Mutation screening of ASMT, the last enzyme of the melatonin pathway, in a large sample of patients with Intellectual Disability
- Cecile Pagan1, 2, 3,
- Hany Goubran Botros1, 2,
- Karine Poirier3, 4,
- Anne Dumaine5,
- Stéphane Jamain5, 6,
- Sarah Moreno1, 2,
- Arjan de Brouwer7,
- Hilde Van Esch8,
- Richard Delorme1, 2,
- Jean-Marie Launay3, 9,
- Andreas Tzschach10,
- Vera Kalscheuer10,
- Didier Lacombe11,
- Sylvain Briault12, 13,
- Frédéric Laumonnier14, 15, 16,
- Martine Raynaud14, 15, 16, 17,
- Bregje W van Bon7,
- Marjolein H Willemsen7,
- Marion Leboyer5, 6, 18, 19,
- Jamel Chelly3, 4 and
- Thomas Bourgeron1, 2, 20Email author
© Pagan et al; licensee BioMed Central Ltd. 2011
Received: 19 October 2010
Accepted: 20 January 2011
Published: 20 January 2011
Intellectual disability (ID) is frequently associated with sleep disorders. Treatment with melatonin demonstrated efficacy, suggesting that, at least in a subgroup of patients, the endogenous melatonin level may not be sufficient to adequately set the sleep-wake cycles. Mutations in ASMT gene, coding the last enzyme of the melatonin pathway have been reported as a risk factor for autism spectrum disorders (ASD), which are often comorbid with ID. Thus the aim of the study was to ascertain the genetic variability of ASMT in a large cohort of patients with ID and controls.
Here, we sequenced all exons of ASMT in a sample of 361 patients with ID and 440 controls. We then measured the ASMT activity in B lymphoblastoid cell lines (BLCL) of patients with ID carrying an ASMT variant and compared it to controls.
We could identify eleven variations modifying the protein sequence of ASMT (ID only: N13H, N17K, V171M, E288D; controls only: E61Q, D210G, K219R, P243L, C273S, R291Q; ID and controls: L298F) and two deleterious splice site mutations (IVS5+2T>C and IVS7+1G>T) only observed in patients with ID. We then ascertained ASMT activity in B lymphoblastoid cell lines from patients carrying the mutations and showed significantly lower enzyme activity in patients carrying mutations compared to controls (p = 0.004).
We could identify patients with deleterious ASMT mutations as well as decreased ASMT activity. However, this study does not support ASMT as a causative gene for ID since we observed no significant enrichment in the frequency of ASMT variants in ID compared to controls. Nevertheless, given the impact of sleep difficulties in patients with ID, melatonin supplementation might be of great benefit for a subgroup of patients with low melatonin synthesis.
Intellectual disability (ID), is defined as IQ < 70 and is associated with functional deficits in adaptive behavior, such as daily-living skills, social skills and communication. It affects 1-3% of the general population and results from heterogeneous environmental, chromosomal and monogenic causes . Besides the cognitive deficit, patients with ID often present with sleep disorders, which are persistent and a burden for the patients and their families. The most commonly reported disorders are delayed settling to sleep and frequent waking at night, with frequencies ranging from 58 up to 86% [2–4]. Several therapeutic strategies have been considered for treating sleep disturbances in ID. Among them, pharmacological use of melatonin was demonstrated to be efficient in several studies, recently reviewed in a meta-analysis : exogenous melatonin appeared to decrease sleep latency and number of wakes per night, and increase total sleep time in individuals with ID. This positive effect of melatonin treatment could suggest that, in some patients with ID, endogenous melatonin level may not be sufficient to adequately set the sleep-wake cycles.
Melatonin is considered as a major biological signal of day-night rhythms, and thus a major endogenous "Zeitgeber" (time-giver). It is synthesized in the dark in the pineal gland from serotonin, first acetylated by aryl alkylamine N-acetyltransferase (AA-NAT) and then converted into melatonin by acetyl serotonin methyl transferase (ASMT also known as hydroxyindole O-methyltransferase or HIOMT). Besides sleep induction and circadian rhythms regulation, melatonin is also involved in various other physiologic functions, including immune response, antioxydative defense, metabolic regulations and memory [6–8]. Abnormal melatonin synthesis or signaling was reported as a risk factor for diverse medical conditions such as diabetes, circadian and psychiatric disorders [9–14]. Among these, autism spectrum disorders (ASD) - which are also often associated with ID and with sleep disorders - have been associated with low melatonin levels in at least four independent studies [15–18]. Melke et al  showed that melatonin deficit in patients with autism is correlated with low activity of the ASMT enzyme, and, in some patients, associated with mutations in the ASMT gene. This study provided the first insight into a molecular mechanism for melatonin deficit associated with neurodevelopmental disorders.
We hypothesized that patients with ID could carry deleterious ASMT mutations. If this was the case, these mutations might act as risk factors for sleep/circadian disorders and subsequently exacerbate the effect of independent genetic/environmental causes of ID. To address this question, we first screened the ASMT gene for rare variants in 361 patients with ID and 440 controls. For patients carrying ASMT mutations, we then measured the ASMT activity in B lymphoblastoid cell lines (BLCL) and if available provided information on sleep.
In this study, we tested 361 clinically characterized male patients with established or putative X-linked ID, collected by the European XLMR Consortium (France, Belgium, Germany and the Netherlands). This panel included 182 established X-linked ID families characterized by at least two boys affected in two different generations and 113 brother-pair families with two or more affected brothers. Of 66 families, the exact number of affected males is not known, but linkage to the X chromosome was highly suspected. To study the frequency of ASMT mutations, we did not exclude from this cohort 68 previously described families with established X-linked mutations. The majority of the patients were from European ancestry. All samples were obtained after receiving informed consent. CGG expansions for fragile X syndrome, assessed by Southern blot analysis using DNA digested with EcoRI/EagI endonucleases and an StB12-3 probe corresponding to FRAXA, were excluded. Unrelated healthy controls of French origin (n = 220, 155 males, 65 females) were recruited among blood donors in two French university hospitals (Pitié-Salpêtrière and Henri-Mondor hospitals, Paris, France). Unrelated Swedish participants from the general population (n = 220, 142 males, 78 females) were recruited through advertisements. The local research ethics boards reviewed and approved the study. Informed consent was obtained from all participants.
Screening of the ASMTgene for rare variations
DNA was extracted from blood cells by the phenol/chloroform method. All PCRs were performed with Qiagen HotStar Taq kit. Primers and PCR conditions have been described previously . PCR products were sequenced with the BigDye Terminator Cycle Sequencing Kit (V3.1, Applied Biosystems) and then subjected to electrophoresis, using an ABI PRISM genetic analyzer (Applied Biosystems). For all non-synonymous mutations, genotyping was confirmed by sequencing of an independent PCR product. The nomenclature of genetic variations was determined according to reference protein sequence ENSP00000370627 in Ensembl database (345 aa). In silico functional predictions were assessed using PolyPhen (http://genetics.bwh.harvard.edu/pph/) and SIFT (http://sift.jcvi.org/) algorithms.
Measurement of ASMT enzyme activity in BLCL
BLCL were established from EBV-transformed lymphocytes according to standard protocol, and grown at 37°C in RPMI-1640 medium (Life Technologies Inc.) supplemented with undialysed fetal calf serum, 2 mM glutamine, 2,5 mM sodium, 100 mg/mL streptomycin and 100 IU/mL penicillin, under standard conditions. ASMT enzyme activities were determined on BLCL pellets, at least in duplicate, by radioenzymology, as described previously , after lysis with 100 hemolytic units of a purified SH-activated toxin (streplolysin O, generously provided by Prof. J. Alouf, Institut Pasteur, Paris).
Non-synonymous variants in the ASMTgene
ASMT mutations identified in 361 patients with ID and 440 controls.
ID Patients (n = 377)
Controls (n = 440)
Functional prediction (Polyphen/SIFT)
Possibly damaging/affects protein function
IVS5+2T > C
IVS7+1G > T
ID and Controls
Possibly damaging/affects protein function
Probably damaging/affects protein function
Probably damaging/affects protein function
Probably damaging/affects protein function
Clinical observations and ASMT activity in B lymphoblastoid cell line of patients with ID and ASMT mutations.
ASMT activity (pmol/mg prot/30 min)
Other known genetic anomalies
Mild ID. No other abnormalities or autistic features.
IQ:76, hyperkinesis, language delay, attention deficit and impulsivity, no epilepsy, no dysmorphic features.
Moderate ID, spasticity and severe language delay. No sleep-wake anomaly.
Some autistic features; some compulsive behavior. Normal sleep pattern, although sleeps lightly and is easily wakened.
Moderate ID, hyperactivity, attention deficit. No dysmorphic features. No evidence for sleeping problems or autistic features.
Mild ID, epilepsy, dysmorphic features, scoliosis, strabismus, epilepsy, corpus callosum agenesis, subdural hygroma, hypermetry. Normal sleep pattern, although sleeps lightly. Some autistic features, with relatively low expressive communication and interpersonal relations, and compulsive behavior.
Severe ID, a few words. Dysmorphic features, hypotonia. Abnormal EEG, moderately enlarged lateral ventricles. No evidence for autistic features ('friendly behavior').
Severe ID and no speech, however good social and eye contact. Never walked. No evidence for sleeping problems, or autistic features.
Controls (n = 31) median (range)
3.8 (0.2 - 9.5)
Six variants were found only in controls and not in patients, and one variant (L298F) was found both in the ID group and in the control group. One variant, N17K (rs17149149), is mentioned in the SNP database at an allelic frequency of 6.7% in the Han Chinese population. When considering all the identified rare variations, we could not detect an enrichment of mutations in patients with ID compared to the control group (8/361 vs 8/440 or 2.2% vs 1.8%; p = 0.88; OR = 1.25 (0.46-3.46)).
Impact of non-synonymous genetic variations on ASMT enzyme activity
On average, ASMT activity in BLCL of ID patients carrying variants was much lower as compared to control subjects without coding mutations of ASMT (1.4 ± 0.2 pmol/mg protein/30 min and 4.1 ± 0.4 pmol/mg protein/30 min, respectively, Wilcoxon test: p = 0.004). These data suggest that most variants identified in patients, although heterozygous, are associated with a low ASMT activity ex vivo.
Alterations of the melatonin pathway have been suggested as susceptibility factors to developmental disorders, and especially to ASD [15–17, 19–23]. The mechanisms leading to ASMT/melatonin deficit in humans are most likely diverse, including genetic/epigenetic alterations. The impact of melatonin deficit on sleep and on the susceptibility to developmental disorders (such as ASD or ID) also remains unclear. It may involve its role as a circadian synchronizer and sleep inducer, its effects on synaptic plasticity, and/or its antioxidant properties [8, 14, 19, 24, 25]. Melatonin deficit may also alter and/or desynchronize many physiological processes, and indirectly exacerbate other pathological processes.
We could identify predicted deleterious variants in a subgroup of patients with ID, including two deleterious splice site variants of ASMT found only in patients with ID. The splice site mutation in intron 7 (IVS7+1G>T) was never observed before. The splice site mutation in intron 5 (IVS5+2T>C) was previously identified in patients with ASD and reported to be more frequent in patients compared to controls (6/749 vs 1/861; p = 0.04) [17, 20, 22]. In addition, biochemical studies indicated that several of the variants, although present at the heterozygous state, were associated with low ASMT activity and might thus impair melatonin synthesis in vivo. These results were consistent with the biochemical studies performed by Melke et al. on families carrier of the L298F and IVS5+2T>C mutations and presenting with a dramatic decrease in ASMT activity and blood melatonin concentration . Nevertheless, despite these interesting findings, we could not detect ASMT mutation enrichment in patients with ID compared to the controls. Our results were similar to those previously reported in patients with ASD, for whom no significant enrichment in ASMT rare variants was found [17, 20, 22], although melatonin deficit is very frequently associated with this condition [15, 16], and is correlated with low ASMT activity in vivo . Low ASMT activities were also observed in BLCL of some controls subjects who did not carry a coding mutation of ASMT. Low ASMT activity can thus be observed even in the absence of coding mutations. For example, SNPs within the promoter were associated with low ASMT mRNA levels [17, 21].
Several limitations exist in this study. First, the sample of patients with ID was initially collected for the identification of X-linked genes (e.g. families with multiple affected males). Therefore, this population is not representative of the broad diversity of patients with ID and is negatively biased for the identification of mutations in the ASMT gene, located on the pseudo-autosomal region 1 (PAR1) shared by the X and Y chromosomes. Another limitation is the sparse information that we could collect about sleep disorders. Further studies will be required to establish the precise link between ASMT variants, melatonin levels and sleep disorders.
This study does not support ASMT as a causative gene for ID since we observed no significant enrichment in the frequency of ASMT variants in ID compared to controls. Nevertheless, we could identify patients with deleterious ASMT mutations as well as decreased ASMT activity. Given the importance of sleep difficulties in patients with ID, for this subgroup of patients, melatonin supplementation might be beneficial.
Autism Spectrum Disorders
Acetyl-serotonin Methyl Transferase
B Lymbhoblastoid cell lines
Single Nucleotide Polymorphism
We thank the cell bank of Cochin hospital (J Chelly), the Plateforme de Ressource Biologique (B. Ghaleh), the Clinical Investigation Centre 006 (P. Le Corvoisier) of Mondor-Chenevier hospitals, the blood donor centre (J.L. Beaumont and B. Mignen, EFS, Creteil), and the Genetic Unit of the University Hospital of Tours (B Jauffrion) for technical assistance. We thank C. Bouchier and S. Duthoy for the use of sequencing facilities at the Génopole Pasteur, B. Costes and N. Martin at the Genomic platform of the IMRB (M. Gossens). We thank E. Abadie, J. Deshommes and K. Le Dudal for their assistance. This work was supported by the Pasteur Institute, INSERM, Assistance Publique des Hôpitaux de Paris, Agence Nationale pour la Recherche (ANR NEURO2006 - Project Manage_BPAD), EU grant QLG3-CT- 2002-01810 (EURO-MRX), and the RTRS Santé Mentale (Foundation FondaMental).
- Chelly J, Khelfaoui M, Francis F, Cherif B, Bienvenu T: Genetics and pathophysiology of mental retardation. Eur J Hum Genet. 2006, 14: 701-713. 10.1038/sj.ejhg.5201595.View ArticlePubMed
- Didde R, Sigafoos J: A review of the nature and treatment of sleep disorders in individuals with developmental disabilities. Res Dev Disabil. 2001, 22: 255-272. 10.1016/S0891-4222(01)00071-3.View Article
- Quine L: Sleep problems in children with mental handicap. J Ment Defic Res. 1991, 35 (Pt 4): 269-290.PubMed
- Richdale A, Francis A, Gavidia-Payne S, Cotton S: Stress, behaviour, and sleep problems in children with an intellectual disability. J Int Dev Dis. 2000, 25: 147-161. 10.1080/13269780050033562.View Article
- Braam W, Smits MG, Didden R, Korzilius H, Van Geijlswijk IM, Curfs LM: Exogenous melatonin for sleep problems in individuals with intellectual disability: a meta-analysis. Dev Med Child Neurol. 2009, 51: 340-349. 10.1111/j.1469-8749.2008.03244.x.View ArticlePubMed
- Simonneaux V, Ribelayga C: Generation of the melatonin endocrine message in mammals: a review of the complex regulation of melatonin synthesis by norepinephrine, peptides, and other pineal transmitters. Pharmacol Rev. 2003, 55: 325-395. 10.1124/pr.55.2.2.View ArticlePubMed
- Rawashdeh O, de Borsetti NH, Roman G, Cahill GM: Melatonin suppresses nighttime memory formation in zebrafish. Science. 2007, 318: 1144-1146. 10.1126/science.1148564.View ArticlePubMed
- Claustrat B, Brun J, Chazot G: The basic physiology and pathophysiology of melatonin. Sleep Med Rev. 2005, 9: 11-24. 10.1016/j.smrv.2004.08.001.View ArticlePubMed
- Lyssenko V, Nagorny CL, Erdos MR, Wierup N, Jonsson A, Spegel P, Bugliani M, Saxena R, Fex M, Pulizzi N, et al: Common variant in MTNR1B associated with increased risk of type 2 diabetes and impaired early insulin secretion. Nat Genet. 2009, 41: 82-88. 10.1038/ng.288.PubMed CentralView ArticlePubMed
- Bouatia-Naji N, Bonnefond A, Cavalcanti-Proenca C, Sparso T, Holmkvist J, Marchand M, Delplanque J, Lobbens S, Rocheleau G, Durand E, et al: A variant near MTNR1B is associated with increased fasting plasma glucose levels and type 2 diabetes risk. Nat Genet. 2009, 41: 89-94. 10.1038/ng.277.View ArticlePubMed
- Henningsson S, Jonsson L, Ljunggren E, Westberg L, Gillberg C, Rastam M, Anckarsater H, Nygren G, Landen M, Thuresson K, et al: Possible association between the androgen receptor gene and autism spectrum disorder. Psychoneuroendocrinology. 2009, 34: 752-761. 10.1016/j.psyneuen.2008.12.007.View ArticlePubMed
- Arendt J: Importance and relevance of melatonin to human biological rhythms. J Neuroendocrinol. 2003, 15: 427-431. 10.1046/j.1365-2826.2003.00987.x.View ArticlePubMed
- Brzezinski A: Melatonin in humans. N Engl J Med. 1997, 336: 186-195. 10.1056/NEJM199701163360306.View ArticlePubMed
- Barnard AR, Nolan PM: When clocks go bad: neurobehavioural consequences of disrupted circadian timing. PLoS Genet. 2008, 4: e1000040-10.1371/journal.pgen.1000040.PubMed CentralView ArticlePubMed
- Nir I, Meir D, Zilber N, Knobler H, Hadjez J, Lerner Y: Brief report: circadian melatonin, thyroid-stimulating hormone, prolactin, and cortisol levels in serum of young adults with autism. J Autism Dev Disord. 1995, 25: 641-654. 10.1007/BF02178193.View ArticlePubMed
- Tordjman S, Anderson GM, Pichard N, Charbuy H, Touitou Y: Nocturnal excretion of 6-sulphatoxymelatonin in children and adolescents with autistic disorder. Biol Psychiatry. 2005, 57: 134-138. 10.1016/j.biopsych.2004.11.003.View ArticlePubMed
- Melke J, Goubran Botros H, Chaste P, Betancur C, Nygren G, Anckarsater H, Rastam M, Stahlberg O, Gillberg IC, Delorme R, et al: Abnormal melatonin synthesis in autism spectrum disorders. Mol Psychiatry. 2008, 13: 90-98. 10.1038/sj.mp.4002016.PubMed CentralView ArticlePubMed
- Kulman G, Lissoni P, Rovelli F, Roselli MG, Brivio F, Sequeri P: Evidence of pineal endocrine hypofunction in autistic children. Neuroendocrinol Lett. 2000, 21: 31-34.PubMed
- Bourgeron T: The possible interplay of synaptic and clock genes in autism spectrum disorders. Cold Spring Harb Symp Quant Biol. 2007, 72: 645-654. 10.1101/sqb.2007.72.020.View ArticlePubMed
- Toma C, Rossi M, Sousa I, Blasi F, Bacchelli E, Alen R, Vanhala R, Monaco AP, Jarvela I, Maestrini E: Is ASMT a susceptibility gene for autism spectrum disorders? A replication study in European populations. Mol Psychiatry. 2007, 12: 977-979. 10.1038/sj.mp.4002069.View ArticlePubMed
- Galecki P, Szemraj J, Bartosz G, Bienkiewicz M, Galecka E, Florkowski A, Lewinski A, Karbownik-Lewinska M: Single-nucleotide polymorphisms and mRNA expression for melatonin synthesis rate-limiting enzyme in recurrent depressive disorder. J Pineal Res. 2010, 48: 311-317. 10.1111/j.1600-079X.2010.00754.x.View ArticlePubMed
- Jonsson L, Ljunggren E, Bremer A, Pedersen C, Landen M, Thuresson K, Giacobini M, Melke J: Mutation screening of melatonin-related genes in patients with autism spectrum disorders. BMC Med Genomics. 2010, 3: 10-10.1186/1755-8794-3-10.PubMed CentralView ArticlePubMed
- Chaste P, Clement N, Mercati O, Guillaume JL, Delorme R, Botros HG, Pagan C, Perivier S, Scheid I, Nygren G, et al: Identification of pathway-biased and deleterious melatonin receptor mutants in autism spectrum disorders and in the general population. PLoS One. 2010, 5: e11495-10.1371/journal.pone.0011495.PubMed CentralView ArticlePubMed
- El-Sherif Y, Tesoriero J, Hogan MV, Wieraszko A: Melatonin regulates neuronal plasticity in the hippocampus. J Neurosci Res. 2003, 72: 454-460. 10.1002/jnr.10605.View ArticlePubMed
- Wan Q, Man HY, Liu F, Braunton J, Niznik HB, Pang SF, Brown GM, Wang YT: Differential modulation of GABAA receptor function by Mel1a and Mel1b receptors. Nat Neurosci. 1999, 2: 401-403. 10.1038/8062.View ArticlePubMed
- Burgess HJ, Fogg LF: Individual differences in the amount and timing of salivary melatonin secretion. PLoS One. 2008, 3: e3055-10.1371/journal.pone.0003055.PubMed CentralView ArticlePubMed
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/17/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.