AU2016351311B9 - SCAP gene mutant and the application thereof - Google Patents

Abstract

The present disclosure relates to an SCAP gene mutant and the application thereof, and particularly to an isolated nucleic acid encoding an SCAP mutant, an isolated polypeptide, a 5 method for screening a biological sample predisposed to premature myocardial infarction, a system for screening a biological sample predisposed to premature myocardial infarction and a kit for screening a biological sample predisposed to premature myocardial infarction. The isolated nucleic acid encoding an SCAP mutant has a c.3035C>T mutation as compared to SEQ ID NO: 1.

Description

SCAP GENE MUTANT AND THE APPLICATION THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

This application claims a priority to and benefits of Chinese Patent Application Serial No. 201510980242.6, filed with the State Intellectual Property Office of P. R. China on December 23, 2015, the entire content of which is incorporated herein by reference.

FIELD

The present disclosure relates to an SCAP gene mutant and application thereof, and more specifically to an isolated nucleic acid encoding an SCAP mutant, to an isolated polypeptide, to a method for screening a biological sample predisposed to premature myocardial infarction, to a system for screening a biological sample predisposed to premature myocardial infarction and to a kit for screening a biological sample predisposed to premature myocardial infarction, a construct and a recombinant cell.

BACKGROUND

Myocardial Infarction (MI) is a severe disease causing sudden death due to myocardial necrosis resulting from vascular blockage and myocardial ischemia-anoxia in corresponding vascular blockage region, which are induced from a rupture of a fibrous cap of coronary artery atherosclerotic plaque. Among others, MI occurring in a population showing clinical features such as onset in early age (<50 (Male), <60 (Female)) and familial aggregation is defined as Premature Myocardial Infarction (PMI). It has been shown that heritability of PMI is up to 63%. Because a first symptom presents the sudden death caused by acute myocardial necrosis, patients suffering PMI may have exhibited no symptoms related to coronary heart disease and the risk factors thereof before, and thus being in a great danger as compared to those having non-Premature Myocardial Infarction. Therefore, it is important to perform a risk prediction before occurrence of lethal symptoms and thus achieve a “primary prevention” for PMI by early detection of the genetic information of patients. Pathogenesis of PMI is so far unclear, as well as the pathogenic gene and pathogenic mutation, and the genetic marker thereof is not enough to identify a PMI patient.

Therefore, further researches regarding the pathogenic gene and pathogenic mutation of

PIDC1154344PAU

2016351311 15Nov2018

PMI is still required.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

SUMMARY

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

The present invention relates to a method for effectively screening a biological a method for effectively screening a biological sample predisposed to premature myocardial infarction.

The present disclosure is accomplished based on the following works. By means of high-throughput exome sequencing and verification of a candidate mutant gene, the present inventors have determined that the SCAP gene mutant is the pathogenic gene for PMI, and specifically the C.3035OT mutation in exon 18 of the SCAP gene is the pathogenic mutation for PMI.

In a first aspect, the present disclosure provides in some embodiments an isolated nucleic acid encoding an SCAP mutant. In some embodiments, the nucleic acid, compared to SEQ ID NO: 1, has a c.3035C>T mutation, i.e., the SCAP gene mutant of the present disclosure has Thymine (T) mutated from Cytosine (C) at base 3035, as compared to the SCAP gene in wild type. In some embodiments, the present inventors have determined that the SCAP gene and the SCAP mutant are closely correlated with PMI onset. Therefore, it can be effective to determine whether a biological 25 sample is predisposed to PMI by detecting an existence of the SCAP mutant in the biological sample.

In a second aspect, the present disclosure provides in some embodiments an isolated polypeptide. In some embodiments, the isolated polypeptide, compared to SEQ ID NO: 2, has a p.A1012V mutation, i.e., such a mutation is caused by a nonsense mutation of c.3035C>T.

2016351311 15Nov2018

Specifically, such a mutation indicates that the isolated polypeptide has Valine (V) mutated from Alanine (A) at amino acid 1012 as compared to the SCAP gene in wild type. It can be effective to determine whether a biological sample is predisposed to PMI by detecting whether the isolated polypeptide is expressed in the biological sample.

In a third aspect, the present disclosure provides in some embodiments a method for screening a biological sample predisposed to PMI. In some embodiments, the method includes: extracting a nucleic acid sample from the biological sample; and determining a nucleic acid sequence of the nucleic acid sample, wherein the nucleic acid sequence having a c.3035C>T mutation as compared to SEQ ID NO: 1 is an indicator of predisposition for the biological sample

2a to premature myocardial infarction. It may be effective to screen a biological sample predisposed to PMI by the method for screening the biological sample predisposed to PMI provided in some embodiments of the present disclosure.

In a fourth aspect, the present disclosure provides in some embodiments a system for screening a biological sample predisposed to PMI. In some embodiments, the system includes: a nucleic acid extracting device for extracting a nucleic acid sample from a biological sample; a nucleic acid sequence determining device, connected to the nucleic acid extracting device, for analyzing the nucleic acid sample to determine a nucleic acid sequence thereof; a determining device, connected to the nucleic acid sequence determining device, for determining whether the biological sample is predisposed to PMI based on whether the nucleic acid sequence has a c.3035C>T mutation as compared with SEQ ID NO:1. The method for screening the biological sample predisposed to PMI can be effectively performed by such a system, allowing screening the biological sample predisposed to PMI effectively.

In a fifth aspect, the present disclosure provides in some embodiments a kit for screening a biological sample predisposed to premature myocardial infarction. In some embodiments, the kit includes: a reagent suitable for detecting an SCAP gene mutant, wherein the SCAP gene, compared to SEQ ID NO: 1, has a c.3035C>T mutation. It may be effective to screen a biological sample predisposed to PMI by the kit according to the embodiment of the present disclosure.

In a sixth aspect, the present disclosure further provides in some embodiments a construct. In some embodiments, the construct includes the isolated nucleic acid encoding an SCAP mutant described above. It may be thus effective to screen a drug for PMI therapy with a recombinant cell which is transformed from a receptor cell by the construct of the present disclosure.

In a seventh aspect, the present disclosure further provides in some embodiments a recombinant cell. In some embodiments, the recombinant cell is obtained by transforming a receptor cell by the construct described above. In some embodiments, it can be effective to screen a drug for PMI therapy with the recombinant cell of the present disclosure.

Additional aspects and advantages of embodiments of the present disclosure will be given in part in the following descriptions, become apparent in part from the following descriptions, or be learned from the practice of the embodiments of the present disclosure.

PIDC1154344PAU

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects and advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following descriptions made with reference to the drawings, in which:

Fig. 1 is a block diagram showing a system and constituent parts thereof for screening a biological sample predisposed to PMI according to an embodiment of the present disclosure, in which

Fig. 1A is a block diagram showing a system for screening a biological sample predisposed to PMI according to an embodiment of the present disclosure,

Fig. IB is a block diagram showing a nucleic acid extracting device according to an embodiment of the present disclosure,

Fig. 1C is a block diagram showing a nucleic acid sequence determining device according to an embodiment of the present disclosure;

Fig. 2 is a schematic view showing a PMI pedigree according to an embodiment of the present disclosure;

Fig. 3 is representative SCAP gene profiles, verified by Sanger sequencing, from a patient suffering PMI and a healthy subject within the PMI pedigree as well as a health subject outside the PMI pedigree according to an embodiment of the present disclosure, in which the SCAP gene of the patient suffering PMI has a c.3035C>T mutation.

DETAILED DESCRIPTION

Reference will be made in detail to embodiments of the present disclosure. The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present disclosure. The embodiments shall not be construed to limit the present disclosure. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions.

SCAP gene mutant

In a first aspect, the present disclosure provides in some embodiments an isolated nucleic acid encoding an SCAP mutant. In some embodiments, the nucleic acid, compared to SEQ ID NO: 1, has a c.3035C>T mutation. The expression “a nucleic acid encoding an SCAP mutant” used herein refers to a nucleic acid substance corresponding to a gene encoding the SCAP mutant, i.e., the

PIDC1154344PAU nucleic acid is not particularly limited to any type, and may be any deoxyribonucleotides and/or ribonucleotides polymers corresponding to the gene encoding the SCAP mutant, including but not necessarily limited to DNA, RNA or cDNA. In a specific embodiment, the nucleic acid encoding the SCAP mutant described above is DNA. In some embodiments, the present inventors have determined that SCAP gene and the SCAP mutant are closely correlated with PMI onset. Therefore it can be effective to determine whether a biological sample or an organism is predisposed to PMI by detecting an existence of the SCAP mutant in the biological sample or the organism.

It would be appreciated that the nucleic acid, as mentioned in the description and claims of the present disclosure, includes any one, or two, of a complementary DNA double-strand. In the description and claims of the present disclosure, only one strand is provided in most cases for convenience, but the disclosure includes the other one strand of the complementary DNA double-strand. For example, when referring to SEQ ID NO: 1, it includes a complementary sequence thereof. It would be also understood that one strand can be determined using the other one strand of the complementary DNA double-strand, vice versa.

The nucleic acid encoding the SCAP mutant is determined to be a pathogenic gene for PMI containing a pathogenic mutation by means of high-throughput exome sequencing and verification of a candidate mutant gene. Such a mutant site has not been reported in prior art.

Among others, cDNA of the SCAP gene in wide type has a nucleotide sequence shown as follow:

agaggtgaag	gggcgggcac	ccggcggcca		ggagggcgcc	acgcaccgga	60
ctgcgggccg	agagcgcgca	cgccgcgctc	cgcccctgct	gccgcccccg	tcgccgccgc	120
cgccgccgcc	gcagcttggg	aggtgctgcc	accacaggta	cctgcacatg	ttgttctttg	180
tcagtgctgt	caagtgtgtg	ccagggtgat	ccatggtcac	tttccgggat	ggcagcaagg	240
tgacttcggc	tgaggatgac	cctgactgaa	aggctgcgtg	agaagatatc	tcgggccttc	300
tacaaccatg	ggctcctctg	tgcatcctat	cccatcccca	tcatcctctt	cacagggttc	360
tgcatcttag	cctgctgcta	cccactgctg	aaactcccct	tgccaggaac	aggacctgtg	420
gaattcacca	cccctgtgaa	ggattactcg	cccccacctg	tggactctga	ccgcaaacaa	480
ggagagccta	ctgagcagcc	tgagtggtat	gtgggtgccc	cggtggctta	tgtccagcag	540
atatttgtga	agtcctcagt	gtttccctgg	cacaagaacc	tcctggcagt	agatgtattt	600
cgttcacctt	tgtcccgggc	attccaactg	gtggaggaga	tccggaacca	cgtgctgaga	660
gacagctctg	ggatcaggag	cttggaggag	ttgtgtctgc	aagtgaccga	cctgctgcca	720
ggccttagga	agctcaggaa	cctactccct	gagcatggat	gcctgctgct	gtcccctggg	780
aacttctggc	agaatgactg	ggaacgcttc	catgctgatc	ctgacatcat	tgggaccatc	840
caccagcacg	agcctaaaac	cctgcagact	tcagccacac	tcaaagactt	gttatttggt	900
gttcctggga	agtacagcgg	ggtgagcctc	tacaccagga	agaggatggt	ctcctacacc	960
atcaccctgg	tcttccagca	ctaccatgcc	aagttcctgg	gcagcctgcg	tgcccgcctg	1020
atgcttctgc	accccagccc	caactgcagc	cttcgggcgg	agagcctggt	ccacgtgcac	1080

PIDC1154344PAU

	ttcaaggagg tttgcctaca gccctggctg ctcttcggcc	agattggtgt tctacttctc ccgtggtcac tgacgcccac	cgctgagctc cacgcggaag agtgctcagc cctcaatggc	atcccccttg tgaccaccta	catcatcttg gtgggggctg actctgcaca tgtggtggtt	1140 1200 1260 1320
atcgacatgg tcgctgctca ggcgagattt	tcaagtccaa tgtctgtggg tcccctacct
5	attgggttag	agaatgtgtt	ggtgctcacc	aagtctgtgg	tctcaacccc	ggtagacctg	1380
	gaggtgaagc	tgcggatcgc	ccaaggccta	agcagcgaga	gctggtccat	catgaagaac	1440
	atggccacgg	agctgggcat	catcctcatc	ggctacttca	ccctagtgcc	cgccatccag	1500
	gagttctgtc	tctttgctgt	cgtggggctg	gtgtctgact	tcttccttca	gatgctgttt	1560
	ttcaccactg	tcctgtccat	tgacattcgc	cggatggagc	tagcagacct	gaacaagcga	1620
10	ctgccccctg	aggcctgcct	gccctcagcc	aagccagtgg	gacagccaac	gcgctacgag	1680
	cggcagctgg	ctgtgaggcc	gtccacaccc	cacaccatca	cgttgcagcc	gtcttccttc	1740
	cgaaacctgc	ggctccccaa	gaggctgcgt	gttgtctact	tcctggcccg	cacccgcctg	1800
	gcacagcgcc	tcatcatggc	tggcaccgtt	gtctggattg	gcatcctggt	atacacagac	1860
	ccagcagggc	tgcgcaacta	cctcgctgcc	caggtgacgg	aacagagccc	attgggtgag	1920
15	ggagccctgg	ctcccatgcc	cgtgcctagt	ggcatgctgc	cccccagcca	cccggaccct	1980
	gccttctcca	tcttcccacc	tgatgcccct	aagctacctg	agaaccagac	gtcgccaggc	2040
	gagtcacctg	agcgtggagg	tccagcagag	gttgtccatg	acagcccagt	cccagaggta	2100
	acctgggggc	ctgaggatga	ggaactttgg	aggaaattgt	ccttccgcca	ctggccgacg	2160
	ctcttcagct	attacaacat	cacactggcc	aagaggtaca	tcagcctgct	gcccgtcatc	2220
20	ccagtcacgc	tccgcctgaa	cccgagggag	gctctggagg	gccggcaccc	tcaggacggc	2280
	cgcagtgcct	ggcccccacc	ggggcccata	cctgctgggc	actgggaagc	aggacccaag	2340
	ggcccaggtg	gggtgcaggc	ccatggagac	gtcacgctgt	acaaggtggc	ggcgctgggc	2400
	ctggccaccg	gcatcgtctt	ggtgctgctg	ctgctctgcc	tctaccgcgt	gctatgcccg	2460
	cgcaactacg	ggcagctggg	tggtgggccc	gggcggcgga	ggcgcgggga	gctgccctgc	2520
25	gacgactacg	gctatgcgcc	acccgagacg	gagatcgtgc	cgcttgtgct	gcgcggccac	2580
	ctcatggaca	tcgagtgcct	ggccagcgac	ggcatgctgc	tggtgagctg	ctgcctggca	2640
	ggccacgtct	gcgtgtggga	cgcgcagacc	ggggattgcc	taacgcgcat	tccgcgccca	2700
	ggcaggcagc	gccgggacag	tggcgtgggc	agcgggcttg	aggctcagga	gagctgggaa	2760
	cgactttcag	atggtgggaa	ggctggtcca	gaggagcctg	gggacagccc	tcccctgaga	2820
30	caccgccccc	ggggccctcc	gccgccttcc	ctcttcgggg	accagcctga	cctcacctgc	2880
	ttaattgaca	ccaacttttc	agcgcagcct	cggtcctcac	agcccactca	gcccgagccc	2940
	cggcaccggg	cggtctgtgg	ccgctctcgg	gactccccag	gctatgactt	cagctgcctg	3000
	gtgcagcggg	tgtaccagga	ggaggggctg	gcggccgtct	gcacaccagc	cctgcgccca	3060
	ccctcgcctg	ggccggtgct	gtcccaggcc	cctgaggacg	agggtggctc	ccccgagaaa	3120
35	ggctcccctt	ccctcgcctg	ggcccccagt	gccgagggtt	ccatctggag	cttggagctg	3180
	cagggcaacc	tcatcgtggt	ggggcggagc	agcggccggc	tggaggtgtg	ggacgccatt	3240
	gaaggggtgc	tgtgctgcag	cagcgaggag	gtctcctcag	gcattaccgc	tctggtgttc	3300
	ttggacaaaa	ggattgtggc	tgcacggctc	aacggttccc	ttgatttctt	ctccttggag	3360
	acccacactg	ccctcagccc	cctgcagttt	agagggaccc	cagggcgggg	cagttcccct	3420
40	gcctctccag	tgtacagcag	cagcgacaca	gtggcctgtc	acctgaccca	cacagtgccc	3480
	tgtgcacacc	aaaaacccat	cacagccctg	aaagccgctg	ctgggcgctt	ggtgactggg	3540
	agccaagacc	acacactgag	agtgttccgt	ctggaggact	cgtgctgcct	cttcaccctt	3600
	cagggccact	caggggccat	cacgaccgtg	tacattgacc	agaccatggt	gctggccagt	3660
		atggggccat	ctgcctgtgg	gatgtactga	ctggcagccg	ggtcagccat	3720
45	gtgtttgctc	accgtgggga	tgtcacctcc	cttacctgta	ccacctcctg	tgtcatcagc	3780
	agtggcctgg	atgacctcat	cagcatctgg	gaccgcagca	caggcatcaa	gttctactcc	3840

PIDC1154344PAU

attcagcagg	acctgggctg	tggtgcaagc	ttgggtgtca	tctcagacaa	cctgctggtg	3900
actggcggcc	agggctgtgt	ctccttttgg	gacctaaact	acggggacct	gttacagaca	3960
gtctacctgg	ggaagaacag	tgaggcccag	cctgcccgcc	agatcctggt	gctggacaac	4020
gctgccattg	tctgcaactt	tggcagtgag	ctcagcctgg	tgtatgtgcc	ctctgtgctg	4080
gagaagctgg	actgagcgca	gggcctcctt	gcccaggcag	gaggctgggg	tgctgtgtgg	4140
gggccaatgc	actgaacctg	gacttggggg	aaagagccga	gtatcttcca	gccgctgcct	4200
cctgactgta	ataatattaa	acttttttaa	aaaaccatat	catcatctgt	caggc	4255 (SEQ

ID NO: 1), and the protein encoded has an amino acid sequence shown as follow:

MTLTERLREK	ISRAFYNHGL	LCASYPIPII	LFTGFCILAC	CYPLLKLPLP	GTGPVEFTTP	60
VKDYSPPPVD	SDRKQGEPTE	QPEWYVGAPV	AYVQQIFVKS	SVFPWHKNLL	AVDVFRSPLS	120
RAFQLVEEIR	NHVLRDSSGI	RSLEELCLQV	TDLLPGLRKL	RNLLPEHGCL	LLSPGNFWQN	180
DWERFHADPD	IIGTIHQHEP	KTLQTSATLK	DLLFGVPGKY	SGVSLYTRKR	MVSYTITLVF	240
QHYHAKFLGS	LRARLMLLHP	SPNCSLRAES	LVHVHFKEEI	GVAELIPLVT	TYIILFAYIY	300
FSTRKIDMVK	SKWGLALAAV	VTVLSSLLMS	VGLCTLFGLT	PTLNGGEIFP	YLWVIGLEN	360
VLVLTKSWS	TPVDLEVKLR	IAQGLSSESW	SIMKNMATEL	GIILIGYFTL	VPAIQEFCLF	420
AVVGLVSDFF	LQMLFFTTVL	SIDIRRMELA	DLNKRLPPEA	CLPSAKPVGQ	PTRYERQLAV	480
RPSTPHTITL	QPSSFRNLRL	PKRLRWYFL	ARTRLAQRLI	MAGTWWIGI	LVYTDPAGLR	540
NYLAAQVTEQ	SPLGEGALAP	MPVPSGMLPP	SHPDPAFSIF	PPDAPKLPEN	QTSPGESPER	600
GGPAEVVHDS	PVPEVTWGPE	DEELWRKLSF	RHWPTLFSYY	NITLAKRYIS	LLPVIPVTLR	660
LNPREALEGR	HPQDGRSAWP	PPGPIPAGHW	EAGPKGPGGV	QAHGDVTLYK	VAALGLATGI	720
VLVLLLLCLY	RVLCPRNYGQ	LGGGPGRRRR	GELPCDDYGY	APPETEIVPL	VLRGHLMDIE	780
CLASDGMLLV	SCCLAGHVCV	WDAQTGDCLT	RIPRPGRQRR	DSGVGSGLEA	QESWERLSDG	840
GKAGPEEPGD	SPPLRHRPRG	PPPPSLFGDQ	PDLTCLIDTN	FSAQPRSSQP	TQPEPRHRAV	900
CGRSRDSPGY	DFSCLVQRVY	QEEGLAAVCT	PALRPPSPGP	VLSQAPEDEG	GSPEKGSPSL	960
AWAPSAEGSI	WSLELQGNLI	WGRSSGRLE	VWDAIEGVLC	CSSEEVSSGI	TALVFLDKRI	1020
VAARLNGSLD	FFSLETHTAL	SPLQFRGTPG	RGSSPASPVY	SSSDTVACHL	THTVPCAHQK	1080
PITALKAAAG	RLVTGSQDHT	LRVFRLEDSC	CLFTLQGHSG	AITTVYIDQT	MVLASGGQDG	1140
AICLWDVLTG	SRVSHVFAHR	GDVTSLTCTT	SCVISSGLDD	LISIWDRSTG	IKFYSIQQDL	1200
GCGASLGVIS	DNLLVTGGQG	CVSFWDLNYG	DLLQTVYLGK	NSEAQPARQI	LVLDNAAIVC	1260
NFGSELSLVY	VPSVLEKLD				127 9 (SEQ ID NO: 2).

The inventors have found that the SCAP mutant, compared to SEQ ID NO: 1, has a c.3035C>T mutation, i.e., the SCAP gene mutant of the present disclosure has Thymine (T) mutated from Cytosine (C) at base 3035, as compared to the SCAP gene in wild type. Accordingly, the encoded product, compared to the SCAP gene in wild type, has a p.Al012V mutation caused by a nonsense mutation of c.3035C>T. Specifically, such a mutation indicates that the isolated polypeptide has Valine (V) mutated from Alanine (A) at amino acid 1012 as compared to the SCAP gene in wild type.

It should be noted that the SCAP gene is located at chromosome 3 which consists of 23 exons and contains 1279 amino acids. It serves as a “central regulatory factor” for lipid synthesis and

PIDC1154344PAU intake in mammal. For example, the SCAP gene changes its conformation as intracellular cholesterol level decreases, then achieves a cascade of transcriptional regulations through activation of an SREBP gene by enzyme digestion and subsequent nuclear translocation of the SREBP gene, and up-regulates key enzymes for lipid metabolisms including LDLR and HMGCoA to facilitate cholesterol formation; In contrast, the SCAP gene stops enzyme digestion when detects excessively high intracellular cholesterol level as intracellular cholesterol level increases. Therefore, the SCAP gene is an important negative feedback for cholesterol metabolism. It was reported that a point mutation located at the SCAP gene may cause decrease in sensitivity to intracellular cholesterol level change, such that it will not be inhibited by increasing cholesterol level, resulting in excessively increase of intracellular cholesterol level. This process when occurring in main cholesterol synthesis tissues of organism such as liver cells may result in hypercholesterolemia. This process when occurring in macrophage and smooth muscle cells may result in formation of “foam cells”, which serves as an important mechanism and a cytological marker for the development of myocardial infarction. The present inventors have further found and determined that the SCAP gene mutant is the pathogenic gene for PMI, and specifically the c.3035C>T mutation in exon 18 of the SCAP gene is the pathogenic mutation for PMI. However, the mutant site c.3035C>T at the SCAP gene has not been reported in prior art so far.

In a second aspect, the present disclosure provides in some embodiments an isolated polypeptide. In some embodiments, the isolated polypeptide, compared to the SCAP gene in wild type, has a p.A1012V mutation, i.e., such a mutation is caused by a nonsense mutation of c.3035C>T. Specifically, such a mutation indicates that the isolated polypeptide has Valine (V) mutated from Alanine (A) at amino acid 1012 as compared to the SCAP gene in wild type. In some embodiments, the polypeptide is encoded by the isolated nucleic acid encoding the SCAP mutant described above. It can be effective to determine whether a biological sample or an organism is predisposed to PMI by detecting expression of the polypeptide in the biological sample or the organism..

In a third aspect, the present disclosure provides in some embodiments a method for screening a biological sample predisposed to PMI. In some embodiments, the method includes extracting a nucleic acid sample from the biological sample; and determining a nucleic acid sequence of the nucleic acid sample.

In some embodiments, the nucleic acid sample is extracted from the biological sample which

PIDC1154344PAU is not restricted to any specific type, as long as the nucleic acid sample reflecting the existence of the SCAP mutant can be extracted therefrom. In some embodiments, the biological sample may be selected from at least one of human blood, skin and subcutaneous tissue, preferably the biological sample is peripheral blood, such that it is convenient for sampling and detection, thus further improving efficiency of screening the biological sample predisposed to PMI. In various embodiments, the term “nucleic acid sample” used herein should be understood broadly, which can be any sample capable of reflecting the existence of the SCAP mutant in the biological sample, such as whole genomic DNA directly extracted from the biological sample, or part of the whole genome containing an encoding sequence for the SCAP gene, or total RNA extracted form the biological sample, or mRNA extracted form the biological sample. In some embodiments, the nucleic acid sample is whole genomic DNA. Thus, a source of the biological sample is expanded and a plurality of information for the biological sample can be determined, thereby improving efficiency of screening the biological sample predisposed to PMI. Moreover, in some embodiments, extracting the nucleic acid sample, where RNA served as the nucleic acid sample, from the biological sample further includes: extracting an RNA sample, preferably mRNA, from the biological sample; and obtaining a cDNA sample by reverse transcription from the extracted RNA sample, wherein the cDNA sample constitutes the nucleic acid sample, such that it may further improve efficiency of screening the biological sample predisposed to PMI with RNA as the nucleic acid sample..

Subsequently, the nucleic acid sample obtained is analyzed for determining a nucleic acide sequence. In some embodiments, a method and device for determining the nucleic acid sequence of the nucleic acid sample are not subject to any specific restrictions. In some embodiments, the nucleic acid sequence of the nucleic acid sample can be determined by a sequencing approach. In some embodiments, a method and device for sequencing are not subject to any specific restrictions. In some embodiments, the Second-generation sequencing technology is used, as well as the third-, fourth-generation sequencing technology or sequencing technologies of more advanced. In some embodiments, at least one of Illumina HiSeq4000, SOLiD, 454 and single molecule sequencing device may be used for sequencing the nucleic acid sequence. With the latest sequencing technology, a higher sequencing depth for a single site can be achieved and detection sensitivity and accuracy may be largely improved. Due to high-throughput and deep sequencing features of the sequencing technology, efficiency of detection and analysis for the nucleic acid is further

PIDC1154344PAU improved, and accuracy and precision for subsequent analysis of sequencing data are thus improved. In some embodiments, determining a nucleic acid sequence of the nucleic acid sample further includes: constructing a library for sequencing the nucleic acid sample; and sequencing the library to obtain a sequencing result consisting of a plurality of sequencing data. In some embodiments, the library is sequenced by at least one selected from Illumina HiSeq4000, SOLiD, 454 and single molecule sequencing device. Furthermore, in some embodiments, the nucleic sample is screened and an SCAP exon is enriched before, during and after constructing the library. In some embodiments, constructing the library further includes: amplifying the nucleic acid sample by PCR with a primer specific for an exon of an SCAP gene; and constructing the library for sequencing the nucleic acid sample, such that efficiency of screening the biological sample predisposed to PMI can be improved by PCR amplification and enrichment of SCAP exon, especially exon 18. In some embodiments, a sequence of the primer specific for the exon of the SCAP gene is not specific restricted. In some embodiments, these primers specific for the exon of the SCAP gene (targeting exon 18 of the SCAP gene) have nucleic acid sequence as shown in SEQ ID NO: 37 and 38:

SCAP-18F: gactccccaggctatgact (SEQ ID NO: 37);

SCAP-18R: acagcagttgaagagaaccag (SEQ ID NO: 38).

The present inventors have surprisingly found that the exon of the SCAP gene can be effectively amplified with such primers in the PCR reaction system. It should be noted that the nucleotide sequence shown in SEQ ID NO: 37 and SEQ ID NO: 38 are obtained accidently through tough efforts and labors of the present inventors.

Methods and procedures of constructing the library for sequencing the nucleic acid sample may be chosen by those skilled in the art depending on sequencing technology used. Details of the procedure may follow protocol provided by manufacturers such as Illumina Co., Ltd, for example Multiplexing Sample Preparation Guide (Part# 1005361; Feb 2010) or Paired-End SamplePrep Guide (Part# 1005063; Feb 2010) of Illumina Co., Ltd, the entire contents of which are incorporated herein by reference. In some embodiments, the method and device for extracting nucleic acid sample from the biological sample are not specifically restricted and may be performed by a commercial nucleic acid extracting kit.

It should be noted that the term “nucleic acid sequence” used herein should be understood broadly, which can be complete nucleic acid sequence information obtained after assembly of the io

PIDC1154344PAU sequencing data of the nucleic acid sample, or can be sequencing data (i.e., reads) obtained by sequencing the nucleic acid sample, as long as corresponding encoding sequence to the SCAP gene is included by the nucleic acid sequence.

At last, the determined nucleic acid sequence of the nucleic acid sample is compared to SEQ ID NO: 1. The existence of the C.3035OT mutation in nucleic acid sequence is an indicator of predisposition for the biological sample to PMI. Therefore, the method provided in some embodiments of the present disclosure can effectively screen out the biological sample predisposed to PMI. In some embodiments, a method and procedure for comparing the nucleic acid with SEQ ID NO: 1 are not restricted specifically, and any software known in the art can be used for the purpose. In an embodiment, SOPA software is used for comparison.

In some embodiments, the premature myocardial infarction is autosome inheritable myocardial infarction.

It should be noted that the use of “a method for screening a biological sample predisposed to premature myocardial infarction” is not restricted specifically, for example in non-diagnostic purposes.

System and kit for screening biological sample predisposed to premature myocardial infarction

In a fourth aspect, the present disclosure provides in some embodiments a system for screening a biological sample predisposed to PMI.

With Reference to Fig. 1, in some embodiments, the system 1000 for screening a biological sample predisposed to PMI includes: a nucleic acid extracting device 100, a nucleic acid sequence determining device 200 and a determining device 300.

In some embodiments, the nucleic acid extracting device 100 is used for extracting a nucleic acid sample from a biological sample. In some embodiments, the nucleic acid sample, as mentioned above, is not restricted to any specific type. When RNA serves as the nucleic acid sample, the nucleic acid extracting device further includes an RNA extracting unit 101 for extracting an RNA sample from the biological sample, and an RNA reverse transcription unit 102, connected to the RNA extracting unit 101, for obtaining a cDNA sample by reverse transcription from the extracted RNA sample, in which the cDNA sample constitutes the nucleic acid sample.

In some embodiments, the nucleic acid sequence determining device 200, connected to the nucleic acid extracting device 100, analyzes the nucleic acid sample to determine a nucleic acid

PIDC1154344PAU sequence of the nucleic acid sample. The nucleic acid sequence of the nucleic acid sample can be determined by sequencing as described above. In some embodiments, the nucleic acid sequence determining device 200 may further includes: a library construction unit 201 for constructing a library for sequencing the nucleic acid sample and a sequencing unit 202, connected to the library construction unit 201, for sequencing the library to obtain a sequencing result consisting of a plurality of sequencing data. As described above, an exon of an SCAP gene is enriched by PCR amplification, thereby further improving efficiency of screening the biological sample predisposed to PMI. Thus, the library construction unit 201 may further include a PCR amplification module (not shown in drawings). The PCR amplification module is provided with a primer specific for an exon of an SCAP gene for amplifying the nucleic acid sample. In some embodiments, the primers specific for the exon of the SCAP gene (targeting exon 18 of the SCAP gene, have the nucleic acid sequence as shown in SEQ ID NO: 37 and 38. In some embodiments, the sequencing unit 202 may include at least one of ILLUMINA HISEQ4000, SOLiD, 454 and single molecule sequencing device. With the latest sequencing technology, a higher sequencing depth for a single site can be achieved and detection sensitivity and accuracy may be largely improved. Due to high-throughput and deep sequencing features of the sequencing technology, efficiency of detection and analysis for the nucleic acid is further improved, and accuracy and precision for subsequent analysis of sequencing data are thus improved.

In some embodiments, the determining device 300, connected to the nucleic acid sequence determining device 200, is used for comparing the nucleic acid sequence of the nucleic acid sample to determine whether the biological sample is predisposed to PMI by comparing the nucleic acid sequence of the nucleic acid sample with SEQ ID NO: 1, specifically based on whether a c.3035C>T mutation exists in the nucleic acid sequence. As described above, in an embodiment, the nucleic acid sequence of the nucleic acid sample having the C.3035OT mutation as compared to SEQ ID NO: 1 is an indicator of predisposition for the biological sample to premature myocardial infarction. As described above, in some embodiments, a device for comparing the nucleic acid with SEQ ID NO: 1 are not restricted specifically, and any software known in the art can be used for the purpose. In a specific embodiment, SOPA software is used for comparison.

Thus, the method for screening the biological sample predisposed to PMI is effectively performed by such a system, allowing improvement in screening the biological sample

PIDC1154344PAU predisposed to PMI.

In a fifth aspect, the present disclosure provides in some embodiments a kit for screening a biological sample predisposed to premature myocardial infarction. In some embodiments, the kit includes: a reagent suitable for detecting an SCAP gene mutant, in which the SCAP gene has a c.3035C>T mutation compared to SEQ ID NO: 1. It may be effective to screen a biological sample predisposed to PMI by the kit provided in some embodiments of the present disclosure. The term “a reagent suitable for detecting an SCAP gene mutant” used herein, should be understood broadly, and may refer to a reagent for detecting an SCAP encoding gene or a reagent for detecting an SCAP mutant polypeptide, such as an antibody for recognizing a specific site. In some embodiments, the reagent is a nucleic acid probe or a prime. Preferably, the nucleic acid probe or the prime has a nucleotide sequence as shown in SEQ ID NO: 37-38. The biological sample predisposed to PMI thus can be effectively screened.

It should be noted, the features and advantages describe in the method for screening the biological sample predisposed to premature myocardial infarction are also applied to the system and kit for screening the biological sample predisposed to premature myocardial infarction. No details would be given for brevity.

Construct and recombinant cell

In a sixth aspect, the present disclosure further provides in some embodiments a constmct. In some embodiments, the constmct includes the isolated nucleic acid encoding an SCAP mutant described above, namely the SCAP gene mutant of the present disclosure. It may be thus effective to screen a drug for PMI therapy with a recombinant cell which is transformed from a receptor cell by the constmct of the present disclosure. The receptor cell is not limited to any specific type. For example, the receptor cell may be an Escherichia coli cell, or a mammalian cell; preferably the receptor cell derived from a mammal.

The term “constmct” used in present disclosure refers to a genetic vector containing a specific nucleic acid sequence and capable of transferring a targeting nucleic acid sequence into a host cell to obtain a recombinant cell. In various embodiments, the constmct is not specifically limited in any form. In some embodiments, the constmct can be at least one of plasmid, bacteriophage, artificial chromosome, cosmid and vims, preferably plasmid. As a genetic vector, the plasmid is easy to deal with and can carry larger fragment. The plasmid is also not specifically limited in any form and can be a circular plasmid or linear plasmid, single-strand or double-strand, which can be

PIDC1154344PAU selected by a person skilled in the art depending on actual requirement. The term “nucleic acid” used herein can be any polymer containing deoxyribonucleotides or ribonucleotides, including but not necessarily limited to modified or unmodified DNA and RNA, and shall has no specific limits to its length. The nucleic acid, for the construct for constructing the recombinant cell, preferably is DNA as it’s more stable and easier for operation compared to RNA.

In a seventh aspect, the present disclosure further in some embodiments provides a recombinant cell. In some embodiments, the recombinant cell is obtained by transforming a receptor cell by the construct mentioned above. The recombinant cell thus can express the SCAP gene mutant carried by the construct. In some embodiments, it can be effective to screen a drug for PMI therapy with the recombinant cell of the present disclosure. In some embodiments, the receptor cell is not specifically limited to any type and may be for example an Escherichia coli cell, or a mammalian cell, preferably the cell derived from a mammal.

Reference will be made in detail to examples of the present disclosure. It would be appreciated by those skilled in the art that the following examples are explanatory, and cannot be construed to limit the scope of the present disclosure.

If the specific technology or conditions are not specified in the examples, a step will be performed in accordance with the techniques or conditions described in the literature in the art (for example, referring to “Molecular Cloning: A Laboratory Manual, 3rd Ed., Science Press or in accordance with the product instructions. If the manufacturers of reagents or instruments are not specified, the reagents or instruments may be commercially available. Unless defined otherwise, all technic and processes used herein are commonly known in the art. Both the manufactures and trade names of the reagents, as well as the ingredients necessarily listed thereof, are indicated as first appeared. Unless defined otherwise, the reagent interpreted previously has the same meaning as indicated previously.

Embodiment 1 Determination of the pathogenic mutation for PMI

1. Sample collection

A five-generation Chinese Premature Myocardial Infarction (or PMI in short in the text) patient pedigree was collected by present inventors, shown in Fig. 2. As shown in Fig. 2, φ represents a female patient, J represents a male patient, Q represents a female healthy represents a deceased female, represents a deceased male; and an arrow represents a proband. Four generations, 19

PIDC1154344PAU members in total, were survived in the pedigree with 3 PMI patients (2 males and 1 female).

All patients were given a physical, blood biochemical and imaging examination by present inventors. All clinical and excluded diagnosis were based on a gold standard for MI, namely: coronary angiography positive is the clinical diagnosis for myocardial infarction and coronary CT angiography negative was excluded diagnosis for myocardial infarction based on the typical myocardial infarction symptoms and the electrocardiogram manifestation of the patients. As shown in Fig. 2, the proband PMI1-1 was a middle-aged woman and suffered acute myocardial infarction at the age of 39. Intermittent symptom of typical effort angina had developed and progressively aggravated during 3 years before onset of the myocardial infarction. The patient was diagnosed with myocardial infarction by coronary angiography when she was 39 and 4 stents in total were subsequently implanted in by elective operation. Her past medical history included 4 years of hypertension and hyperlipemia with irregularly medication. Besides, hyperlipemia and premature corcnary heart disease were found in her family history.

The present inventors collected samples from 3 living patients and 16 non-PMI patients (i.e. the healthy subject in the pedigree) in the PMI patient pedigree.

2. Whole exome sequencing

Sequencing for samples from 3 patients (PMI1-1, PMI 1-3 and PMI 1-8) and 16 healthy subjects in the PMI patient pedigree was performed with PE 150 sequencing strategy using Agilent SureSelect Human All ExonV5 kit and on illumina HiSeq4000 platform. Specific steps for the sequencing are shown as follows.

2.1 DNA extraction

Peripheral blood was collected from 3 patients and 16 healthy subjects in the PMI patient pedigree shown in Fig. 2. Genomic DNA of each member in the PMI patient pedigree was extracted from the peripheral blood sample by salting-out method. The purity or concentration of DNA were measured by spectrophotometer, wherein OD260/OD280 of each genomic DNA were between 1.7 to 2.0, the concentration of each genome DNA was not less than 200 ng/pL and the total amount of each genomic DNA was not less than 3 pg.

2.2 Exon capture and sequencing

Each genomic DNA sample was fragmented by ultrasonicator (CovarisS2, Massachusetts, USA) to obtained fragments having a length of 150-200bp. Each fragment was ligated with

PIDC1154344PAU an adaptor at both end thereof for constructing a DNA library following the manufacturer’s instruction (Seen in Illumina/Solexa Library Construction provided by http://www.illumina.com/, the entire content of which is incorporated herein by reference).

The DNA library obtained above was hybridized(95 °C for 5min, 65 °C for 24 h) with a biotin labeled RNA probe using the Agilent SureSelect Human All ExonV5 kit. The DNA-RNA mixture was captured by streptavidin coated magnetic beads and DNA was eluted using the Qiagen purification kit. The DNA captured was amplified with Agilent PCR primers for 12 cycles. Accordingly, a DNA sequence having a length of 50 Mb and containing 334,378 exons of 20,965 genes were captured.

Furthermore, quality inspection for the library was performed by DNA chip on Agilent bioanalyzer, followed by sequencing if qualified, to obtain raw sequencing data. The sequencing platform was Illumina Hiseq 4000, a read length was 90bp and the average sequencing depth for each sample was 150*.

3. Data quality control, variation detection, annotation and variation filtration

3.1 Data quality control

The raw data obtained by sequencing using Hiseq4000 was saved as a file in fastq form (filename: *fq). The raw data contains information of used adaptor, low-quality bases and undetected base (referred by N). The information was removed before analysis due to the server interference for following analysis. The data thus obtained was the valid data.

Specifically, raw data obtained above was filtrated using Illumina basecalling Software 1.7 according to conditions provided as follows:

A. filtrating reads containing an adaptor sequence;

B. removing a read containing more than 10% N over all bases when sequenced by single-end sequencing;

C. removing a read containing more than 50% low-quality bases (with a base quality value lower than 5) over all bases when sequenced by single-end sequencing.

High-quality clean data was thus obtained by strict filtration for the sequencing data.

Statistics had been made for obtained clean data, such as the number of sequenced reads, data volume, error rate of sequencing, Q20 content, Q30 content and GC content.

3.2 Sequence alignment

Furthermore, the clean sequencing data was aligned to a UCSC human reference genome

PIDC1154344PAU (hgl9, build 37.1, http://genome.ucsc.edu/) using SOAPaligner/SOAP2 (seen in Li R, Li Y, Kristiansen K, et al, SOAP: short oligonucleotide alignment program. Bioinformatics 2008, 24(5):713-714; Li R, Yu C, Li Y, ea al, SOAP2:an improved ultrafast tool for short read alignment. Bioinformatics 2009, 25(15):1966-1967, the entire content of which is incorporated herein by reference) to obtain a uniquely-aligned sequence.

Specifically, certain bias was introduced due to PCR amplification during library construction,

i.e., some sequences may be excessively amplified. As such, the identical excessively amplified sequence may be mapped to the same location of the genome. But these excessively amplified reads cannot serve as a proof in mutation detection as they were not the intrinsic sequence of the genome. Duplicates formed by PCR amplification should be removed as far as possible, which can be performed by picard. These duplicates, showing no influence toward the results, can be neglected at late stage of GATK analysis. A large amount of base mismatched during alignment appeared near insert-deletion (indel), which was unfavorable to recalibration and mutation detection for base quality. As a result, reads aligned near indel required re-alignment to reduce the mismatch to minimum. In GATK analysis, Realigner Target Creator was used for determining regions requiring re-alignment and Indel Realigner was used for alignment of such region.

3.3 Variation detection and functional annotation

A genotype of a targeting region was determined by SOAPsnp (seen in Li R, Li Y, Fang X, Yang H, et al, SNP detection for massively parallel whole-genome resequencing.Genome Res 2009, 19(6):1124-1132, the entire content of which is incorporated herein by reference).

Specifically, the final BAM file was subjected to SNP/INDEL detection by HaplotypeCaller module of GATK. The variation result was annotated by ANNOVAR. Predictions for variation location, variation type and conservative region were made based on dbSNP and 1000 Genome database. Prediction for variation in an exon region was made based on CDS, RefSeq, database and USCS.

3.4 Variation filtration

Filtration was performed using public database such as dbSNP database (http://hgdownload.cse.ucsc.edu/goldenPath/hgl9/database/snpl35.txt.gz·), HapMap database (ftp:// ftp .ncbi.nlm.nih. gov/hapmap), 1000 Genomes database (ftp://ftp.1000genomes.ebi.ac.uk/voll/ftp), Yanhuang database (http://yh.genomics.org.cn/) and 1000 Genome (1000 Genomes Project Consortium) to remove known variation and clinical

PIDC1154344PAU irrelevant sites; retain a site near an exon region or a digested site, which may lead to changes of corresponding amino acid; remove synonymous mutation and retain non-synonymous mutation; and thus predict conservation of the amino acid by PolyPhen and SIFT. Accordingly, 249 SNP mutant sites and 361 insertion/deletion mutant sites were obtained.

Furthermore, based on the features of the mutant sites: (a) a mutation in a common gene, at a common site or in a common type among three patients were chosen; (b) Screening was performed based on the type of autosomal dominant inheritance (incomplete dominance) of the disease in the pedigree; (c) Sites speculated to be ΜΙ-related were further screened according to literatures and gene function.

As a result, a point mutation C.3035OT (p.A1012V) located in exon 18 of the SCAP gene was determined to be the potential pathogenic gene mutant. All the three patients carried the above mutation (c.3035C>T) which was a heterozygotic mutation whereas no mutant was found at this site in other healthy subjects.

Investigation was made on the SCAP gene in the PIM patient pedigree shown in Fig. 2, namely performing Sanger sequencing to determine nonsense mutation C.3035OT in the SCAP gene. The results showed that a co-segregation with phenotypes was existed in the pedigree.

The SCAP gene was thus determined to be the pathogenic gene for PIM. The nonsense mutation C.3035OT (p.A1012V) was determined to be a pathogenic cause for the PMI patient in the pedigree, namely the mutation C.3035OT (p.A1012V) was determined to be the pathogenic mutant for PMI.

Embodiment 2 Sanger sequencing

The SCAP gene from the 3 three patients (PMI1-1, PMI 1-3 and PMI 1-8) and 16 healthy subjects from the PMI pedigree described in embodiment 1, 70 PMI patients from Chinese Han population and 200 healthy subjects outside the PMI pedigree were analyzed, respectively. Primer was designed for the SCAP gene; an SCAP related sequence was obtained by successively PCR amplification, product purification and sequencing. The relevance of PMI to the SCAP gene and the mutation c.3035C>T (p.A1012V) of the SCAP gene was confirmed according to obtained sequencing result.

Specific step of sequencing includes steps as follows.

1. DNA extraction

Genomic DNA was extracted from peripheral blood of subjects according to DNA

PIDC1154344PAU extraction described in embodiment 1.

2. Primer design and PCR amplification

Primers specific for an exon of an SCAP gene shown as SEQ ID NO: 3-48 were designed according to human genome database GRCh38.p2, seen in following table.

Primer name	Primer sequence (5’-3’)
SCAP-IF	catacttccctccggtgtcc(3)
SCAP-1R	aacccaaacagcaccaaagc(4)
SCAP-2F	gtggccttttagcattgcct(5)
SCAP-2R	ttagctaaccaggccaggac(6)
SCAP-3F	ccagttgggcttcttcgtaag(7)
SCAP-3R	tacagtcccagctaatcggg(8)
SCAP-4F	gtgacatcggcaagctcatt(9)
SCAP-4R	ggctatgtgtgtgccatctg( 10)
SCAP-5F	tagtacacttgcccgtcctc(l 1)
SCAP-5R	gggtgatggtgtaggagacc( 12)
SCAP-6F	agcctaaaaccctgcagact( 13)
SCAP-6R	caagagagaccagaggaggc(14)
SCAP-7F	ttgtgattttcccagtgccc( 15)
SCAP-7R	aagctcctaagaccctgctg( 16)
SCAP-8F	cctgtccctttcctttggtg( 17)
SCAP-8R	tatacacacccagcccacaa(18)
SCAP-9F	gatgcaggtcttgttgggtg( 19)
SCAP-9R	tgacacatgaaccaggacga(20)
SCAP-1 OF	ccacactctgcttcttccct(21)
SCAP-1 OR	gctgtagtgatctgcttgcc(22)
SCAP-1 IF	ttggatcaggccttcagtcc(23)
SCAP-HR	tc gcttgttc aggtctgcta(24)
SCAP-12F	tttcaccactgtcctgtcca(25)
SCAP-12R	tgcttctcccacaggcttat(26)
SCAP-13F	gcaagtgttggcaggtgtta(27)
SCAP-13R	gggaaaggggatggtgagtt(28)
SCAP-14F	gacctacctctgtccccaac(29)
SCAP-14R	cagcagcaccaagacgatg(3 0)
SCAP-15F	gagacgtcacgctgtacaag(31)

PIDC1154344PAU

SCAP-15R	ggcacaaaggaggaaaggg(32)
SCAP-16F	catcgtcttggtgctgctg(33)
SCAP-16R	tgagaaggggcaagggaaaa(34)
SCAP-17F	Ttttcccttgccccttctca(35)
SCAP-17R	cacgctcaccttttgtccaa(36)
SCAP-18F	gactccccaggctatgact(37)
SCAP-18R	acagcagttgaagagaaccag(38)
SCAP-19F	gctttcctcttggccatgtc(39)
SCAP-19R	agcacccaagagacaagaca(40)
SCAP-20F	tgcagtttagaggtcggagg(41)
SCAP-20R	acctggtcaatgtacacggt(42)
SCAP-21F	tgtcttgtctcttgggtgct(43)
SCAP-21R	tgagcaaacacatggctgac(44)
SCAP-22F	atgtttgctgactcctggga(45)
SCAP-22R	caaaaggagacacagccctg(46)
SCAP-23F	gtcagccatgtgtttgctca(47)
SCAP-23R	gcctgacagatgatgatatggt(48)

Genomic DNA samples for PCR amplification were prepared and PCR amplification was then carried out:

A total volume of the reaction system was 25 pl and 0.2 ml Eppendorf centrifuge tubes were used. After being well-mixed and subjected to transient centrifugation, the system, containing 9.5 5 pl of ddH2O, 12.5 pl of Mix, lpl of forward primer, lpl of reverse primer and 1 pl of template, was subjected to PCR amplification performed on PCR instrument.

Touchdown PCR protocol was performed as follows: pre-denaturing at 95 °C for 5 min, denaturing at 95 °C for 30 s, annealing at 68 °C for 30 s and extending at 72 °C for 30s (annealing temperatures decreased by 1 °C for each circle and decreased to 58 °C after 10 circles), followed 10 by 30 circles of denaturing at 95 °C for 30 s, annealing at 60 °C for 30 s and extending at 72 °C for 30 s, after the last cycle of which Extension was performed at 72 °C for 10 min.

Thus, PCR amplification products of each sample were obtained.

3. Sequencing

The PCR products obtained from step 2 were purified by membrane under vacuum with a

MultiScreen-PCR Plate (Millipore, Billerica, MA, USA). Sequencing was conducted by Sanger method with a BigDye Terminator DNA Sequencing kit (version3.1) and 3730XL sequencer

PIDC1154344PAU (Applied Biosystems, Foster City, CA, USA). All suspicious mutations were identified by reverse sequencing. Purification and sequencing were done by BIG sequence Co., Ltd..

Fig. 3 is representative SCAP gene profiles, verified by Sanger sequencing, from a patient suffering PMI and a healthy subject within the PMI pedigree as well as a health subject outside the PMI pedigree, in which the SCAP gene of the patient suffering PMI has a c.3035C>T mutation. It can be seen form Fig. 3, all patients from the PMI pedigree carry the c.3035C>T mutation in exon 18 of the SCAP gene whereas both the healthy subjects from the PMI pedigree and the non-PIM pedigree do not carry such a mutation. Besides, one patient in the 70 PMI patients from Chinese Han population carries a point mutation p.V468A (c.1403 T>C) in exon 9 of the SCAP gene, which is not found in other subjects of both the healthy subjects and patients in the PIM pedigree and non-PIM pedigree.

Further bioinformatics analysis shows that the c.3035C>T (p.A1012V) mutation and p.V468A (c.1403 T>C) mutation were negative in 28,000 East Asian and kept highly conserved between species.

In summary, the SCAP gene was identified to be the pathogenic gene for PMI and the c.3035C>T (p.A1012V) mutation in SCAP gene was identified to be the pathogenic mutation for PMI.

Embodiment 3 Detection Kit

A detection kit, including primers capable of detecting a c.3035C>T mutation (in exon 18), for screening a biological sample predisposed to PMI was prepared. Such primers were a primer specific for an exon of an SCAP gene, namely exon 18 of the SCAP gene. The sequences of such primers are shown as SEQ ID NO 37-38 in embodiment 1.

Steps of screening the biological samples predisposed to PMI includes: extracting a DNA sample from a subject to be detected according to step 1 of embodiment 2, amplifying extracted the DNA sample (as a template) with the primer specific for the exon of the SCAP gene by PCR reaction; purifying obtained PCR products by the method commonly known in the art; and sequencing purified product, in which an existence of a c.3035C>T mutation in obtained sequence by sequencing is an effective indicator for detection whether the SCAP gene mutant of the present disclosure exists in the DNA sample of the subject to be detected, thereby determining whether the subject to be detected was predisposed to PMI and further screening out the biological sample predisposed to PMI from all the subjects.

PIDC1154344PAU

Specifically, the SCAP gene from the 3 three patients (PMI1-1, PMI 1-3 and PMI 1-8) and 16 healthy subjects from the PMI pedigree described in embodiment 1, and 200 healthy subjects were detected for the C.3035OT mutation. As a result, the C.3035OT mutation existed in all patients from the PMI pedigree but was not found in all healthy subjects in the PMI pedigree as well as from non-PMI pedigree.

Industrial applicability

The SCAP gene mutant according to present disclosure, compared to SEQ ID NO: 1, has a c.3035C>T mutation. It is effective to determine whether a biological sample is predisposed to PMI by detecting the existence of the mutation in the biological sample.

Referring to the following descriptions and drawings, these and other aspects of the embodiments of the present disclosure will be apparent. In these descriptions and drawings, some specific approaches of the embodiments of the present disclosure are provided, so as to show some ways to perform the principle of the embodiments of the present disclosure, however it should be understood that the embodiment of the present disclosure is not limited thereby. Instead, the embodiments of the present disclosure comprise all the variants, modifications and their equivalents within the spirit and scope of the present disclosure as defined by the claims.

Reference throughout this specification to “an embodiment”, “some embodiments”, “one embodiment”, “another example”, “an example”, “a specific example” or “some examples” means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as “in some embodiments”, “in one embodiment”, “in an embodiment”, “in another example”, “in an example”, “in a specific example” or “in some examples” in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims (1)

1. What is claimed is:

   1. An isolated polypeptide having a p.A1012V mutation as compared to SEQ ID NO: 2.

   1000

   A

   100

   B

   200

   library construction unit sequencing unit 201 202

   Fig. 1