Association of dopamine β-hydroxylase polymorphism rs1611115 and serum levels with psychiatric disorders in Pakistani population

Abstract Aim Dopamine β-hydroxylase (DBH) is a copper-containing enzyme that has an important role in maintaining the cellular homeostasis between the two neurotransmitters, dopamine (DA) and nor-adrenaline (NA). DBH functional polymorphisms are associated with multiple neuro-psychiatric conditions and are found to alter the DBH protein levels in serum affecting DBH enzymatic activity. The current study was conducted to determine the genetic and serum levels association of DBH rs1611115 functional polymorphism with major depressive disorder (MDD), bipolar disorder (BD) and schizophrenia (SHZ) in the Pakistani population. Methods In total n = 1097 subjects including MDD (n = 427), BD (n = 204), SHZ (n = 134) and healthy controls (n = 332), were screened for the functional polymorphism by polymerase chain reaction-restriction fragment length polymorphism. Univariate logistic regression analysis was applied and the results were adjusted for age and sex. The DBH levels in serum were determined through enzyme-linked immunosorbent assay (ELISA) and the Mann Whitney U test was applied. Results The minor allele (-1021 C > T) was found to be significantly associated with a higher risk of developing BD and SHZ in both univariable and multivariable analyses. The overall total serum concentration of DBH was comparatively raised in MDD, however, in cross-comparison DBH serum levels were found markedly higher in CC homozygotes compared to TT homozygotes within the BD group. Conclusion The present study suggested a significant association of DBH rs1611115 with BD and SHZ and also the effect of rs1611115 on DBH serum levels in MDD and BD for the first time in the Pakistani population.


Introduction
Dopamine β-hydroxylase (DBh) is a copper-containing enzyme of the dopaminergic system, which catalyzes the dopamine (Da) conversion into nor-adrenaline (Na) [ 1 , 2 ]. the dopaminergic system regulates mood, attention, motivation, reward system, decision making and psychomotor activity of the brain and Da is one of the important stimulatory neurotransmitters in this pathway [ 3 ]. the gene encoding DBh resides on chromosome 9q34, it has 12 exons and is approximately 23 kb in size [ 4 , 5 ]. the DBh is expressed in the locus-coeruleus part of the brain [ 6 ], and is localized in synaptic vesicles, of peripheral (noradrenergic) and central (noradrenergic and adrenergic) neurons and is also found in the neuro-secretory cells of the adrenal medulla [2 , 7 ].Upon adrenergic stimulation, the DBh and Na are released from vesicles through exocytosis from the sympathetic neurons and the adrenal medulla and then enters into the blood circulation, where DBh can be detected in plasma/serum [ 8 ].
however, the role of DBH sNPs in common psychiatric diseases including MDD, BD and shZ in individuals of Pakistani descent has not been established yet.the present study was, therefore, conducted to investigate the role of DBH rs1611115 functional polymorphism and the effect of the risk allele on serum DBh levels in psychiatric disorder cohort (MDD, shZ, and BD) compared to healthy controls in the Pakistani population.

Subject recruitment
a total of 1097 participants were recruited for this study, including MDD (n = 427), BD (n = 204), shZ (n = 134) and healthy controls (n = 332).all the subjects participating in the study were recruited from the Pakistan institute of Medical sciences (PiMs) hospital, islamabad, Pakistan.the participants were diagnosed by qualified registered psychiatrists of PiMs, according to the international classification of Diseases, tenth Revision (icD-10) guidelines (F20-F29 schizophrenia, F30-F39 bipolar disorder and depression) [ 29 ],.
Following the guidelines, any psychiatry patient with comorbidities such as neurological disorders (Parkinson's, alzheimer's and stroke), thyroid disorder, malignancies or contagious infectious disease were excluded from the study.For healthy controls, if the participant had a history of any psychiatric disorder or had a positive family history of psychiatric disorders, or stroke, or had serious brain injuries, or are drug abusers, they were also excluded from the studied cohort ( Figure 1 ). the clinical and demographic features of each participant were also documented.

Sample collection and processing
approximately 5 ml of blood sample was collected from each participant, of which 3 ml blood was added in ethylenediaminetetraacetic acid (eDta) tube (1 Becton Drive, Franklin lakes, NJ) for genomic DNa extraction according to the standard phenol-chloroform DNa extraction protocol.While 2 ml blood was added in gel activator containing tubes (1 Becton Drive, Franklin lakes, NJ), for serum separation.these tubes were subjected to centrifugation for 10 min at 2,000 RPM at 4 °c.extracted DNa and serum samples were stored at −20 °c till further use.

Genotyping of rs1611115
Genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (PcR-RFlP) technique using a previously reported set of primers [16], with modified reaction and thermal profile.the 25 μl PcR reaction mixture contained, 10X taq buffer, 2 mM dNtPs, 3 mM Mgcl 2 , 0.5 μM of each primer, 2.5 U of taq polymerase and 2.0 μl (40-50ng/ μl) of template DNa. the thermal profile for amplification consisted of initial denaturation at 95 °c for 6 mins, followed by 35 cycles of 95 °c for 35 sec, 56 °c for 1 min, 72 °c for 1 min and a final extension at 72 °c for 10 mins.PcR products (131 bp) were subjected to restriction enzyme digestion with 1 U of HhaI (thermo scientific tM , catalogue number: eR1851) at 37 °c for 16 hrs.Fragments containing the c allele (wild type) were digested by HhaI giving two fragments of 109 bp and 22 bp, whereas fragments with the t allele (mutant) remained undigested.after digestion, the fragments were analyzed on a 3% agarose gel.

Serum DBH level assessment
a subset from each case group (MDD, BD and shZ) and controls were selected based on genotype (with an almost equal number of males and females in each subset group) and were subjected to serum DBh level quantification.human DBh (Dopamine beta hydroxylase elisa kit, elaB science, catalogue number: e-el-h1226 (human serum), tX) kit was used as per the manufacturer's instructions for the detection of DBh levels in the serum samples.

Statistical analysis
all the statistical analyses were done using R studio version 4.00.to compare age, gender and demographic features between the cases and controls, the Fisher t-test was used for continuous variables and the χ 2 test was applied for discrete data.the χ 2 test was also used to determine whether the rs1611115 genotype distribution in the control group was in hardy-Weinberg equilibrium (hWe).Power calculations for case-control association analysis were also performed via Quanto-v.1.2.4 (see supplementary information, table s1).Genotypes were coded additively (additive model) and logistic regression analysis was used to determine the differences in the distribution of the genotypes of the sNP in cases (MDD, BD and shZ) as compared to controls (univariate analysis).the results were adjusted for age, sex and tobacco use (multivariate analysis).heterozygosity analysis was also done to determine the risk allele effect.the sNP data were also analyzed in comparison to the 1000 genome project data of the Punjab population (n = 96) from lahore (PJl) to determine the differences.the serum DBh levels were compared between the cases (MDD, BD and shZ) and controls using the Mann-Whitney U test. the level of significance was kept at a P value/α ≤ 0.05 in all statistical tests.

Descriptive analysis of cohort
Descriptive features of the studied cohort are given in table 1 .Most of the participants in the current study were inhabitants of the Punjab province of Pakistan and their average age (years) ± sD were 33.09 ± 11.52 in MDD, 30.7 ± 11.70 in BD, and 29.63 ± 9.88 in shZ and 23.79 ± 5.72 in controls.the cohort had 38.2% males in MDD, 58.3% males in BD, 70.9% males in shZ and 53.0%males in controls.the studied case-control cohort was not age and sex-matched (p < 0.05).it was also observed in the cohort that more tobacco users were in cases, shZ (P = 8.38e-14), BD (P = 8.904e-10), MDD (P = 5.15e-03) as compared to the controls.

Comparison with 1000 genome project PJL data
the genotype count and allele frequencies of sNP rs1611115 from the 1000 Genomes Project PJl data (Punjabi descent from Pakistan), were also retrieved and compared with the current study data.Marked differences in allele [χ 2 (P)=12.85(0.0003)] and genotype count [χ 2 (P)=18.82(<0.001)] were observed despite the same ethnicity of the present study cohort and 1000 Genome PJl data (table 2).

Serum analysis of DBH concentration
Descriptive features and the total serum concentrations of the selected samples (cases and controls) from the studied cohort are given in table 3 .the descriptive statistics of all the genotype-based studied sub-cohorts are represented in (see supplementary information, tables s2 and s3). the serum levels of Dopamine β hydroxylase (DBh) were found not to be normally distributed (see supplementary information, Figure . s1), therefore, a non-parametric Mann-Whitney U test was applied. in the initial analysis, we investigated the association of the DBh levels with the phenotypes compared to controls.the total DBh serum levels were found elevated (p = 0.008) in the MDD group as  compared to the control group ( Figure 2 ). in the second analysis, the DBh serum levels were also analyzed within each group concerning the genotypes (cc, tc and tt) to determine the association of DBh serum levels with the sNP.significant differences in DBh concentrations were observed in the BD group.serum DBh levels were observed significantly higher in BD (p = 0.008) as compared to the controls, in cc homozygotes.the lower serum DBh levels were found in tt homozygotes in BD as compared to the reference cc homozygotes of the BD group (p = 0.014) and also compared to the tt homozygotes of the MDD group (p = 0.038).however, after multiple testing Bonferroni's corrections (adjusted p = 0.003), the P valve did not remain significant for the BD (cc) vs coN (cc) and BD (tt) vs MDD(tt) findings, ( table 4 & see supplementary information, table s4).

Discussion
in the current study the association of the DBH-rs1611115 and its effect on the DBh serum levels in different psychiatric phenotypes MDD, BD and shZ was investigated in comparison to controls in the Pakistani population. in genetic association analysis, we observed a significant association of the risk allele (t) of rs1611115 with a higher risk in BD and shZ and the heterozygosity testing suggested that a single copy of the t allele would be sufficient to contribute to BD and shZ susceptibility in Pakistani population. in comparison to our study, there are contradictory findings regarding the role of rs1611115 in psychiatric patients in different populations worldwide.a study on the turkish population reported no association of rs1611115 with BD [20 ], while the same sNP revealed an association with shZ susceptibility in the North indian population [ 30 ]. in one of the studies on chinese MDD patients, rs1611115 disease risk association was observed, while in the present study no significant association of rs1611115 was observed in the MDD cohort, which complies with another chinese study on West chinese MDD patients [ 31 , 32 ]. the minor allele (t) frequency of the studied controls was also compared to the 1000 Genome Project PJl ethnic group from Pakistan and it was found to be significantly different.it might suggest geographical differences to be a possible reason [ 33 ]. as the 1000 genome project has represented data of only the lahore-Punjab population (PJl) which is geographically separated about 400-450 km apart from our sample's geographical location which is mostly from islamabad and Rawalpindi.secondly, the 1000 genome has a better quality of genotyping data, and nonetheless, the 1000 genome had a smaller sample size (n = 96),  as compared to the current study sample (controls) size (n = 332), which was larger than the 1000 genome PJl data set.as in the current study, no comparative elisa-based serum estimation of the DBh levels has been conducted before for MDD, BDP and shZ.however, the determination of DBh activity in serum and plasma of psychiatric patients through different biochemical techniques has revealed reduced plasma DBh activity that appeared to be due to diminished DBh levels in the circulation in carriers of the t allele in different psychiatric conditions [14 ,15 , 34 , 35 ]. in the present study, irrespective of the genotypes (cc/ct/tt ), the serum-based expression profiling of DBh determined higher serum DBh levels only in the MDD cohort.DBh, being an important regulatory enzyme in the dopaminergic system that converts Da into Na and the higher concentrations of serum DBh can thus potentially disturb the homeostasis between Da and Na, which then alter the sympathetic neuronal responses [ 36 , 37 ].thus the elevated DBh levels result in reduced Da in the circulation leading to altered neuronal activity, which is a hallmark feature in psychiatric disorders and therefore considered to be one of the contributing factors in MDD aetiology as reported previously [ 38 ].When the serum DBh levels in the sub-cohort were grouped by genotype, significant differences in expression were observed.the tt homozygotes showed significantly lower serum DBh levels, while cc homozygotes had comparatively higher DBh levels within the BD group.however, in cross-comparison between the groups, apparently, the serum-DBh levels were seemed lower in the tt homozygotes in BD than in MDD (tt homozygotes) and seemed higher than in controls (cc homozygotes), but these findings were not statistically remained significant after multiple testing Bonferroni's corrections.in an earlier study (15,] observed reduced DBh serum expression in the tt homozygotes that resulted in diminished DBh activity, in turn, disrupts the Da to Na homeostasis and subsequently raises the levels of Da in the circulation [6], thus making the risk allele carriers susceptible to various neurological and psychiatric conditions [15]. Various functional studies of DBH promoter concerning the influence of genetic changes and promoter activity, with the subsequent effect on DBh expression, have reported the role of cis-regulatory motifs, containing cReB and additional Phox2a/2b binding sites present in domain ii of the DBh promoter.the genetic changes in domain ii resulted in inactivated promoter indicating its crucial role in the DBh promoter activity along with the main domain iV of the DBh promoter [ 39 , 40 ]. the studied polymorphism rs1611115 is among the functional sNPs residing in domain ii and is also a part of the haplotype block (between -600bp to -1100bp) of the DBH promoter, located at position −1021 up-stream from the start codon, thus significantly affecting the DBh gene expression [ 41 ].
the studies on haplotype block analyses of promoter region sNPs of DBH have reported a significant role of rs1611115 in the variation in DBh plasma levels and activity as compared to any other sNP in the same haplotype block [13 , 42 ].these authors also reported the individual sNP effect, as well as the haplotype block effect, where rs1611115 accounted for the largest proportion of variance in DBh plasma activity in populations from diverse geographic origins including african-americans, east-asian, american-europeans, Germans and Japanese [14,15].a study similar to these authors' work on genotype as well as haplotype association of the DBH sNPs with plasma DBh activity has been reported in unrelated individuals from the east-indian population [34].these reported studies, therefore, suggest a population-specific role, of the haplotype block containing rs1611115, in DBH promoter regulation and its protein serum levels and therefore explain the differential expression of DBh in different psychiatric phenotypes in the current study.
there are also few studies [ 43 , 44 ] that have reported the contribution of non-genetic risk factors on the serum DBh levels, where Nelson and Davis [44] reported the association of dysregulation of the hypothalamic-pituitary axis (hPa) with variations in DBh plasma levels, thus indicating the role of hPa in the regulation of DBh protein in plasma.While Mustapic et al. [43] on the other hand conducted a related study on war veterans with or without posttraumatic stress disorder (PtsD) in croatian caucasians and found variation in plasma DBh levels, depending upon stress exposure irrespective of which allele (c/t) of rs1611115 was present.the contribution of non-genetic risk factors might be the reason that no significant differences were observed in DBh serum levels within MDD and shZ cohorts with respect to the genotype (cc/ct/tt) in the current study.
the present and previous findings of the association of rs1611115 genetic susceptibility with psychiatric conditions and DBh serum levels thus suggest a significant population-based variation of this polymorphic locus.the GWas and Meta-GWas studies data on DBh polymorphisms have also revealed the role of rs1611115 in phenotypes from different ethnicities (see supplementary information, table s3). the meta-analyses showed the rs1611115 significant association with Parkinson's disease (PD) in southern china ethnic group and chinese population [ 45 ], while another meta-GWas study [ 46 ], on larger data set of european and asian (including indian and chinese) population, determined no association with PD. however, the GWas study [ 47 ] on the population of european ancestry and Native americans revealed the significant association of rs1611115 in PtsD patients, moreover, this study also includes the first GWas data on plasma DBh activity showing the influence of rs1611115 on plasma DBh activity.these paradoxical results therefore highlight and suggest the complex nature of the psychiatric conditions and a significant inter-ethnic variation of this polymorphic locus in different psychiatric phenotypes.
the limitations of the current study included insufficient sample size for statistical measurements as the sample size in the current study was relatively small in BD and shZ groups resulting in current study to be underpowered.therefore, there is a need for thorough investigations in comparison to overlapping phenotypes involving larger cohorts to decide on a genetic association study in multifactorial disorders, due to the diversity of population genetics.in addition, there is also a lack of previous research on the role of DBH rs1611115 and its effect on serum level in manifestation of BD, shZ and MDD phenotypes in different ethnicities worldwide.Despite the above limitations, the rs1611115 was found in hWe in our study which suggests very low chances of genotyping error in the current study.Moreover, in-vitro and in-vivo functional characterization is also needed to determine the population-specific role of the studied sNP in the Pakistani population.

Conclusion
in conclusion, the present study on the role of DBh polymorphism rs1611115 revealed the genetic association of DBH rs1611115 risk allele t with BD and shZ phenotypes and the influence of the t allele on DBh serum levels in BD and, in MDD phenotype irrespective of the allele (c/t) in Pakistani population.the current study also explains that divergent genetic backgrounds, as well as different environmental niches of the different ethnic populations, could not be ruled out in causing greater genetic as well as clinical heterogeneity in psychiatric illnesses.

Figure 1 .
Figure 1.flow chart of selection criteria of studied cohort.sections f20-f29 and f30-f39 were the guidelines followed for the diagnosis and selection of patients.MDD, Major depressive disorder; BD, Bipolar disorder; sHZ, schizophrenia.

Figure 2 .
Figure 2. Bar charts representing a comparison of serum DBH levels in cases and controls.the first bar chart represents the overall serum DBH concentration in each group, second bar chart represents serum DBH levels in each group, with respect to alleles (cc, tc, tt).P valve from non-parametric Mann-Whitney U test.con = controls, MDD = Major depressive disorder, BD = Bipolar disorder, sHZ = schizophrenia).

Table 1 .
Demographic characteristics of the cohort (cases and controls).

Table 3 .
Descriptive features of subset-cohort for elIsa.

Table 2 .
association of DBH rs1611115 with BD, sHZ and MDD.

Comparison of Genotype and Allele frequencies from 1000 genomes PJL data and the Control data from our study
adjusted for age and sex, **adjusted for age, sex and tobacco users, the P, P*, P** values from Pearson's chi-squared χ2 test with Yates' continuity correction.

Table 4 .
comparative analysis of serum DBH protein concentration with respect to genotype in each group.