The association analysis of GWAS and candidate gene loci belonging to dopaminergic system genes, in the current psychiatric cohort of the Pakistani population revealed a significant association for CACNA1c polymorphisms (rs1006737, rs2238056), DRD1-rs4532 DRD2-rs1799732, DRD4-120bp, and DRD5-rs10033951. Among CACNA1c polymorphisms, rs1006737 was associated with a higher risk in both MDD and BD, while, rs2238056 was found to be associated with BD susceptibility only. However, the association did not survive for rs1006737 in MDD when the P-value was adjusted for age and sex, suggesting the possible impact of these demographic variables in this association with MDD, while the results remained significant in BD.
Similar to the current findings previously reported studies by He et al, (2014) in Chinese Han and Green et al, (2010) in the British populations, reported the rs1006737 association with MDD (Green et al., 2010; He et al., 2014), and in the European-British population, the association of both the polymorphisms; rs1006737, rs2238056 were reported with BD (Ferreira et al., 2008; Gershon et al., 2014; Green et al., 2010; Starnawska et al., 2016). However, studies conducted on Americans(Frazier et al., 2014), Germans(Kloiber et al., 2012), Europeans (Cross-Disorder Group of the Psychiatric Genomics, 2013) and Swedish population (Lavebratt et al., 2010) found contrasting results for rs1006737 association with MDD and in Southern Taiwanese population with BD (Jan et al., 2014). Whereas, a study of SHZ by Nie et al, (2015), reported the association of rs1006737 in European and Asian populations (Nie et al., 2015), while, the present study failed to establish a significant association of CACNA1c polymorphisms in SHZ, which points to the inconsistent association results of CACNA1c polymorphism in different populations and ethnicities.
CACNA1c is a calcium channel gene that encodes the alpha subunit (α1C) of the L-type voltage-gated calcium channel (LTCC). The function of the alpha subunit is to determine the kinetics, conductance, voltage dependence and pharmacology of the LTCC (Heyes et al., 2015). LTCC is involved in various aspects of neuronal development, signalling, and also in the establishment of maintenance of connectivity during and after neuronal development (Spitzer, 2006). The role of CACNA1c polymorphisms as a pathophysiological factor in the progression of psychiatric disorders has been reported by Yoshimizu et al, (2015) and Starnawska et al, (2016) in clinical and functional studies (Starnawska et al., 2016; Yoshimizu et al., 2015). Both polymorphisms (rs1006737 and rs2238056), are located in intron 3 of CACNA1c, which has important gene expression-regulatory functions, through interaction with the transcription start site. This intron also carries CpG islands which underwent a DNA methylation shift due to hypermethylation in the presence of rs2238056 (C > T), (Starnawska et al., 2016). This explains the impact of the polymorphisms (rs1006737 and rs2238056), and the role of intron 3 in the expression of CACNA1c, by regulating the 3D genomic architect associated with the chromosomal looping, in the presence of risk alleles of rs1006737 (A) and rs2238056 (T), (Starnawska et al., 2016). Thus the altered LTCC are unable to regulate the calcium influx and efflux which are important steps in neurotransmission, as calcium ions are also needed for the fusion of neurotransmitter vesicles to release neurotransmitters (dopamine, acetylcholine etc.) in the synaptic cleft (Imbrici et al., 2013), suggesting the contribution of CACNA1c polymorphisms in the genetic aetiology of psychiatric conditions by altering the functional activity of LTCC in different brain circuits and CACNA1c expression.
DRD1 is an extensively studied gene, yet very few reported association studies are available regarding rs4532, which is located on the 5′ UTR (untranslated region) (Pan et al., 2014). This locus has been reported to be associated with the therapeutic response to antipsychotics (Potkin et al., 2003), the positive symptoms of SHZ (psychotic symptoms), and with the prefrontal cortex (PFC), related cognitive functions (Rybakowski et al., 2005; Williams & Castner, 2006). In the initial analysis, DRD1-rs4532 and DRD2-rs1799732 were found to be associated with the risk of MDD in the current cohort, however, the association did not survive for any of these SNPs in MDD, after applying multiple test corrections. The reported association studies in different populations have reported contrasting results to our current findings. A study in the Estonian population has reported no association of rs4532 with MDD (Koks et al., 2006), while, a study of the Sardinian population, reported a significant association of rs4532 with BD (Severino et al., 2005). Whereas in SHZ, the role of rs4532 is still controversial and its role in the manifestation of SHZ is yet to be established in different populations, however, a meta-analysis study conducted in 2014, reported no association of rs4532 in SHZ (Pan et al., 2014), which supports the current findings in SHZ of the present study.
DRD2 SNP rs1799732 is a functional polymorphism in a promoter region of the DRD2 gene, involving the insertion (INS)/deletion (DEL) of a cytosine (-141C INDEL), which affects the receptor density by modulating the transcription and transcription-binding-factors (TBF), and reduces DRD2 expression 20–40% (Arinami et al., 1997). This polymorphism has been reported for its association with MDD in the Chinese Han population (He et al., 2013), while a study in Caucasian populations reported contradictory results in MDD (Furlong et al., 1998; Leszczynska-Rodziewicz et al., 2005). In BD several association studies have reported a lack of risk association of rs1799732 in different ethnicities including Polish (Leszczynska-Rodziewicz et al., 2005), Asian (Han Chinese), and Caucasians (Furlong et al., 1998; Li et al., 1999; Stober et al., 1998). A meta-analysis by Zou et al, (2012), has also reported no association between rs1799732 and mood disorders (BD & MDD) in Caucasians (Zou et al., 2012). Whereas, studies in SHZ have reported risk association of this SNP, rs1799732 in Chinese Han (Xiao et al., 2013), British Caucasian (Breen et al., 1999), and Brazilian population (Cordeiro et al., 2009). Though an ethnicity-based meta-analysis for SHZ, revealed the significant association of rs1799732 with a higher risk of SHZ susceptibility in Asians (Wang et al., 2016), and in Chinese Han (Zhao et al., 2016), but not in Caucasian, Japanese and Indian populations. (Arinami et al., 1997; Zhao et al., 2016).
In the present study, we have also observed that 2 of the studied variants (DRD4-120bp repeat and DRD5-rs10033951), were found to be associated with a protective effect. DRD4-120bp (2R = longer repeat or 120bp duplication), showed a significantly lower risk association with MDD and BD susceptibility, while the association was not found with SHZ. The DRD4 encodes dopamine receptor 4 (D4), which is involved in inhibitory neurotransmission (Andersen et al., 1990; Civelli et al., 1993; Missale et al., 1998; Niznik & Van Tol, 1992). The DRD4-120bp repeat is a promoter region tandem duplication polymorphism (functional polymorphism), having consensus binding sites for transcription factors. This suggests the role of the duplication sequence in conferring differential transcriptional activity (Seaman et al., 1999), by enhancing the binding capacity of certain transcription factors such as Sp1 (specificity protein 1) in the duplicated form (2R), (Ronai et al., 2004). The Sp1 transcription factor binds to a GC-rich region and can activate or repress the transcriptional activity in response to the physiological or/and pathological stimuli (Infantino et al., 2011). However, functional studies by McCracken et al, (2000)d Souza et al, (2004) reported the lower transcriptional activity of longer repeat (2R), than the shorter allele (1R), suggesting that the duplication allele might have a regulatory role in DRD4 expression providing an underlying biological mechanism in the aetiology of neuropsychiatric disorders (D'Souza et al., 2004; McCracken et al., 2000).
The DRD4-120bp repeat sequence is known to have a difference in allele frequencies in different populations. Significant risk association of duplication sequence (2R), in MDD, has been reported in the Chinese population (Lai et al., 2010) and in SHZ in the Chinese and North Indian populations (Li et al., 2004; Srivastava et al., 2006; Xing et al., 2003), while the non-significant results were reported in Danish SHZ patients (Olsen et al., 2005). The association of DRD4-120bp tandem duplication polymorphism has not been reported in case-control association studies of BD, however, an association study on novelty seeking (NS) behaviour analysis in BD patients has reported the association of shorter repeat (homozygous 1R) with higher NS scoring in BD (Rogers et al., 2004). In parallel, the DRD5 variant, rs10033951 was found as a protective factor in all the studied groups, (MDD, BD and SHZ), in the present study. The literature on the DRD5 variant is very limited and this locus has not been well studied in different ethinicities for MDD, BD and SHZ. However this variant has been explored in different antipsychotics analysis in psychiatric conditions (Hwang et al., 2012). In addition, DRD genetic variants specifically DRD4-48bp VNTR had shown differences in allelic distribution in different ethnic groups from Pakistan (Mansoor et al., 2008). Therefore further replication studies of DRD4-120bp and DRD5 rs10033951 for psychiatric conditions in different population is suggested. On the contrary, the DRD3 polymorphism rs6280 which is a missense variant (Ser9Gly), showed no risk association in either of the disorders (MDD, BD and SHZ), in our population. However, an association study by Chang et al., (2013) in Chinese BD patients, comorbid with or without anxiety disorders reported a significant association of rs6280 in BD comorbid with an anxiety disorder (Chang et al., 2013). Whereas association studies in SHZ did report a significant association of rs6280 with SHZ, however, all meta-analyses conducted for this variant failed to confirm a significant risk association with SHZ in different ethnicities (www.szgene.org, 2010), which comply with the current findings in SHZ.
Furthermore, 4 variants (POU3F2-rs2388334, PACS1-rs10896090, TRANK1-rs9834970, FSTL5-rs11724116), that were recently identified to be associated with BD cohorts of European, North American and Australian populations (Stahl et al., 2019), were screened in the present BD cohort of the Pakistani population. We observed that rs10896090 showed no risk association with BD in the present study, while it was reported to have a significant risk association with BD in a GWAS (Stahl et al., 2019). On the other hand, rs9834970, rs11724116 and rs2388334, were found with lower risk (protective-effect) in BD in the same GWAS (Stahl et al., 2019), however, in the present BD cohort of the Pakistani population, only rs2388334 was found to have a protective effect in BD susceptibility, which supports the previously reported GWAS findings (Stahl et al., 2019).
In the comparative analysis of the genotype and allele frequencies of all the studied genetic variants in the current study to the 1000 Genomes (PJL), significant differences were observed in genotype frequencies or allele frequencies of 6 variants (rs2238056, rs4532, rs6280, rs10033951, rs9834970, rs11724116 and rs2388334). All these variants were found in HWE except, rs10033951, rs11724116 and rs2388334. Similar differences were observed in comparison to ALFA SAS data, except rs4532, which did not show allele frequency differences with 1000 Genome Project PJL data but showed marked deviation in comparison to ALFA SAS allele frequency data. The deviation from 1000 Genome PJL reported frequencies might be due to the smaller sample size (n = 96), of that study as compared to the current study cohort size (n = 390) which was 3–4 folds larger than the 1000 genome PJL Pakistani data set, in addition, PJL included samples of Punjabi origin only from Lahore, which is g geographically separated (about 400–450 km apart) from Islamabad and Rawalpindi. As opposed to this the ALFA SAS reported frequencies were based on a larger sample size and included data of all South Asian populations but unlike the 1000 Genome, it did not describe the geographical location of the ethnicities they have included from Pakistan. In the present study, the majority of the participants, geographically belong to Rawalpindi and Islamabad, while the rest (20–33%) belongs to other geographical areas of Pakistan. Thus sample size and geographical differences might be the possible reasons for the observed deviation and differences.
The present study also had a few limitations, first, the majority of the participants of the current study were of Punjabi ethnic background, secondly, the control group that was collected was from the same geographical location as of the patients, therefore there are chances of overlooking the population stratification because three variants (rs10033951, rs11724116 and rs2388334) showed deviation from HWE of the control population. There is a chance of differences in the origins of the studied population. Thirdly, the sample size was relatively small in BD and SHZ groups. It is always very challenging with smaller cohort sizes, to decide on a genetic association study in multifactorial disorders, due to the diversity of population genetics. Since both the genetic and environmental backgrounds vary for different populations and ethnicities, thus the generalization of these current findings to other populations is limited. Despite all the reported association of the studied variants with the phenotypes, it must be admitted that most of the genetic association analyses in the present study were underpowered (See Supplementary Table 3), which might explain why some of the variants did not replicate the risk association observed in previous studies. Thus, the present study only provides preliminary explanations for the observed associations of the studied variants with MDD, BD and SHZ as these variants were screened for the first time in these psychiatric conditions in the Pakistani population. Future replication studies with larger cohort sizes in multiple ethnic populations are warranted to confirm these findings and functional studies may lead to a better understanding of the underlying genetic architectures of these psychiatric conditions (MDD, BD, and SHZ).
In conclusion, the present study was a replication study of the GWAS and association studies identified variants, in MDD, BD and SHZ in the Pakistani population. The present study explains evidence of some degree of genetic overlap between MDD, BD and SHZ, the underpinnings of susceptibility to these psychiatric conditions. We observed BD associated risk allele at CACNA1c polymorphisms (rs1006737), which also confers the risk of MDD susceptibility, whereas the risk allele at DRD5 polymorphism (rs10033951), found to act as a protective factor in all the 3 psychiatric conditions (MDD, BD and SHZ), in the current Pakistani cohort. However, the role of studied variants in SHZ susceptibility remains elusive due to the contradictory findings on the association of these SNPs in SHZ. We did observe deviation of our results from previously reported findings thus explaining the contribution of divergent genetic and geographical backgrounds of the different populations in clinical heterogeneity in psychiatric illnesses.