Association of the CHGA gene polymorphism in patients with hemorrhagic stroke and/or aneurysm

Introduction: Cerebrovascular diseases have been associated with several genes. Chromogranin A ( CHGA ) has been used as maker in cardiovascular disease. Therefore, evaluating the polymorphism and verifying its association with this pathology is very important to better understand this disease. Objective: The aim of this study was to identify the association between coding region polymorphism in -264 position of the CHGA gene ( Glu264Asp ) and hemorrhagic stroke (HS)/aneurysm in the Federal District, Brazil. Methods: This is a population-based case-control, involving 45 cases with HS and/or aneurysm. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method is used for genotyping these samples. A significance level of 5% was adopted. Results: The absence of the CC genotype the Glu264Asp CHGA polymorphism in the study participants and the significant presence of the GC heterozygote genotype were observed in this study. However, the distribution of genotypes did not differ statistically in the groups. Conclusion: The Glu264Asp CHGA polymorphism does not seem to contribute to the genesis of the CHGA protein expression in this patients group, but to understand whether or not there is a possible association of the pathology in question and whether the mutation will contribute in the gene therapy and thus to improve patients’ quality of life.

intrOductiOn Cerebrovascular accident (CVA), also known as stroke, consists of an acute neurological dysfunction of vascular origin, followed by sudden occurrence of signs and symptoms related to impairment of focal areas in the brain, where symptoms persist for more than 24 hours (1) . According to the Brazilian Society of Cerebrovascular Diseases, 2016, this pathology is the leading cause of death in Brazilians: each year, 17 million individuals have a stroke worldwide; 6.5 million die; and 26 million are permanently incapacitating (2) .
Cerebral aneurysm can be defined as an abnormal arterial dilation at a point in the arterial wall of the circle of Willis. In the United States, 27,000 new cases of subarachnoid hemorrhage occur from aneurysm rupture, and 5% to 15% develop into hemorrhagic stroke (HS) (3,4) .
In general, stroke has as its main feature the lack of blood supply in a particular brain region, which may be caused by a blockage of an artery or when a blood vessel bursts. It is divided into two categories: ischemic and hemorrhagic, which have similarities, but differ in physiology, etiology and treatment (5) .
The ischemic stroke (thrombosis) is characterized by interruption of blood flow in a particular brain region. This interruption usually occurs due to the presence of a thrombus, that is, an inappropriate clotting process in the artery or vein, with bacterial aggregates and inflammatory cells (6) . The HS is the result of vascular fragility that can lead to rupture of a vessel and consequently to blood leakage in brain regions (7) ; it presents systemic arterial hypertension (SAH) as the main associated pathology (8) .
Currently, the diagnosis of this pathology is based on patient's clinical history and the aid of computed tomography (CT). Symptoms may vary and are related to different areas of the brain. It is common that neurological disorders associated interfere with speech, vision, cognition, sensitivity, balance and movement (9) . As well as the various chronic non communicable diseases (NCDs) commonly found in the world population, the hemorrhagic stroke is a multifactorial disease that is related to a number of factors that influence its environmental and genetic development (10,11) . Therefore, identifying the causes, with the help of some health technologies, is important to understand such pathology and to provide better quality of life to the patient, since this disease requires some physical dependence. This type of dependence generates social, emotional and economic changes, and isolation, causing neuropsychiatric disorders (12,13) .
Chromogranin A (CHGA) is an acid secreting glycoprotein located on chromosome 14q32.12. This glycoprotein has been used as a biomarker of tumors in neuroendocrine tissues, in addition to neurodegenerative and neuropsychiatric diseases, hypertension, cardiovascular diseases, and renal and liver failure (14) . Evaluating the polymorphism of any gene is a major challenge, especially in relation to the CHGA gene, as there are few studies that relate its mutation to any pathology. Among the different regions of this gene, the G/A base exchange in CHGA-264 has been shown to be a decreasing factor in the expression of this protein.
Based on the above, the objectives of this study are to verify the frequency of CHGA-264 G/A polymorphism in a Brazilian population sample of individuals with HS and/or cerebral aneurysm, as well as to investigate a potential association between CHGA G/A polymorphism, in the coding region, with different clinical manifestations. Additionally, to analyze this association in the prognosis of patients in relation to the pathology studied.

Composition
The study of 97 patients was conducted at the Faculdade de Ceilândia, Universidade de Brasília (UnB), Brazil. The samples that comprise this case-control study were obtained at the Base Hospital for two years ( January 2011 to December 2012). A total of 45 individuals diagnosed with HS and/or cerebral aneurysm (32 women and 13 men; mean age 52 ± 1 year) were recruited for this study. All patients presented the clinical signs described in the World Health Organization (WHO) definition of stroke; these signals were also confirmed by imaging [CT or magnetic resonance imaging (MRI)].
The control group consisted of 52 individuals (31 women and 21 men; mean age 51 ± 1 year) considered healthy and grouped by age and sex. All were volunteers who accompanied patients at the general outpatient clinic. The sample was calculated by estimating a 2% prevalence of HS/aneurysm in the adult population; 5% sampling error; 95% confidence interval (CI) in the 112 patients evaluated according to the criteria described above. A total of 24 participants were included. We considered the sample of 45 patients with HS to compensate for any loss.
The consent form was obtained from all participants before collecting the information. The Institutional Ethics Committee approved the study. Participants younger than 18 years with no diagnosis of HS and/or aneurysm, or those who presented any kind of blood relationship to the control group were excluded.

Clinical features
Detailed history and clinical evolution were tracked. Patients were asked if they had hypertension (blood pressure was measured) or diabetes, if they were smokers and consume alcohol. Scales were used to measure severity, disease stages and disability of patients: Rankin Scale (RS) to check motor functional capacity; Barthel index (BI) for mobility and personal care; and Glasgow Scale to evaluate patient's level of consciousness. In addition, biochemical tests, such as glucose, creatinine and platelets, were obtained through the patient's clinical records.

Laboratory methods
Each individual in this study, case and control, provided a 5 ml sample of venous blood. Deoxyribonucleic acid (DNA) was extracted from the blood collected using Invisorb Spin Blood Mini Kit (250) of the Invitek (catalog #CA10-0005, lot #1031100300). CHGA-264 G/A polymorphism genotyping was performed using the polymerase chain reaction (PCR) technique combined with the restriction analysis of the fragment studied in the polymorphism. The primers used for CHGA-264 G/A amplification were 5'-AGGGTGGCAGGCAAAGAG-3' sense and 5'-AAGGTGGAATGAGGTTATGG-3' antisense. Amplification was performed based on the following thermocycling conditions: 94⁰C for 5 minutes (initial denaturation), followed by 30 cycles of denaturation at 94⁰C for 1 minute, followed by 56ºC for 1 minute and 72⁰C for 1 minute for primers annealing. Finally, the extension process was performed at 72⁰C for 7 minutes. Two hundred thirty-five pb products of PCR were incubated for 90 minutes at 37⁰C with the DPN1 enzyme. Allele G was not cleaved by the enzyme, thus yielding a 235 pb fragment. Allele A presented two DNA fragments, one 106 pb and one 129 pb. The fragments were visualized on 3% agarose gel with ethidium bromide and exposed to ultraviolet light.

Statistical analysis
The frequency of genotypes and alleles in patients with HS and/or aneurysm was compared with the control group using the Chi-square test in the recessive and dominant models. The Armitage test was performed to check for dosage effects on the risk allele for HS/aneurysm. The association with clinical characteristics and each genotype was analyzed by Chi-square, adopting a significance level of 5%. To analyze the difference in the mean of the laboratory exams in the different genotypes, a Student's t test was performed, since normality assumptions were observed.

results
In Table 1, genotypic frequencies were described. The absence of CC genotype in both groups was observed, but a high prevalence of heterozygous individuals (GC) in the sample was verified. Thus, it was observed that there was no significant association between the pathology studied with the Glu264Asp CHGA mutation. Regarding the association between the presence of the polymorphic allele and the HS, no relationship was verified either (p > 0.05). e1412020 Tables 2 and 3 describe the associations between the clinical laboratory characteristics and the prognosis of patients with the polymorphism studied. No association of mutation with the study variables was verified (p > 0.05).

discussiOn
Stroke is a disorder of great social impact that causes a neurological deficit of vascular origin; affects adults and the elderly. This disease is the leading cause of death in Brazil and is the second leading cause worldwide, right after the ischemic heart disease (15,16) . In this study, the incidence of women (71.1%) -age approximately 52 years -was higher than that of men (28.9%) in the HS and/or aneurysm group.
Genetic research involving CHGA polymorphisms associated with some diseases has already been described in the literature. In this study, an analysis of the association between the presence of the Glu264Asp CHGA polymorphism and patients with HS and/or aneurysm who were undergoing treatment at a health facility in the Federal District, Brazil, was performed.
Genetic variation studies indicate that of all stroke cases, about 38% are hereditary. They also state that most of the genetic contribution to stroke is polygenic and reflects effects of multiple genes, each of them exerting small effects (17) . Regarding HS, few literature reviews have been performed, when compared with the ischemic outcome of the disease.
CHGA is part of a group of proteins present in various neuroendocrine tissues; it has been considered as a marker for several pathologies, such as hypertension, neuropsychiatric diseases, renal and hepatic failure (14,18,19) . In addition, this protein stands out in the heart failure (HF) marker group because it is included as part of the mechanism of neuroendocrine dysfunction. Braunwald Classification of Biomarkers for HF, grouped into seven categories, identifies chromogranins as biomarkers for prognostic and cardiovascular risk assessment (20) . Thus, studies on the CHGA genetic polymorphism may contribute to a better understanding of the pathogenesis and prognosis of cardiovascular diseases, such as HS. However, this study did not indicate any association of CHGA-264 G/A polymorphism with the occurrence of HS and/or aneurysm. Few studies have identified this polymorphism in a group of individuals. The study Association of the CHGA gene polymorphism in patients with hemorrhagic stroke and/or aneurysm proposed by Salem et al. (2008) (21) identified that polymorphisms in the CHGA gene were associated with the presence of hypertensive kidney disease, but did not observe the relevance of Glu264Asp CHGA polymorphism for the pathogenesis of the disease (21) .
Another study, by Kogawa et al. (2016) (22) , found that the polymorphism was not associated with the presence of diabetes, the same was observed in the present study, when it was aimed to investigate this association between patients with diabetes in the case group and those in the control group.
Regarding the prognosis, in this study it was also not found that CHGA polymorphism was associated with patient profile measured by neurological assessment scales. It can be observed that HS and/or aneurysm is a broad-spectrum disease in the population. However, as a limitation of this study, the sample size was very small when considering the magnitude of the pathology in question. Therefore, further research on specific populations should be performed to help understanding the disease associated with the genetic mutation, as Brazil is represented by a group of miscegenated individuals.
Understanding the genes responsible for normal or pathological traits allows us to apply the foundations of genomic medicine that will modify gene therapy. The principle of this technology involves the application of genes responsible for proteins that may be beneficial to the individual in the patient with genetic diseases, i.e., the application of a normal gene may reverse the clinical condition of the carrier. This procedure could contribute to the improvement of the patients' quality of life and possibly to reduce the incidence rates, not only for this pathology, but also for several others (23) .

cOnclusiOn
The Glu264Asp CHGA polymorphism does not seem to contribute to the genesis of CHGA protein expression in this group of patients, so it is not the ideal basis for understanding the reasons why this protein is a HS biomarker.
Understanding the genetic mutations of this pathology contributes to the development of a gene therapy, a technology of great importance to health. Therefore, further studies should be carried out to improve the understanding of the disease and improve quality of life for patients. This study showed no association, but checking for potential association in populations from different countries would be an interesting investigation.