A method for detecting pathogenic/susceptible genes of pulmonary arterial hypertension

A pulmonary arterial hypertension and susceptibility gene technology, applied in the field of genetic engineering and molecular genetics, can solve the problems of lagging progress, high incidence, heavy economic burden, etc., and achieve low cost, strong practicability, and many detection sites. Effect

Active Publication Date: 2021-02-05
GENERAL HOSPITAL OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The second-generation high-throughput sequencing technology (next-generation sequencing technology) has the advantages of fast, accurate, and low cost, and can detect various types of mutations in multiple genes at the same time, and has been widely used in the etiology detection and molecular analysis of genetic defects. Genetic diagnosis, however, so far, there have been no high-throughput probes, chips or kits specifically for the detection of pathogenic and / or susceptibility genes for pulmonary arterial hypertension. The high level remains high, genetic counseling and necessary prenatal interventions cannot be implemented, and patients with this disease that should have been discovered early, identified the cause early, intervened early, and even partially cured cannot receive early attention, which not only brings huge losses to patients and their families. The pain and heavy economic burden of the country have also seriously hindered the substantial improvement of the overall quality of the population in our country.

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  • A method for detecting pathogenic/susceptible genes of pulmonary arterial hypertension
  • A method for detecting pathogenic/susceptible genes of pulmonary arterial hypertension
  • A method for detecting pathogenic/susceptible genes of pulmonary arterial hypertension

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Embodiment 1 Design and preparation of the probe set of the present invention

[0075] 1. Screening of pathogenic and / or susceptibility genes

[0076] The pathogenic and / or susceptible genes in this example are: BMPR2, SMAD9, CAV1, KCNK3, ABCA3, SARS2, HTR2B, THBS1, TOPBP1, EIF2AK4, CPS1, KRT18, KRT8, ABCD4, PIEZO2, NFU1, BOLA3, ATP5A1 , ARHGAP31, DOCK6, EOGT, RBPJ, DLL4, ENG, ACVRL1, GDF2, and SMAD4.

[0077] Whole exome sequencing was carried out on 100 patients with pulmonary arterial hypertension (the patients were clinically diagnosed with pulmonary arterial hypertension in the hospital, each patient signed an informed and voluntary agreement, and was approved by the hospital's medical ethics committee), covering and testing the human genome. All exons of more than 30,000 genes are known. Among them, the mutated genes of patients with pulmonary arterial hypertension are highly concentrated. Although the remaining genes (accounting for the vast majority) of the gen...

Embodiment 2

[0083] Embodiment 2: Composition, preparation and use of the kit of the present invention.

[0084] The kit for detecting the pathogenic and / or susceptibility genes of pulmonary arterial hypertension described in this embodiment is to carry out the molecular genetic diagnosis of the individual under examination by detecting the mutations of the above-mentioned 27 pathogenic and / or susceptibility genes Or a kit for morbidity risk prediction.

[0085] 1. The composition of the kit

[0086] The components contained in the kit are: the probe set obtained in Example 1 (160 μL, 150 ng / μL), enrichment buffer (208 μL), hybridization buffer (800 μL), binding buffer (3.2 mL), washing Solution 1 (9mL), rinse solution 2 (45mL), NaOH solution (0.1M, 1mL), Tris-HCl buffer (1M, pH 7.5, 1.2mL), PCR reaction solution (580μL), TE buffer (800μL, 10mM Tris-HCl, 1mM EDTA, adjust the pH to 8.0, and adjust the volume to 500mL with water). Wherein each buffer composition is as follows:

[0087] (...

Embodiment 3

[0138] Example 3 The use effect verification of the kit of the present invention

[0139] Using the kit of the present invention (prepared and obtained in Example 2) to detect 72 samples, the results confirm that the capture rate of the target disease-causing and / or susceptibility genes is satisfactory, and more than 97.7% of the original short sequences can be compared back to the target For the reference sequence of the region, the average effective sequencing data volume of the target region reaches 343.78Mb, and the average sequencing depth of the target region is 596X (see Table 1), which is much higher than the general genetic disease diagnosis requirements (generally 20X).

[0140] Table 1 The quality of sequencing data for pathogenic and / or susceptibility genes

[0141]

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Abstract

The invention provides application of a reagent for detecting the pathogenic and / or susceptibility genes of pulmonary arterial hypertension in the preparation of products for diagnosing the pulmonaryarterial hypertension. The reagent can capture various pathogenic and / or susceptibility genes of the pulmonary arterial hypertension and can detect 27 related genes at most. The reagent and products containing the same can explain the causes of the pulmonary arterial hypertension, evaluate the risks and risk factors of the pulmonary arterial hypertension and provide reference for individual patient molecular genetic diagnosis, high-risk people screening, high-risk family member screening, corresponding genetic counselling and prenatal diagnosis and intervention so as to reduce the incidence rate of the pulmonary arterial hypertension. A probe set, a chip and a kit in the invention are efficient, many in detection sites, accurate, simple to operate, good in specificity, high in sensitivity,fast, high in practicality and low in cost.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and molecular genetics, in particular to a probe set and kit for individualized capture and sequencing of pathogenic and / or susceptible genes of pulmonary arterial hypertension. Background technique [0002] Pulmonary arterial hypertension (PAH) is the most common pulmonary vascular disease, with progressive increase in pulmonary artery pressure, diffuse pulmonary arteriolar spasm and pulmonary vascular remodeling as the main pathological changes, eventually leading to death of patients due to right heart failure . Pulmonary hypertension is defined as mean pulmonary arterial pressure (mPAP) ≥ 25 mmHg (1 mmHg = 0.133 kPa) at rest, while pulmonary capillary wedge pressure (PCWP) or left atrial pressure (LAP) ≤ 15 mmHg. Pulmonary hypertension is currently divided into (5th Pulmonary Hypertension Conference in Nice, 2013): arterial pulmonary hypertension (including idiopathic, hereditary...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6809
Inventor 何昆仑伍建石金龙贾倩贾志龙赵晓静于康王依慧马俊峰张润敏
Owner GENERAL HOSPITAL OF PLA
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