Glutamate receptor genetic variants affected peripheral glutamatergic transmission and treatment induced improvement of Indian ADHD probands

Attention deficit hyperactivity disorder (ADHD), a childhood-onset neurobehavioral disorder, often perturbs scholastic achievement and peer-relationship. The pivotal role of glutamate (Glu) in learning and memory indicated an influence of Glu in ADHD, leading to the exploration of Glu in different brain regions of ADHD subjects. We for the first time analyzed GluR genetic variations, Glu levels, as well as expression of Glu receptors (GluR) in the peripheral blood of eastern Indian ADHD probands to find out the relevance of Glu in ADHD prognosis. After obtaining informed written consent for participation, peripheral blood was collected for analyzing the genetic variants, Glu level, and expression of target genes. Since ADHD probands are often treated with methylphenidate or atomoxetine for providing symptomatic remediation, we have also tested post-therapeutic improvement in the ADHD trait scores in the presence of different GluR genotypes. Two variants, GRM7 rs3749380 “T” and GRIA1 rs2195450 “C”, exhibited associations with ADHD (P ≤ 0.05). A few GluR genetic variants showed significant association with higher trait severity, low IQ, lower plasma Glu level, down-regulated GluR mRNA expression, and poor response to medications. This indicates that down-regulated glutamatergic system may have an effect on ADHD etiology and treatment efficacy warranting further in-depth investigation.


Quantitative trait analysis (QTA)
Influences of the genetic variants on different traits were analyzed by quantitative trait analyses.

Behavioral problem (BPr)
ADHD probands with rs2284411 and rs1422884 'C' alleles and 'CC' genotypes exhibited higher scores for BPr and ADHD Index (AI) (Table 1; P ≤ 0.02) in comparison to those with the T allele/ TT genotype.A higher AI score was also detected in the presence of rs3792452 'T' allele and 'TT' genotype as compared to those with the rs3792452 'C' allele and 'CC' genotype (Table 1; P ≤ 0.05).
ADHD probands with rs2284411 'T' allele and 'TT' genotypes (Table 1; P ≤ 0.01) showed lower scores for oppositional defiant disorder (ODD) in reference to those with the rs2284411 'C' allele and 'CC' genotypes.On the other hand, the scores for the same traits were higher in the presence of rs1422884 'C' allele/'CC' genotype with respect to rs1422884 'T' allele/'TT' genotype (Table 1; P ≤ 0.02).The score for Parental Account of Children's Symptoms (PACS) was lower in the presence of rs2067011 'GG' genotype (P = 0.04) as compared to the rs2067011 ' AA' genotype.

Discussion
The present analysis on the Indian ADHD probands for the first time showed significantly lower circulating Glu levels, reduced expression of Glu receptor genes in the peripheral blood, and higher behavioural abnormalities as well as executive deficit in the presence of few mGluR and iGluR genetic variants.Adequate glutamatergic signalling is necessary for maintaining the synaptic plasticity crucial for proper learning and memory 33 .In both the central and peripheral nervous system, Glu is present in all types of neurons in the sensory ganglia and is released from the axon terminals as well as the cell bodies 25,26 .The synaptic NMDARs play   www.nature.com/scientificreports/crucial roles in excitatory synaptic transmission and plasticity, thereby modulating learning, memory, and higher cognitive functions 26,33 .On the other hand, the synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, acting as a cation channel, are fundamental for synapse maturation and plasticity 34 .
Association studies and GWAS performed on the Caucasoid subjects revealed a link between the GluR genetic variants and ADHD 28,30,35 .Association with symptom severity was also reported in the Caucasoid ADHD probands 16,27,36 .An earlier investigation on a limited number of Caucasoid children/adolescents with ADHD (N = 9) showed increased Glu levels in the striatum and frontal regions 17 .On the other hand, proton magnetic resonance spectroscopy of European Caucasoid adults with ADHD (N = 40) revealed lower Glu levels in the basal ganglia 37 .A lower Glu level was also reported in the left mid-frontal region of Norwegian adults with ADHD (N = 29) as compared to the controls (N = 38) 38 .A vital role of GRIN2B and GRM7 in responses to MPH treatment was observed by clinical pharmacological studies of ADHD subjects 39,40 .The increased glutamatergic tone in the frontal and striatal regions of ADHD subjects was found to be normalized after treatment with stimulants as well as non-stimulants like ATX 41 .On the contrary, MPH-induced increase in the mesocortical glutamatergic pathways was also evidenced by proton magnetic resonance spectroscopy, possibly through the inhibition of DA reuptake or through a direct effect on the NMDA receptors.This investigation for the first time documented lower circulating Glu levels in the ADHD probands.
Amongst the metabotropic receptors, Glu receptor 5 (GRM5), triggering a variety of signaling pathways in the neurons and glial cells, was reported to have an association with intellectual disability and Autism 42 .In Australian autistic subjects, gene pathway analysis identified GRM5 rs905646 as a protective SNP, while rs11020772 was recognized as a risk variant 43 .The present pilot study for the first time documented down-regulated GRM5 expression and lower circulating Glu levels in the peripheral blood of eastern Indian ADHD probands carrying GRM5 rs905646 "A" and rs11020772 "T" variants.Frequencies of rs905646 "A" and rs11020772 "T" were higher in the Indian population as compared to the other Asian populations.The two sites showed strong LD indicating higher chances of being present together.Though no statistically significant associations of the studied variants were detected with individual traits, MDR analysis exhibited a significant independent effect of rs11020772 in ADHD.ATX treatment improved IA rs11020772 'GG' genotypes.Based on these observations we conclude that further analysis involving functional genetic variants may help in elucidating the actual role of GRM5 in ADHD.
Two other mGluR variants, Glu receptor 6 (GRM6) rs762724 and rs2067011, were reported to have an association with higher myopia in the Han Chinese population 44 .The present study revealed strong LD between these two markers in the ADHD probands.In the presence of rs762724 'T' and rs2067011 'G' alleles, the ADHD probands exhibited reduced IQ score, less EF deficit, and reduced GRM6 expression.The differential impact observed for rs762724 'T' and rs2067011 'G' alleles on IQ and EF could be due to the procedural differences used for testing the two parameters; while IQ assessment depends on the momentary performance requiring sustained attention of the participant, EF is assessed from the input provided by the parents or caregivers and may not be influenced by the inattention problem of the proband.As a result, while IA of the probands may reduce the IQ score, it may not be reflected on the EF score.GRM6 variants did not show any significant association with other traits.MPH treatment was beneficial for the probands with GRM6 rs762724 'CC' , rs2067011 ' AA' , genotype.The observed impact of the studied GRM6 variants on GRM6 expression warrants further investigation on the role of GRM6 in the etiology of ADHD using more functional variants.
The mGluR 7 (GRM7), widely expressed in the cerebral cortex, hippocampus, and cerebellum, was speculated to affect anxiety, fear responses, and working memory 45 .In the Korean ADHD probands, the biased parental transmission of GRM7 rs3792452 'C' allele was reported 35,40 .Poor performance during the Continuous Performance Test was also observed in the presence of this variant 35 .The present study on Indian ADHD probands revealed marginally higher scores for IA and AI in the presence of the rs3792452 'T' allele.In the presence of GRM7 rs3792452 'CC' genotype, both medicines effectively reduced the HA score.Another GRM7 variant, rs3749380 is a C to T transition at codon 74 (C222T), resulting in a missense substitution (Leu74Ser), and the 'T' allele was reported to increase the risk of alcohol consumption 46 .The 'T' allele and 'TT' genotype also showed an association with schizophrenia 47 .Our pilot study revealed a higher occurrence of the rs3749380 'TT' in the ADHD probands, mild negative impact of the "T" variant on all the trait scores, circulating Glu levels, and GluR expression.On both medications, all the traits were improved in the presence of rs3749380 'CC' genotype.The data obtained indicates an influence of GRM7 variants on the down-regulation of glutamatergic transmission which requires further confirmation.
NMDA receptor variants, GRIN2A rs2229193, and GRIN2B rs2284411, were identified to confer an increased risk of attention impairment in Korean ADHD patients 27 ; the probands exhibited fewer errors in the presence of the rs2229193 'CC' genotype 27 .However, no significant differences in the allele/genotype frequencies for rs2284411 were detected in the Korean ADHD subjects by earlier investigators 35 .In silico analysis revealed that rs2229193 is a C to T transition at codon 425 (C1275T), resulting in a synonymous change (Leu425), while rs2284411 is a T to C transition at the intron 7. The present genetic analysis showed biased paternal transmission of the rs2229193 'C' allele to the female ADHD probands.Trait scores and executive deficit of the ADHD probands were higher in the presence of the rs2284411 'CC' genotype.Probands having rs2229193 "C" and rs2284411 "C" variants exhibited significantly reduced circulating Glu level as well as GRIN2A and GRIN2B expression.In the presence of rs2284411 'TT' , scores for BPr and HA were improved after MPH and ATX treatment.On the other hand, Korean ADHD patients with rs2284411 'CC' genotype were reported to show significantly better treatment response 39 .This difference in response to treatment in the presence of a particular genotype could be attributed to differences in food habits or other environmental factors in different ethnic groups, which merits further exploration.
We have also analyzed two regulatory AMPA variants, GRIA1 rs1422884 (feature type-enhancer) and rs2195450 (feature type-promoter), for the first time in the ADHD probands.An earlier study in the Italian population revealed an association of GRIA1 rs1422884 with schizophrenia susceptibility 48 .However, a meta-analysis www.nature.com/scientificreports/later confirmed a significant association of GRIA1 rs2195450 C > T with the risk of migraine in the Asian population only 49 .The present investigation on the Indo-Caucasoid ADHD subjects revealed an increase in the trait scores in the presence of GRIA1 rs1422884 'CC' genotype, while in the presence of the ''CC' genotype the probands showed reduced Glu level, less executive deficit, and remarkable improvement in HA score after ATX treatment.We have also observed a marginally higher frequency of GRIA1 rs2195450 'C/CC' in the ADHD probands, the biased paternal transmission of the 'C' allele, and lower trait scores in the presence of the 'CC' genotype.Expression of GRIA1 was significantly down-regulated in the probands group.We conclude from the data obtained that GRIA1 may influence ADHD severity by affecting different traits which needs further exploration in the field.The studied genetic variants exhibited various levels of independent as well as interactive effects in the ADHD probands as compared to the ethnically matched control group, as is evident from the MDR analysis.Further analyses, considering individual phenotypes (data not presented for brevity), revealed that BPr score was affected by the synergistic interactions between GRIA1-GRM6 (rs1422884-rs762724), and GRM5-GRM7 (rs905646-rs3749380) variants.The trait IA was also found to be affected by synergistic interactions between GRM5-GRM6 (rs11020772-rs20670110) and GRM5-GRM7 (rs762724-rs3749380).The behavioral traits, ODD and PACS, were synergistically influenced by GRM5 variants (rs905646-rs11020772).The trait scores for EF were found to be affected by the synergistic interaction between GRM6-GRM7 (rs2067011-rs3749380).Based on these observations we concluded that GRM5, GRM6, GRM7, and GRIA1 may affect ADHD etiology in an interactive manner.
During the past decade, pharmacogenomic research on the efficacy of medication in improving ADHD related traits has largely focused on the response to MPH and ATX 15 .Both these medications affect the dopaminergic transmission and are widely prescribed for symptomatic remediation, even in India [50][51][52] Besides the direct action on the dopaminergic system, MPH also influences other neurotransmitters including the glutamate receptors 53 .Pharmacological studies on ADHD revealed that GRIN2B and GRM7 play a vital role in MPH treatment 39,40 .Proton magnetic resonance spectroscopic studies indicated an MPH-induced increase in the mesocortical glutamatergic activity through the inhibition of DA reuptake or a direct effect on the NMDA receptors 54 .Experimental rat models showed influence of MPH on NMDA-receptor-mediated excitatory synaptic transmission 55 .However, some patients were found to develop acute side effects following intervention with MPH and ATX leading to treatment discontinuation 50,[56][57][58] .Pharmaceutical intervention, through targeted genotyping, was thus thought of as an useful method for subjects requiring long-term intervention 50,59,60 .It is evident from our study that in the presence of the ancestral alleles, treatment outcome was better.Our investigation also revealed that the post-treatment improvements were dependent on the GluR genetic variants as well as the medicine used; while MPH therapy improved the BPr of the probands having the GRIN2B rs2284411 'TT' , ATX treatment improved BPr in the presence of GRM7 rs3749380 'CC' .We infer from this data that pharmacogenomic investigations may aid in predicting the therapeutic success, thereby minimizing the number of individuals discontinuing pharmacotherapy.
The major limitations of the present investigation are (1) analysis of different glutamatergic components only in the peripheral system, (2) analysis of limited number of genetic variants, (3) limitation in the number of female probands, and (4) analysis of only prime ADHD traits.However, the association of GluR genotypes with reduced circulating Glu level, down-regulated GluR mRNA expression, higher trait scores, IQ deficit, as well as treatment outcome of the Indian ADHD probands indicates that an inadequate glutamatergic transmission may be, at least partially, responsible for the severity of the behavioral traits and inappropriate treatment response.Furthermore, this investigation on the glutamatergic system was performed entirely on the peripheral system, and hence, further in-depth analysis in the central nervous system is warranted to understand the actual role of Glu in the etiology of ADHD.

Recruitment of subjects
Nuclear families with ADHD probands (n = 279; mean age 9.50 ± 3.46; male: female ratio 7.72:1) were recruited based on the Diagnostic and Statistical Manual of Mental Disorders IV-text revised (DSM-IV-TR) 61 and DSM-5 criteria 1 .Probands exhibiting hyperactivity (HA), impulsivity (Imp), or inattention (IA) due to other neuropsychiatric disorders like Pervasive developmental disorders, Intellectual disability, Fragile X syndrome, Down's syndrome, Prader-Willi syndrome, etc., were excluded from this study.The majority of the recruited ADHD probands belonged to the combined subtype (72.0%), while the HA/Imp (11%) and IA (17%) subtypes were only a few.Parents (n = 416; 192 fathers and 224 mothers) of the probands were included in the study for family-based genetic association analysis.An ethnically-matched control group (n = 352; mean age 21.52 ± 2.13; male: female ratio 1:1.27) was recruited for population-based genetic association analysis.The participants were asked to fill up a questionnaire to collect the demographic details and medical histories, symptoms of IA, HA, and Imp, etc.For participation in the study, informed written consent was obtained from the parents or caregivers of the probands and control subjects.The study protocol (No.PR-003-17) was approved by the Manovikas Ethics Committee on Human Subjects, with Scientists, Psychiatrists, Psychologists, Advocates, and Social workers as members.

Assessment of traits of the ADHD subjects
The ADHD-associated traits BPr, IA, HA, and AI were assessed by Conner's Parent and Teacher Rating Scalerevised (CPRS-R) 62 .The Wechsler Intelligence Scale was used to evaluate IQ 63 .The BDEF-CA, a practical tool for evaluating executive function, was used to assess the executive deficit 64 .PACS was used to determine the behavioral problems 65 .Co-morbid ODD was evaluated by DSM 1,61 .

Selection of markers and analysis of target sites
In the present study, 10 markers from 6 candidate genes (GRM5, GRM6, GRM7, GRIN2A, GRIN2B, GRIA1) were selected based on their functional relevance, minor allele frequency in other ethnic groups, and earlier reports of association with neuropsychiatric disorders, including ADHD (Supplementary Table S1).

Measurement of plasma glutamate (Glu) levels
Peripheral blood samples were collected in a pre-cooled vacutainer from the ADHD probands and age-matched control subjects fasting for 12 h.Plasma Glu levels were measured by competitive enzyme-linked immunosorbent assay (ELISA) following the manufacturer's protocols (MyBioSource, San Diego, California USA) and the optical density of the end products was measured at 450 nm in an ELISA plate reader (Genetix, Biotech Asia Pvt Ltd).

Analysis of GluRs mRNA expression
Expressions of GRM5, GRM6, GRM7, GRIN2A, GRIN2B, and GRIA1 in the peripheral blood were examined in the ADHD probands (n = 23) and age-matched control subjects (n = 19).Briefly, RNA was isolated from the peripheral blood by the TRIzol method (TRIzol Reagent User Guide; Pub.No.MAN0001271B.0)and reverse transcribed into complementary DNA (cDNA), which was later used as the real-time PCR or quantitative PCR (qPCR) template to perform gene expression analysis.The data was normalized against Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, serving as an internal control.

Pharmaceutical intervention
ADHD probands (n = 84), who had adhered to the pharmaceutical intervention and were available for posttreatment follow-up, were included in this analysis.The probands with age-inappropriate HA, residing in the urban areas, and < 10 years of age (n = 52) were prescribed MPH at a dose of 0.3 mg/kg body weight/day for two months, followed by 0.6 mg/kg body weight/day for another 4 months.Probands (n = 32) with significant IA, > 10 years of age, and residing in rural areas where access to MPH is limited, were prescribed ATX at a dose of 0.8 mg/kg body weight/ day for two months, followed by 1.2 mg/kg body weight/day for another four months.All the probands were re-assessed by the CPRS-R after treatment completion.

Statistical analysis
Hardy Weinberg Equilibrium (HWE) was calculated using the online software (http:// www.oege.org/ softw are/ hwe-mr-calc.shtml/) to determine the pattern of the studied variants genotypic frequencies.Population-based comparative analysis and family-based transmission analyses were performed using the UNPHASED version 3.1.7 67, after 1000 permutations, which takes care of the multiple corrections.Quantitative trait (QT) analysis was performed to identify the association between the genetic variants and ADHD-associated trait scores using the UNPHASED version 3.1.7 67.Odds ratio (OR) and confidence intervals (CI) were calculated using the Odds Ratio calculator (http:// www.hutch on.net/ Confi dORnu lhypo.htm).The relative risk (RR) of the studied variants was calculated using the relative risk calculator.The Power of the significant observations was calculated using Piface software 68 .Pairwise LD between the variants was measured using the Haploview program v4.2 69 .MDR v3.0.2 program 70 was used to identify the effect of studied variants on ADHD using the case-control data.Confounded effect of other covariates like age, sex, and IQ were not considered for this pilot study.Case-control comparative analysis on the GluRs mRNA expression and plasma Glu levels was performed by the Unpaired T-test using Prism 9.0 (GraphPad Software, Inc).Genotype-based stratified analysis on the Glu levels of the controls and probands were analyzed using the Mann-Whitney statistics.Expression of mRNA in the presence of different GluR genotypes was evaluated using the Kruskal-Wallis test, followed by multiple comparisons using the Dunn's test.Improvement in the trait scores after pharmaceutical interventions was calculated by 1-Tn/To (To = initial trait score, Tn = Post-treatment trait score) as detailed in a previous article 50 and is presented as the improvement index.The association between GluR gene variants and treatment-induced changes in the trait scores, measured based on the improvement index, was analyzed by the Mann-Whitney (M-W) U test using the Prism 9.0 software.The data from the test are presented as Mean ± Standard error of the mean (SEM).

Figure 1 .
Figure 1.Pair-wise measures of LD, such as normalized LD coefficient (D') and correlation coefficient (r 2 ), were estimated using the Haploview program v4.2.The numbers represent the D' value expressed as a percentile.All control (A,G); all ADHD probands (B,H); male control (C,I); male probands (D,J); female controls (E,K); and female probands (F,L).

Figure 2 .
Figure 2. Multidimensionality Reduction analysis on the case-control dataset to identify the association between genetic variants and ADHD; (A) total ADHD cases, (B) male ADHD cases, (C) female ADHD cases.Nodal values (% Information Gain, IG) indicate the independent effect of each SNP, while the connecting lines indicate pairwise interactive effects.Connections with positive values indicate synergistic interaction between the markers (red line), and lines with negative IG values indicate redundancy or lack of any synergistic interaction between the markers (blue line).

Figure 3 .
Figure 3. Comparative analysis on the plasma Glu levels of age-matched controls and ADHD probands.The middle line and the whisker in the box plot represent the median value and the data range respectively.

Traits Variant Allele/genotype Add Value χ 2 (p) 95%CI
ContinuedVol:.(1234567890) Scientific Reports | (2023) 13:19922 | https://doi.org/10.1038/s41598-023-47117-5www.nature.com/scientificreports/Post-therapeutic improvement in the presence of genetic variants Out of 84 ADHD probands available for post-therapeutic follow-up, 52 received MPH, while 32 were treated with ATX based on the age at presentation, presenting symptoms, and availability of the medicine, and the treatment efficacy was tested in the probands having different GluR genotypes.Both MPH and ATX treatments improved Conner's Parent and Teacher Rating Scale-revised (CPRS-R) trait scores of ADHD probands carrying

Table 1 .
Quantitative Trait analysis to identify the association between genetic variants and ADHD associated traits.DSM diagnostic and statistical manual of mental disorder, CPRS Conner's parent rating scale, BPr behavioral problem; IA inattention, HA hyperactivity, AI ADHD Index, ODD oppositional defiant disorder, IQ intelligence quotient, Χ 2 Chi square, p P value ≤ 0.05, CI confidence interval.

Table 2 .
Genotype-based stratified analysis on the Glu levels of controls and ADHD probands.Data are expressed as mean ± standard error mean, Mann-Whitney U test statistic; significant differences are presented in bold.

Table 3 .
Analysis of the impact of the studied variants on treatment-induced changes in the trait scores.