Application of NR3C1 gene BclⅠ site polymorphism in the evaluation of refractory septic shock in neonates

Abstract

The present invention provides an application of NR3C1 gene BclⅠ site polymorphism in the evaluation of refractory septic shock in neonates. The present invention finds that NR3C1 gene BclⅠ site polymorphism can be used to evaluate the severity of neonatal septic shock; further, NR3C1 gene BclⅠ site polymorphism can be used to evaluate the risk and prognosis of refractory septic shock in neonates. Compared with NR3C1 gene BclⅠ site G allele, children with NR3C1 gene BclⅠ site C allele have a higher risk of developing refractory septic shock and a worse prognosis. The present invention provides a new specific indicator for evaluating the severity of neonatal septic shock and the risk of children developing refractory septic shock, provides an early warning for refractory septic shock, and provides guidance for the formulation of clinical treatment plans for neonatal septic shock.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular diagnosis, and specifically relates to the application of NR3C1 gene BclⅠ site polymorphism in the evaluation of refractory septic shock in neonates. Background Art

[0002] Septic shock, also known as infectious shock, is the most critical stage of sepsis progression. It is caused by circulatory, cellular and metabolic disorders caused by microorganisms and their toxins. Microorganisms and their toxins, cell wall products, etc. in the infection focus invade the blood circulation and activate various cells and fluid systems of the host; cytokines and endogenous mediators are produced, which act on various organs and systems of the body, leading to tissue hypoperfusion, cardiovascular dysfunction, and even multiple organ failure.

[0003] Septic shock is the leading cause of neonatal death, accounting for 45% of deaths in neonatal intensive care units. In addition, the mortality rate of refractory septic shock (RSS) can be as high as 80%. Studies have shown that RSS is the leading cause of death in children with septic shock. Therefore, the successful diagnosis and treatment of RSS is an important means to reduce the mortality rate of children with septic shock. In 2016, the European Society of Paediatrics and Neonatal Intensive Care proposed a septic shock score (SSS) to facilitate the early diagnosis and treatment of RSS. Based on whether the child has hyperlactatemia, high doses of vasoactive drugs and myocardial dysfunction, the SSS was proposed to guide the diagnosis and treatment of children with RSS, and it was pointed out that bSSS ≥ 2 and cSSS ≥ 3.5 were significantly correlated with mortality.

[0004] The polymorphism of the NR3C1 gene BclⅠ (rs41423247) site is the wild-type GG genotype (called the G allele), the mutant GC genotype, and the CC genotype (called the C allele). There are some related studies on the relationship between the NR3C1 gene BclⅠ (rs41423247) site polymorphism and disease. For example, premature infants with hypotension carrying the NR3C1 gene BclⅠ site C allele require higher doses of vasoactive drugs and hydrocortisone; COVID-19 patients carrying the NR3C1 gene BclⅠ site C allele have a higher risk of death. However, there are no reports on the NR3C1 gene BclⅠ site polymorphism in neonatal septic shock. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide an application of the NR3C1 gene BclⅠ site polymorphism in the evaluation of refractory septic shock in neonates, so as to achieve early diagnosis and treatment of refractory septic shock and reduce neonatal mortality.

[0006] To achieve the above object, the present invention adopts the following technical solution.

[0007] The first aspect of the present invention provides the use of a reagent for detecting polymorphism of the BclⅠ site of the NR3C1 gene in the preparation of a kit for assessing the severity of septic shock in neonates.

[0008] In some embodiments, the severity of septic shock in neonates with the C allele at the Bcl I site of the NR3C1 gene is higher than that in neonates with the G allele at the Bcl I site of the NR3C1 gene.

[0009] The second aspect of the present invention provides the use of a reagent for detecting the polymorphism of the BclⅠ site of the NR3C1 gene in the preparation of a kit for assessing the risk of refractory septic shock in neonates.

[0010] In some embodiments, a neonate with the C allele at the Bcl I site of the NR3C1 gene has a higher risk of developing refractory septic shock than a neonate with the G allele at the Bcl I site of the NR3C1 gene.

[0011] The third aspect of the present invention provides the use of a reagent for detecting polymorphism of the BclⅠ site of the NR3C1 gene in the preparation of a kit for assisting in the prognosis assessment of neonatal septic shock.

[0012] The fourth aspect of the present invention provides the use of a reagent for detecting the polymorphism of the BclⅠ site of the NR3C1 gene in the preparation of a kit for assisting in the evaluation of a treatment regimen for neonatal septic shock.

[0013] In some embodiments, the reagent is a reagent used for detection by PCR method, qPCR method, sequencing method, PCR-RFLP method, TaqMan probe method, or gene chip method.

[0014] In some embodiments, the reagents include specific amplification primers for the NR3C1 gene.

[0015] In some preferred embodiments, the primers include an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2.

[0016] A fifth aspect of the present invention provides a kit for assessing the severity and / or prognosis of neonatal septic shock, the kit comprising a reagent for detecting the polymorphism of the BclⅠ site of the NR3C1 gene.

[0017] In some embodiments, the kit is a kit for detection by PCR method, qPCR method, sequencing method, PCR-RFLP method, TaqMan probe method, or gene chip method.

[0018] In some embodiments, the kit includes specific amplification primers for the NR3C1 gene.

[0019] In some preferred embodiments, the primers include an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2.

[0020] The present invention finds that the polymorphism of the BclⅠ site of the NR3C1 gene can be used to assess the severity of neonatal septic shock, and the severity of septic shock in neonates with the C allele of the BclⅠ site of the NR3C1 gene is higher than that in neonates with the G allele of the BclⅠ site of the NR3C1 gene. Further, the polymorphism of the BclⅠ site of the NR3C1 gene can be used to assess the risk and prognosis of refractory septic shock in neonates. Compared with the G allele of the BclⅠ site of the NR3C1 gene, the children with the C allele of the BclⅠ site of the NR3C1 gene have a higher risk of developing refractory septic shock and a worse prognosis. The present invention provides a new specific indicator for assessing the severity of neonatal septic shock and the risk of children developing refractory septic shock, provides an early warning for refractory septic shock, and provides guidance for the formulation of clinical treatment plans for neonatal septic shock. DETAILED DESCRIPTION

[0021] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0022] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0023] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0024] The following describes the invention in conjunction with specific embodiments.

[0025] In the present invention, a newborn is defined as an infant within 28 days after birth from the time the umbilical cord is tied.

[0026] Example 1

[0027] This example studies the association between the polymorphism of the BclⅠ site of the NR3C1 gene and septic shock in patients with neonatal septic shock.

[0028] 1. Sample information included in the study

[0029] Inclusion criteria: Patients met the diagnostic criteria for neonatal septic shock, that is, cardiovascular dysfunction caused by severe infection.

[0030] Exclusion criteria: congenital heart disease, suspected chromosomal disease or inherited metabolic disease, diaphragmatic hernia, and fetofetal transfusion syndrome.

[0031] According to the above inclusion and exclusion criteria, a total of 41 children were included.

[0032] 2. Detection method of polymorphism of BclⅠ site of NR3C1 gene

[0033] 1. When each participant is enrolled, 1 mL of venous blood is collected and placed in an EDTA tube and stored at -20°C. DNA is extracted from 300 μl of peripheral blood samples using the TIANamp Blood DNA Kit, and the operation is performed strictly according to the instructions.

[0034] 2. Take 20ng DNA for PCR+Sanger sequencing.

[0035] Specific primers for NR3C1 gene amplification are as follows:

[0036] NR3C1-F:5'-AGG TTA CGG GGT AGA GAA AT-3' (SEQ ID NO. 1);

[0037] NR3C1-R: 5'-AGC AAT GCA GTG AAC AGT GT-3' (SEQ ID NO. 2).

[0038] The following PCR amplification system was configured: 2.5mmol / L dNTP (1ul), 10*PCR buffer (2.5ul), 25mmol / LMgCl2, 10umol / L NR3C1-F (1ul), 10umol / L NR3C1-R (1ul), Taq DNA polymerase (2.5ul), and template DNA (5ul).

[0039] The PCR amplification system is carried out according to the following procedures: (1) high temperature denaturation: the DNA melting reactor is heated to about 93°C and maintained for a certain period of time, and the double-stranded DNA of the template DNA or the double-stranded DNA formed by PCR amplification is separated into single strands to facilitate binding with the primers; (2) low temperature annealing: the primers bind to the template DNA and the DNA molecules are heated to denature into single strands, and then the temperature is reduced to about 55°C, and the primers are paired with the complementary sequences of the template DNA single strand; (3) medium temperature extension: when the DNA synthesis reaches a suitable temperature (about 70°C), the DNA template-primer complex is subjected to the action of Taq DNA polymerase, with dNTP as the reaction raw material and the target sequence as the template, and according to the principle of base complementary pairing and semiconservative replication, a new semiconservative replication chain complementary to the template DNA chain is synthesized; (4) the following three steps are repeated for multiple cycles: high temperature denaturation → low temperature annealing → medium temperature extension.

[0040] The PCR amplification products were sent to the Institute of Women and Children's Health, Guangdong Provincial Women's and Children's Health Hospital for Sanger sequencing.

[0041] According to the sequencing results, among the 41 children's samples, 22 carried the BclⅠC allele (CC genotype and GC genotype) of the NR3C1 gene, and 19 carried the G allele (GG genotype).

[0042] 3. Result Analysis Method

[0043] According to the sequencing results, the children were divided into the NR3C1 gene BclⅠ site C allele group and the NR3C1 gene BclⅠ site G allele group.

[0044] 1. To analyze the following indicators of each group of children: low cardiac index, positive blood culture, epinephrine use rate, 72-hour cumulative fluid overload percentage, intensive care unit (h), and evaluate the severity of the disease in different allele groups.

[0045] 2. Analyze the situation of refractory septic shock in children in each group.

[0046] The present invention adopts the septic shock score (SSS) proposed by the European Association of Paediatrics and Neonatal Intensive Care in 2016 to judge refractory septic shock and non-refractory septic shock. The applicant can refer to the following research results: Zhong Junjuan et al.; The evaluation value of different shock scoring methods for the prognosis of refractory septic shock in neonates; Chinese Journal of Neonatology; Vol. 36, No. 6, November 2021. The details are as follows:

[0047] The SSS score is divided into computed septic shock scores (cSSS) and bedside septic shock scores (bSSS). It involves vaso-inotrope score (VIS), LAC, cardiac index (CI) and ejection fraction (EF), and is calculated according to the following formula:

[0048] ①VIS = dopamine dose + dobutamine dose + [(norepinephrine dose + epinephrine dose) × 100] + (milrinone dose × 10) [the dose of vasoactive drugs is in μg / (kg·min)];

[0049] ②CI = stroke volume × heart rate / [(0.035 × body weight + 0.1) × 1000];

[0050] ③cSSS=1.001 VIS +1.1 LAC(mmol / L) +18×[cardiac arrest or EF < 25% or CI < 2.2 L / (min·m 2 )]. Considering that this formula is not convenient for clinical calculation, it is improved to cSSS = 1.001 × VIS + 1.1 × LAC (mmol / L) + 18 × [cardiac arrest or EF < 25% or CI < 2.2 L / (min·m 2 ), if one or more items are met, the value is “1”, if none of them are met, the value is “0”];

[0051] ④bSSS: 1 point for VIS>200, 1 point for LAC>8mmol / L, 1 point for cardiac arrest or EF<25% or CI<2.2L / (min·m 2 ) gets 3 points.

[0052] cSSS ≥ 3.5 or bSSS ≥ 2 points was considered as refractory septic shock.

[0053] 3. SPSS 22.0 was used for statistical analysis. The measurement data were expressed as median (quartile) [M (P25, P75)], and the inter-group comparison was performed using the Mannn-Whitney U test; the enumeration data were expressed as frequency and rate (%), and the inter-group comparison was performed using the Chi-square test. P < 0.05 was considered statistically significant.

[0054] IV. Results

[0055] 1. Disease severity results

[0056] The results are shown in Table 1:

[0057] Table 1

[0058]

[0059] The results showed that compared with the NR3C1 gene BclⅠ site G allele group, the NR3C1 gene BclⅠ site C allele group had significantly higher incidence of low cardiac index, positive blood culture rate, adrenaline use rate, 72-hour cumulative fluid overload percentage, and intensive care unit time (h) (P < 0.05). In addition, the 28-day all-cause mortality of the NR3C1 gene BclⅠ site G allele group and C allele group was 1 case (5.3%) and 5 cases (22.7%), respectively. This indicates that the disease severity of the NR3C1 gene BclⅠ site C allele group was higher than that of the NR3C1 gene BclⅠ site G allele group, and the mortality rate was higher.

[0060] The odds ratio (OR) was further used to analyze the risk of low cardiac index (an evaluation index of the severity of septic shock) in the two groups of children. The results are shown in Table 2:

[0061] Table 2

[0062]

[0063] The results showed that compared with the children with the G allele at the BclⅠ site of the NR3C1 gene, the children with the C allele at the BclⅠ site of the NR3C1 gene had a 4.0-fold increased risk of low cardiac index, and the difference was statistically significant (P<0.05). This indicates that the disease severity of the children with the C allele at the BclⅠ site of the NR3C1 gene is higher than that of the children with the G allele.

[0064] 2. Outcomes of Refractory Septic Shock

[0065] The results of refractory septic shock, cSSS ≥ 3.5 and bSSS ≥ 2 in the two groups are shown in Table 3:

[0066] Table 3

[0067]

[0068] The results showed that the number of newborns with the C allele at the BclⅠ site of the NR3C1 gene who developed refractory septic shock was significantly higher than that with the G allele at the BclⅠ site of the NR3C1 gene, suggesting that children with the C allele had a higher risk of developing refractory septic shock.

[0069] Further odds ratio (OR) analysis was performed, and the results are shown in Table 4:

[0070] Table 4

[0071]

[0072] The results showed that the risk of refractory septic shock in children with the C allele at the BclⅠ site of the NR3C1 gene was 3.7 times that of children with the G allele at the BclⅠ site of the NR3C1 gene, which was significantly higher than that of children with the G allele at the BclⅠ site of the NR3C1 gene, and the difference was statistically significant (P<0.05).

[0073] In summary, the present invention found that the polymorphism of the BclⅠ site of the NR3C1 gene can be used to assess the severity of neonatal septic shock and to assess the risk of refractory septic shock in neonates.

[0074] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0075] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a reagent for detecting polymorphism of the BclⅠ site of the NR3C1 gene in the preparation of a kit for assessing the severity of septic shock in neonates, characterized in that: The severity of septic shock in neonates with the C allele at the BclⅠ site of the NR3C1 gene was higher than that in neonates with the G allele at the BclⅠ site of the NR3C1 gene.

2. Use of a reagent for detecting polymorphism of the BclⅠ site of the NR3C1 gene in the preparation of a kit for assessing the risk of refractory septic shock in neonates, characterized in that: Neonates with the C allele at the BclⅠ site of the NR3C1 gene have a higher risk of developing refractory septic shock than those with the G allele at the BclⅠ site of the NR3C1 gene.

3. The use according to any one of claims 1 to 2, characterized in that: The reagent is a reagent used for detection by PCR method, qPCR method, sequencing method, PCR-RFLP method, TaqMan probe method or gene chip method.

4. The use according to any one of claims 1 to 2, characterized in that: The reagents include specific amplification primers for the NR3C1 gene.

5. The use according to claim 4, characterized in that: The primers include an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2.

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