Background:Serotonin (5-hydroxytryptamine or 5-HT) is a monoamine neurotransmitter synthesized and released by serotonergic neurons in the raphe nuclei of the central nervous system. The physiological function of the serotonergic synapses largely depends on serotoninin reuptake in the synaptic cleft. The serotonin transporter encoded by the solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 (SLC6A4) gene at locus 17q11.2, also known as 5-HT transporter (5-HTT), modulates serotonin balance at the synapsis, thus playing a pivotal role in serotonergic neurotransmission. The 5-HTT-linked polymorphic region (5-HTTLPR) identifies a VNTR polymorphism (14-16 copies of 20-23 imperfect repeat sequences) in the promoter region of SLC6A4 gene that has been suggested to influence SLC6A4 gene expression. Functionally, in comparison to the long (L) allele (16 repeat) the short (S) allele (14 repeat) has been associated with a lower level of transcriptional efficiency of the 5-HTT promoter, leading to reduced expression and 5-HT reuptake [1]. Aims: Identification and functional characterization of two novel 5-HTTLPR alleles that are longer than the common L allele (extra long XL alleles, 18 repeats). An XS variant allele, already described [2], that is shorter by 23 base pairs than the S allele, was also characterized. Materiali: Genomic DNAs were isolated from 4 ml of peripheral blood by means of the salting-out method Metodo: The 5-HTTLPR VNTR was genotyped using PCR as previously described [3]. Each alleles was cloned into a reporter vector and the sequence subsequently verified. Functional analysis was made by reporter gene expression assay both in human placental choriocarcinoma cell line JAR and rat neuronal cell line RN46A Results: Preliminary analysis in JAR cells indicates an appreciable decrease of expression with the XS allele in comparison with both S and L alleles. No significant difference in basal transcriptional activity was observed with both XL alleles when compared with L allele. The production of reporter protein was shown to be promoter dependent by cloning each VNTR into the vector in the reverse orientation. Discussion: The 5HTTLPR polymorphisms seems to influence SLC6A4 gene expression. Conclusions: Whereas our results confirm a functional role for 5-HTTLPR polymorphism on the expression of SLC6A4 protein, further studies are needed to investigate a dose effect for 5-HTTLPR repeats in SLC6A4 gene expression. References 1. Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S, Benjamin J, Müller CR, Hamer DH, Murphy DL. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 1996;274:1527-1531. 2. Frisch A, Finkel B, Michaelovsky E, Sigal M, Laor N, Weizman R. A rare short allele of the serotonin transporter promoter region (5-HTTLPR) found in an aggressive schizophrenic patient of Jewish Libyan origin. Psychiatr Genet 2000;10:179-183. 3. Seripa D, Panza F, D'onofrio G, Paroni G, Bizzarro A, Fontana A, Paris F, Cascavilla L, Copetti M, Masullo C, Pilotto A. The Serotonin Transporter Gene Locus in Late-Life Major Depressive Disorder. Am J Geriatr Psychiatry 2012 (doi 10.1097/JGP.0b013e31823e2caf).
Identification and functional characterization of novel alleles in the promoter region of the serotonin transporter gene.
Sandra Iurescia;Monica Rinaldi;
2012
Abstract
Background:Serotonin (5-hydroxytryptamine or 5-HT) is a monoamine neurotransmitter synthesized and released by serotonergic neurons in the raphe nuclei of the central nervous system. The physiological function of the serotonergic synapses largely depends on serotoninin reuptake in the synaptic cleft. The serotonin transporter encoded by the solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 (SLC6A4) gene at locus 17q11.2, also known as 5-HT transporter (5-HTT), modulates serotonin balance at the synapsis, thus playing a pivotal role in serotonergic neurotransmission. The 5-HTT-linked polymorphic region (5-HTTLPR) identifies a VNTR polymorphism (14-16 copies of 20-23 imperfect repeat sequences) in the promoter region of SLC6A4 gene that has been suggested to influence SLC6A4 gene expression. Functionally, in comparison to the long (L) allele (16 repeat) the short (S) allele (14 repeat) has been associated with a lower level of transcriptional efficiency of the 5-HTT promoter, leading to reduced expression and 5-HT reuptake [1]. Aims: Identification and functional characterization of two novel 5-HTTLPR alleles that are longer than the common L allele (extra long XL alleles, 18 repeats). An XS variant allele, already described [2], that is shorter by 23 base pairs than the S allele, was also characterized. Materiali: Genomic DNAs were isolated from 4 ml of peripheral blood by means of the salting-out method Metodo: The 5-HTTLPR VNTR was genotyped using PCR as previously described [3]. Each alleles was cloned into a reporter vector and the sequence subsequently verified. Functional analysis was made by reporter gene expression assay both in human placental choriocarcinoma cell line JAR and rat neuronal cell line RN46A Results: Preliminary analysis in JAR cells indicates an appreciable decrease of expression with the XS allele in comparison with both S and L alleles. No significant difference in basal transcriptional activity was observed with both XL alleles when compared with L allele. The production of reporter protein was shown to be promoter dependent by cloning each VNTR into the vector in the reverse orientation. Discussion: The 5HTTLPR polymorphisms seems to influence SLC6A4 gene expression. Conclusions: Whereas our results confirm a functional role for 5-HTTLPR polymorphism on the expression of SLC6A4 protein, further studies are needed to investigate a dose effect for 5-HTTLPR repeats in SLC6A4 gene expression. References 1. Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S, Benjamin J, Müller CR, Hamer DH, Murphy DL. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 1996;274:1527-1531. 2. Frisch A, Finkel B, Michaelovsky E, Sigal M, Laor N, Weizman R. A rare short allele of the serotonin transporter promoter region (5-HTTLPR) found in an aggressive schizophrenic patient of Jewish Libyan origin. Psychiatr Genet 2000;10:179-183. 3. Seripa D, Panza F, D'onofrio G, Paroni G, Bizzarro A, Fontana A, Paris F, Cascavilla L, Copetti M, Masullo C, Pilotto A. The Serotonin Transporter Gene Locus in Late-Life Major Depressive Disorder. Am J Geriatr Psychiatry 2012 (doi 10.1097/JGP.0b013e31823e2caf).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
