RU2627643C2 - Method for prediction of risk of subarachnoidal bleeding due to brain vessels aneurism rupture in persons of asian race - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: DNA is isolated from the peripheral venous blood lymphocytes of the examinee, followed by determination of the A1166C polymorphism genotype of AGTR1 gene (rs 5186). Prediction of an increased risk of subarachnoid bleeding due to cerebral aneurism rupture in persons of the Asian race is accomplished by genomic typing of A1166C polymorphism of AGTRl gene (rs 5186). In the presence of the C allele and AC genotype of A1166S polymorphism of the AGTR1 gene (rs 5186), increased risk of aSAC is predicted.

EFFECT: use of the method improves the accuracy of prediction of the risk of cerebral vessels aneurism development and rupture, leading to subarachnoid bleeding in persons of the Asian race.

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Description

The invention relates to medicine, namely to molecular medicine, neurology, and can be used to predict an increased risk of subarachnoid hemorrhage due to rupture of cerebral aneurysm and personalized prevention of this disease.

State of the art

Aneurysms of the cerebral vessels are local protrusions of the walls of the vessels of the brain and are the most common cause of non-traumatic aneurysmal subarachnoid hemorrhages (ASAC). Despite recent advances in the diagnosis and surgical treatment of ruptured aneurysms, ASAC remains a common disease with a high mortality rate. According to population studies, mortality reaches 66.7% (DJ Nieuwkamp, LE Setz, A. Algra [et al.]. Changes in case fatality of aneurysmal subarachnoid haemorrhage over time, according to age, sex, and region: a meta- analysis // Lancet Neurol. - 2009. - No. 8. - P. 635-642). The prevalence of cerebral aneurysms in the population is estimated at about 5-10% (F. Caranci, F. Briganti, L. Cirillo [et al.]. Epidemiology and genetics of intracranial aneurysms // Eur J Radiol. - 2013. - V. 82 (10) .- P. 1598-1605).

Due to the high prevalence of asymptomatic aneurysms of the cerebral vessels, on the one hand, and adverse outcomes of ASAKs, on the other hand, the problem of diagnosing cerebral aneurysms with a high risk of rupture and, accordingly, the risk of developing ASACs is urgent. In this case, a promising task is to identify aneurysms at the stage before their rupture and before the development of severe cerebrovascular complications.

Screening for the detection of asymptomatic unbroken aneurysms in the general population using invasive methods (e.g. cerebral angiography or magnetic resonance angiography) is not currently supported (Guidelines for the management of aneurysmal subarachnoid hemorrhage: a guideline for healthcare professionals from the American Heart Association / American Stroke Association / ES Connolly, AA Rabinstein, JR Carhuapoma [et al.] // Stroke. - 2012. - V. 43 (6). - P. 1711-1737).

There are still no diagnostic tests for specific genetic risk factors to identify patients at high risk for ASAK due to rupture of aneurysm of the cerebral vessels. The search for genetic markers of the development and rupture of aneurysms is intensively carried out in many populations, but progress in this direction remains moderate. A knowledge of the genetic determinants can be useful in order to understand the mechanism of aneurysm formation, as well as to obtain diagnostic tools for identifying individuals at increased risk for ASAK (Caranci F, Briganti F, Cirillo L, Leonardi M, Muto M. Epidemiology and genetics of intracranial aneurysms. Eur J Radiol., 2013, Oct; 82 (10): 1598-605. Doi: 10.1016 / j.ejrad.2012.12.026. Epub 2013 Feb 8).

Various approaches can be used to identify the genetic factors responsible for complex diseases (that is, those in which many genetic and external factors are involved), such as intracranial aneurysms.

One of them is an approach based on hypotheses about the functional characteristics of genes (the so-called functional candidate genes, for example, the elastin gene). These studies are widely represented, associations between aneurysms and specific alleles in functional candidate genes are analyzed between groups of patients with aneurysms and control. The disadvantage of this method is that the genes involved in the development of aneurysms through unknown pathogenesis remain unexplored (Y.M. Ruigrok; G.J.E. Rinkel. Genetics of Intracranial Aneurysms. Stroke. 2008; 39: 1049-1055. Doi: 10.1161 / STROKEAHA.107.497305).

Another method to avoid this drawback is a hypothesis-free method through genome-wide screening. The disadvantage of this method is that disease genes are not detected if aneurysms are caused by rare variants in the genes (Y.M. Ruigrok, G.J.E. Rinkel. Genetics of Intracranial Aneurysms. Stroke. 2008; 39: 1049-1055. Doi: 10.1161 / STROKEAHA.107.497305).

A known method for the diagnosis of subarachnoid hemorrhage using genetic markers (patent No. JP 2005151854 (A) C07K 14/47; C07K 16/18; C12N 15/09; C12P 21/08; C12Q 1/68; G01N 33/53; G01N 37 / 00; (IPC1-7): С07К 14/47; С07К 16/18; C12N 15/09; С12Р 21/08; C12Q 1/68; G01N 33/53; G01N 37/00. MARKER FOR DIAGNOSING HUMAN SUBARACHNOID HEMORRHAGE AND USE OF THE MARKER). The method provides for the diagnosis of subarachnoid hemorrhage using a polypeptide marker and a marker gene. The disadvantage of this invention is that this method provides for the determination of specific injuries of subarachnoid hemorrhage, but does not reveal the risk of developing cerebral aneurysm and its rupture, that is, it cannot be used to prevent subarachnoid hemorrhage.

Closest to the claimed invention is a method for diagnosing the risk of cerebral aneurysms using genetic markers (patent No. JP 2002238577 (A) С07К 14/78; C12N 15/09; C12Q 1/68; (IPC 1-7): C12N 15 / 09; C12Q 1/68 CEREBRAL ANEURYSM-SENSITIVE GENE). The method provides a diagnosis of the risk of cerebral aneurysm by identifying the genetic polymorphism of the human elastin gene. The disadvantage of this invention is that this method diagnoses the risk of developing aneurysm, but not breaking it. Most cerebral aneurysms are not broken, which is confirmed by the fact that the frequency of ASAK in the population is significantly lower than the prevalence of unexploded aneurysms (F. Caranci, F. Briganti, L. Cirillo [et al.]. Epidemiology and genetics of intracranial aneurysms // Eur J Radiol . - 2013. - V. 82 (10). - P. 1598-1605). Thus, this method cannot be applied to detect aneurysms of cerebral vessels, leading to the development of subarachnoid hemorrhages.

Our proposed method for genotyping the A1166C polymorphic locus of the AGTR1 gene (rs 5186) provides a diagnosis of the risk of cerebral aneurysm and subarachnoid hemorrhage and, thus, allows for individualized prevention of the disease before severe cerebrovascular complications develop.

Task and technical result

The objective of the invention is to develop a method for predicting an increased risk of developing cerebral aneurysm, leading to subarachnoid hemorrhage in people of the Asian race.

The technical result when using the invention is to improve the accuracy of predicting the risk of developing cerebral aneurysm and its rupture in people of the Asian race, leading to subarachnoid hemorrhage, due to the receipt of effective prediction criteria, and the possibility of carrying out personalized prevention of subarachnoid hemorrhage in people of the Asian race.

The implementation of the invention

The specified technical result is achieved by the fact that in the method for predicting the risk of subarachnoid hemorrhage due to rupture of cerebral aneurysm in people of the Asian race, genotyping of the polymorphic A1166C locus of the AGTR1 gene (rs 5186) is used as genetic markers of the disease.

Based on the presence of carriers of the A1166C polymorphism of the AGTR1 gene in people of the Asian race, the degree of predisposition to the risk of subarachnoid hemorrhage due to rupture of the cerebral aneurysm is determined.

The proposed method for predicting the risk of ASAA due to rupture of the cerebral aneurysm is as follows.

The patient receives peripheral venous blood from the ulnar vein in an amount of 5.0 ml.

DNA is isolated from the patient’s blood using a modified method using extraction with a phenol-chloroform mixture.

Genomic typing is carried out by methods based on polymerase chain reaction (PCR) (a method for analyzing the length polymorphism of restriction fragments of PCR products (PCR-RFLP method), where a single nucleotide substitution is part of a specific restriction enzyme recognition site and genotypes are determined by the presence or absence of restriction fragments; and PCR method using allele-specific primers (PCR-SSP method), where genotypes are determined by the presence or absence of an amplification product in the case of a primer specific for a particular allele). Genomic typing of the A1166C polymorphism of the AGTR1 gene (rs 5186) is carried out.

Carrier genotypes of A1166C polymorphism of the AGTR1 gene are determined. In the presence of the carriage of the C allele and the allelic combination of AS A1166C polymorphism of the AGTR1 gene, an increased risk of developing subarachnoid hemorrhage due to rupture of cerebral aneurysm in people of the Asian race is predicted.

Experiment description

A brief description of the experiment:

Stage 1. The alleles and genotypes frequencies of polymorphic gene loci were studied: Gln12Ter polymorphism of the AMPD1 gene (rs 146422068), SELP gene Thr715Pro polymorphism, AGTR1 gene polymorphism (rs 5186), ACE gene I / D polymorphism (rs 4646994rgMT (rs 4646994r17), polymorphism 4762), AGT gene Met235Thr polymorphism (rs 699) in the ASAK group and in the control group, comparable by sex, age and ethnicity.

2 stage. A comparative analysis of the carriage frequencies of alleles and genotypes of polymorphic loci of the studied genes in the ASAK patient group and in the control group.

3 stage. A study of the prognostic value of identified genetic markers to determine the risk of cerebral aneurysm and its rupture, leading to ASAC.

Experiment Description:

A case-control study was carried out in ASAK patient groups (n = 77) and in the control group consisting of healthy individuals without a history and history of acute cerebrovascular accident (n = 200).

The characteristic of the 1st group (ASAK patients): 77 patients of the Regional Vascular Center (Republican Hospital No. 2-CEMP, Yakutsk) hospitalized in the acute stage of the disease in the period 2012-2013. The average age is 49.2 years. Men - 36 people (46.8%), women - 41 (53.2%). Ethnicity: the indigenous ethnic group of the Asian race is 77 people (100%).

Description of the 2nd group (control group): 200 people without clinical and medical history for acute cerebrovascular accident. Average age: 46.9 years. Men - 66 (33%), women - 134 (67%). Ethnicity: Indigenous ethnic group of the Asian race - 200 people (100%).

Clinical examination of patients with ASAA included: a neurological examination with a history (100%), general clinical tests (100%), CT (MRI) of the brain (100%), coagulation (100%), ultrasound (Doppler and duplex) of brachiocephalic vessels (100%), subtraction cerebral angiography (100%).

Molecular genetic research was carried out in the educational laboratory of Genomic Medicine, Clinic of the Medical Institute, NEFU M.K. Ammosov.

Blood was drawn from the ulnar vein into a tube with EDTA. Genomic DNA was isolated from peripheral blood leukocytes by phenol-chloroform extraction [Maniatisetal. 1982].

Genotyping of the Thrl74Met and Met235Thr polymorphic marker of the AGT gene, I / D of the ACE gene, A1166C of the AGTR1 gene and Gln12Ter of the AMPD1 gene was carried out using kits (LLC Testgen), in accordance with the manufacturer’s instructions on the amplifier with the possibility of fluorescence analysis using the iQ5 endpoint iCycler "(BioRad, United States).

The polymorphic marker Thr715Pro of the SELP gene was genotyped using the allele-specific polymerase chain reaction (PCR) method using kits (LLC NPF Litekh, Moscow), in accordance with the instructions of the manufacturer. PCR was performed in an S1000 ThermalCycler amplifier (Bio-Rad, USA). Allele-specific PCR products were analyzed by electrophoresis in 3% agarose gel (Helikon Company LLC, Moscow).

Research results

Stage 1. A molecular genetic study of the risk of developing subarachnoid hemorrhage due to rupture of cerebral aneurysm in the indigenous ethnic group of the Asian race of Yakutia between a group of patients (n = 77) and a healthy control group (n = 200) without anamnestic and clinical signs of acute cerebrovascular accident. A study was carried out on the possible association of Gln22Ter polymorphism of the AMPD1 gene, Thr715Pro polymorphism of the SELP gene, A1166C polymorphism of the AGTR1 gene, I / D polymorphism of the ACE gene, Thr74Met polymorphism of the AGT gene, AGT gene metamorphism Met235Thr with risk-based statistical methods. A comparative analysis of the frequency of occurrence of alleles in homozygous or heterozygous forms and the carrier frequency of the genotypes of the studied polymorphisms in groups of patients and control was carried out.

2 stage. The results of a comparative analysis of the frequency distribution of the AMPD1 gene alleles and genotypes Gln12Ter in the ASAK and control groups are presented in Table 1. As a result of the study of the biallelic candidate locus of the AMPD1 gene, no significant differences were found in the carrier frequency of the alleles and genotypes of this polymorphism between the studied groups.

Genotyping of the Thr715Pro polymorphism of the SELP gene did not reveal differences in the frequency of carriage of alleles and genotypes of this polymorphism between the studied groups (table 2).

The results of a comparative analysis of the frequency distribution of alleles and genotypes of A1166C polymorphism of the AGTR1 gene are shown in Table 3. Significant differences were found in the carrier frequency of both alleles and genotypes of this polymorphism between groups of patients with ASAK and the group of healthy control. So, the frequency of the C allele is significantly higher in the patient group (p = 0.015; OR = 1.63; 95% CI: 1.08-2.44). Accordingly, the number of carriers of this allele is significantly higher in the patient group compared with the control group (79.2% versus 57.5%) (p = 0.001, OR = 2.81; 95% CI: 1.46-5.49) . The frequency of the allele A was significantly lower in the group of patients compared with the control (59.7% versus 70.7%) (p = 0.015; OR = 0.61; 95% CI: 0.41-0.92). But the number of carriers of the A allele between the two groups did not differ and amounted to 98.7% and 99%, respectively. Significant differences in the carrier frequency of genotype AA were obtained between the two compared groups (20.8% versus 42.5%) (p = 0.01; OR = 0.35; 95% CI 0.18-0.68). The frequency of AS genotype carriage was significantly higher in the group of patients (77.9% versus 56.5%) (p = 0.01; OR = 2.71; 95% CI: 1.43-5.22). Relative carriage of the SS genotype was not obtained reliable results due to the small number of carriers in the groups of patients and control (1 and 2 people, respectively) (table 3).

As a result of the study of the biallelic candidate locus of the ACE gene, no significant differences were found in the frequency of carriage of alleles and genotypes of I / D polymorphism between the examined groups (table 4).

Genotyping of two AGT gene polymorphisms (Thr174Met and Met235Thr) was performed. Allele and genotype distribution frequencies did not have significant differences between the groups of patients and the control for both AGT Thr174Met polymorphism (Table 5) and AGT Met235Thr polymorphism (Table 6).

Thus, significant differences in the distribution frequency of alleles and genotypes of the studied 6 polymorphisms were revealed only for the A1166C polymorphism of the AGTR1 gene. Based on the data obtained, it can be concluded that the carriage of the C allele and the AS genotype of the Al166C polymorphism of the AGTR1 gene increases the risk of ASAK development.

3 stage. At the third stage of the study, the prognostic value of the identified genetic marker A1166C AGTR1 was evaluated: sensitivity and specificity, prognostic value of a negative result and the prognostic value of a positive result for the test were calculated. Markers with specificity and / or sensitivity greater than 70.0 were considered significant.

As can be seen from table 7, the carriage of the C11 allele of the A1166C polymorphism of the AGTR1 gene is characterized by high sensitivity with respect to the risk of disease (79.2). Carriage of this allele is characterized by low specificity (42.5).

For the allelic combination of AS polymorphism A1166C of the AGTR1 gene, a sensitivity of 77.9 was established, and specificity was 43.5.

Thus, tests for the carriage of the C allele and the allelic combination of A1166C A polymorphism of the AGTR1 gene are highly sensitive to the risk of ASAK due to rupture of the aneurysm and provide a high probability of disease with a positive diagnostic test result (test).

If the test is positive, a high risk of subarachnoid hemorrhage due to rupture of the aneurysm is predicted, and special measures are taken to prevent the disease (invasive and / or non-invasive methods for detecting cerebral aneurysms (magnetic resonance angiography, computed tomographic angiography, cerebral subtraction angiography)).

The proposed method is illustrated by the following examples.

Example 1

Patient L., 42 g., Yakut. In the anamnesis: there are no diseases of the cardiovascular system, no bad habits. He became acutely ill on 05.01.2013, suddenly developed a severe headache, repeated vomiting. A CT scan of the brain diagnosed a massive subarachnoid hemorrhage, with a breakthrough into the ventricles of the brain. On cerebral angiography from 01/06/2013. revealed saccular aneurysm of the right internal carotid artery. In objective status: meningeal syndrome. Genotyping results: AS genotype of A1166C polymorphism of the AGTR1 gene. The operation of clipping aneurysm of the right internal carotid artery was performed. Recommended: Stage 1 - examination of relatives of the first line of kinship for the carriage of polymorphism A1166C of the AGTR1 gene; Stage 2 - with a positive result of genotyping - examination using minimally invasive methods for detecting cerebral aneurysms (CT angiography, MRI angiography); Stage 3 - when identifying aneurysms - invasive diagnostic methods (cerebral angiography) and removal of the aneurysm (before its rupture).

Example 2

Patient S., 35 years old, Yakut. History: suffers from arterial hypertension. There are no bad habits. He became acutely ill on 10/28/2012., Suddenly lost consciousness, repeated vomiting, developed right-sided hemiparesis, speech disorders. When examining a CT scan of the brain, subarachnoid hemorrhage, intracerebral hematoma in the temporoparietal parts of the left hemisphere, up to 20.0 cm ³ , were diagnosed against a background of rupture of the aneurysm of the left middle cerebral artery, with a shift of the middle structures to the right up to 1 mm. On cerebral angiography revealed a large saccular aneurysm of the fork of the right middle cerebral artery, multicameral aneurysm of the middle cerebral artery on the left. In objective status: right-sided hemiplegia, motor aphasia. Genotyping results: AS genotype of A1166C polymorphism of the AGTR1 gene. Surgical treatment of a ruptured aneurysm of the left middle cerebral artery was performed. Recommended: Stage 1 - planned surgical treatment of unexploded aneurysm of the right middle cerebral artery; Stage 2 - examination of relatives of the first line of kinship for the carriage of A1166C polymorphism of the AGTR1 gene Stage 3 - with a positive result of genotyping - examination using minimally invasive methods for detecting cerebral aneurysms (CT angiography, MRI angiography); Stage 4 - when identifying aneurysms - invasive diagnostic methods (cerebral angiography) and removal of the aneurysm (before its rupture).

Example 3

T., 35 years old, Yakut. History: arterial hypertension. The sister suffered a subarachnoid hemorrhage due to rupture of the cerebral aneurysm. Genotyping results: AS genotype of A1166C polymorphism of the AGTR1 gene. Given the hereditary burden and carriage of the genetic risk factor for the ASAK genotype of the AS polymorphism A1166C of the AGTR1 gene, it is recommended: Stage 1 - examination using minimally invasive methods for detecting cerebral aneurysms (CT angiography, MRI angiography); Stage 2 - when detecting aneurysm - invasive diagnostic methods (cerebral angiography) and removal of the aneurysm (before its rupture); Stage 3 - examination of relatives of the first degree of kinship (genotyping A1166C of the AGTR1 gene, with a positive result - non-invasive methods for the diagnosis of cerebral aneurysm).

Using the proposed method will improve the accuracy of predicting an increased risk of subarachnoid hemorrhage due to rupture of aneurysm of cerebral vessels in people of the Asian race, which will, in turn, carry out personalized prevention of the disease by selecting persons subject to invasive and non-invasive methods for the diagnosis of cerebral aneurysms in the period before rupture of the aneurysm with the development of subarachnoid hemorrhage.

Claims (1)

A method for predicting the risk of subarachnoid hemorrhage due to rupture of cerebral aneurysm in people of Asian race, including the extraction of DNA from lymphocytes of the peripheral venous blood of the subject, molecular genetic studies and the detection of AGTR1 polymorphism A1166C predict the risk of subarachnoid hemorrhage due to rupture of aneurysm.