MINISTRY OF DEFENCE MINISTRY OF EDUCATION AND TRAINING VIETNAM MILITARY MEDICAL UNIVERSITY
DANG TIEN TRUONG
RESEARCH ON BRAIN MORPHOLOGY AND
GENE POLYMORPHISMS IN SCHIZOPHRENIA
Speciality: Biomedical science
Code: 9720101
SUMMARY OF MEDICAL DOCTORAL THESIS
HANOI - 2019
This research was carried out in
Vietnam Military Medical University
Supervisors:
1. Tran Hai Anh, M.D., Ph.D., Assoc.Prof.
2. Nguyen Duy Bac, M.D., Ph.D., Assoc.Prof.
Reviewer 1: Ngo Xuan Khoa, M.D., Ph.D., Assoc.Prof.
Reviewer 2: Tran Van Cuong, M.D., Ph.D., Assoc.Prof.
Reviewer 3: Tran Van Khoa, M.D., Ph.D., Assoc.Prof.
This thesis was defended in doctoral examination council of Vietnam
Military Medical University at … o’clock on ….
This thesis is available at:
1. National Library
2. Library of Vietnam Military Medical University
3. ………………………………………………….
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INTRODUCTION
1. Imperativeness
Schizophrenia (SCZ) is a severe mental disorder, progress slowly, changing devastatingly personal characteristics. Nowadays, 70 million peoples affected worldwide, accounting for 1% of the population with an increasing rate of 0.15% yearly. In Vietnam, the rate is 0.3–0.8% Vietnamese population and surge up 0.1-0.15 each year.
Up to now, pathology of schizophrenia is still unclear in some aspects. The global research showed that: there were changes in molecular level in neural activity mechanism in schizophrenia patients, and changes in gross level, such as size and morphology of the brain.
On the other hand, the research on the changes in the molecular, genetics in the disease had been seen very early. Notably, the gene changing in the schizophrenia was focused by many research on different aspects. However, results were not consistent, particular in the different population. Some gene has associated with the disease in the European people, but not associated with the Asian.
frontal
In Vietnam, there have been many studies on schizophrenia, but most of these have focused on a description of symptoms, the progress, and effect of therapy of the disease. The research on pathology and etiology have been limited. Recently, MRI system equipped with better resolution, some primary research have been acted in the aspect mechanism of schizophrenia, such as the study on the morphological thalamus, corpus callosum, lobe, hippocampus; on the variation of dopamine concentration in psychiatric patients. However, these had not assessed gray matter, white matter and regional brain with the particular function of related cognitive disorders as prefrontal cortex; causing delusions, hallucinations as temporal lobe, the disorder of cognition, emotion, and memory in schizophrenia. Furthermore, there has been any public about genetics variants in Vietnamese patients.
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Thus, this thesis titled “research on the morphology of brain and gene polymorphisms in schizophrenia” was carried out for the following purposes: 1. Determining characteristics of brain morphology using MRI images in schizophrenia in Vietnamese patients. 2. Determining polymorphic variants of DISC1 and COMT genes in schizophrenia in Vietnamese patients.
2. Scientific significance The thesis has provided data about changes in brain volume and gray thickness of brain regions in MRI image in schizophrenia. The results of the reduction of brain volume of frontal, temporal regions, subcortical structures provided information on functional disorders caused by structural abnormalities of the brain in schizophrenia.
Furthermore, the thesis provided information on ratio and distributions of rs821616/DISC1 and rs4680/COMT in schizophrenia in Vietnam. This has been the first data in Vietnamese patients serving as a base for other studies on genetics variants of schizophrenia in Vietnam.
3. Practical significance
The results of the reduction of regional brain volume in schizophrenia could be used as criteria in the objective assessment of specialty symptoms in schizophrenia.
4. Structure of the thesis The thesis consists of 114 pages. Introduction 2 pages; Chapter 1 (Documentary Overview) 23 pages; Chapter 2 (Subject and methods) 19 pages; Chapter 3 (Results) 27 pages; Chapter 4 (Discussion) 22 pages; Conclusions 2 page and Recommendations 1 page. The thesis has 34 tables, 29 figures (16 appendix figures) and 117 references (8 Vietnamese and 109 English references).
Chapter 1. DOCUMENTARY OVERVIEW
1.1. Schizophrenia
The term "Schizophrenia" is derived from the Greek word "schizo" which means "separation," and "phrenia" which means mind and spirit. The disease is quite popular. According to the World
3 Health Organization, the prevalence of schizophrenia is from 0.6 to 1.5% of the population, regardless of race or ethnicity. The disease is mainly onset in young people aged from 15 to 25 years old, the age of learning and working. The rate of male and female are similar. It was researched centuries ago. Today, many aspects of the disease, the pathology and etiology are gradually being clarified. Pathology and pathogenesis are governed by many factors. The factors are divided into two major groups that are genetic and environmental factors. Pathological Morphology is one of the basic of the pathophysiology of pathological series in general pathology.
1.2. Brain abnormalities in schizophrenia
Schizophrenia (SZ) is associated with structural changes and biochemical processes in the brain in the acute phase. Brain morphological changes were found in 50% of patients. Studies using psychiatric test and image technology of the brain such as functional MRI to evaluate functional changes in brain activities have shown that the most common change is in frontal lobes, temporal lobes, and hippocampus. Reduction of brain volume in sz less than in Alzheimer's is reported: frontal cortex and temporal lobe cortex. These changes are associated with the lack of cognition in schizophrenia.
Many CT imaging studies showed that the ventricular dilatation in patients with schizophrenia. MRI studies also showed similar results. Shenton et al., (2001) summarized 55 MRI studies in schizophrenia, 80% of which confirmed that ventricular dilatation, 73% confirmed 3rd ventricular dilatation, and had structural changes of the medial temporal gyrus, hippocampus, and temporal cortical structures, and some evidence of change in the frontal lobe, special in the prefrontal cortex. 1.3. DISC1 and COMT gene in schizophrenia
The theory of genetic, gene changes in schizophrenia has been mentioned very early. Studies focused on genes such as COMT and DISC1. However, the results of the studies have been not consistent with many aspects, especially regarding race. Moreover, there have had no published genetic changes in Vietnamese patients.
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1.3.1. Rs821616 in schizophrenia
DISC1 encodes the DISC1 protein. DISC1 protein plays an important role in the development of the nervous system and the maturation of the brain. The American Mental Health Institute had found evidence of the relation between DISC1's genotypes and SZ, in which rs821616 is showing the strongest signal. Nucleotide T is replacing nucleotide A of rs821616 cause serine substitution with cysteine at position 704 of DISC1 protein. Rs821616 was also known as S704C or Ser704Cys. Many studies have demonstrated the association of rs821616 with disease, symptoms, drug resistance and morphologic changes in the brain. Callicott et al., (2005) found that the T allele of rs821616 increased the risk of schizophrenia and reduced hippocampal volume. However, some studies gave the opposite result. Kim et al., (2007) did not find correlations of rs821616 polymorphism with schizophrenia but were associated with their ability of concentration. Borough and et al., found no association with the disease in the Iranian patients. Results of the studies on the relationship between rs821616 and schizophrenia are not consistent. In particular, there has been no report of rs821616 in Vietnamese patients. 1.3.2. Rs4680 in schizophrenia
is in involved the degeneration of
COMT (Catechol-O-methyl transferase) codes enzyme COMT. The enzyme important neurotransmitters such as dopamine, epinephrine, norepinephrine. Lachman et al. (1996) found a variant of COMT gene that encodes enzymes altering the activity of the COMT enzyme three to four times, rs4680 polymorphism. The results of the studies showed that there was a relation between COMT and the pathogenesis of schizophrenia. However, the results have not been yet consistent across different races, the different studies and between different approaches.
In conclusion, the studies of the changes in brain morphology, DISC1 gene, COMT gene in schizophrenia has been far published, but there has had a lack of agreement among different races. There
5 have not had reports of polymorphism of DISC1 and COMT genes in the Vietnamese.
A number of studies have initially assessed brain morphology in schizophrenia in Vietnam, but only in a limited number of regions of brain, not yet specific brain region associated with functional disorders in schizophrenia; not yet assessed gray and white matter; There have had no detailed assessment of specific region related to mental disorders such as the frontal cortex causing hallucinations, delusions and memory disorders in schizophrenia.
Chapter 2. SUBJECTS AND METHODS
2.1. Subjects Two hundred participants, twenty-five including
112 schizophrenia patients and 113 persons without schizophrenia. Blood samples were collected. 80 MRI image met standards were used.
* Brain morphology research: 80 subjects consisted of two groups: the schizophrenia group of 39 patients diagnosed with schizophrenia according to the DSM IV criteria, the American Psychiatric Association. The control group included 41 volunteers, without schizophrenia and other chronic neurological diseases. * Gene polymorphic variant research: 225 subjects, including two groups: 112 patients with schizophrenia were diagnosed with schizophrenia according to the DSM IV criteria of the American Psychiatric Association; The control group included 113 volunteers, without schizophrenia, without any related chronic neuropathy. Venous blood samples were drawn from subjects by standards procedures, preserved and analyzed polymorphisms. 2.2. Methods 2.2.1. Research design
Cross-sectional study with analyses.
2.2.2. Materials and equipment
* Brain morphology research
system FreeSurfer MRI 1,5 Philips; and Mango 5.3 v.4.0.1
6 Tesla, (http://surfer.nmr.mgh.harvard.edu/) (http://www.uthscsa.edu/) are installed workstation computer.
* Rs821616/DISC1 and rs4680/COMT variants research - Materials for genetics variants research for following steps: DNA extraction (QIAGEN Blood Mini kit, QIAGEN, Germany); reagents for PCR: Dream Tag polymerase and dNTPs from Thermo Fisher Scientific (USA); primer from IDT (USA); Electrophoresis: agarose, TBE 1X; Reagents for DNA purification; Sequencing: Bigdye Terminator 3.1 from Applied Biosystems (USA) and other primary material provided by beloved companies.
- Equipment: PCR thermocycler from Eppendorf; Electrophoresis system from Scie-places (England); Chemidoc XRS from Bio-rad (USA); ABI 3130XL sequencer and other essential equipment in Molecular lab. 2.2.3. Research procedure Subjects were selected according to the criteria in the schizophrenia and control groups.
* Brain morphology research: Participants met brain MRI scanned the criteria of the groups on a 1.5-Tesla MRI system of Philips, 3D pulses, T1 Weighted: 1mm in thickness (sagittal), FOV = 256, image size = 256 x 256 pixels. Images were discarded if interference or gross abnormalities detected. Each patient's image data was stored in a CD-ROM, then analysis by the Mango Software v.4.0.1 and Freesurfer 5.3 software package.
Research indexes include: intracranial volume (ICV), total brain volume including ventricular volume, brain volume excluding ventricular volume, cerebral hemisphere volume, left and right hemisphere volume; lateral ventricle, third and fourth ventricles; volume of cerebrospinal fluid; volume of brain stem; volume of cerebellum; cortical gray matter (right, left); cortical white matter (right, left); subcortical; gray matter of the cortex. Gray matter volume, white matter and cortical gray matter thickness of the gyri of the frontal lobes, temporal lobes, cingulate cortex, insula lobes, and subcortical structures.
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polymorphic rs4680/COMT sequence; Agarose
* Genetics variants research: The genetic polymorphism evaluation was carried out through the steps of DNA extraction from blood samples; amplification of regions including rs821616/DISC1 and gel electrophoresis tested products; clean up products of amplification; DNA sequencing using the Bigdye Terminator 3.1 (ABI) kit; purification after Bigdye reaction; sequencing on automatic sequencing system ABI 3130 XL; Identification of genotypes of polymorphisms by SeqScape 2.5 software. Eliminating samples unamplified or bad signal data. Determining the frequency of alleles, distribution of genotypes of polymorphisms between the two groups.
2.2.4. Data analysis
The data were presented in mean, standard deviation, percentage. Comparison of ratios by Chi-Square test; Comparisons of mean values of the two groups, controlled age, gender and intracranial volume control by analysis covariance, on STATA 12.0 software.
2.2.5. Research location
The research was conducted at Laboratory of Physiology, Department of Anatomy, Department of Radiology and Medical Imaging, Department of Psychology, Institute of Biomedicine and Pharmacy in Vietnam Military Medical University.
Chapter 3. RESULTS
3.1. Age and gender of the subjects
There was no difference in age and gender between the two case and control groups in brain morphological and genetics polymorphism groups
3.2. Morphological characteristics of schizophrenia patients Morphological characteristics of
the study subjects were evaluated on indicators including brain volume, white matter volume, cortical gray matter thickness of gyrus and lobes. 3.2.1. Whole brain volume and ventricles in schizophrenic patients The results of whole brain volume analysis showed reductions in whole brain volume including ventricles, whole brain volume
8 without ventricles, brainstem; dilation in lateral ventricle and 3rd ventricles, and increased of cerebrospinal fluid in the case group comparison from control groups with p 0.002, 0.001, 0.020, 0.005, 0.000 and 0.033, respectively. The volume of the cerebellum, the left cerebellum, the right cerebellum, the 4th ventricles were not different between the two groups with p> 0.05.
The results in Table 3.5 showed that reduction of the left and right white matter of hemispheres in the schizophrenia group compared with the control group with p less than 0.001. However, the total gray matter, the gray subcortical matter and the gray matter of both hemispheres were not different between the two groups with p> 0.05. Table 3.5. The volume of white, gray matter in two groups controlled by age, gender, and intracranial volume.
Structure volume (cm3) p F(4, 75) SZ (n=39) (X̅ ± SD) Control (n=41) (X̅ ± SD)
28.5 27.6 29.1 38.4 37.1 39.2 23.2 Global GM Left global GM Right global GM Global WM Left global WM Right global WM Subcortical GM Total GM 438.70 ± 49.83 454.94 ± 52.70 0.113 218.61 ± 25.00 226.77 ± 26.21 0.114 220.09 ± 24.89 228.17 ± 26.55 0.115 498.03 ± 57.05 537.52 ± 51.69 0.000 248.33 ± 28.63 266.67 ± 26.21 0.000 249.71 ± 28.49 270.85 ± 28.90 0.000 58.75 ± 5.30 59.42 ± 5.52 0.709 606.16 ± 62.21 626.34 ± 63.50 32.17 0.083
GM = gray matter; WM = White matter. 3.2.2. Gray matter volume and cortical thickness of the frontal lobe in schizophrenic patients
Table 3.6. The brain volume, white matter, and cortical gray thickness of superior frontal gyrus, adjusted for confounds such as age, gender, and intracranial volume.
The results in Table 3.6 showed that there were reductions of left, right superior frontal gyrus volume, and left WM of superior frontal gyrus and left, right superior frontal gyrus thickness in schizophrenia group with a p-value in turns 0.003/0.017; 0.001; 0,000 /0,000. However, there was no difference in the right white matter volume SFG between the two groups with p=0.063.
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p Superior frontal gyrus (SFG) F(4, 75) SZ (n=39) (X̅ ± SD)
Volume (cm3)
Thickness (mm) Left SFG Left WM Right SFG Right WM SFG Left SFG Right
Control (n=41) (X̅ ± SD) 21.91 ± 2.75 23.55 ± 2.85 17.46 0.003 20.98 ± 2.15 22.72 0.001 19.6 ± 2.21 22.36 ± 3.12 15.82 0.017 21 ± 2.46 20.02 ± 2.24 17.06 0.063 19.1 ± 2.35 2.79 ± 0.14 2.66 ± 0.17 8.64 0.000 2.79 ± 0.16 10.01 0.000 2.65 ± 0.15 Results of middle and inferior frontal gyrus analysis showed a reduction in rostral middle frontal gyrus, rostral and caudal WM of middle frontal gyrus, reduction of thickness of rostral and left caudal middle frontal gyrus; reductions of white matter of pars orbitalis, pars triangularis volume, and thickness of right pars opercularis, left pars orbiters and right pars triangular of inferior frontal gyrus in schizophrenia group (p<0.05). However, there was no significance in reduction of other volume and thickness of inferior and middle frontal gyrus in the case group.
The results of the orbitofrontal analysis show a significant reduction in white matter and brain volume of medial orbitofrontal; increasing thickness of left lateral and left medial orbitofrontal in schizophrenia. There was no significant reduction of WG and brain volume of lateral volume and right lateral, the medial thickness of orbitofrontal gyrus.
Table 3.13. The brain volume, white matter of the frontal cortex and
prefrontal cortex
Volume (cm3)
p
F(4, 75)
SZ (n=39) (X̅ ± SD) Control (n=41) (X̅ ± SD)
Frontal (both sides) lobe Left Frontal lobe Right Frontal lobe
161.59±18.97 168.80± 20.73 81.15 ± 9.87 85.04 ±10.81 83.75 ± 10 80.44 ± 9.19
Prefrontal (both sides) cortex 138.72±16.93 146.61±18.48 74.27 ± 9.64 72.34 ± 8.89
Left Prefrontal cortex Right Prefrontal cortex
69.79 ± 8.95 68.93 ± 8.06
30.7 29.7 30.5 32.4 31.9 31.9
0.041 0.037 0.051 0.010 0.006 0.018
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The results in Table 3.13 showed a reduction in the volume of the prefrontal cortex, the left frontal lobe in the schizophrenia group with p-value 0.01, 0.006, 0.018 and 0.037, respectively. However, there was no significant reduction in right frontal lobe volume in schizophrenia. 3.2.3. Volume and cortical gray thickness of the temporal lobe in schizophrenic patients
The results in Table 3.15 showed a reduction in left superior temporal gyrus, left and right white matter volume, and left gray cortical thickness in the schizophrenia group with p 0.001; 0.002, 0.025 respectively. There was no difference in right superior temporal gyrus volume and right cortical gray thickness in schizophrenia.
Table 3.15. The brain volume, white matter, and temporal gray matter of the temporal cortex, according to the study group
p F(4, 75) Superior temporal gyrus (STG) SZ (n=39) (X̅ ± SD)
Control (n=41) (X̅ ± SD) 10.59 ± 1.45 11.56 ± 1.48 14.91 0.001
8.30 ± 1.10 9.00 ± 1.19 19.42 0.002
Volume (cm3) 10.55 ± 1.46 11.06 ± 1.34 16.46 0.125
7.37 ± 1.00 7.82 ± 0.92 22.4 0.025
Thickness (mm) Left STG White matter left STG Right STG White matter STG (L) Left STG Right STG 2.44 ± 0.17 2.52 ± 0.15 2.50 ± 0.18 2.56 ± 0.18 3.1 5.3
0.050 0.204 In the middle and inferior temporal gyrus, there was a significant reduction in left middle temporal gyrus volume and left white inferior temporal gyrus in schizophrenia — no changes in volume and thickness in another region of middle and inferior temporal gyrus.
The results in Table 3.19 showed that there was a reduction in bilateral PG volume, left gray cortical thickness in the schizophrenia. There was no change in right PG thickness between the two groups.
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Table 3.19. The brain volume, white matter and cortical gray thickness in parahippocampus, according to the study group
p Parahippocampus gyrus (PG) F(4, 75) SZ (n=39) (X̅ ± SD)
Left PG
Volume (cm3) Right PG
Thickness (mm) Control (n=41) (X̅ ± SD) 1.82 ± 0.33 1.97 ± 0.35 White matter PG (L) 1.67 ± 0.30 1.88 ± 0.38 1.73 ± 0.30 1.88 ± 0.32 White matter PG (L) 1.75 ± 0.26 1.94 ± 0.30 2.18 ± 0.28 2.38 ± 0.29 2.21 ± 0.26 2.33 ± 0.27 Left PG Right PG
4.7 0.041 6.9 0.006 3.5 0.071 9.5 0.001 4.7 0.005 4.7 0.117 The results in Table 3.21 showed a significant reduction of bilateral hippocampus volume and right amygdala volume in schizophrenia. However, there was no difference in the volume of right amygdala between the two groups (p> 0.05).
p Structures F(4, 75)
Volume (mm3) Hippocampus Left Right Both sides Amygdala
Volume (mm3) Table 3.21. The gray matter of hippocampus and amygdala SZ (n=39) (X̅ ± SD) 4.13 ± 0.53 4.16 ± 0.45 8.29 ± 0.93 1.40 ± 0.21 1.45 ± 0.20 2.85 ± 0.40 Control (n=41) (X̅ ± SD) 4.47 ± 0.48 4.41 ± 0.40 8.88 ± 0.85 1.46 ± 0.18 1.56 ± 0.23 3.02 ± 0.39 13.3 10.1 13.3 7.2 15.1 12.1 Left Right Both sides
0.002 0.008 0.002 0.093 0.015 0.028 The results of fusiform gyrus analysis showed that a significant reduction in brain volume, white matter volume and bilateral cortical gray thickness in the schizophrenia. 3.2.4. The volume and thickness of the cingulate cortex
There was significant reduction volume in caudal anterior cingulate cortex (cc), reduction of the gray cortical thickness of left rostral anterior cc, bilateral isthmus cc in schizophrenia. However,
12 there was no difference in volume and thickness in other regions of cingulate cortex between the two groups with p> 0.05. 3.2.5. The volume of gray subcortical structures
left pallidum
There was a significant reduction in left thalamus, bilateral caudate, accumbens, in right putamen and schizophrenia. However, there was no difference in right thalamus, left putamen, right pallidum volume between the two groups with p> 0.05. 3.2.6. The volume of parts of the corpus callosum
The results showed a significant reduction in total corpus callosum volume and part II and Part III in schizophrenia with p 0.037, 0.006 and 0.000, respectively. 3.3. Characteristics of genetic polymorphism in patients with schizophrenia 3.3.1. Rs821616 of the DISC1 gene Rs821616 polymorphism was at Hardy Weinberg equilibrium for case and control groups (p> 0.05).
The results in Table 3.27 and 3.28 show no difference in allele frequency and genotypic distribution of rs821616 between the two groups, as well as in male or female with p> 0.05. Table 3.27. the frequency of Allen of rs821616 Allele frequency (n) Groups Allele
SZ (n=100) Control (n=101) 200 202 T 0.06 (12) 0.10 (20)
A 0.94 (188) 0.90 (182) χ2 = 2.09; p = 0.15 Table 3.28. Distribution of genotype of rs821616
n (%) Groups
AA 89 (89.00) 81 (80.20) 170 TT 1 (1.00) 0 (0.00) 1 100 (100) 101 (100) 201 SZ Control Total Number of genotypes (%) AT 10 (10.00) 20 (19.80) 30 p = 0.074 3.3.2. The of rs4680 of COMT gene in schizophrenia
Rs4680 was at Hardy Weinberg equilibrium for case and control groups (p> 0.05). There was no significant difference in allele
13 frequency and genotype distribution of rs4680 between the two groups as well as male and female sex with p>0.05.
Table 3.31. the frequency of allele rs4680 Allele frequency (n) Groups Allele
SZ (n=100) Control (n=101) G (Val) 147 (72.78) 154 (75.49) A (Met) 55 (27.22) 50 (24.51) 202 204 χ2 = 0.39; p=0.53 Table 3.32. Distribution of genotype of rs4680
Groups n (%)
AA 7 (6.86) 8 (7.92) 15 AG 36 (35.30) 39 (38.61) 75 Schizophrenia Control Total 102 (100) 101 (100) 203 Number of genotypes (%) GG 59 (57.84) 54 (53.47) 113 χ2 = 0.403; p = 0.818
Chapter 4. DISCUSSION 4.1. Age and gender characteristics of subjects
There was no difference in age and sex between the control group and the control group (p>0.05) for groups in morphological and genetics research. Changes in brain morphology in schizophrenia patients were due to primary or secondary changes in the pathology of schizophrenia. Gender similarity between the two genetic groups eliminates the effect of gender on the analysis of gene and schizophrenia relation. 4.2. Characteristics of the brain in schizophrenia
to better understand
Schizophrenia is a severe psychiatric illness, characterized by cognitive and emotional function impairment. Studies have been analyzing both structural changes in the brain, identifying changes in specific brain regions associated with functional disorders in the pathogenesis, clinical schizophrenia symptoms, and medications in the treatment of the disease. Many studies have shown that brain volume was influenced by many factors, especially age, gender and intracranial volume (Hulshoff Pol et al., 2001; Chen et al., 2018). These factors were confounded of
14 brain volume, cortical gray matter thickness, and affect the objectivity of the study to determine the pathological effect on brain morphology. Studies have addressed this in some methods in which covariance analysis (ANCOVA) is widely used. This method, which controlled the age, gender, and intracranial volume, will clearly show the effect of disease on brain morphology. In our study, the elimination of the effects of noise factors was applied both at the design and sampling stage, with no difference in age or gender between the two groups. At the same time, covariance analysis was used to control age, gender, and intracranial volume. This eliminates the confounding factors and accurately evaluated the effect of schizophrenia on brain morphology. 4.1.2. Whole brain morphology in schizophrenia 4.2.1.1. Characteristics of intracranial, ventricles, whole brain volume.
The results of total brain volume analysis showed a reduction in total brain volume, brainstem in the schizophrenia group with p<0.05; Total brain volume with p less than 0.01. Significant dilatation of lateral ventricles, third ventricle, and increasing cerebrospinal fluid volume in schizophrenia. Our findings were consistent with many previous studies of total brain volume reduction and ventricular dilatation in schizophrenia patients. Harrison and colleagues reported a reduction in overall brain volume in the schizophrenia. The same findings were also reported by Wright et al. (2000). The results of Olabi et al. also showed a reduction in total brain volume and ventricular dilatation of the lateral ventricle, third ventricle in patients with schizophrenia. In contrast, some reports show no difference in total brain volume between the schizophrenia group and control group by Shenton et al. (2001). The difference in Shenton's (2001) report compared to recent reports might be due to the technological limitations of MRI systems. Most of the studies compiled in Shenton (2001) used old, low- resolution MRI systems. Furthermore, the slice thickness was too thick from 0.5 to 1 cm. This could lead to enormous inaccuracy in the measurement and could not indicate a volume difference of
15 several percents in total brain volume between the two groups. This was also indicated in Shenton’s report (2010). The Ventricles are empty spaces in the brain containing the cerebrospinal fluid. Enlargement of ventricle might also be a sign of brain tissue reduction in surrounding areas or slow development. Thus, the reduction in brain volume was consistent with the widening of the ventricles in our study. In the third ventricle, our results showed that third ventricular dilatation in the schizophrenia group, in line with Becker’s results for third ventricular dilatation in patients with schizophrenia. In contrast, Shenton (1992) et al., Roy et al. (1998) did not see any change in the third ventricle. This difference might be due to the cause of variance of the MRI system used in the studies. Near the third ventricle is the thalamus, so it was possible to dilation of the third ventricle associated with a reduction of thalamus volume in schizophrenia; was due to abnormal development of thalamus or neuro-degeneration of the thalamus. The increase in cerebrospinal fluid volume in our study was consistent with the results of Olabi et al., in-line with the hypothesis of brain volume reduction. 4.2.1.2. Characteristic of the gray and white matter of the brain
The cortex plays the most critical role in performing advanced brain functions such as the ability to use language, logic, ideation, and judgment.
The results of determination of the gray matter, white matter of the brain shown in Table 3.6, showed a reduction in bilateral cortical white matter volume in the schizophrenia with p = 0.000. However, there was no difference in total gray brain volume, gray subcortical structures and cerebral gray matter volume between the two groups with p> 0.05. Although there was no difference, the volume of gray matter in the total gray matter volume, global gray matter, the volume of gray matter was tendency reduction. However, the difference had not reached the level of significance. The results of our study were in line with the results of Olabi et al.; The results of Harrison et al.
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The cortex concentrates densely on the neuronal bodies, forming the surface of the cerebral hemisphere. However, only one-third of the surface area is free, the remainder hidden in the sulcus. Therefore, when determining the volume of gray matter, this characteristic was essential to assess the volume of the brain accurately.
The white matter of the brain consists of axons with Myelin, forming the connective lines of the cerebral cortex. Reducing the volume of white matter was thought to reduce the volume in the axons, explaining the change in the structure of the brain, primarily the white matter and gray matter. Selemon and Goldman-Rakic saw a decrease in the intercellular space, including the axons, dendrites, and synapses, which were located around the typical structure of the neuron. These results have partly elucidated the reduction in the volume of the brain at gross levels. Chance et al. found that a significant reduction in cognitive performance was associated with low cell density due to the reduction of cells in dementia. 4.2.2. Characteristics of the frontal lobe in schizophrenia
The frontal cortex occupies two-thirds of the human brain, which involve cognitive performs many functions, many of which processes, including memory, memory, and decision making.
The frontal lobes with some regions are responsible for the motor function such as the precentral gyrus, and the paracentral gyrus did not show any significant changes.
Our results showed a decrease of volume in superior frontal gyrus, reduction in white matter volume of the middle frontal gyrus, white matter volume of the pars triangular of the inferior frontal gyrus, medial orbitalis gyrus and white matter volume of the frontal pole. This indicated the unequal change in the prefrontal cortex as well as the frontal lobes. It was noticeably that there was a significant reduction in most gray matter areas of the frontal cortex. The clear decrease in the cortical gray matter was evident in the bilateral superior frontal gyrus; in the rostral middle frontal gyrus; some areas of the inferior frontal gyrus; left gray thickness in the orbital gyrus. This suggests that changes in the volume of the frontal
17 cortex were mainly on the reduction in the cortical gray thickness. This result was also clear when assessment in the prefrontal cortex.
temporal lobes in Studies analyzing smaller areas of the frontal lobes indicated a reduction in white matter volume in patients with schizophrenia. Goldstein et al. reported a reduction in inferior frontal gyrus volume from 7 to 15% in schizophrenia. Gur and colleagues showed that prefrontal cortex volume was reduced by about 9% in men and 11% in women. However, Baare et al. reported that there was no change in prefrontal cortex the schizophrenia; However, this study noted a relation of a decrease in gray matter volume in the left and right prefrontal cortex and reduced word memory, visual memory. This difference was able to be related to the use of the Philips 0.5-Tesla MRI system, a slice thickness of 1.2 mm, that not able to determine little changes in volume or gray thickness. The strength of the magnetic field in different MRI systems affects the resolution of the MRI image, and the results are outlined in Shenton (2010). 4.2.3. Morphological characteristics of schizophrenia
The temporal lobe was studied very early in schizophrenia because of the typical symptom of schizophrenia, hallucination, which is thought to be due to temporal abnormal. Southard (1915) also noted abnormalities of the temporal lobe during brain dissection of the cadaver of patients with schizophrenia after death. Our Results showed a decrease in left superior temporal gyrus volume and right white matter of superior temporal gyrus in the schizophrenia (p <0.05). This finding was consistent with many previous MRI studies. This finding was also in line with findings from a reduction in superior temporal gyrus in a cadaver with schizophrenia than previously thought to be illogical. Symptoms of hallucinations and cognitive decline could be controlled by hearing disturbances. Penfield and Perot (1963) showed that the superior temporal gyrus was the brain region just below the Sylvius sulcus. The superior temporal gyrus consisted of is Heschl's in the anterior, which is the auditory cortex. In the posterior, the left is the Wernicke's (41 and 42 of Brodman), which is thought to be the language center.
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in schizophrenia, such as
Barta et al. (1990) firstly reported that superior temporal gyrus volume decreased by 11%, correlated with hallucination. Shenton (1992) reported a 15% reduction in left temporal gyrus volume, correlated with thought disorder and delusion. Then, Barta et al., Hollinger et al., Marsh et al. confirmed similar that reduction. Flaum and colleagues found that positive symptoms were associated with a decrease in superior temporal gyrus volume. These results suggested that changes in the superior temporal gyrus were a particular characteristic of schizophrenia. A review of the superior temporal, temporal changes associated with disorders in schizophrenia was by studies that stimulated the temporal, temporal regions of the brain. Haglund and colleagues reported on thought disorders that occurred in epileptic patients stimulated in the posterior of the superior temporal gyrus. These manifestations were similar to symptoms as criteria impaired memory, mental disorders, and hallucinations. In contrast, some studies did not find a difference in superior temporal gyrus volume between patients with schizophrenia and the control group. Pearlson and colleagues reported that this might be in different methods of measurement.
The reduction of bilateral white matter volume left a cortical thickness of para-hippocampus gyrus in schizophrenia with p-value 0.002, 0008, 0.002, and 0.015 respectively. The reduction in the volume of the hippocampus, right amygdala, para-hippocampus were in with previous results. The reduction of middle temporal gyrus, hippocampus, amygdala, and para-hippocampus were in line with enlargement of lateral ventricles presented in table 3.4. These were in concordance with previous studies on a cadaver, as well as MRI image (Shenton, 2001. Nestor et al. also found a relationship between verbal memory reduction, abstract concept, and a reduction in the volume of both para-hippocampus and superior temporal gyrus. This impairment cognitive was consistent with the function of the brain regions and their role in memory progress, particularly concerning verbal memory, which results showed disorders of the language system.
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4.2.5. Morphological characteristics of subcortical structures
Subcortical structures consist of many different nuclei. Some of these played important roles in the cognition and emotion. Thus, this study conducted a subcortical structure analysis to identify changes that effected on cognitive, memory and emotional functions. The results showed that left thalamus volume, bilateral caudate volume, bilateral accumben, putamen volume, left pallidum with p less than 0.05. Thalamus consists of nuclei that acts as an intermediate station, receives signals from various brain regions, as well as the activated reticular formation, the limbic system. It also acted as the center for processing and filtering information from sensory signals. These functions derive from the connection between the ventral anterior and dorsal medial nuclei of thalamus and prefrontal cortex.
The nucleuses concluding caudate, putamen and pallidum were concentrated research in the schizophrenia because: First, these were the extension of the dopamine to the striatum; Second, the medications were an effective treatment of schizophrenia affecting on dopaminergic; Third was the critical role of these structures in cognition, sensation, and movement. Our results showed an increase in the volume of the bilateral caudate, putamen and pallidum in schizophrenia patients with p<0.05. However, the incumbent volume was decreased in both groups with p <0.05. The increase in the volume of the nuclei in our results was consistent with the results of previous studies by Heckers et al. (1991). Other findings on MRI images in Shelton's reports (2001). In the studies that found basal nucleuses volume increased, an important finding in these subjects was the use of neuroleptics. Chakos and colleagues found a 5.7% increase in pallidum volume after 18 months of follow-up. This suggested that the use of high doses of neuroleptic increased the size of the caudate. This result was confirmed in the report of Keshava et al. (1994). In particular, primary neuroleptic mainly increased the volume of the basal nucleus. In contrast, secondary neuroleptic did not produce this effect (Shenton 2001).
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4.2.6. Characteristics of the corpus callosum
the corpus callosum the enlargement of
Corpus callosum is the most massive white matter, connecting the two cerebral hemispheres, structured by fibers with myelin, forming the cortical connection. Our results showed that the volume of part II and III of the corpus callosum decreased in the schizophrenia group with p less than 0.01. In the corpus callosum, part II connects the pre-motor, part III connects the two moto region. The reduction in the volume of part II and III that connecting the two pre-moto and the motor areas of frontal lobes. These results were consistent with the decrease in white matter volume at these structures with p<0.005 on the left and 0,042 right precentral gyrus; as well as the paracentral; This consistent could be seen in both superior, middle and inferior frontal. Reduction of information processing activity in patients with schizophrenia was associated with abnormalities in the exchange of information between the hemispheres. Post-mortal studies showed in schizophrenia. Seventeen studies had positive results, and ten studies had no difference between the two groups. This difference might be due to the variance between the measurement methods. 4.3. Rs821616 and Rs4680 polymorphisms in schizophrenia 4.3.1. The polymorphism of rs821616 of the DISC1 gene
DISC1 gene encodes the DISC1 protein. DISC1 protein plays an essential role in the development of the nervous system and the maturation of the brain. The American Psychiatric Institute found evidence of the association between DISC1 and all type of alleles, with rs821616 giving the strongest signal (P = 0.004).
In this study, 12 samples from the case group and 12 samples from the control group did not identify genotypes for multiple reasons. Thus, the number of samples that identified the genotypes of rs821616 and the control group were 100 and 101 samples respectively. Hardy Weinberg's equilibrium test results show that
the polymorphism of rs821616 was Hardy Weinberg equilibrium for case and control groups. Rs821616 of the two study groups were not influenced by mutant factors. Our results showed no difference in
21 allele frequency and genotypic distribution of rs821616 between the two groups with p greater than 0.05. Our findings are in line with the previous findings. Kim et al. found no correlation between rs821616 polymorphism and schizophrenia.
Similarly, Luo et al. Norlelawati and colleagues did not find any association between rs821616 and schizophrenia. However, Callicott et al. (2005) found that the association of polymorphisms found rs821616 to be the strongest signal associated with schizophrenia (P = 0.004).
those
Our findings are consistent with other Asian studies, such as Malaysia, Korea, and Iran. In contrast, it was not consistent with some studies, most of which were studies of the Europe race. Gong et al. found the difference between the A allele frequency of rs821616 in the white population (0.300) and Asians (0.068). The different distribution of alleles in different populations might be the cause of differences in the effect of polymorphism in patients with schizophrenia. Also, the genetic linkage analysis of polymorphisms revealed some gene combinations associated with schizophrenia — especially that contain rs821616. Thus, polymorphism rs821616 might play an important role in interactions with other polymorphisms associated with schizophrenia. Also, many studies have identified the association of rs821616 with clinical symptoms, drug resistance, and brain morphological changes such as Vazquez et al.; Hashimoto et al. And Li et al; Hotta et al. However, Mouaffak et al. did not find rs821616 associated with schizophrenia and resistance drug.
In summary, the results of the study had partly reflected the role of rs821616 in the pathogenesis of schizophrenia. This study was a preliminary study in Vietnam on rs821616 polymorphism and schizophrenia; The association between polymorphism and schizophrenia was not found to be consistent with the results of Asian studies. 4.3.2. The polymorphic features rs4680 of the COMT gene
COMT (Catechol-O-methyl transferase) coding enzyme COMT. important the degeneration of involved This enzyme in is
22 neurotransmitters such as dopamine, epinephrine, norepinephrine. In this, the polymorphism of the COMT enzyme was the most potent effect by Val158Met polymorphism (rs4680).
Genotyping of rs4680 had ten samples from the case group, and 12 samples from the control group did not genotype for different reasons. Hardy Weinberg's equilibrium analysis of polymorphism rs4680 in the control group and the control group were at Hardy Weinberg equilibrium (p> 0.05). This result demonstrated the inheritance of polymorphism was random.
The results showed no difference in allelic frequencies and genotypic distribution of rs4680 polymorphism between the control and control groups (p>0.05); There was no correlation between rs4680 polymorphism and schizophrenia in both men and women with p 0.87 and 0.43, respectively.
lobe, negative the Chinese, symptoms in
The results of our correlation with rs4680 and schizophrenia polymorphisms were inconsistent with previous Asian studies such as China and Japan. The Asian study also found similar results. The results were consistent with those of Okochi et al., Regarding the association of polymorphism and schizophrenia in Europeans and not in Asians. However, Costas and colleagues analyzed Meta-analysis, which included 51 studies. Results showed that the rs4680 polymorphism was sensitive to mental disorders. This suggests that too much or too little dopamine might be a risk factor for the disease. Also, studies were showing the role of rs460 in the pathogenesis of schizophrenia. The study found the association of polymorphic rs4680 with mental symptoms; decreased the activity of the prefrontal the microstructure of white matter. Xu et al. found the interaction of rs4680 and rs1344706 to a decrease in gray matter volume in the prefrontal cortex in healthy subjects. Nkam and colleagues also showed that the Val/Val genotype of the rs4680 was associated with an increase in errors in the test. Matsuzaka et al. found that COMT polymorphisms are regulating cognitive performance in patients with schizophrenia. The results of the studies show that there was a relation between COMT and the pathogenesis of schizophrenia.
23 However, the results were not yet consistent across different races, between different studies, and between different approaches.
The results of the study did not show that the association of rs4680 polymorphism with schizophrenia in Vietnamese people. The result was consistent with other studies. However, evaluations of the association of polymorphisms and brain morphologic changes to the mechanisms and effects of polymorphisms in rs4680 in particular and other polymorphisms in schizophrenia should be continued, more deeply. Making a design of studies focuses on each aspect, the pathology mechanism for more clearly.
CONCLUSIONS 1. Morphological characteristics of schizophrenia patients
There was a reduction in total brain volume, brainstem; bilateral third ventricle, and lateral Ventricle, cortical white volume; cerebrospinal fluid volume in the schizophrenia group with p <0.05.
There was a reduction in bilateral superior frontal gyrus, right white in superior frontal gyrus; gray and white matter in some brain region such as rostral and caudal middle frontal gyrus; white of left pars orbitalis, pars triangles and right cortical thickness of pars orbitalis, right pars triangles of inferior frontal gyrus; reduction of brain of medial and white matter medial orbitalis gyrus; reduction of brain in right frontal pole in schizophrenia (p <0.05).
There was a reduction in the volume of the left superior temporal gyrus, bilateral white matter of superior temporal gyrus; reduction of left medial temporal gyrus; reduction in thickness of left middle temporal gyrus; reduction in the volume of the white matter of inferior temporal gyrus in schizophrenia (p<0,05. The reduction of bilateral white matter left a cortical thickness of para-hippocampus in schizophrenia. There was a reduction in the volume of the hippocampus and right amygdala volume in schizophrenia.
Subcortical structures, corpus callosum, and cingulate cortex: There was no difference in the volume of the cingulate cortex between the two groups. However, the cortical thickness of the rostral cingulate cortex decreased in the schizophrenia; Left thalamus
24 volume, bilateral caudate volume, bilateral accumbent volume, right putamen, left caudate decreased in patients with schizophrenia (p <0.05); The volume of the second and third part of the corpus callosum decreased in the schizophrenia (p <0.01). 2. DISC1 and COMT polymorphisms in schizophrenia
- Genetics of rs821616/ DISC1 and rs4680/COMT polymorphism and rs4680 polymorphism of the COMT gene was at Hardy Weinberg law in both groups.
- Frequency of alleles of rs821616 polymorphism in schizophrenia group was 0.94 (A) and 0.06 (T); in the control group was 0.9 (A) and 0.1 (T); The distribution of genotypes of AA, TT and AT in the schizophrenia group was 89%; 1.00% and 10% respectively; In the control group, respectively 80.2%; 0% and 19.8%; There was no difference in the distribution of genotypes and allele frequencies between the two groups (p> 0.05). There was no difference in the distribution of genotypes between the two groups in male and female.
- Frequency of alleles A and G in schizophrenia group was 50 (24.51%) and 154 (75.49%) respectively; in the control group were 55 (27.22%) and 147 (72.78%) respectively; The distribution of AA, GG and AG genotypes in schizophrenia groups were 6.86%, 57.84% and 35.3%, respectively; In the control group, respectively 7.92%; 53.47% and 38.61%. There was no difference in allele frequencies and the gene distribution of rs4680 polymorphism between schizophrenia and control group (p> 0.05). There was no difference in the distribution of genotypes between the two study groups in male and female.
PERSPECTIVES Evaluation of brain morphology in our study focused on cortical thickness, gray matter volume, and white matter volume. It was necessary to understand the relationship of regions with changes in brain morphology. DTI technique will be appropriate for assessing whether or not the number of axons varies between these regions. Also, further studies of gene polymorphism need to be analyzed on more genes, and genomic analysis may be needed.
ARTICLES PUBLISHED THE RESULTS OF THE THESIS
1. Dang Tien Truong, Nguyen Duy Bac, Pham Minh Dam, Tran Hai Anh (2017). No association between rs821616 of disc1 gene and susceptibility to schizophrenia in a Vietnamese population, Journal of Military Pharmaco-medicine, 42 (7): 48-52.
2. Dang Tien Truong, Nguyen Duy Bac, Tran Hai Anh, Tran Ngoc Anh (2017). Gray and white matter reduction in schizophrenia patients, Journal of Military Pharmaco-medicine, 42 (9): 656-661.
3. Dang Tien Truong, Nguyen Duy Bac, Pham Minh Dam, Tran Hai Anh (2017). Val158Met polymorphism in the catechol-o- methyltransferase gene and schizophrenia in a Vietnamese population, Journal of 108 – Clinical medicine and pharmacy, 12 (9): 1-5.
