Abnormal melatonin synthesis in autism spectrum disorders

Abstract

Melatonin is produced in the dark by the pineal gland and is a key regulator of circadian and seasonal rhythms. A low melatonin level has been reported in individuals with autism spectrum disorders (ASD), but the underlying cause of this deficit was unknown. The ASMT gene, encoding the last enzyme of melatonin synthesis, is located on the pseudo-autosomal region 1 of the sex chromosomes, deleted in several individuals with ASD. In this study, we sequenced all ASMT exons and promoters in individuals with ASD (n=250) and compared the allelic frequencies with controls (n=255). Non-conservative variations of ASMT were identified, including a splicing mutation present in two families with ASD, but not in controls. Two polymorphisms located in the promoter (rs4446909 and rs5989681) were more frequent in ASD compared to controls (P=0.0006) and were associated with a dramatic decrease in ASMT transcripts in blood cell lines (P=2 x 10(-10)). Biochemical analyses performed on blood platelets and/or cultured cells revealed a highly significant decrease in ASMT activity (P=2 x 10(-12)) and melatonin level (P=3 x 10(-11)) in individuals with ASD. These results indicate that a low melatonin level, caused by a primary deficit in ASMT activity, is a risk factor for ASD. They also support ASMT as a susceptibility gene for ASD and highlight the crucial role of melatonin in human cognition and behavior.

Figure 1

Non-synonymous ASMT variations in autism spectrum disorders (ASD) families. (a) Pedigree structure of the families carrying the splice–site mutation IVS5+2T>C; reverse transcriptase-polymerase chain reaction (RT-PCR) amplifying exons 4 to 6 of the ASMT cDNA from B lymphoblastoid cell lines of the ASD 1 proband carrying the splice– site mutation IVS5+2T > C (lane 1 +RT, lane 2 −RT) and a control (lane 3 +RT, lane 4 −RT). The insertion (+Ins) of 31 bp in the ASMT transcript originates from a cryptic donor splice–site downstream from exon 5. This insertion should lead to the additional sequence indicated in red and to a frame shift (characters in italics), causing premature truncation of the protein, lacking the methyl-transferase domain. Wt, wild-type. (b) Pedigree structure of the families carrying rare non-synonymous ASMT variations and conservation of the variant amino-acid in different species. Color codes in the pedigrees: autism with mild (orange) or severe (red) mental retardation, Asperger syndrome or high-functioning autism (yellow), attention-deficit/hyperactivity disorder ADHD (light blue) and depression (pink). The proband ASD 3 fulfilled diagnostic criteria for both high functioning autism and ADHD. The proband ASD 7 fulfilled diagnostic criteria for both Asperger syndrome and ADHD. The asterisk and the dot indicate the presence of the mutation and the absence of a DNA sample for analysis, respectively.

Figure 2

Impact of the ASMT mutations on enzyme activity and melatonin concentration. (a) ASMT activity, measured in platelets of the members of families autism spectrum disorder (ASD) 1 and ASD 6 carrying the splice–site IVS5+2T>C and the L326F ASMT mutations, respectively. (b) Blood melatonin concentration in the same individuals. (c) Nocturnal melatonin profile of family ASD 1. The proband (male, 24 years old) and his mother (53 years old) are heterozygous (m/+) for the splice–site mutation IVS5+2T>C. The proband’s father (55 years old) has no ASMT mutation (+/+). Error bars represent s.d. C: controls; F: father; M: mother; P: proband.

Figure 3

Association studies and transcript analyses of the ASMT gene. (a) Haplotype block structure of the ASMT gene. The relative physical position of each single nucleotide polymorphism (SNP) is given in the upper diagram, and the pairwise LD (D0) between all SNPs is given below each SNP combination. (b) Plot of the case–control P-values (−log10) for all variations studied within ASMT. 1: E1A; 2: rs4446909; 3: rs5989681; 4: P1BC; 5:rs6644635; 6: rs6588802; 7: rs28675287; 8: rs6588809; 9: I6A; 10: rs7471973; 11: rs5431942; 12: rs4933063; 13: rs11346829. SNPs located in promoter B are included in the shaded box. P-values for the risk haplotype GGGC are indicated as straight lines with close (cases vs controls) or open circles (transmission disequilibrium test (TDT)). (c) Quantification of ASMT transcripts relative to rs4446909 and rs5989681 genotypes (A represents the individuals with an A/A or A/G genotype; C represents the individuals with C/C or C/G genotype; G represents the individuals homozygous G/G). Black and white circles indicate individuals with autism spectrum disorders (ASD) and controls, respectively. The gray symbols indicate individuals homozygous A/A and C/C for rs4446909 and rs5989681, respectively. Real-time reverse transcriptase (RT)-PCR was performed with B lymphoblastoid cell lines from 38 ASD probands and 29 controls. Horizontal bars indicate medians. No statistical difference was observed between ASD and controls (Mann–Whitney U-test).

Figure 4

ASMT activity and melatonin concentration in patients, parents and controls. (a) Serotonin levels, measured in the platelets of 43 ASD patients (mean±s.d.; 1±0.6 μM), 34 parents (0.8±0.2 μM) and 48 controls (0.4±0.2 μM). (b) ASMT activity, measured in the platelets of 43 ASD patients (0.73±0.43 pmol/10⁹ platelets/30 min), 34 parents (1.20±0.85 pmol/10⁹ platelets/30 min) and 48 controls (1.81±0.68 pmol/10⁹ platelets/30 min). (c) Plasma melatonin concentration in 43 autism spectrum disorders (ASD) patients (73±36 pmol), 34 parents (99±46 pmol) and 48 controls (136±39 pmol). (d) Blood melatonin concentration expressed with respect to ASMT activity in platelets. ASMT activity and melatonin concentration were not correlated in controls (white circles). In contrast, in ASD patients (black circles), and their parents (white triangles), a significant correlation was observed. Spearman’s rho test was used to evaluate the correlation between ASMT activity and melatonin concentration, (e) ASMT activity in B lymphoblastoid cell lines from 53 ASD patients (3.9±3.4) and 33 controls (8.3±2.4). Circles and squares indicate female and male subjects, respectively. Statistical significance was assessed with the Mann–Whitney U-test.