Families with ASD were recruited by the Paris Autism Research International Sibpair study at specialized clinical centers in seven countries (France, Sweden, Norway, Italy, Belgium, Austria, and the United States). Diagnosis was based on clinical evaluation by experienced clinicians, DSM-IV criteria, and the Autism Diagnostic Interview-Revised (ADI-R).25
In Sweden, the Diagnostic Interview for Social and Communication Disorders (DISCO-10)26
was used instead of the ADI-R in some cases. Patients with Asperger syndrome were evaluated with the Asperger Syndrome Diagnostic Interview.27
Patients diagnosed with medical disorders, such as fragile X syndrome or chromosomal anomalies, were excluded from the study.
For mutation screening, the study sample (n=250, 187 men and 63 women) was constituted of 250 independent families (71 subjects from multiplex families and 179 sporadic cases) and included 233 patients with autistic disorder and 11 with Asperger syndrome; six individuals narrowly missed the criteria for autistic disorder and were considered to have atypical autism (pervasive developmental disorder, PDD-NOS). There were 222 Caucasian, nine Black, three Asian, one Hispanic/Latin-American family, and 15 families of mixed ethnicity. For association studies, the ASD sample consisted of 278 patients of Caucasian origin (201 men and 77 women) from 72 multiplex families and 206 sporadic cases. There were 258 patients with autism, 14 with Asperger syndrome and six with atypical autism. The control sample (n=255) comprised 160 French and 95 Swedish individuals. An additional control group of 171 individuals from North Africa was screened for rare variants because one proband carrying the splice-site mutation (IVS5+2T>C) and one proband with the L326F mutation had parents originating from this region.
Blood and platelet biochemical analyses were performed in ASD probands (n=43; 14 female and 29 male patients, 14.8 ± 7 years old), their parents (n=34; 18 females and 16 males: 44 ± 9 years old) and in controls matched for sex and age (n=75; 30 females and 45 males; 27 ± 16 years old). The ASD patients were initially recruited for the analysis of serotonin levels and not chosen on the basis on their ASMT genotype. The controls were recruited at the Department of Orthopedics of the Lariboisière and Robert Debré hospitals in Paris. The control group for the B lymphoblastoid cell lines (BLCL) biochemical analyses comprised 14 French individuals also used in the association study and 19 healthy relatives of patients with Hirschsprung syndrome or mitochondrial diseases. The local research ethics boards reviewed and approved the study. Informed consent was obtained from probands (if possible), parents and controls.
Cell culture, DNA and RNA isolation
BLCL were established from EBV-transformed lymphocytes and grown at 37°C in RPMI-1640 medium (Life Technologies Inc., Grand Island, NY, USA), supplemented with 10% undialyzed fetal calf serum, 2 mM glutamine, 2.5 mM sodium pyruvate, 100 mg/ml streptomycin and 100 IU/ml penicillin, under standard conditions. DNA was extracted by the phenol/chloroform method, and RNA was isolated using the NucleoSpin RNA II kit (Macherey-Nagel, Duren, Germany). DNA/RNA concentrations were determined by measuring absorbance at 260 nm on a biophotometer (Eppendorf, Hamburg, Germany). Human pineal gland cDNAs were obtained from the Incyte cDNA library # LHS1565 (BioCat, Heidelberg, Germany).
Mutation screening and genotyping
Genotyping for the association study and mutation screening were performed by direct sequencing or TaqMan technology. PCR products were sequenced with the BigDye Terminator Cycle Sequencing Kit (V3.1, Applied Biosystems, Foster City, CA, USA). Samples were then subjected to electrophoresis, using an ABI PRISM genetic analyzer (Applied Biosystems). Absence of genotyping errors was controlled by sequencing the PCR product with the opposite primer in a subset of patients. For primers and PCR conditions, see Supplementary Table 1
Association and statistical analyses
The linkage disequilibrium (LD) map for ASMT
was calculated using pairwise LD (D′) between the 13 ASMT
variations in 533 individuals (278 ASD and 255 controls). The LD calculation and the case-control study were performed with Haploview software.28
To detect population stratification bias, individuals with ASD and controls were screened for three single nucleotide polymorphisms (SNPs) (rs2289311, rs4782053, rs1921361), five ALU insertions (Ya5NBC27, Ya5NBC51, YaNBC102, YaNBC109, YbNBC65), and the mtDNA hypervariable region 1 (HVR1). In addition, all mothers from ASD patients were screened for the androgen receptor microsatellite. No significant genotype difference was observed for any of the markers tested (Supplementary Table 2
). The transmission disequilibrium test (TDT) was performed using the family-based association test (FBAT)29
and haplotype-based association test (HBAT)30
using the empirical variance (‘-e’ option). For the TDT, only the four SNPs in promoter B were tested in 278 ASD families in which both parents had been genotyped. All SNPs were at Hardy-Weinberg equilibrium. We evaluated the distribution of the quantitative variables by the Kolmogorov-Smirnov test for Gaussian normality. Because the values for most of the samples were not normally distributed, we used the two-tailed non-parametric Mann-Whitney U
-test to compare two groups and Spearman’s ρ test to evaluate the correlation between ASMT activity and melatonin concentration. We used SPSS version 13 for these tests.
RT-PCR and quantitative RT-PCR
Oligo(dT)-primed cDNA was prepared from 5 μg of BLCL RNA, using Superscript II (Invitrogen, Grand Island, NY, USA), according to the manufacturer’s instructions. The cDNAs were used directly in TaqMan assays, using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Samples were run in duplicate or triplicate on 96-well optical PCR plates (ABgene, Surrey, UK). ASMT mRNA was quantified using commercially available assays. Two different assays were used, one covering the boundary between exon 1B and exon 2 (Hs00946625_ml), and the other covering the boundary between exon 8 and exon 9 (Hs00187839_ml). The two assays gave similar results and only the data obtained with Hs00946625_ml are presented. Relative values of expression were determined for each sample, using the standard curve method (ABI user’s manual), and these values were normalized to the threshold cycle (Ct) values of GAPDH, using the Hs99999905_ml assay. For ASMT and GAPDH, the thresholds were set at 0.2 and 0.25, respectively, within the linear region of the semi-logarithmic plot in all assays (data not shown). The consequence of the splice site mutation was investigated by sequencing the abnormal transcript after cloning the RT-PCR product.
Blood samples were collected in the morning, between 0900 and 1100 h. The procedures for collecting and processing blood samples were designed to prevent release reactions. The anticoagulant was ACD-A (1 vol to 9 vol of whole blood). For the melatonin profile of family ASD 1, blood samples were collected every 2 h, from 1800 h to 1600 h the following day. Overnight, blood samples were collected in dimly lit conditions (<20 lux). Samples were drawn from an indwelling forearm catheter into Becton-Dickinson plastic tubes, centrifuged and frozen at −20°C. Platelets were counted with a Coulter ZBI electronic counter. ASMT enzymatic activities were determined, at least in duplicate, by radioenzymology,31
on the platelet pellet obtained by centrifugation of the platelet-rich plasma at 800 g
for 20 min at room temperature and lysis with 100 hemolytic units of a purified SH-activated toxin (streptolysin O or alveolysin, generously provided by Prof. J. Alouf, Pasteur Institute, Paris). Melatonin content was measured in the resulting supernatant (i.e
., platelet-poor plasma or plasma) by HPLC, with random controls by mass spectrometry.32
The three individuals from family ASD 1 were taking no medication that could interfere with melatonin secretion. Sleep analysis was performed by standard polysomnography (two EEG, two electro-oculograms, one submental electromyogram, two anterior tibialis muscle electromyograms and respiratory signals, Embla N7000, Flaga, Iceland), 1 week before blood was collected for the melatonin assay.