. The mixtures were incubated at 4 C overnight for ligation between the gene fragments and the vectors to obtain plasmids. 5Ll of ligation product (i.e. the plasmids) was taken for liansformation testing. The plasimds were ansformed into E. coli bacteria, and the E. co/i bacteria were cultured on a culture plate containing 2Op.l, 50g11 ampicillin at 37 C overnight.

After selection on the culture plate, the colonies formed on the culture plate were desired. A Mmi-MTM kit (Viogene) was used to purify the plasmids. The purified plasmids were analyzed at the DHPLCs' conditions in Example 2 to obtain a retention time graph of the SMN 1 gene and the SMN2 gene.

The retention time of the SMN1 gene was less than 6 minutes, and the retention time of the SMN2 gene was about 6 minutes. Therefore, two peaks are shown in the retention time graph and the two peaks respectively refer to the SMN1 gene and the SMN2 gene.

An automatic sequencer were used for prepare sequence standard graphs of the SMN1 gene and the SIVfN2 gene. The results are described as follow. When the sample is a hornoduplex DNA with thymine (T) at a specific nucleotide location and shows a single peak, the sample is a SMN2 gene fragment. When the sample is a hoinoduplex DNA with cytosine (C) at the specific nucleotide location and also shows a single peak, the sample is an SMN 1 gene fragment. A person skilled in the art can distinguish the difference from T and C by the peaks.

If a sample has both an SIvfNl gene and an SMN2 gene and is a heteroduplex DNA, the sequence graph shows two peaks at the specific nucleotide location. Also, a skilled person in the art can point out a CIT one point mutation in the sequence graph.

Example 4: Distinguishing the SMN 1 gene from the SMN2 gene The products obtained from Example 2 were sequenced on a PE Biosystems 373A13 100 sequencer by a PE Biosystems Taq DyeDeoxy terminator cycle sequencing kit and obtained results. The results were contrasted with the sequence standard graph obtained from Example 3.

Example 5: Conventional method for distinguishing the SMN 1 gene from the SMN2 gene To further demonstrate that the DHPLC apparatus can distinguish the copy number of the SMN1 gene and the SMN2 gene, a conventional TaqManTM method was used for test.

The TaqManTM method is a real-time PCR method, and the method comprises incubating samples in a 96 wells plate (MicroAmp optical plate, Applied Biosystems), and analyzing the samples by a ABI Prism 700 sequencer.

A forward primer, SEQ ID NO: 3, and a reverse primer, SEQ ID NO. 4, were used to amplify the SMN gene. A minor groove binder probe (MGB probe) with a fluorescent dye, 5'-FAM, was used for distinguishing the sixth base differences inside exon 7 regeion of the SMN1 gene and the SMN2 gene.

The MGB probe for the SMN1 gene was SEQ ID NO: 5, and the MGB probe for the SMN2 gene was SEQ ID NO: 6 DNA amplification by PCR was carried out in a reaction mixture containing 5Ong genomic DNA, 0.3 iM primer pair (SEQ ID NO: 3 and SEQ ID NO: 4), 13 p.1 Platinum qPCR Supermix-UDG (Invitrogene), 0.5 mM ROX (Invitrogene), 2mM MgCI2 and lOOnmol MGB probes (SEQ ID NO: 5 and SEQ ID NO: 6).

DNA amplification was carried out on a thermocycler, and the samples were treated at 50 C for 2 minutes and 95 C for 10 minutes and treated with 40 thermal cycles. Each thermal cycle included denaturation for 15 seconds at 95 C and primer annealing for 1 minute at 60 C.

The results were analyzed with ABI37000SDS software (applied Biosystems).

Example 6: Result from the DHPLC apparatus Different retention times refer to different DNA structures. Different DNA molecules were separated by controlling the heating temperature of the DHPLC apparatus, and the DHPLC apparatus can separated different DNA sequences. Therefore, the DHPLC apparatus can distinguish a difference in a single base of an SIvIN1 gene and an SMN2 gene.

After analyzing many samples with the DHPLC apparatus, two peaks often show in the retention time graphs, and the two peaks in the retention graph means that many samples both have an SMNl gene and an SMN2 gene.

The measure of area under the two peaks of the retention time graph of each sample is equal to the quantitative analysis of the PCR product. Therefore, the method also provides quantitative analysis. Also, the measure of area under each peak referring to the respective gene shows the copy number of the SMN 1 gene or the SMN2 gene. According to the measure of areas of the SIvIN 1 gene and the SMN2 gene, the copy number of the respective gene can be estimated.

When the temperature of the DHPLC apparatus were set at 52.5 C, the copy numbers of the SMTN 1 gene and the SMN2 gene could be distinguished clearly.

For example, when the copy numbers of the SMN1 gene and the SMN2 gene are equal, the measure of area of the two peaks are almost equal.

When the measure of area referring to SMN I gene: the measure of area referring to SMN2 gene is about 2: 1, the copy number of the SMN1 gene: the copy number of the SMN2 gene is about 2: 1. When the measure of area referring to SMN1 gene: the measure of area refeiTing to S2 gene is about 1: 3, the copy number of the SMN1 gene: the copy number of the SMN2 gene is about 1: 3.

Example 7: Clinical test Two SMA families were tested according to the present invention. In family one, the father and the mother are both carriers. Both of their Sons are SMA patients. After analysis with the DHPLC apparatus, the result shows as follow. The father carrier having the copy number of the SMN 1 gene and the SMN2 was about 1 3, the mother carrier having the copy number of the SMN 1 and the SMN2 is about 1: 3. The two boys having the copy number of the SMN1 gene and the SMN2 gene is about 0. 4.

In family two, the father and the mother are both carriers, and they have four children. Two boys of their children are SMA patients, one girl is a carrier, and the other girl is healthy. After analysis with the DHPLC apparatus, the result shows as follow. The father carrier having the copy number of the SMN 1 gene and the SMN2 gene is about 1: 3, and the mother carrier having the copy number of the SMN 1 gene and the SMN2 gene is about L 3. The two boys having the copy number of the SMN I gene and the SMN2 gene is about 0: 4. The girl canier having the copy number of the SMN1 gene and the SMN2 gene is about 1: 3, and the healthy girl having the copy number of the SMN 1 gene and the SMN2 gene is about 2: 2.

Although the invention has been explained in relation to its preferred embodiment, many other possible modifications and variations can be made without departing from the spirit and scope of the invention as hereinafter claimed.

SEQUENCE LISTING

<110> BIONET CORP <120> METHOD OF DETERMINING SUSCEPTIBILITY TO SPINAL MUSCULAR ATROPHY (SMA) AND THE RELEVANT PRIMER PAIRS <130> TAJEC/P3SOO2GB <160> 6 <170> Patentln version 3.1 <210> 1 <211> 22 <212> DNA <213> Artificial <400> 1 tgtcttgtga aacaaaatgc ft 22 <210> 2 <211> 21 <212> DNA <213> Artificial <400> 2 aaaagtctgc tggtctgcct a 21 <210> 3 <211> 27 <212> DNA <213> Artificial <400> 3 aatgcttttt aacatccata taaagct 27 <210> 4 <211> 25 <212> DNA <213> Artificial <400> 4 ccttaattta aggaatgtga gacaa 25 <210> 5 <211> 16 <212> DNA <213> Artificial <400> 5 cagggtttca gacaaa 16 <210> 6 <211> 18 <212> DNA <213> Artificial <400> 6 tgattttgtc taaaaccc 18

Claims

CLAIMS: 1. A method of determining the susceptibility of an individual

to spinal muscular atrophy (S MA), comprising obtaining a DNA sample from an individual to be assessed, analyzing the nucleotides present at the locus of a survival motor neuron (SMN) gene, identifying the copy number of the survival motor neuron gene and, if a survival motor neuron gene mutation is present, the copy number of the mutated gene, and determining the susceptibility of the individual to SMA, wherein the presence of the survival motor neuron gene mutation and the copy number of the mutated survival motor neuron gene are indicative of a greater likelihood of susceptibility to SMA in the individual as compared with an individual without the mutation.

2. A method according to claim 1, wherein the individual to be assessed is an individual at risk for development of SMA.

3. A method according to claim 1, wherein the SMN gene is SMN1.

4. A method according to claim 1, further comprising amplifying the nucleotides present at the locus of a SIvfN gene before analyzing the nuci eotides.

5. A method according to claim 4, wherein the nucleotides are amplified by a polymerase chain reaction (PCR).

6 A method according to claim 4, wherein the nucleotides are amplified using a first primer comprising the sequence of SEQ 1D NO: I and a second primer comprising the sequence of SEQ ID NO:
2.

7. A method according to claim 3, wherein the mutated SIv[N gene is SMN2.

8. A method according to claim 7, wherein the copy numbers of the SMN1 and SMN2 are identified by DHPLC.

9. A method according to claim 8, which further comprises determining the ratio of SMN1 to SMN2.

10. A method according to claim 8, wherein the presence of SMN2 and the copy number of SIvIN2 are indicative of a greater likelihood of susceptibility to SMA in the individual as compared with an individual without the mutation.

11. A primer set consisting essentially of a first primer of the sequence of SEQ ID NO: 1 and a second primer of the sequence of SEQ ID NO: 2.

12. A kit of determining the susceptibility of an individual to spinal muscular atiophy (SMA), which comprises a primer set according to claim ii.

13. A kit according to claim 12, which further comprises a means for DHPLC.