AU2018102033A4 - Use of a molecular marker in early screening for sudden death - Google Patents

Abstract

The present invention provides the use of a molecular marker for early screening for sudden death and a kit for early screening for sudden death. The molecular marker is selected from the RYR2 gene, the RYR3 gene or the KCNQ5 gene.

Description

Title

Use of a molecular marker in early screening for sudden death

FIELD OF THE INVENTION

The present invention relates to the field of biomedicine, in particular to the use of molecular markers in early screening for sudden death and a kit for early screening for sudden death.

BACKGROUND OF THE INVENTION

Sudden death is the biggest challenge in modern cardiovascular medicine. Sudden death refers to a natural, unexpected sudden death. In particular, death within one hour of a patient's condition is defined as sudden death. The World Health Organization (WHO) provides: sudden death within 6 hours after onset of death. In general, unconscious death within 24 hours can also be referred to as sudden death in patients who are healthy or have not been diagnosed with any of these symptoms.

Although the death of some sudden deaths is immediate, most patients have a precursor condition (Reference 1). Most of the causes of sudden death are due to the deceased's cardiovascular disease that causes fatal ventricular arrhythmias (Refs. 2-5). In people over 40 years of age who die suddenly, coronary artery disease is the most important cause of sudden death. However, the proportion of other causes is high in young patients. Although forensic studies have shown that 60% -75% of sudden death in young adults have underlying genetic disorders (references 4-5), there are still a large number of sudden death cases that cannot be explained by clinical evidence.

Current clinical approaches do not provide a definitive diagnosis because most people with sudden unexplained sudden death have no clinically relevant history and do not undergo forensic testing after death (Refs. 3-4). For these sudden death cases, the incidence of sudden death was found to have risen rapidly among relatives of sudden death. Therefore, in order to carry out preventive treatment, genetic studies on sudden death of unknown reason are imminent.

Prior Art Document:

Reference 1: Antonio Baye' s de Lunaa, Roberto Elosuab. Sudden Death. Rev Esp Cardiol. 2012;65(l 1):1039-1052.

Reference 2: Zipes DP, Wellens HJJ. Sudden cardiac death. Circulation. 1998;98:2334-2351.

Reference 3: Bowker TJ, Wood DA, Davies MJ, Sheppard MN, Cary NRB, Burton JDK, Chambers DR, Dawling S, Hobson HL, Pyke SDM, Riemersma RA, Thompson SG. Sudden, unexpected cardiac or unexplained death in England: a national survey. Q J Med. 2003;96:269 -279.

2018102033 09 Dec 2018

Reference 4: Basso C, Calabrese F, Corrado D, Thiene G. Postmortem diagnosis in sudden cardiac death victims: macroscopic, microscopic and molecular findings. Cardiovasc Res. 2001;50:290 -300.

Reference 5: Drory Y, Turetz Y, Hiss Y, Lev B, Fisman EZ, Pines A, Kramer MR. Sudden unexpected death in persons less than 40 years of age. Am J Cardiol. 1991;68:1388-1392.

SUMMARY OF THE INVENTION

In order to solve one or more of the above problems, the present invention provides the use of a molecular marker selected from the RYR2 gene, the RYR3 gene or the KCNQ5 gene in early screening for sudden death.

Among them, the RYR2 gene SNP site A2387T. Among them, the RYR3 SNP locus is Q1989R.

Among them, the KCNQ5 gene SNP site is VI11G.

The present invention also provides a kit for early screening for sudden death that includes one or more binding moieties of a protein that selectively binds to a molecular marker selected from the group consisting of a RYR2 gene, a RYR3 Gene or KCNQ5 gene. Among them, the RYR2 gene SNP locus A2387T, RYR3 SNP locus Q1989R, KCNQ5 SNP locus VI11G.

According to the present invention, an important scientific basis for early screening for sudden death can be provided.

DESCRIPTION OF DRAWINGS

Figure 1 shows a family of proband's family as a research subject according to an embodiment of the present invention.

DESCRIPTION OF PREFERRED EMBODIMENT

The inventor tried to use the study of sudden death. Second-generation sequencing is a high-throughput, low-cost method (see references 6 and 7). Specifically, the inventors conducted an early screening study of sudden death using whole exome sequencing as the most cost-effective second generation sequencing technology. By detecting the genetic variation of the sudden-death pedigrees, we found for the first time the mutation sites that have positive significance for the diagnosis of sudden death and further applied these genes and loci to the early screening of sudden death. In the following, in order to make the objectives, technical solutions and advantages of the present invention more comprehensible, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not intended to limit the present invention. It should be noted that, unless otherwise specified, the experimental methods in the part of the embodiments are all conventional methods, and details are not described herein again.

materials and methods

1.1 research object

2018102033 09 Dec 2018

Probate (male, 15 years old) and their parents

The proband in 2012 due to sudden death and family history of dizziness, fatigue went to cardiovascular medicine clinic. After admission to their resting ECG, exercise ECG, 24h Holter, Doppler echocardiography and cardiac MRI methods were examined, the results showed that were normal. The electrophysiological test results show that with premature ventricular premature beats (for Is) symptoms. Immediately after discharge from the hospital, the proband died suddenly after walking slowly. The age of death is 17 years old. Post mortem autopsy of the proband, the results showed no obvious cardiovascular system organic disease.

As shown in the family tree of probands in Figure 1, the probationer's brother, Z, who died suddenly attempting to seize the horizontal bars of the playground, was 18 years old. The family, X and Q were sudden death at the age of 35 and 18, no history of syncope. The proband's cousin W now has repeated symptoms of syncope.

1.2 The main reagent

Genomic Extraction and Purification Kit (Qiagen, Germany), DNA Sample Pretreatment Kit (llumina, USA).

1.3 The main equipment

High-throughput second-generation sequencer (Hiseq 2000, llumina, USA).

1.4 whole exome sequencing

Reference to the manufacturer's instructions, the kit (TruSeq Exome Enrichment kit, Illumina, USA) probands and their parents of genomic DNA were processed. After processing, sequencing was performed using a sequencer (Hiseq 2000), ensuring at least 60-fold coverage of each sample during sequencing. The raw data is processed using software (Illumina basecalling software 1.7) and the parameters are set to software defaults.

1.5 data processing

DNA sequence data was processed using the software Burrows-Wheeler Aligner (Reference 8) and SOAPaligner v2.21 (Reference 9), a reference database for the NCBI human genome reference assembly build 37 (http://hgdownload.cse.ucsc.edu / goldenPath / hgl9 / bigZips /), the parameters using software defaults.

Single nucleotide polymorphisms (SNPs) data and insertion / deletion data are also analyzed. SOAPsnp software is used to match the heterogeneity of DNA sequences and to differentiate genotypes in the target region (Ref. 10). Annovar (Ref. 11) software is used to annotate the effects of mutations. All included SNPs were filtered using the Single Nucleotide Polymorphism database (dbSNP 132, http://www.ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi) database, filtering out all known SNPs. SIFT software (http://sift.jcvi.org) was used to assess the potential threat of mutation. The processed raw data is considered to be related to the occurrence of sudden death.

2, the result

A total of 600,897,152 raw read and write data were obtained through experiments. The average misreading rate was only 0.21%, with an average coverage of 99.34%. After strict quality control and data processing, three candidate mutation sites were screened out as diagnostic molecular markers for sudden death.

2018102033 09 Dec 2018

Specifically, based on the pedigrees, the recessive genetic model was used to analyze the screening sites. The results showed that the single nucleotide mutation at position 237804240 on chromosome 1 of RYR2 gene was detected, and the mutation pattern was A2387T. The SIFT predicted result was dangerous Mutation; RYR3 gene chromosome 33988524 on chromosome 15 single-base mutation, the mutation in the form of Q1989R, bioinformatics prediction for the risk of mutation (DAMAGING); KCNQ5 gene on chromosome 673332249 V111G single base mutation occurs, the result is a dangerous mutation (Table 1).

Table 1: SNPs using for early screening of sudden death

Gene	Chromosomal location	ref	codon	replacement	SIFT prediction
RYR2	chrl_237804240	G	GCG7159ACG	A2387T	DAMAGING
RYR3	chrl5_33988524	G	CAG5966CGG	Q1989R	DAMAGING
KCNQ5	Chr5_673332249	A	GTG332GGG	V111G	DAMAGING

Therefore, the A2387T site of RYR2 gene, the Q1989R site of RYR3 gene and the V111G gene of KCNQ5 have a strong correlation with the occurrence of sudden death, and can be used as molecular markers for screening for sudden death. The discovery of these markers can provide an important scientific basis for the early screening of sudden death and the development of diagnostic kits. At the same time, it provides an important laboratory basis for revealing the molecular biology and genetics mechanism of sudden death.

3, verification test

Patients who take the case group 600, 600 to adopt healthy persons as a control, DNA extracted from peripheral per person, and using PCR primers designed technology, including the mutation site was amplified DNA sequences in vitro, and then use restriction The method of enzymatic digestion, DNA fragments were cut to determine the genotype.

The obtained genotype data for statistical analysis, as shown in Table 2. Table 2:analysis results of relationship between SNP and sudden death „ _R case control odds ratio (95% P

SNPkS genotype group group CI) value

N (%) N (%)

RYR2

A2387T

GG 378(63%) 340(56.7%)

GA 196(32.7) 218(36.3%)

AA 26(4.3%) 42(7%)

AVSG

AAVS GG

1.29(1.07,1.56) 0.009

1.80(1.08,2.99) 0.023

2018102033 09 Dec 2018

	AA VS (AG+GG)	~ ~ 1.30(1.03,1.64)	0.025
	(AA+AG) VS GG			1.66(1.01,2.75))	0.046
RYR3
Q1989R	AA	276(46%)	238(39.7%)
	GA	272(45.3 %)	285(47.5%)	—	—
	GG	52(8.7%)	77(12.8%)	—	—
	AVSG			1.26(1.07,1.50)	0.006
	AAVS GG			1.72(1.16,2.54)	0.007
	AA VS (AG+GG)			1.30(1.03,1.63)	0.027
	(AA+AG) VS GG			1.55(1.07,2.25)	0.020
KCNQ5 V111G	TT	383(63.8 %)	349(58.2%)
	GT	197(32.8 %)	206(34.3%)
	GG	20(3.3%)	45(7.5%)
	TVSG			1.33(1.10,1.61)	0.004
	TT VS GG			2.47(1.43,4.26)	< 0.001
	TT VS (TG+GG)			1.27(1.01,1.60)	0.044
	(TT+TG) VS GG			2.35(1.37,4.03)	0.002

The test showed that in the homozygous, allelic, overdominant and invisible gene models, the genotype distributions of the three mutation sites were significantly different between the control group and the case group ( P <0.05).

In summary, in order to carry out preventive treatment for sudden death, the present invention uses advanced second-generation sequencing technology to study the disease and found that RYR2 gene A2387T site, RYR3 gene Q1989R site and KCNQ5 gene V111G and Sudden death occurs with a strong correlation, which will be used as a molecular marker of sudden death screening. The discovery of these markers can provide an important scientific basis for the early screening of sudden death and the development of diagnostic kits. At the same time, it provides an important laboratory basis for revealing the molecular biology and genetics mechanism of sudden death.

The above is only the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention. Any modification or equivalent

2018102033 09 Dec 2018 substitution of the present invention without departing from the spirit and scope of the present invention should be covered by the following claims Among the scope of protection.

Claims (8)

1. Use of a molecular marker in early screening for sudden death, which is the molecular marker, is selected from the group consisting of RYR2 gene, RYR3 gene or KCNQ5 gene.

2. The use according to claim 1, which is the SNP site of the RYR2 gene is A2387T.

3. The use according to claim 1, which is the RYR3 gene SNP site is Q1989R.

4. The use according to claim 1, which is the KCNQ5 gene SNP site is V111G.

5. A kit for early screening for sudden death, which is the kit comprises one or more binding moieties for a protein selectively bound to a molecular marker selected from the group consisting of RYR2 Gene, RYR3 gene or KCNQ5 gene.

6. The kit according to claim 5, which is the SNP site of the RYR2 gene is A2387T.

7. The kit according to claim 5, which is the SNP site of the RYR3 gene is Q1989R.

8. The kit according to claim 5, which is the SNP site of the KCNQ5 gene is V111G.