<p>Supplementary Material</p><p>Article title: Role of the 5-HTTLPR and SNPs promoter polymorphisms on serotonin transporter gene expression: a closer look at genetic architecture and in vitro functional studies of common and uncommon allelic variants</p><p>Journal name: Molecular Neurobiology</p><p>Authors name: Sandra Iurescia1≠, Davide Seripa, Monica Rinaldi1≠</p><p>≠ Corresponding authors</p><p>Affiliation and e-mail address of the corresponding authors: 1Institute of Translational</p><p>Pharmacology (IFT), National Research Council (CNR), via Fosso del Cavaliere 100, 00133 Rome,</p><p>Italy; [email protected] (S. Iurescia); [email protected] (M. Rinaldi)</p><p>Supplementary Material</p><p>Patients </p><p>Patients #2407, #2408, #C.M.8, #1042 and #2406 were recruited in the frame of a previous study investigating the role of the SLC6A4 gene locus in late-life major depressive disorder [1]. Genomic DNA was purified from fresh/frozen blood samples following the salting-out method [2]. </p><p>Genotyping analysis 5-HTTLPR genotypes were determined as previously described [1]. Briefly, genomic DNA samples from patients were amplified by polymerase chain reaction (PCR) by using the forward primer 5’ > ATG CCA GCA CCA GCA CCT AAC CCC TAA TGT > 3’ and the reverse primer 5’ > GGA CCG CAA GGT GGG CGG GA > 3’. Genotype identification was made by running on a 2.5 % (1xTBE) agarose gel electrophoresis (AGE) the PCR products. DNA for sequencing was purified from AGE by using the Illustra GFX PCR DNA and GEL BAND purification kit (GE HealthCare, Life Science, Piscataway, NJ, USA). The same forward and the reverse primers were used to set-up the sequence reaction by using the BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies Ltd, Paisley, UK). The Applied Biosystems 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) was used to analyze reaction products. Patients 5-HTTLPR genotyping resulted in #2407: genotype L/L; #2408: genotype S/S; #C.M.8: genotype L/XL1; #1042: genotype L/XL2; #2406: genotype S/XS1.</p><p>Reporter plasmids construction Genomic DNA samples extracted from the individuals carrying alleles S, L, XS1, XL1, XL2 were used as templates to amplify the corresponding 5-HTTLPR fragments. The primer sequences were the following: forward primer 5’-GGCGTTGCCGCTCTGAATGC-3’ and reverse primer 5’- GAGGGACTGAGCTGGACAACCAC-3’. The PCR products contained a 5-HTTLPR region larger than that obtained in the genotyping amplification and were sequenced in both directions by means of the forward and reverse primers used in the amplification reactions. The 5-HTTLPR</p><p>® fragments (S, L, XS1, XL1 and XL2) were subsequently subcloned into the Gene BLAzer-TOPO TA reporter vector (Invitrogen by Life Technologies, Monza, Italy) upstream of the -lactamase gene and transformed into One Shot TOP10 competent Escherichia coli cells according to the manufacturer’s instructions. Recombinant clones were screened for the presence of the 5-HTTLPR insert in the correct orientation using the restriction enzyme PstI. DNA constructs were subsequently verified by sequencing using the T7 primers specific to the pGene BLAzer-TOPO®. Additionally, reporter constructs carrying each insert in the reverse orientation with respect to lactamase gene were used to control for transcriptional effects due to consensus 5-HTTLPR sequences. </p><p>Sequence alignment tools Alignment of the 5-HTTLPR nucleic acid sequences was performed by MultiAlin software available at multialin.toulouse.inra.fr/multialin [3] following the Nakamura approach. L and S 5-HTTLPR variants were compared with the reference sequence (i.e. the L allele GenBank AF117826) through a pairwise alignment. XS1, XL1 and XL2 variants were compared with the reference sequence and with the S variant to detect the missing and the extra repeats, respectively. A multiple sequence alignment from all 5-HTTLPR variants was further performed. Individual elements were manually matched with repeat sequences described so far by Nakamura and by Murdoch to identify composition and arrangement of repeat elements for each 5-HTTLPR allelic variants. Thus, the patient #1042 carries a new XL allele, the XL2, containing a novel repeat unit (’) in the 7th position of the VNTR structure. (Acc. Number KM054529, available in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/).</p><p>Transfection and gene reporter assay Plasmid DNA with a suitable quality for transfection was made using a Plasmid DNA Midi preparation kit (Qiagen S.r.l. Milan, Italy). The 5-HTTLPR- lactamase reporter constructs were transfected into human placental choriocarcinoma cell line JAR kindly provided by Dr. Di Simone N., Università Cattolica del Sacro Cuore, Rome, Italy. The JAR cells were cultured in complete Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) with appropriate supplements. Transient transfections were done using 4,5 x 105 cells/60mm plate, 8 µg of reporter plasmid and Lipofectamine® 2000 (Invitrogen by Life Technologies, Monza, Italy) according to the manufacturer's guidelines. -lactamase activity was determined 24 hours after cells transfection using the In Vitro GeneBLAzer™ Detection kit (Invitrogen by Life Technologies, Monza, Italy). Transfection of JAR cells with the human ubiquitin promoter construct was routinely performed as positive control. Relative -lactamase activity was determined by dividing the normalized activity of cells transfected by each reporter plasmid by the basal activity of cells transfected with 5- HTTLPR S allele plasmid. Statistical significance was measured by Student’s t-test, assuming a value of significance  < 0.005.</p><p>References</p><p>1. Seripa D, Panza F, D'Onofrio G, Paroni G, Bizzarro A, Fontana A, Paris F, Cascavilla L, Copetti M, Masullo C, Pilotto A (2013) The serotonin transporter gene locus in late-life major depressive disorder. Am J Geriatr Psychiatry 21, 67-77 2. Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16, 1215 3. Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16, 10881-10890</p>