aryl hydrocarbon receptor nuclear translocator - like ( arntl ) protein is a transcription factor and a core component of mammalian circadian rhythms regulatory network . convergent functional genomics approach , integrating the genetics with functional genomics in human as well as animal models identified the arntl gene as the top candidate associated with bd . several polymorphisms in arntl gene are reported to be associated with bd and sad in different ethnic groups . as the family members in the study were experiencing sad , this study was undertaken to delineate the role of arntl gene in sad . we attempted to decipher the five single nucleotide polymorphisms ( snps ) ( rs2279287 , rs1982350 , rs7126303 , rs969485 , and rs2290035 ) in arntl gene [ figure 1 ] and correlated it to differential seasonal behavior . however , the interaction between photoperiodic mechanisms ( light - dark cycle ) and the circadian system in the onset of sad is obscure . distribution of the five single nucleotide polymorphisms in aryl hydrocarbon receptor nuclear translocator - like gene the subjects comprised 30 members of close - knit family with sad ( dsm-5 criteria was used in diagnosis ) and age- and sex - matched 30 controls of the same caste with no prior history of psychiatric illness . in addition , 30 age- and sex - matched controls with no prior history of psychiatric illness belonging to 17 different castes formed as a negative control . the human ethical committee of bharathiar university , tamil nadu , india approved the study . the clinical investigations of all the study participants were carried out in kg hospital , coimbatore , tamil nadu , india . dna was isolated from the blood samples using hi - pura mini blood dna isolation kit ( himedia , india ) . five snps across the arntl gene namely rs2279287 ( a / g ) , rs1982350 ( c / t ) , rs7126303 ( c / t ) , rs969485 ( a / g ) , and rs2290035 ( a / t ) were selected to test for their association as the risk factor in sad . briefly , polymerase chain reaction ( pcr ) reactions were run in a total volume of 5 l , containing 10 ng dna , 2.5 l 1  pcr buffer , and 0.125 l 40  allelic discrimination primer - probe mix . reactions were run in abi 7500 real - time system ( applied biosystems , usa ) with the following cycle parameters : 95c for 10 min ; followed by 40 cycles at 95c for 15 s and 60c for 30 s. the post - pcr run was done at 60c for 1 min . statistical significance was assessed by more powerful quasi - likelihood score test ( mqls)-xm , a program written in c , that performs single - snp , case - control association testing on the autosomal chromosomes in samples with related individuals . the program is applicable to association studies with completely general combinations of related and unrelated individuals , where the relationship among the sampled individuals is assumed to be known . for each snp , for autosomal snps , the three test statistics computed are mqls , wqls and corrected . the link to the statistical software used is https://galton.uchicago.edu/~mcpeek/software/mqls_xm/index.html . the subjects comprised 30 members of close - knit family with sad ( dsm-5 criteria was used in diagnosis ) and age- and sex - matched 30 controls of the same caste with no prior history of psychiatric illness . in addition , 30 age- and sex - matched controls with no prior history of psychiatric illness belonging to 17 different castes formed as a negative control . the human ethical committee of bharathiar university , tamil nadu , india approved the study . the clinical investigations of all the study participants were carried out in kg hospital , coimbatore , tamil nadu , india . dna was isolated from the blood samples using hi - pura mini blood dna isolation kit ( himedia , india ) . five snps across the arntl gene namely rs2279287 ( a / g ) , rs1982350 ( c / t ) , rs7126303 ( c / t ) , rs969485 ( a / g ) , and rs2290035 ( a / t ) were selected to test for their association as the risk factor in sad . briefly , polymerase chain reaction ( pcr ) reactions were run in a total volume of 5 l , containing 10 ng dna , 2.5 l 1  pcr buffer , and 0.125 l 40  allelic discrimination primer - probe mix . reactions were run in abi 7500 real - time system ( applied biosystems , usa ) with the following cycle parameters : 95c for 10 min ; followed by 40 cycles at 95c for 15 s and 60c for 30 s. the post - pcr run was done at 60c for 1 min . statistical significance was assessed by more powerful quasi - likelihood score test ( mqls)-xm , a program written in c , that performs single - snp , case - control association testing on the autosomal chromosomes in samples with related individuals . the program is applicable to association studies with completely general combinations of related and unrelated individuals , where the relationship among the sampled individuals is assumed to be known . for each snp , the three test statistics computed are mqls , wqls and corrected . the link to the statistical software used is https://galton.uchicago.edu/~mcpeek/software/mqls_xm/index.html . the polymorphic count and allele frequency distribution of different snps in arntl gene in cases and controls of the same caste are given in table 1 . the polymorphic count and allele frequency distribution of different snps in arntl gene in cases and controls of different castes are given in table 2 . among the five snps of arntl gene , there was a significant variation in snp rs2279287 between the cases and controls of same caste [ table 1 ] . the allelic frequency of the mutant allele ( g ) was found to be 0.75 in cases . genotyping of the cases for this snp showed that 50% of the cases are homozygous , and the remaining individuals are heterozygous for the mutant allele . none of the cases were found to be wild . in the control group belonging to the same caste , 20% were homozygous , 47% were heterozygous , and 33% were wild type . in the control group of different castes , the other 4 snps ( rs1982350 , rs7126303 , rs969485 , and rs2290035 ) were almost equally distributed both in the cases and controls [ tables 1 and 2 ] . among the 90 subjects ( 30 cases and 30 controls of the same caste and 30 controls of different castes ) , only one individual is wild for all the five snps studied . polymorphic count and allele frequency distribution of different db - single nucleotide polymorphisms in arntl gene in cases and controls of same caste polymorphic count and allele frequency distribution of different db - single nucleotide polymorphisms in arntl gene in cases and controls of different castes snp rs2279287 , which is a significant marker in the family under study is located in the promoter region of arntl gene and hence the polymorphism in this region could affect the clock regulated processes . the controls in this study carried the mutant alleles , and the same finding was reported by other studies . the significant marker rs2279287 in the present study is a part of the most significant haplotype of arntl gene of caucasians similar to this study , association of arntl gene variation with a seasonal pattern in bd has been reported in different populations recently . people with wild - type genotype of snp rs2290035 in arntl gene are associated with less seasonal variation in energy level . about 96% of patients with the mutant genotype of arntl gene have a routine seasonal variation of energy level . the functionality of the arntl protein in terms of homozygous , heterozygous , and wild polymorphic nature is not elucidated still . a fully functional arntl protein is hypothesized to inhibit mania by inactivating dopamine through monoamine oxidase a and thus inhibiting the promanic effects of dopamine and hence any defect in arntl gene ( homozygous / heterozygous ) is expected to promote mania . arntl protein has period - arnt - single - minded domains which regulate biological responses to light and hence mutations in the arntl gene could alter the sensitivity to light which is observed in sad subjects . thus , one can not rule out the essential nature of fully functional arntl gene . similar to previous reports , the present study also identified a potentially functional polymorphism , rs2279287 in arntl gene in an indian family diagnosed with sad . conclusively , we propose that polymorphisms in arntl gene disrupt the circadian rhythms causing sad , and genetic predisposition becomes more deleterious in the presence of adverse environment . this is the first report on arntl gene mutations aggravated by environment associated with sad in indian population .