Blood serotonin levels in autism spectrum disorder: A systematic review and meta-analysis
Introduction
The term “autism spectrum disorder” (ASD) defines a complex and heterogeneous group of neurodevelopmental conditions, characterized by impaired social and communication skills, as well as stereotyped behaviors and restricted patterns of interests (American Psychiatric Association, 2013). Despite many advances in our understanding of the neurobiological and developmental processes underlying ASD (Chugani, 2012, Freitas et al., 2014, State and Levitt, 2011), our knowledge remains limited and its translational impact into the clinics is still insufficient. Most importantly, behavioral abnormalities are typically not evident until approximately 12–18 months of age (Mitchell et al., 2011, Wan et al., 2013). Furthermore, ASD individuals vary enormously in clinical presentation, severity, developmental trajectory, and treatment response. This complexity is spurring an intensive search to identify biological markers able to aid clinicians in achieving earlier diagnoses and in predicting clinical prognosis as well as treatment response (Walsh et al., 2011). A biomarker can be defined as a biological variable associated with the disease of interest across and within individuals, measurable directly in a given patient or in his/her biomaterials using sensitive and reliable quantitative procedures. Elevated blood serotonin (5-hydroxytryptamine or 5-HT) was the first biomarker identified in autism research (Hanley et al., 1977, Schain and Freedman, 1961), and is still one of the quantitative traits most consistently associated with the disease in a sizable subgroup of ASD patients, as reviewed elsewhere (Anderson et al., 1987, Cook and Leventhal, 1996, Veenstra-VanderWeele and Blakely, 2012). In particular, most studies have described significantly higher mean 5-HT blood levels in approximately 30% autistic individuals on average, as compared to typically developing controls. Elevated 5-HT blood levels are seemingly autism-specific, as they are not present in cognitively impaired individuals, and are equally distributed in different Pervasive Developmental Disorder subtypes (i.e., Autistic Disorder vs Pervasive developmental Disorder Not Otherwise Specified) (Mulder et al., 2004).
Several studies have addressed the mechanisms underlying 5-HT blood elevation in ASD. Physiologically, peripheral 5-HT is produced by enterochromaffin cells in the gut (Gershon, 2004). Approximately 99% of blood 5-HT is sequestered inside platelets by the antidepressant-sensitive 5-HT transporter (SERT) and then inside vesicles by the vesicular monoamine transporter (VMAT2) (Zalsman et al., 2011), while only 1% of total 5-HT remains free in the plasma and thus exposed to the action of catabolic enzymes (Anderson et al., 1987). The regulation of SERT activity and trafficking at the plasma membrane of platelet represents an important and well investigated mechanism in the regulation of 5-HT blood levels. The protein complex involved in the SERT regulatory network includes αIIbβ3 integrin, protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), the 5-HT2A receptor and undergoes PKC-, PKG-, p38 MAPK-mediated regulation (Veenstra-VanderWeele and Blakely, 2012). An initial study by Katsui et al. (1986) found increased 5-HT blood levels associated with increased SERT density on the platelet membrane (Vmax), while the affinity (Kd) of SERT for 5-HT was unchanged. This initial study was later replicated by Marazziti et al. (2000). Hence SERT trafficking may be skewed in favor of SERT externalization in individuals showing elevated 5-HT blood levels, although the existence of additional factors cannot be excluded. The molecular mechanisms favouring SERT externalization involve different genes in males and females (Mei et al., 2007, Weiss et al., 2005), including for example common variants at ITGB3 (Napolioni et al., 2011, Weiss et al., 2004, Weiss et al., 2005, Weiss et al., 2006a, Weiss et al., 2006b) and rare variants at SLC6A4 (Prasad et al., 2005, Veenstra-VanderWeele et al., 2012). It is also apparently modulated by sex hormones, since 5-HT blood levels are especially elevated in some autistic children, whereas after puberty this excess becomes less pronounced (McBride et al., 1998).
Abnormal neurodevelopment underlies autistic behaviors (DiCicco-Bloom et al., 2006). Interestingly, 5-HT plays a key role not only as a neurotransmitter in the adult brain, by regulating a variety of behavioral, autonomic and cognitive functions (Murphy and Lesch, 2008), but also as a trophic factor during prenatal neurodevelopment (Di Pino et al., 2004, Persico, 2009). According to recent animal models, during pregnancy embryonic 5-HT is initially produced by the placenta using maternal tryptophan, whereas subsequently 5-HT synthesis is undertaken by serotonergic neurons located in raphe nuclei and extending their projections to the cortex, basal ganglia, amygdala, hippocampus and hypothalamus (Bonnin et al., 2011). Abnormalities in brain 5-HT systems were also reported in ASD, including an altered developmental trajectory of 5-HT turnover (Chugani et al., 1999) and reduced binding of 5-HT receptors and SERT (Murphy et al., 2006, Nakamura et al., 2010). Remarkably, proteins regulating 5-HT homeostasis peripherally and in the central nervous system (CNS) are largely conserved: the SLC6A4 and HTR2A genes encode the same SERT and 5-HT2A receptor, respectively, both in platelets and brain (Cook et al., 1994, Lesch et al., 1993). Several genes involved in SERT trafficking, especially SLC6A4 and ITGB3, were mapped to autism linkage regions, found to host rare variants causing ASD, or were associated with autism through common variants (Cantor et al., 2005, Coutinho et al., 2004, Napolioni et al., 2011, Stone et al., 2004, Weiss et al., 2004, Weiss et al., 2006a). Therefore, the molecular mechanisms involved in blood 5-HT elevation may not only tag a pathogenetically homogeneous subgroup of autistic patients, but also underlie at least in part the neurodevelopmental abnormalities present in the CNS of these patients.
The consistency of the association between elevated 5-HT blood levels and autism, as well as the relevance of 5-HT roles in neurodevelopment, collectively make 5-HT blood levels a primary candidate for comprehensive ASD biomarker panels currently under scrutiny. However, studies evaluating peripheral 5-HT in autistic patients and controls differ in sampling demographics (i.e., age range, sex, ethnicity), patient characteristics, selection of controls, 5-HT measurement protocol, technologies and biomaterials. We thus undertook a systematic review of all studies assessing 5-HT blood levels in ASD patients and controls, followed by a series of meta-analyses in order to provide an overall estimate of the effect size and between-study heterogeneity for the association between elevated 5-HT blood levels and autism, while verifying whether and to what extent differences in biomaterials, experimental protocols and technologies influence results.
Section snippets
Literature search strategy
Studies eligible for this systematic review and meta-analysis were identified through a strategy employing an initial a priori search protocol, followed by reiterative modifications aimed at progressively maximizing search efficiency by yielding increased numbers of pertinent publications. Our final search terms were as follows:
(Autism OR autistic OR ASD OR pervasive developmental disorder OR Asperger) AND (serotonin OR 5-HT OR 5-hydroxytryptamine) AND (serotonergic OR levels OR peripheral OR
Study characteristics
The Literature search yielded 551 records, including 469 records from PubMed, 76 records from Scopus, and 6 additional records from Eric. Applying our exclusion criteria (Figure 1), 22 studies were selected for review and meta-analysis (Table 1), whereas 529 were excluded (listed as Suppl. Refs.). These 22 papers were published between 1970 and 2012; thirteen studies were conducted in United States, 6 were from Europe, 2 from Asia and 1 from Australia. Sample sizes varied widely, ranging from 7
Discussion
We conducted a systematic review and selection of studies measuring 5-HT blood levels in autistic and control samples using different biomaterials and methodologies. Data from selected studies were then meta-analyzed, yielding results expressed as overall percentage of ASD patients displaying elevated 5-HT blood levels, and as global mean odds ratios. The procedure employed here to systematically detect published papers on 5-HT blood levels in autism was broad-based and thorough. Our strict
Role of the funding source
This work was supported by the Italian Ministry for University, Scientific Research and Technology (PRIN n.2006058195 and n.2008BACT54_002), the Italian Ministry of Health (RFPS-2007-5-640174 and RF-2011-02350537), the Fondazione Gaetano e Mafalda Luce (Milan, Italy), Autism Aid ONLUS (Naples, Italy), Autism Speaks (Princeton, NJ), the Autism Research Institute (San Diego, CA), and the Innovative Medicines Initiative Joint Undertaking (EU-AIMS, n. 115300). The authors declare that these funding
Contributors
SG performed the systematic review, extracted data from papers and contributed to meta-analyses. RS performed and supervised statistical analyses. AMP designed the study. SG and AMP wrote the manuscript. All authors approved the final version of the manuscript.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
We wish to thank all the investigators who kindly provided additional information regarding their papers for appropriate data management.
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