AU2018101290A4 - A method for predicting genetic risk of salt-sensitive hypertension based on next-generation sequencing - Google Patents

A method for predicting genetic risk of salt-sensitive hypertension based on next-generation sequencing Download PDF

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AU2018101290A4
AU2018101290A4 AU2018101290A AU2018101290A AU2018101290A4 AU 2018101290 A4 AU2018101290 A4 AU 2018101290A4 AU 2018101290 A AU2018101290 A AU 2018101290A AU 2018101290 A AU2018101290 A AU 2018101290A AU 2018101290 A4 AU2018101290 A4 AU 2018101290A4
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snps
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Jinge Cao
Jialao Chen
Ruichuan Li
Tang Li
Jianzhi Niu
Ziqi ZHOU
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Cao Jinge Miss
Li Tang Miss
Niu Jianzhi Miss
Zhou Ziqi Miss
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Li Tang Miss
Niu Jianzhi Miss
Zhou Ziqi Miss
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Abstract

This invention aims to predict the genetic risk of salt-sensitive hypertension. It consists of 3 fundamental technical steps: panel design, gene sequencing and standardized bioinformatics analysis. First, through the NCBI database and related literature searches, the research group selected 47 single nucleotide polymorphisms (SNPs) that are significantly associated with salt-sensitive hypertension, and the variants at these positions would induce the disease to various degrees. Then, with the Burrows-Wheeler Aligner (BWA), Picard-tools, GATK, Annovar and other arithmetic operations, the genome are mapped within the BWA. Subsequently, the data attained are sorted, deduped, realigned, calibrated, filtered, and annotated. Afterward, an annotated file with the data of variant positions, the gene types, the chromosome number of the SNPs, the types of mutation, and the functions of the reference gene is generated. Finally, Perl is programmed to extract the SNPs of the test subjects, and compare them with the SNPs in the panel designed. If a person has more variants included in the panel than the average level of normal samples, he may have a high risk of afflicting salt-sensitive hypertension.

Description

invention aims to predict the genetic risk of salt-sensitive hypertension. It consists of 3 fundamental technical steps: panel design, gene sequencing and standardized bioinformatics analysis. First, through the NCBI database and related literature searches, the research group selected 47 single nucleotide polymorphisms (SNPs) that are significantly associated with salt-sensitive hypertension, and the variants at these positions would induce the disease to various degrees. Then, with the Burrows-Wheeler Aligner (BWA), Picard-tools, GATK, Annovar and other arithmetic operations, the genome are mapped within the BWA. Subsequently, the data attained are sorted, deduped, realigned, calibrated, filtered, and annotated. Afterward, an annotated file with the data of variant positions, the gene types, the chromosome number of the
SNPs, the types of mutation, and the functions of the reference gene is generated. Finally, Perl is programmed to extract the SNPs of the test subjects, and compare them with the SNPs in the panel designed. If a person has more variants included in the panel than the average level of normal samples, he may have a high risk of afflicting salt-sensitive hypertension.
2018101290 04 Sep 2018
DESCRIPTION
Title
A method for predicting genetic risk of salt-sensitive hypertension based on next-generation sequencing
FIELD OF THE INVENTION
The invention relates to the field of genetic diagnosis, with a focus on salt-sensitive hypertension. This method is unquestionably more efficient than the traditional clinical diagnosis, regarding the time required and patient's comfort level. This invention presents a method for predicting genetic risk of salt-sensitive hypertension.
BACKGROUND OF THE INVENTION
Salt sensitivity is an increase in blood pressure induced by a high amount of salt intake. Sodium ion transport mechanism, renin-angiotensin-aldosterone system (RAAS) mechanism, sympathetic activation, renal sodium excretion and inflammation, endothelial dysfunction, and endocrine mechanism are involved in the pathogenesis of salt-sensitive hypertension/11 D Gu conducted a genome-wide association study of BP responses in 1906 Han Chinese which is the most extensive in China. Additionally, salt-sensitive normotensive subjects have higher mortality and morbidity rate than salt-resistant normotensive subjects121; however, accurate testing of salt sensitivity is not only laborious but also expensive, and with low patient compliance.
2018101290 04 Sep 2018
More importantly, patients who have normal blood pressure but are salt-sensitive cannot be diagnosed in an office setting, and there are no laboratory tests for salt sensitivity131. To deal with this medical dilemma, precision medicine with a focus on genome sequencing is applied.
Precision Medicine, also known as personalized therapy, is an advanced therapeutic method considering individual genotype, environment, and individual lifestyle. With the molecular diagnosis, the doctor could treat the patients more accurately and minimize the side effects.
Genome sequencing technology, a key driver for precision medicine, has improved substantially in accuracy, speed, and cost141. Nowadays, scientists are striving to combine targeted sequencing technology with big clinical data to assist medication.
The candidate genes are selected from the paper of D Gu. and inputted to the computer programs of GATK and Perl.171 To manoeuver this mechanism, the researchers conduct gene sequencing on the allelic variants found. Through the sequencing, the correlation between these genes and salt-sensitive hypertension is established with valid evidence.
This application of the gene database and genome sequencing raises people's awareness of salt-sensitive hypertension and expedites the diagnosis and treatment of it. Thus, theoretically, this invention can facilitate the doctors to cure the patients more accurately and without
2018101290 04 Sep 2018 the abuse of medication.
Reference:
[1] Yi L. Advances in Salt Sensitivity Hypertension [J]. Adv Cardiovasc Dis,
2013, 34(5):644-647.
[2] Strazzullo P, D'Elia L, Kandala N B, et al. Salt intake, stroke, and cardiovascular disease: meta-analysis of prospective studies[J]. Bmj,
2009, 137(7733):1296-1296.
[3] Felder R A, White M J, Williams S M, et al. Diagnostic tools for hypertension and salt sensitivity testing[J]. Current Opinion in
Nephrology & Hypertension, 2013, 22(1):65.
[4] Feero W G, Wicklund C A, Veenstra D. Precision Medicine, Genome
Sequencing, and Improved Population Health[J]. Jama, 2018, 319(19).
[5] Sanada H, Jones J E, Jose P A. Genetics of salt-sensitive hypertension.[J]. Current Hypertension Reports, 2011,13(1):55-66.
[6] Strazzullo P, Galletti F. Genetics of salt-sensitive hypertension[J].
Current Hypertension Reports, 2007, 9(1):25-32.
[7] Guo L, Liu F, Chen S. Common variants in the Na(+)-coupled bicarbonate transporter genes and salt sensitivity of blood pressure: the
GenSalt study.[J]. Journal of Human Hypertension, 2015, 30(sl):208-209.
[8] Moyer A M, Caraballo P J. The challenges of implementing pharmacogenomic testing in the clinic[J]. Expert Review of
Pharmacoeconomics & Outcomes Research, 2017,17(6):567.
2018101290 04 Sep 2018
SUMMARY OF THE INVENTION
Salt-sensitive hypertension refers to the mechanism of one's body responding to high salt-intake with blood pressure fluctuations. With this invention, the researchers can determine whether a patient is likely to be afflicted with salt-sensitive hypertension. By reading relative literature, the research group found SNPs related to salt-sensitive hypertension among Han Chinese population. The SNPs in the panel should meet the following criteria: first, the SNPs are significant, which suggests a p value under 0.05; secondly, there should be no gender difference; lastly, the
SNPs should be related to Pathogenesis of salt-sensitive hypertension.
The SNPs with all the criteria met are considered filtered and ready for the panel.
Eventually, 47 gene sites related to salt-sensitive hypertension were selected into the designed panel. Computing programs such as BWA,
Picard-tools, GATK, and Annovar were applied to predict the genetic risk of salt-sensitive hypertension. The individual variation of salt-sensitive hypertension can be obtained by the following steps.
1. mapping stage
The essence of mapping is to place a set of molecular gene markers on their gene positions, so it will facilitate the observation and related record processing.
2. Preprocessing stage
2018101290 04 Sep 2018
Sort the sequence in ascending order and mark the duplicates of the sequence, and then re-align and re-calibrate the sequence.
3. Variants calling stage
The process by which we identify variants from sequence data, so we could detect and identify SNPS and Indels.
4. Variants filtration stage
Filter SNPs and Indels according to different standards to achieve the purpose of calibration.
5. Annotation stage
Annotation is used to mark the location and frequency of variations, whether the amino acids change, and which DNA fragments are changed by SNP also whether the mutations cause salt-sensitive hypertension.
Ultimately, based on the self-designed Perl program , it would be identified whether the person has high frequency of mutations causing salt-sensitive hypertension so that genetic risk of salt-sensitive hypertension would be predicted.
DESCRIPTION OF DRAWING
The following drawings are only for the purpose of description and explanation but not for limitation, where in:
Figure.1 Process of selecting SNPs
Figure.2 Distribution of mutations on chromosomes
Figure.3 Distribution of mutations on func.Ref
2018101290 04 Sep 2018
Figure.4 The relation of pathogenesis and mutations.
Figure.5 procedures
DESCRIPTION OF PREFERRED EMBODIMENTS
Designing of the panel
The ultimate objective of this panel is to establish a method for predicting genetic risk of salt-sensitive hypertension via genetic sequencing on some specific mutation sites. The panel designing is based on the salt-sensitivity hypertension incidence rates of a mutation site. In cases of each polymorphism on table x, all of those mutation sites mathematically induce a higher risk for experimental objects. The reference documentation mainly focused on the genes relating blood pressure experiments, GenSalt study by Dongfeng Gu on Chinese Han nationality since 21century. There are 47 mutation sites in total from the references, and they played different roles in the mechanism of salt-sensitivity hypertension. Moreover, the distribution feature of the corresponding genes is not concentrated on a specific chromosome but diverse. Parts of detailed information for some mutation sites are not comprehensive, and they are completed based on the database from
PubMed, an institute of biomedical information.
SNPs we found shows in tablel.
Risk Variants
Chr Gene Snpl38
2018101290 04 Sep 2018
1 ECE1 rs213011
1 rsl69884
1 rs84853
1 rs213012
1 rs213014
1 rs213018
1 rs213025
1 HSD11B1 rs932335
1 PRMT6 rsl330225
1 SELE rs4656704
1 rs6427212
1 rs5368
1 rs3917436
2 ARL4C rsll887188
2 CDCA7 rsl0930597
2 TRPM8 rs7577262
3 AGTR1 rs75367686
4 NR3C2 rsl879829
4 rs2070951
4 rs7694064
4 rs6856803
4 SLC4A4 rs4254735
2018101290 04 Sep 2018
4 rsl0022637
6 IRAK1BP1 rsl6890334
7 FBXL13 rsl7135875
12 SCNN1A rsll064153
12 rsll614164
12 rsll064153
13 PIBF1 rs8002688
14 BDKRB2 rsll846625
16 SALL1 rs2030114
16 SCNN1B rsl004749
16 rs250567
16 SCNN1G rs4073930
16 rs4073291
16 rS7404408
16 rs4299163
16 rs4499238
16 rs4073930
16 rs4073291
16 rS7404408
16 rs5735
16 rs4299163
16 rs4499238
2018101290 04 Sep 2018
16 rs4401050
X ACE2 rsl84874220
X RENBP rs78377269
Tablel
Procedures of Genome Comparison
The panel data from exome of people with salt-sensitive hypertension, of which single-nucleotide variants (SNVs), multi-nucleotide polymorphism (MNPs), small insertions and deletions (INDELs), and the combination of
SNPs and INDELs at a single position (MIXED) are arrayed as raw reads in
FASTQ format.
Quality evaluation
A software FastQC was used in this process, which could evaluate the sequence quality across all bases (Sanger / illumine 1.9 encoding).
1. Mapping
Burrows-Wheeler Aligner (BWA) was to create a reference index and then get the alignment of the sample sequence to the reference sequence, which created by FastQC. The information contains: number of samples sequence, number of references sequence, flag, the position of genes, quality, CIGAR, number of references sequence for the next fragment comparison, position of the next fragment comparison, length of templet, sequence information and quality information of sequence.
2018101290 04 Sep 2018
2. Preprocessing
This step was to standardise the file and fix any possible errors, involves sorting, marking duplicating, realigning and recalibrating.
1) Sorting and duplicates marking
The order sorting of chromosomes and duplicates marking was processed by SortSam and MarkDuplicate (Picard) respectively.
2) Realigning
RealignerTargetCreator (GATK) was a tool that marks the regions which need to be compared again. In this case, there was necessary to acquire some known mutation sites. Those regions need to be re-aligned with
IndelRealigner (GATK) to get the result.
3) Recalibrating
The realigned document was used to establish a calibration model with known mutation database via BaseRecalibrator (GATK). The quality of reported data was be calibrated with the empirical quality via PrintReads (GATK).
3. Calling
Calling is a process using HaplotypeCaller (GATK) to detect and identify
SNPs, Indels and some SNVs variants of salt-sensitive hypertension. It could determine potential variants region and potential haplotypes variants, construct a deBrujin assembly of the region, calculate haplotype similarity and allele frequency distribution. As a result, it can
2018101290 04 Sep 2018 provide quality, filter information, detailed information for variants and genotype of the sample.
4. Filtering
The filtering process sifted out false-positive SNPs and INDELs resulting from sequencing errors and alignment errors. SelectVariants (GATK) and
Variantfiltration (GATK) were used to select target variants and then filter by specific filtering rules of SNPs and INDELs respectively. These two results were combined into one filter information document with
CombineVariants (GATK).
5. Annotation
There are few functions involved in Annovar software: annotating SNVs,
INDELs, gene-based, region-based, filter-based variants or even custom annotations. Those variants related to salt-sensitivity hypertension could be annotated during this process.
2018101290 04 Sep 2018

Claims (3)

Claims
1) build an index
1) sort and dedup the sequence Picard
2) re-align and re-calibrate the sequence GATK filter false-positive SNPs and Indels caused by sequencing and aligning errors
Part 6 Extract Target Region extract the salt-sensitive related information by
L J
Part Ί Mapping
1. mapping stage
The essence of mapping is to place a set of molecular gene markers on their gene positions, so it will facilitate the observation and related record processing;
2. Preprocessing stage
Sort the sequence in ascending order and mark the duplicates of the sequence, and then re-align and re-calibrate the sequence;
3. Variants calling stage
The process by which we identify variants from sequence data, so we could detect and identify SNPS and Indels;
4. Variants filtration stage
Filter SNPs and Indels according to different standards to achieve the purpose of calibration;
5. Annotation stage
Annotation is used to mark the location and frequency of variations, whether the amino acids change, and which DNA
2018101290 04 Sep 2018 fragments are changed by SNP also whether the mutations cause salt-sensitive hypertension;
Ultimately, based on the self-designed Perl program , it would be identified whether the person has high frequency of mutations causing salt-sensitive hypertension so that genetic risk of salt-sensitive hypertension would be predicted.
2018101290 04 Sep 2018
Figure 1
2018101290 04 Sep 2018
chr1 ECE1 HSD11B1 PRMT6 SELE ARL4C Ichr2 CDCA7 TRPM8 chr3 AGTR1 |chr4 NR3C2 SLC4A4 chrS 1RAK1BP1 chr7 IFBXL13 chr12 SCNN1A dir13 pibfi chrl4 | BDKRB2 SALL1 chr16 SCNIN1B 1SCNN1G lx ACE2 x RENBP
Figure 2 intergenic 
15 prime UTR variant 3'UTR | Exonic
Intronic ARL4C CDCA7 IRAKI BP1 PRMT6 SALL1 TRPM&
AGTR1 |NR3C2 RENBP ISCNN1B |SCNN1G | SELE ACE2 BDKRB2 ECE1 FBXL13 HSD11B1 PIBF1 SCNIN1A SLC4A4
Figure3
2018101290 04 Sep 2018 | About ENaC About Endothelial function
About RAAS unknown SCNN1A SCNN1B SCNN1G ECE1 SELE ACE2 AGTR1 NR3C2 RENBP ARL4C BDKRB2 CDCA7 FBXL13 HSD11B1 IRAKI BP1 PWIBF1 PRMT6 SALL1 SLC4A4 TRPM8
Figure4
Part 2 Preprocessing
1. An method of for detecting genetic risk of salt-sensitive hypertension,in which 47 Single Nucleotide Polymorphisms were selected after analysis in which we used BMA, Picard-tools, GATK,
ANNOVAR and a Perl program we designed.
2. As claim 1 said method, which includes following steps:
2) align the sample sequence
BWA .
Part
3 Calling detect and identify the saltsensitive related variant
Part 5 Annotation add gene-based, regionbased and filter-based annotation
Annovar j
Figure5
AU2018101290A 2018-09-04 2018-09-04 A method for predicting genetic risk of salt-sensitive hypertension based on next-generation sequencing Ceased AU2018101290A4 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109750101A (en) * 2019-02-15 2019-05-14 中国医学科学院阜外医院 Detect gene panel and its application of monogenic inheritance hypertension

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109750101A (en) * 2019-02-15 2019-05-14 中国医学科学院阜外医院 Detect gene panel and its application of monogenic inheritance hypertension

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