A pioneering study indicate role of GABRQ rs3810651 in ASD severity of Indo-Caucasoid female probands

Alteration in gamma aminobutyric acid (GABA), the principal inhibitory neurotransmitter, is speculated to be a potential risk factor for Autism Spectrum Disorder (ASD) due to an altered expression in the brain. Sensory, social, and emotional deficits of subjects with ASD were reported to be caused by an imbalance between excitatory and inhibitory neurotransmission as well as GABAergic dysfunction caused by inadequate receptor function. We for the first time studied association between ASD and a missense coding variant rs3810651 (I478F) in the GABRQ gene, encoding for one of the subunits of GABAA receptors. Stratified analysis on families with ASD probands (N = 251) and ethnically matched control subjects (N = 250) revealed marginally higher frequency of “A” allele and “AA” genotype in female ASD probands as compared to gender matched controls. Female probands demonstrated higher severity for Verbal communication (χ2 = 5.75, P = 0.01), Activity level (χ2 = 7.26, P  = 0.007), as well as Level and consistency of intellectual response (χ2 = 7.83 P = 0.005) in presence of “A/AA” warranting further in-depth investigation on the role of rs3810651 in ASD.


Discussion
In silico analysis on X-linked synaptic genes predicted GABRQ A > T: Ile478Phe (i.e. rs3810651) to be nonpathogenic 13 . Genomic DNA samples of ASD patients showed truncating mutations in GABRQ and GABRA3 in two different families with ASD probands 8 though no association between rs3810651 and ASD was reported 8,13 .  www.nature.com/scientificreports/ On the other hand, rs3810651 was found to be a potential candidate for a common neurological disorder migraine and carriers of rs3810651 "AT" genotype showed increased risk of migraine, which was significant for female subjects 9 . Frequency of the "A" allele was appreciably higher in the subgroup of female migraine patients with ≤ 15 years age of onset and subjects with "AA" genotype showed a lower mean age of onset of migraine attacks 10 . rs3810651 "T" allele exhibited association with improvement in essential tremor 11 . In Major depressive disorder patients, rs3810651 "A" allele showed significant positive association with antidepressant drug response 14 .
No significant association of the studied variant was noticed with restless leg syndrome 12 . Genotype of female participants recruited for the present study followed the Hardy-Weinberg equilibrium. Assumption taken into consideration while calculating Hardy-Weinberg equilibrium was (1) non-consanguineous marriage of parents, (2) same ethnicity, and (3) no genetic drift.
Our investigation for the first time revealed association of this variant with phenotypes of female ASD probands indicating a role of GABRQ rs3810651 ' A' allele in the severity of ASD associated traits of female probands. In spite of having reasonable number of male probands, we failed to notice any significant association of GABRQ rs3810651 in this subgroup. An earlier study on a large dataset showed that female ASD probands with less than 70 IQ had overall more impairments than males with ASD in terms of social-communicative abilities, cognitive and adaptive abilities, and externalizing behaviors 15 . Girls diagnosed with ASD were also reported to have a higher mean total problem score (hyperactivity, anxiety, and conduct, peer, and prosocial problems) and higher frequency of low IQ than boys with ASD 16 . Comparative analysis on neurobehavioral profile exhibited poorer inhibition in females with ASD as compared to males 17 . Females with ASD showed significantly increased grey matter volume in the right anterior cingulate cortex and right cerebellum 18 , brain regions with neuronal activity typically mediated by GABA 19 .
We infer from the present investigation that GABRQ rs3810651 may have a specific role in ASD severity which is dependent on gender. Our study has three major drawbacks; (1) limitation in number of female ASD probands. However, it has already been noticed that from human studies to model systems, limited availability of female www.nature.com/scientificreports/ ASD samples and limited attention to gender as a biological variable have hindered our ability to describe as well as to understand the importance of sex difference in ASD pathology 20 . Hence, further extensive research including larger female ASD samples is warranted to identify the importance of GABRQ gene in the etiology of ASD in sex balanced sample sets. (2) absence of high functioning (HF) ASD patients, i.e. with IQ > 80, in our study group. Our subjects were homogenous for age (i.e. 5.96 years ± 3.31), low IQ (< 80) and limited verbal ability. This could be one of the reasons for our observed higher trait scores in female probands, as low IQ have already been reported to be associated with poorer traits in female ASD patients 15,16 . Having a comparative analysis on subjects with broader ASD phenotypes characterized by a more diverse sample in terms of high functioning vs. low-functioning would help to identify the precise association between GABRQ rs3810651 and ASD phenotype.
(3) assessment of traits using CARS2-ST. Analysis of association between GABRQ rs3810651 and ASD using diagnostic criteria mentioned in the DSM-5 and scores obtained through other ASD assessment tools like Autism Diagnostic Observation Schedule along with the Autism Diagnostic Interview-Revised may aid in strengthening our findings. Under the current scenario, we conclude that to identify the importance of GABRQ gene in the etiology of ASD further extensive research is warranted.

Materials and methods
Subject selection and sample collection. Genotyping and statistical analysis. Genotyping of coding region variant, rs3810651 of GABRQ was done by TaqMan-based allelic discrimination assay (Assay ID C__27492635_10, Thermo Fisher Scientific, Singapore). Population-and family-based analyses were performed using UNPHASED v 3.1.7 24 . Quantitative Trait (QT) analysis, to identify association between rs3810651 and phenotypic domains incorporated under the CARS as well as cumulative CARS score, was also performed using UNPHASED (v.3.1.7) program 24 .