Heritability of the melatonin synthesis variability in autism spectrum disorders

Autism Spectrum Disorders (ASD) are heterogeneous neurodevelopmental disorders with a complex genetic architecture. They are characterized by impaired social communication, stereotyped behaviors and restricted interests and are frequently associated with comorbidities such as intellectual disability, epilepsy and severe sleep disorders. Hyperserotonemia and low melatonin levels are among the most replicated endophenotypes reported in ASD, but their genetic causes remain largely unknown. Based on the biochemical profile of 717 individuals including 213 children with ASD, 128 unaffected siblings and 376 parents and other relatives, we estimated the heritability of whole-blood serotonin, platelet N-acetylserotonin (NAS) and plasma melatonin levels, as well as the two enzymes arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT) activities measured in platelets. Overall, heritability was higher for NAS (0.72 ± 0.091) and ASMT (0.59 ± 0.097) compared with serotonin (0.31 ± 0.078), AANAT (0.34 ± 0.077) and melatonin (0.22 ± 0.071). Bivariate analyses showed high phenotypic and genetic correlations between traits of the second step of the metabolic pathway (NAS, ASMT and melatonin) indicating the contribution of shared genetic factors. A better knowledge of the heritability of the melatonin synthesis variability constitutes an important step to identify the factors that perturb this pathway in individuals with ASD.

observed in patients compared to relatives and controls, including hyperserotonemia, deficits in AANAT and ASMT platelet activity, increased platelet NAS and melatonin deficit 10,15 . These alterations were also observed to a lesser extent, in the relatives of patients with ASD, compared to controls 10,15 , but familial correlations and heritability have never been assessed for both steps of the melatonin synthesis pathway in families with ASD.
Serotonin is involved in a wide range of central processes, such as brain development, emotion, learning, memory or cognitive functions 16,17 . NAS is an agonist of melatonin receptors and activates TrkB in mice, a receptor of the brain-derived neurotrophic factor (BDNF), in a selective and circadian manner 18,19 . Melatonin is a pleiotropic neuroendocrine molecule essential for synchronizing circadian and seasonal rhythms, as well as sleep/ wake cycles, but also displays antioxidant, neuroprotective, or immunomodulatory effects [20][21][22] . Biochemical alterations of this pathway could thus be related to the core symptoms of autism or the comorbidities such as cognitive problems, epilepsy, sleep and gastrointestinal disorders observed in ASD 17,[23][24][25] .
In order to identify shared underlying genetic factors between an endophenotype and a disease, the endophenotype must be heritable with either high broad sense heritability (H 2 ), defined as the ratio of total genetic variance (additive, dominance and epistasis) to phenotypic variance, or high narrow sense heritability (h 2 ), reflecting only the additive part (the average effect of the alleles on the trait) of the genetic contribution to the phenotypic variance (Supplementary Note). Several studies in humans and in animal models have demonstrated relatively high heritability for serotonin [26][27][28] and melatonin [29][30][31][32] . In humans, serotonin narrow sense heritability estimates range from 0.2 to 0.51 26,27,33 and urinary melatonin narrow sense heritability estimate was 0.53 in families with acute intermittent porphyria 32 . To our knowledge, NAS, AANAT and ASMT heritability have never been investigated. In this study, we estimated the narrow sense heritability of all five quantitative traits (whole blood serotonin, platelet AANAT and ASMT activities, platelet NAS and morning plasma melatonin) in 717 individuals including 213 children with ASD, 364 parents, 128 unaffected siblings and 12 other relatives (Supplementary Fig. S1) 10,15 . We also evaluated the correlations of genetic and environmental factors between pairs of traits. The assessment of additive factors contribution to the inter-individual phenotypic variability should provide crucial information for further quantitative genetic investigations of this pathway in families with ASD.

Results
Biochemical ascertainment of the melatonin synthesis pathway. The study sample included 717 participants (182 patients with ASD, 364 parents, 128 unaffected siblings, 31 affected siblings and 12 other relatives) with a mean age of 31.3 years and 59.3% males (425/717). As frequently observed in ASD 34,35 , the sex ratio for the patients in our cohort was one female for 4.5 males (39 females and 174 males) (Table 1), whereas the number of males and females was similar in the unaffected siblings (M/F = 62/66 = 0.94). As previously reported for this cohort 10,15 , there were significant biochemical differences between patients and their relatives (parents or unaffected siblings) for each biochemical parameter: hyperserotonemia, increased platelet NAS, and deficit in platelet ASMT, platelet AANAT and plasma melatonin (Table 1).
Regarding the distribution of the biochemical traits within pedigrees, different categories of families could be determined (Fig. 1a). We observed that for some families, affected and/or unaffected children displayed values within the range defined by their corresponding parental values, while in other families, children biochemical values were outside this range. In most cases, children with ASD had the most extreme values compared to unaffected children. To quantify the difference between parents and affected or unaffected children, we used quartet families including one affected child and one unaffected child (Fig. 1b). For each trait, the distance between children and average parental values were compared. We observed that affected children values were significantly more distant (P < 0.005) from their corresponding average parental values than unaffected children. We therefore performed detailed familial correlation and heritability analyses of each step of the melatonin synthesis pathway. Heritability of the melatonin synthesis variability. We first calculated the correlation coefficients for parent-offspring (P-O), sibling-sibling (S-S) and father-mother (F-M) pairs (Table 2). When all individuals of the families were taken into account (affected and unaffected), the P-O correlation coefficients were significantly different from 0 for all traits after Bonferroni correction for multiple testing, except for melatonin (uncorrected p-value = 0.027), and the correlation was particularly high for NAS compared with other parameters (NAS: 0.38;  Table 2). No S-S correlations remained significant after Bonferroni correction. Interestingly, F-M correlations were significant for AANAT and melatonin. We then estimated the heritability of each trait. When all individuals were taken into account, the narrow sense estimates of heritability (reflecting additive effects) were all significant (Bonferroni corrected p-value < 0.05; Fig. 2 Table S1). The heritability estimates ranged from 0.22 for melatonin to 0.72 for NAS (serotonin: 0.31; AANAT: 0.34; NAS: 0.72; ASMT: 0.59; melatonin: 0.22) providing support for a significant genetic contribution to the melatonin synthesis variability. Proportions of variance due to all covariates included were relatively low, ranging from 0.029 to 0.072 (Supplementary Table S2). When the sample was stratified by ASD status, all heritability estimates tended to be higher for unaffected children than for children with ASD ( Fig. 2 and Supplementary Table S1). For example, both serotonin and melatonin heritability estimates were not significant for children with ASD, after Bonferroni correction for multiple testing.

and Supplementary
To investigate the relationship between each pair of traits and evaluate the proportion of heritability of one trait that is attributable to the other trait, we calculated bivariate trait correlation coefficients for ASD and unaffected children separately (Fig. 3a). In both groups, we observed phenotypic correlations ρ P > 0.4 for ASMT-melatonin, ρ P < −0.8 for NAS-ASMT and a ρ P < −0.2 for NAS-melatonin. These results confirmed a high correlation between the three parameters of the last step of the melatonin biosynthesis pathway (NAS, ASMT and melatonin), as previously reported 15 . When only children with ASD were included, these phenotypic correlations were significantly different from 0 (NAS-ASMT: ρ P = −0.84; ASMT-melatonin: ρ P = 0.57; NAS-melatonin: ρ P = −0.42) (Fig. 3a). For     (Fig. 3a). These genetic correlations were not significantly different from 1 or −1. There were also significant common environmental influences for NAS-ASMT (ρ E = −0.70) and for ASMT-melatonin (ρ E = 0.48) in children with ASD (Fig. 3a). In conclusion, these results showed an overlap of additive genetic effects as well as shared environmental factors influencing phenotypic variability in families with ASD, particularly for NAS, ASMT and melatonin (Fig. 3b).

Discussion
Very few heritability estimates for serotonin and melatonin are available for humans in the literature (Table 3) and this study is the first to characterize the heritability of the melatonin synthesis variability in a large sample of families with ASD. We first calculated familial correlations of the five traits (serotonin, AANAT, NAS, ASMT and melatonin). Using all family members, we observed significant P-O correlations except for melatonin, but no S-S correlations remained significant after correction for multiple testing, probably due to the relatively small number of S-S pairs compared to P-O pairs. Unexpectedly, we also observed significant correlations between fathers and mothers for AANAT and melatonin. AANAT activity and melatonin production display marked circadian variations in the pineal gland 15,36 . These processes are closely related to light-dark cycle [37][38][39] and their regulation might depend on environmental factors, such as sleep-wake cycle and artificial light exposure. Thus, synchronization could be observed between parents that share daily habits and life rhythms. We then estimated the heritability of the five traits. In the literature, the first estimate of blood serotonin heritability was obtained by Abney et al. 27 in an inbred founder population of Hutterite families. They found respectively 0.51 and 1.0 for narrow and broad sense heritability, suggesting a very strong genetic influence with both additive and dominance components. Our estimation of serotonin narrow sense heritability was lower (0.31) than the one reported by Abney et al., but very similar to the one obtained in a large cohort of 866 monozygotic and 878 dizygotic unaffected twin pairs (0.33; Table 3) 26 . For melatonin, our estimation of the heritability (0.22) ranged between two previous estimates 31,32 . Wetterberg et al. (1983) estimated melatonin heritability to 0.53 in 107 families with acute intermittent porphyria 32 . In contrast, a twin study measuring late night plasma melatonin in nine monozygotic and 11 dizygotic twin pairs, reported a high monozygotic correlation (r MZ = 0.928) and a high dizygotic correlation (r DZ = 0.867) 31 . Using these correlations and the Falconer's formula (H 2 = 2(rMZ -rDZ)), the heritability for melatonin would be 2(0.928-0.867) = 0.12.
All the five traits studied here are not expected to be independent since they belong to the same metabolic pathway. Using a bivariate approach, we estimated pairwise phenotypic, genetic and environmental correlations in samples including only children with ASD or only unaffected children. In both groups, phenotypic correlations appeared to be particularly high for the last step of melatonin biosynthesis pathway (NAS, ASMT and melatonin) (Fig. 3b). Therefore, considering these traits as dependent might be useful for further association studies exploring variants associated with these phenotypes. Interestingly, although ASMT and NAS were phenotypically correlated with melatonin, their heritability differed, particularly in children with ASD (high for ASMT and NAS, low for melatonin). In children with ASD, this discrepancy could be explained for ASMT and melatonin by the fact that phenotypic correlation between these traits was mainly attributable to shared environment while genetic factors seemed to be more divergent (Fig. 3a). However, for NAS and melatonin, genetic correlation was high and environmental correlation was not significant. These results suggest that even if there are probably common genetic mechanisms in the regulation of these three traits, environmental factors such as light exposure or drugs interacting with cytochromes, might act differentially on NAS and melatonin levels.
There are several limitations of this study such as the absence of data on circadian rhythm. However, previous studies showed that the deficit in melatonin in ASD was also observed in blood and urine samples collected during the night 12,13,40 . Another limitation is the absence of analyses in the pineal gland. Finally, our estimations for unaffected children might not reflect the heritability the melatonin synthesis variability in the general population. Nevertheless, for serotonin and melatonin, our heritability estimates are very similar from those obtained in previous studies (Table 3) 26,27,32,33 .
In summary, our results revealed that the resemblance between unaffected children and parents seems to be mostly due to additive genetic effects, while patients resemblance to their parents, which was lower, probably includes additional factors such as de novo events, dominant effects, or non-shared environmental influences related to ASD condition. Another interesting finding is that NAS displayed the highest heritability estimate within the melatonin synthesis pathway. In contrast to serotonin and melatonin, NAS received much less interest from the scientific and clinical community 10,41 . This is unfortunate, especially since NAS could have its own biological functions, such as TrkB activation, immunomodulation or analgesia 19,[41][42][43][44] . We thus propose to use NAS, in addition to serotonin and melatonin, as a suitable endophenotype for further quantitative genetic studies in ASD. Further studies using larger study samples and molecular genetic analyses such as genome-wide association studies are now required to identify the variants involved in melatonin synthesis variability in the general population and in patients suffering from sleep disorders and circadian rhythms abnormalities.

Subjects and Methods
Ethics statement. The local Institutional Review Boards (IRB) at the Institut Pasteur in Paris (France) approved the study. Methods were performed in accordance with the relevant guidelines and regulations. Written informed consents were obtained from all participants. For the patients who were unable to consent for themselves, a parent or legal guardian consented to the study on their behalf.

Subjects and clinical evaluations.
Clinical evaluations of patients with ASD, their relatives and control subjects have been detailed previously 10 . All the subjects were recruited into the Paris Autism Research International Sib-pair (PARIS) study. The ASD diagnosis was based on clinical expert assessment including the Autism Diagnostic Interview -Revised (ADI-R) 45 and the Autism Diagnostic Observation Schedule (ADOS) 46 . Intellectual Quotient (IQ) was measured using an age-appropriate Wechsler scale. For the most severe and/or non-verbal patients, the Raven's Standard Progressive Matrices and the Peabody Picture Vocabulary test were used.
Biochemical measurements. Blood samples were collected in the morning, between 8:30 and 10:30 as described previously 10,14 . Subjects were asked to avoid food with high content of tryptophan and/or serotonin two days before blood sampling and individuals receiving exogenous melatonin were not included in this study. Melatonin was measured in plasma using a radio-immunoassay (RK-MEL, Bühlmann, Switzerland) according to the manufacturer's instructions. Whole-blood serotonin (5-hydroxytryptamine, 5-HT) was measured by high-performance liquid chromatography 47 . NAS, as well as enzyme activities of AANAT and ASMT were determined in platelets by radio-enzymology 48 . Patients who were receiving melatonin for treatment of sleep disorders were not included in the analyses. Only pedigrees with both parents and at least one child were included in the following analyses. The overlap between the biochemical data for serotonin, AANAT, NAS, ASMT and melatonin is illustrated in Supplementary Fig. S2. A detailed study of the differences between groups has been published elsewhere 10,15 . Statistical methods. Data management and graphs were performed using JMP Pro 12 (SAS, USA) and R software 49 . Because some of the studied traits were not normally distributed, nonparametric statistical tests (Wilcoxon rank-sum test) were preferred to compare groups of individuals. For familial correlation and heritability analyses, serotonin, ASMT and melatonin values were logarithmically transformed in order to conform more closely to a normal distribution (Supplementary Fig. S3). Since melatonin and serotonin are known to be age and sex dependent 50-53 , they were included in the models using residuals from regression models obtained with the program Sequential Oligogenic Linkage Analysis Routines (SOLAR) 7.2.5 (Southern Foundation for Biomedical Research, San Antonio, TX, USA) 54,55 , when their effect as covariates (age, sex) were significant at the threshold of P < 0.1. Individuals at more than three standard deviations (SD) from the mean were considered as outliers and were removed. Depending on the biochemical trait and the subgroup considered, the number of outliers was always very low, ranging from 0 to 7 (Supplementary Table S3). A total of 717 individuals (182 ASD patients and 535 relatives) were included in this study. There were 185 families, including from three to nine relatives (Supplementary Table S4): 68 trios, 87 quartets, 22 quintets, four sextets, one septet, one nonet and two extended pedigrees. For three families, biochemical measurements could be obtained for the parents and unaffected children, but not for the ASD probands and these families were included in the analyses. For each analysis, uncorrected p-values are shown, but due to multiple testing, the significance was assessed after Bonferroni correction.   father-mother (F-M) familial correlations with their asymptotic standard errors. S.A.G.E then tests the difference of the correlation coefficients from 0 using Fisher's z-transformation. Narrow sense heritability was estimated using the variance component-based program SOLAR 7.2.5 with a maximum likelihood estimation method and polygenic models. When the effect of the covariates (age, sex) was significant (P < 0.1), relative proportion of variance explained by known covariates was estimated and they were included in the model. SOLAR can extend the variance component model to a bivariate analysis that maximizes the model for two dependent traits 57 . The trait pairs included all pairwise combinations and the same covariates (age and sex) significant at the threshold of P < 0.1 were used. Three parameters were estimated for each pair of traits: the additive genetic correlation (ρ G ), the shared environmental correlation (ρ E ) and the total phenotypic correlation (ρ P ) (Supplementary Note).
Familial correlations and univariate heritability analyses were first performed on all individuals, including parents and their affected children as well as their unaffected children (Supplementary Fig. S4). Secondly, stratified analyses were conducted on parents with their affected children and on parents with their unaffected children ( Supplementary Fig. S4).