Arylalkylamine N-acetyltransferase Gene Research Articles

Functional Characterization of Arylalkylamine N-Acetyltransferase, a Pivotal Gene in Antioxidant Melatonin Biosynthesis from Chlamydomonas reinhardtii.

Arylalkylamine N-acetyltransferase (AANAT) is a pivotal enzyme in melatonin biosynthesis that catalyzes the conversion of serotonin to N-acetylserotonin. Homologs of animal AANAT genes are present in animals, but not in plants. An AANAT homolog was found in Chlamydomonas reinhardtii, but not other green algae. The characteristics of C. reinhardtii AANAT (CrAANAT) are unclear. Here, full-length CrAANAT was chemically synthesized and expressed in Escherichia coli. Recombinant CrAANAT exhibited AANAT activity with a Km of 247 μM and Vmax of 325 pmol/min/mg protein with serotonin as the substrate. CrAANAT was localized to the cytoplasm in tobacco leaf cells. Transgenic rice plants overexpressing CrAANAT (CrAANAT-OE) exhibited increased melatonin production. CrAANAT-OE plants showed a longer seed length and larger second leaf angle than wild-type plants, indicative of the involvement of brassinosteroids (BRs). As expected, BR biosynthesis- and signaling-related genes such as D2, DWARF4, DWARF11, and BZR1 were upregulated in CrAANAT-OE plants. Therefore, an increased endogenous melatonin level by ectopic overexpression of CrAANAT seems to be closely associated with BR biosynthesis, thereby influencing seed size.

Suppression of MT1 and Melatonin Treatment Improves Liver Phenotypes in Mdr2‐/‐ mice

Background & AimPrimary Sclerosing Cholangitis (PSC) is a cholestatic liver disease characterized by hepatic fibrosis and portal inflammation. Melatonin is synthesized by arylalkylamine N‐acetyltransferase (AANAT) in the pineal gland, as well as extrapineal sites such as cholangiocytes. We previously found that: (i) the MT1 receptor is primarily expressed in cholangiocytes with low and absent expression in hepatic stellate cells and hepatocytes, respectively; (ii) melatonin reduces biliary proliferation via MT1 receptor signaling; and (iii) melatonin treatment for 1 wk decreases biliary proliferation and liver fibrosis in bile duct ligated rats by downregulation of MT1 and clock genes (PER1, CRY1, CLOCK and BMAL1). We aimed to evaluate the beneficial effects of long‐term melatonin treatment and MT1 signaling on biliary phenotypes, liver fibrosis and portal inflammation in Mdr2‐/‐ mice (a model of PSC).MethodsMale FVB/NJ and Mdr2‐/‐ mice had access ad libitum to drinking water with/without melatonin for 3 months. We evaluated: (i) ductular reaction (DR) by immunohistochemistry (IHC) for CK19, (ii) liver fibrosis by Sirius Red staining and (iii) portal inflammation by IHC for F4/80. MT1, AANAT, and clock genes immunoreactivity was evaluated by immunofluorescence (IF) co‐stained with CK19 and qPCR in isolated cholangiocytes. Male C3H‐Hej (WT for MT1‐/‐), FVB/NJ (WT for Mdr2‐/‐), MT1‐/‐, Mdr2‐/‐ mice and MT1‐/‐/Mdr2‐/‐ (DKO) mice were euthanized at 12 wk of age. We analyzed: (i) MT1 expression by IF and mRNA expression in total liver by qPCR to validate our model; (ii) liver damage by H&E, DR by IHC for CK19 and liver fibrosis by Sirius Red staining and (iii) portal inflammation by IHC for F4/80 and expression of the inflammatory markers by qPCR in total liver.ResultsLong‐term melatonin treatment reduces DR, liver fibrosis and portal inflammation in Mdr2‐/‐mice. Prolonged administration of melatonin in Mdr2‐/‐mice improves liver phenotype by decreasing the immunoreactivity of MT1, AANAT and clock genes in Mdr2‐/‐ mice, which suggests the chronobiotic action of melatonin on biliary circadian rhythm. DKO mice have no MT1 expression and display ameliorated liver phenotype compared to Mdr2‐/‐ mice.ConclusionWe demonstrated that chronic melatonin treatment improves liver histology and restores the biliary circadian rhythm by interaction with MT1. Suppression of MT1 receptor in Mdr2‐/‐mice ameliorates biliary/liver phenotypes through changes in clock genes and AANAT. Restoration of the circadian rhythm by modulation of melatonin/MT1 signaling may be key for the management of cholangiopathies.

Gene Duplication and Loss of AANAT in Mammals Driven by Rhythmic Adaptations.

Arylalkylamine N-acetyltransferase (AANAT) plays a crucial role in synchronizing internal biological functions to circadian and circannual changes. Generally speaking, only one copy of AANAT gene has been found in mammals, however, three independent duplications of this gene were detected in several cetartiodactyl lineages (i.e., Suidae, Hippopotamidae, and Pecora), which originated in the middle Eocene, a geological period characterized with the increased climate seasonality. Lineage-specific expansions of AANAT and the associated functional enhancement in these lineages strongly suggest an improvement in regulating photoperiodic response to adapt to seasonal climate changes. In contrast, independent inactivating mutations or deletions of the AANAT locus were identified in the four pineal-deficient clades (cetaceans, sirenians, xenarthrans, and pangolins). Loss of AANAT function in cetaceans and sirenians could disrupt the sleep-promoting effects of pineal melatonin, which might contribute to increasing wakefulness, adapting these clades to underwater sleep. The absence of AANAT and pineal glands in xenarthrans and pangolins may be associated with their body temperature maintenance. The present work demonstrates a far more complex and intriguing evolutionary pattern and functional diversity of mammalian AANAT genes than previously thought and provides further evidence for understanding AANAT evolution as driven by rhythmic adaptations in mammals.

Association of Melatonin Pathway Gene's Single-Nucleotide Polymorphisms with Systemic Lupus Erythematosus in a Chinese Population.

Objectives This study was to investigate the association of melatonin (MTN) pathway gene's single-nucleotide polymorphisms (SNPs) with susceptibility to systemic lupus erythematosus (SLE). Methods We recruited 495 SLE patients and 493 healthy controls, 11 tag SNPs in MTN receptor 1a (MTNR1a), MTNR1b, and arylalkylamine N-acetyltransferase (AANAT) genes were genotyped and analyzed. Serum MTN concentration was determined by enzyme-linked immunosorbent assay (ELISA) kits. Results Two SNPs of AANAT gene (rs8150 and rs3760138) associated with the risk of SLE; CC carriers of rs8150 had a lower risk as compared to GG (OR = 0.537, 95% CI: 0.361, 0.799), whereas GG carrier in rs3760138 had an increased risk (OR = 1.823, 95% CI: 1.154, 2.880) compared to TT. However, we did not find any genetic association between the other nine SNPs with SLE risk. Case-only analysis showed associations of rs2165667 and rs1562444 with arthritis, rs10830962 with malar rash, rs3760138 with immunological abnormality, and rs8150 with hematological abnormality. Furthermore, a significant difference between plasma MTN levels with different genotypes of rs1562444 was observed. Haplotype analyses revealed that haplotype of CCTAT, CTAGT, and GGG was significantly associated with the increased risk in SLE susceptibility, but TCTAT and CTG appeared to be a protective haplotype. Conclusions The present study supported the genetic association of MTN pathway genes with SLE susceptibility and specific clinical manifestations, suggesting the potential role of MTN pathway genes in the pathogenesis and development of SLE.

Rumen-Protected 5-Hydroxytryptophan Improves Sheep Melatonin Synthesis in the Pineal Gland and Intestinal Tract.

BackgroundBased on the extensive biological effects of melatonin (MLT), it is beneficial to increase the MLT content in the bodies of animals at a specific physiological stage. This study was conducted to investigate the effect of a diet supplemented with rumen-protected (RP) 5-hydroxytryptophan (5-HTP) on the pineal gland and intestinal tract MLT synthesis of sheep.Material/MethodsEighteen Kazakh sheep were assigned randomly to 3 diet groups: control group (CT, corn-soybean meal basal diet), CT+111 group (111 mg/kg BW RP 5-HTP), and CT+222 group (222 mg/kg BW RP 5-HTP). The gene expressions of aromatic amino acid decarboxylase (AADC), arylalkylamine N-acetyltransferase (AA-NAT), hydroxyindole-O-methyltransferase (HIOMT), monoamine oxidase A (MAOA), and the intermediates of MLT synthesis were observed from the pineal gland and intestinal tract by the reverse transcription (RT)-PCR method. The 5-HTP, 5-HT, N-acetylserotonin (NAS), MLT, and 5-hydroxyindole acetic acid (5-HIAA) contents in the pineal gland and intestinal tract were analyzed by ultra-high-performance liquid chromatography–tandem mass spectrometry.ResultsThe study showed that the pineal gland HIOMT expression (P<0.05), MLT (P<0.05) and 5-HIAA (P<0.05) levels in the 222 mg/kg group significantly increased compared to those in the CT and CT+111 mg/kg groups. In addition, the AADC (P<0.01) and AA-NAT (P<0.05) gene expression levels in the duodenum and jejunum were increased by the supplementation of RP 5-HTP.ConclusionsRumen-protected 5-hydroxytryptophan promoted melatonin synthesis in the pineal gland and intestinal tract during the natural light period.

Genetic polymorphism in melatonin receptor 1A and arylalkylamine N-acetyltransferase and its impact on seasonal reproduction in Egyptian sheep breeds.

This study was carried out to detect polymorphisms in the melatonin receptor 1A (MTNR1A) and arylalkylamine N-acetyltransferase (AA-NAT) genes and their association with reproductive traits. Blood samples of 126 animals from three Egyptian sheep breeds were collected. DNA was extracted and subjected to PCR restriction fragment length polymorphism (RFLP) analysis using the RsaI and SmaI enzymes. Two alleles (C and T) and three genotypes (CC, CT and TT) for MTNR1A and for AA-NAT (A and G; GG, GA and AA) were detected. The alleles C and A and the genotypes CT and GA showed the highest frequencies for the MTNR1A and AA-NAT genes, respectively. Association analysis of the MTNR1A single nucleotide polymorphism (SNP) with ewe reproductive traits revealed significant associations in the Ossimi and Rahmani breeds with age at first lambing, and the C allele seemed to be the favorable allele. The results for the AA-NAT SNP demonstrated significant correlations in Ossimi with age at first lambing and litter size and in Rahmani with lambing interval; the G allele seemed to be the desirable allele. In the first conception season, ewes carrying CT exhibited a significantly lower age of first lambing in the unfavorable season. Additionally, GG ewes exhibited a significantly lower age of first lambing in the early favorable season, followed by the unfavorable season. To the best of our knowledge, this is the first study of these associations in Egyptian sheep breeds. In conclusion, the polymorphisms revealed in this study could be used as genetic markers to improve reproductive efficiency during the unfavorable season, and the obtained desirable genotypes could be considered in new genetic selection schemes.

A new arylalkylamine N-acetyltransferase in silkworm (Bombyx mori) affects integument pigmentation.

Dopamine is a precursor for melanin synthesis. Arylalkylamine N-acetyltransferase (AANAT) is involved in the melatonin formation in insects because it could catalyze the transformation from dopamine to dopamine-N-acetyldopamine. In this study, we identified a new AANAT gene in the silkworm (Bombyx mori) and assessed its role in the silkworm. The cDNA of this gene encodes 233 amino acids that shares 57 % amino acid identity with the Bm-iAANAT protein. We thus refer to this gene as Bm-iAANAT2. To investigate the role of Bm-iAANAT2, we constructed a transgenic interference system using a 3xp3 promoter to suppress the expression of Bm-iAANAT2 in the silkworm. We observed that melanin deposition occurs in the head and integument in transgenic lines. To verify the melanism pattern, dopamine content and the enzyme activity of AANAT were determined by high-performance liquid chromatography (HPLC). We found that an increase in dopamine levels affects melanism patterns on the heads of transgenic B. mori. A reduction in the enzyme activity of AANAT leads to changes in dopamine levels. We analyzed the expression of the Bm-iAANAT2 genes by qPCR and found that the expression of Bm-iAANAT2 gene is significantly lower in transgenic lines. Our results lead us to conclude that Bm-iAANAT2 is a new arylalkylamine N-acetyltransferase gene in the silkworm and is involved in the metabolism of the dopamine to avoid the generation of melanin.

Identification, characterization and analysis of expression of genes encoding arylalkylamine N‐acetyltransferases in the pea aphid Acyrthosiphon pisum

Most organisms exhibit some kind of rhythmicity in their behaviour and/or physiology as an adaptation to the cyclical movements of the Earth. In addition to circadian rhythms, many organisms have an annual rhythmicity in certain activities, such as reproduction, migration or induction of diapause. Current knowledge of the molecular basis controlling seasonal rhythmicity, especially in insects, is scarce. One element that seems to play an essential role in the maintenance of both circadian and seasonal rhythms in vertebrates is the hormone melatonin. In vertebrates, the limiting enzyme in its synthesis is the arylalkylamine N-acetyltransferase (AANAT). Melatonin is also present in insects but the precise biochemical pathway and the enzymes involved in its synthesis are unknown. Insects possess phylogenetically distant arylalkylamine N-acetyltransferases but their involvement in melatonin synthesis still needs to be fully demonstrated. Aphids have a seasonally rhythmical life cycle, reproducing parthenogenetically by viviparity in favourable seasons but, in unfavourable seasons, they produce a single generation of sexual individuals. The length of the photoperiod is the main environmental factor that controls the mode of reproduction in aphids. Taking advantage of the availability of the genome of the aphid Acyrthosiphon pisum, we searched for genes encoding aphid arylalkylamine N-acetyltransferase homologues that could be candidates for participation in seasonal rhythmicity. We identified four AANAT genes, of which at least two (Ap-AANAT1 and Ap-AANAT3) showed highly significant variation in transcription levels depending on the photoperiod conditions. These results are discussed in the context of how seasonality can be controlled in aphids.

Association between AA-NAT gene polymorphism and reproductive performance in sheep

Arylalkylamine N-acetyltransferase (AA-NAT) is critical enzyme in Melatonin (MLT) biosynthesis for MLT regulating the animal seasonal breeding. In this study, DNA sequencing methods were applied to detect the polymorphisms of the AA-NAT gene in 179 Chinese sheep belonging to two non-seasonal reproduction breeds and two seasonal reproduction breeds. One mutation at exon 3 (NM_001009461:c.486A > G) was firstly described at the sheep AA-NAT locus. Hence, we described the Sma I PCR-RFLP method for detecting EX3 486A > G mutation, frequencies of the AA-NAT-G allele varied from 0.871 to 0.908 in two non-seasonal reproduction breeds and 0.517 to 0.578 in two seasonal reproduction breeds. The associations of Sma I polymorphism with estrus traits was analyzed in non-seasonal reproduction breeds sheep and seasonal reproduction breeds sheep, the significant statistical results were found between them, the GG genotype frequencies was higher in non-seasonal reproduction breeds (p G mutation could facilitate association analysis and serve as a genetic marker for Chinese sheep breeding and genetics.

Melatonin pathway genes and breast cancer risk among Chinese women

Previous studies suggest that melatonin may act on cancer growth through a variety of mechanisms, most notably by direct anti-proliferative effects on breast cancer cells and via interactions with the estrogen pathway. Three genes are largely responsible for mediating the downstream effects of melatonin: melatonin receptors 1a and 1b (MTNR1a and MTNR1b), and arylalkylamine N-acetyltransferase (AANAT). It is hypothesized that genetic variation in these genes may lead to altered protein production or function. To address this question, we conducted a comprehensive evaluation of the association between common single nucleotide polymorphisms (SNPs) in the MTNR1a, MTNR1b, and AANAT genes and breast cancer risk among 2,073 cases and 2,083 controls, using a two-stage analysis of genome-wide association data among women of the Shanghai Breast Cancer Study. Results demonstrate two SNPs were consistently associated with breast cancer risk across both study stages. Compared with MTNR1b rs10765576 major allele carriers (GG or GA), a decreased risk of breast cancer was associated with the AA genotype (OR = 0.78, 95% CI = 0.62-0.97, P = 0.0281). Although no overall association was seen in the combined analysis, the effect of MTNR1a rs7665392 was found to vary by menopausal status (P-value for interaction = 0.001). Premenopausal women with the GG genotype were at increased risk for breast cancer compared with major allele carriers (TT or TG) (OR = 1.57, 95% CI = 1.07-2.31, P = 0.020), while postmenopausal women were at decreased risk (OR = 0.58, 95% 0.36-0.95, P = 0.030). No significant breast cancer associations were found for variants in the AANAT gene. These results suggest that common genetic variation in the MTNR1a and 1b genes may contribute to breast cancer susceptibility, and that associations may vary by menopausal status. Given that multiple variants in high linkage disequilibrium with MTNR1b rs76653292 have been associated with altered function or expression of insulin and glucose family members, further research may focus on clarifying this relationship.

Production of transgenic Nicotiana sylvestris plants expressing melatonin synthetase genes and their effect on UV-B-induced DNA damage

We have obtained transgenic plants of Nicotiana sylvestris expressing an arylalkylamine N-acetyltransferase (AANAT) gene and a hydroxyindole-O-methyltransferase (HIOMT) gene using Agrobacterium tumefaciens-mediated transformation. Both AANAT and HIOMT are key enzymes in melatonin synthesis. Transgenic plants of N. sylvestris were characterized by polymerase chain reaction (PCR) and reverse transcription PCR analyses. The content of melatonin was significantly higher in transgenic plants than in nontransgenic plants. The highest melatonin content of transgenic plant leaves reached 50.4 μg/g dry weight, while almost no melatonin could be detected in the nontransformed plants. To investigate effects of the expression of the AANAT and HIOMT genes on melatonin function in plants, isolated protoplasts of N. sylvestris were exposed to ultraviolet (UV)-B radiation for different durations. DNA damage was evaluated by single-cell gel electrophoresis, which showed that the TailDNA percentage value in transgenic protoplasts was lower than that in the nontransformed protoplasts in the range of 0–30 s of UV-B radiation. DNA damage caused by UV-B was therefore reduced in transgenic N. sylvestris plants.

Ethanol consumption and pineal melatonin daily profile in rats

It is well known that melatonin participates in the regulation of many important physiological functions such as sleep-wakefulness cycle, motor coordination and neural plasticity, and cognition. However, as there are contradictory results regarding the melatonin production diurnal profile under alcohol consumption, the aim of this paper was to study the phenomenology and mechanisms of the putative modifications on the daily profile of melatonin production in rats submitted to chronic alcohol intake. The present results show that rats receiving 10% ethanol in drinking water for 35 days display an altered daily profile of melatonin production, with a phase delay and a reduction in the nocturnal peak. This can be partially explained by a loss of the daily rhythm and the 25% reduction in tryptophan hydroxylase activity and, mainly, by a phase delay in arylalkylamine N-acetyltransferase gene expression and a 70% reduction in its peak activity. Upstream in the melatonin synthesis pathway, the results showed that noradrenergic signaling is impaired as well, with a decrease in β1 and α1 adrenergic receptors' mRNA contents and in vitro sustained loss of noradrenergic-stimulated melatonin production by glands from alcohol-treated rats. Together, these results confirm the alterations in the daily melatonin profile of alcoholic rats and suggest the possible mechanisms for the observed melatonin synthesis modification.

Disruption of an N-acetyltransferase gene in the silkworm reveals a novel role in pigmentation

The pigmentation of insects has served as an excellent model for the study of morphological trait evolution and developmental biology. The melanism (mln) mutant of the silkworm Bombyx mori is notable for its strong black coloration, phenotypic differences between larval and adult stages, and its widespread use in strain selection. Here, we report the genetic and molecular bases for the formation of the mln morphological trait. Fine mapping revealed that an arylalkylamine N-acetyltransferase (AANAT) gene co-segregates with the black coloration patterns. Coding sequence variations and expression profiles of AANAT are also associated with the melanic phenotypes. A 126 bp deletion in the mln genome causes two alternatively spliced transcripts with premature terminations. An enzymatic assay demonstrated the absolute loss of AANAT activity in the mutant proteins. We also performed RNA interference of AANAT in wild-type pupae and observed a significant proportion of adults with ectopic black coloration. These findings indicate that functional deletion of this AANAT gene accounts for the mln mutation in silkworm. AANAT is also involved in a parallel melanin synthesis pathway in which ebony plays a role, whereas no pigmentation defect has been reported in the Drosophila model or in other insects to date. To the best of our knowledge, the mln mutation is the first characterized mutant phenotype of insects with AANAT, and this result contributes to our understanding of dopamine metabolism and melanin pattern polymorphisms.

Disruption of an N-acetyltransferase gene in the silkworm reveals a novel role in pigmentation

The pigmentation of insects has served as an excellent model for the study of morphological trait evolution and developmental biology. The melanism (mln) mutant of the silkworm Bombyx mori is notable for its strong black coloration, phenotypic differences between larval and adult stages, and its widespread use in strain selection. Here, we report the genetic and molecular bases for the formation of the mln morphological trait. Fine mapping revealed that an arylalkylamine N-acetyltransferase (AANAT) gene co-segregates with the black coloration patterns. Coding sequence variations and expression profiles of AANAT are also associated with the melanic phenotypes. A 126 bp deletion in the mln genome causes two alternatively spliced transcripts with premature terminations. An enzymatic assay demonstrated the absolute loss of AANAT activity in the mutant proteins. We also performed RNA interference of AANAT in wild-type pupae and observed a significant proportion of adults with ectopic black coloration. These findings indicate that functional deletion of this AANAT gene accounts for the mln mutation in silkworm. AANAT is also involved in a parallel melanin synthesis pathway in which ebony plays a role, whereas no pigmentation defect has been reported in the Drosophila model or in other insects to date. To the best of our knowledge, the mln mutation is the first characterized mutant phenotype of insects with AANAT, and this result contributes to our understanding of dopamine metabolism and melanin pattern polymorphisms.

Resequencing and association analysis of arylalkylamine N-acetyltransferase (AANAT) gene and its contribution to major depression susceptibility

Circadian rhythms disruptions, including abnormalities of circadian phase position and melatonin secretion, have been described in major depression (MD). Arylalkylamine N-acetyltransferase (AANAT) is a key enzyme of the melatonin pathway involved in circadian oscillations of melatonin levels. We assessed the contribution of AANAT gene variability to susceptibility to MD considering common and rare genetic variations through a sequential sequencing and single nucleotide polymorphism (SNP)-based genotyping approach in a sample of 445 unrelated patients with MD (257 unipolar MD, 188 bipolar depression) and 440 community-based screened control subjects. We identified 17 sequence changes, thirteen of which represented novel sequence variations. We did not observe an over-representation of patients carrying rare variants compared with the healthy controls. Common variants (MAF > 2%) were included in a case-control association analysis that showed significant association after multiple testing correction of two SNPs located in the promoter region of AANAT with MD: rs3760138 (P = 0.00006) and rs4238989 (P = 0.005). Multimarker analysis found significant associations between two three-marker protective haplotypes and a susceptibility three-marker haplotype containing the rare alleles of rs3760138-rs4238989-rs8150 and MD. We present evidence of the association of genetic variability in the AANAT gene with susceptibility to MD. Our results support the hypothesis that the melatonin-signaling pathway and circadian clock mechanisms may contribute to the pathophysiology of MD.

Chronic fluoxetine treatment increases daytime melatonin synthesis in the rodent

Circadian rhythm disturbances can occur as part of the clinical symptoms of major depressive disorder and have been found to resolve with antidepressant therapy. The pineal gland is relevant to circadian rhythms as it secretes the hormone melatonin following activation of the cyclic adenosine monophosphate (cAMP) signaling cascade and of arylalkylamine N-acetyltransferase (AA-NAT), the rate-limiting enzyme for its synthesis. Cyclic AMP is synthesized by adenylate cyclases (AC) and degraded by phosphodiesterases (PDEs). Little is known about the contribution of the PDE system to antidepressant-induced alterations in pineal cAMP signaling and melatonin synthesis. In the present study we used enzyme immunoassay to measure plasma melatonin levels and pineal cAMP levels and as well as quantitative real-time polymerase chain reaction to measure pineal expression of PDE, AC, and AA-NAT genes in rats chronically treated with the prototypic antidepressant fluoxetine. We found elevated melatonin synthesis with increased pineal AA-NAT gene expression and daytime plasma melatonin levels and downregulated cAMP signaling with increased PDE and unchanged AC pineal gene expression, and decreased content of pineal cAMP. We conclude that chronic fluoxetine treatment increases daytime plasma melatonin and pineal AA-NAT gene expression despite downregulated pineal cAMP signaling in the rodent.

Association Study of Tryptophan Hydroxylase 1 and Arylalkylamine N-Acetyltransferase Polymorphisms With Adolescent Idiopathic Scoliosis in Han Chinese

A genetic association study of tryptophan hydroxylase 1 gene (TPH1) and arylalkylamine N-acetyltransferase gene(AANAT) with adolescent idiopathic scoliosis (AIS) in Han Chinese. To access whether TPH1 and AANAT polymorphisms are associated with the predisposition, gender, and/or severity of AIS. Studies have shown that AIS is a multifactorial inheritance disease, but the etiology is still unknown. In addition, several lines of evidence show that melatonin deficiency is closely associated with AIS, although there are still doubts and debates. Some polymorphisms in TPH1 and AANAT, the genes of 2 critical enzymes involved in melatonin biosynthesis, may contribute to variability of melatonin production in pineal glands. We genotyped 16 reported single nuclear polymorphisms (SNPs) present in TPH1 and AANAT in 103 AIS patients and 108 controls with matched sex and age. The data of 6 SNPs with minor allele frequence (MAF) above 5% were analyzed by the allelic and genotypic association analysis, the genotype-phenotype (gender and Cobb angle) association analysis, and the haplotype analysis. The single SNP analysis showed that rs10488682, located in the promoter region of TPH1, was related with the occurrence of AIS (P < 0.05). No SNP was found to be correlated with gender or Cobb angle. Two makers (rs8176799 and rs2108977) in TPH1 were found to be in strong LD [ D' = 1.0 (95% CI, 0.9-1.0), gamma = 0.501, LOD = 18.93] in the controls. Both global haplotype analysis and individual haplotype analysis showed that there was no haplotype significantly associated with AIS in this LD block. TPH1 polymorphisms were associated with AIS but not with gender and Cobb angle in AIS patients. AANAT polymorphisms were not associated with AIS. These results suggested that TPH1 was an AIS predisposition gene, and there was a close relationship between the dyssynthesis of melatonin and AIS.

Arylalkylamine N-Acetyltransferase (AANAT) Genotype as a Personal Trait in Melatonin Synthesis

The melatonin rhythm is arguably the best marker for the phase of the endogenous “biological clock.” Arylalkylamine N-acetyltransferase (AANAT) is known to catalyze the acetylation of serotonin, a rate-limiting process in melatonin synthesis. Different single-nucleotide polymorphisms (SNPs) in the AANAT gene were identified recently in the Japanese population, and one of the genes was significantly associated with the delayed sleep phase syndrome. Thus, 54 healthy Caucasian males were genotyped to investigate whether these SNPs in the AANAT gene affected melatonin levels. The endogenous melatonin levels were analyzed in saliva under standardized experimental conditions (“constant routines”) by radioimmunoassay. Despite the broad temporal variation of the human nocturnal melatonin profiles, none of the investigated SNPs were found in the AANAT gene in this study. These findings point to ethnic differences with respect to these SNPs, rather than time of day termed “morningness.” In summary, SNPs in the AANAT gene identified thus far cannot explain the observed interindividual differences for nocturnal melatonin profiles in the subjects investigated.

Daily oscillation of gene expression in the retina is phase-advanced with respect to the pineal gland

The photoreceptive retina and the non-photoreceptive pineal gland are components of the circadian and the melatonin forming system in mammals. To contribute to our understanding of the functional integrity of the circadian system and the melatonin forming system we have compared the daily oscillation of the two tissues under various seasonal lighting conditions. For this purpose, the 24-h profiles of the expression of the genes coding for arylalkylamine N-acetyltransferase ( AA-NAT), nerve growth factor inducible gene-A ( NGFI-A), nerve growth factor inducible gene-B ( NGFI-B), retinoic acid related orphan receptor β ( RORβ), dopamine D4 receptor, and period2 ( Per2) have been simultaneously recorded in the retina and the pineal gland of rats under short day (light/dark 8:16) and long day (light/dark 16:8) conditions. We have found that the cyclical patterns of all genes are phase-advanced in the retina, often with a lengthened temporal interval under short day conditions. In both tissues, the AA-NAT gene expression represents an indication of the output of the relevant pacemakers. The temporal phasing in the AA-NAT transcript amount between the retina and the pineal gland is retained under constant darkness suggesting that the intrinsic self-cycling clock of the retina oscillates in a phase-advanced manner with respect to the self-cycling clock in the suprachiasmatic nucleus, which controls the pineal gland. We therefore conclude that daily rhythms in gene expression in the retina are phase-advanced with respect to the pineal gland, and that the same temporal relationship appears to be valid for the self-cycling clocks influencing the tissues.

Daily Profile in Melanopsin Transcripts Depends on Seasonal Lighting Conditions in the Rat Retina

The retinal photopigment melanopsin (Opn4) mediates photoentrainment of the circadian system. In the present study, seasonal regulation of the melanopsin gene was investigated in comparison with the arylalkylamine N-acetyltransferase (AA-NAT) gene as an indicator of retinal pacemaker output. For this purpose, the daily profiles in the amount of melanopsin mRNA and AA-NAT mRNA were monitored under 8 : 16 h light/dark, 12 : 12 h light/dark and 16 : 8 h light/dark photoperiods using real-time polymerase chain reaction analysis. We found that, under all of the lighting regimes, melanopsin and AA-NAT expression oscillated with a peak around dark onset and the middle of the dark phase, respectively. The lighting regime influenced both genes, but in an opposing manner. Under long photoperiods, the duration of peak expression was prolonged for melanopsin, whereas it was shortened for AA-NAT. Under constant darkness, the rhythm of mRNA was abolished for melanopsin, but persisted for AA-NAT whereas, under constant light, the rhythm of mRNA was abolished for both genes. Our findings suggest that, in contrast to the AA-NAT gene, the daily and photoperiod-dependent regulation of the melanopsin gene does not rely on a circadian oscillator but is directly illumination-dependent.