The present study provides evidence that TPH2 and GLO1 gene variants do not contribute significantly to autism pathogenesis in our sample, while GLO1 could seemingly exert a protective effect in unaffected siblings. Our conclusions are strengthened by the consistency of multiple statistical approaches and by the parallel assessment of some of the most reliable biochemical and morphological endophenotypes described in autism research to this date, namely macrocephaly, hyperserotoninemia and enhanced peptiduria [20–23]. These heritable traits are believed to be more directly linked to the genetic make-up of an individual than complex clinical symptoms, and could thus possibly identify subgroups of autistic patients characterized by relatively homogenous pathogenetic processes. These parameters were thus chosen on this ground, and not following a-priori hypotheses. In particular, 5-HT blood levels do not reflect TPH2 activity, as peripheral 5-HT largely comes from the digestive tract and is thus produced by TPH1 [9–11]. Therefore, the non-significant trend towards an association between TPH2 gene variants and 5-HT blood levels either represents a spurious finding, or necessarily stems from an indirect pathophysiological link which will require further investigation.
Some differences must be drawn in the interpretation of the data pertaining to TPH2 and GLO1. Our results exclude causal TPH2 roles in our sample, and do not support the hypothesis that TPH2 gene variants may exert a major influence on repetitive and stereotypic behaviors in autistic patients. This conclusion confirms the outcome of a recent study, reporting no association either with autism, or with obsessive-compulsive and self-stimulatory behaviors in autistic patients . Collectively, these findings, although negative, are important due to the relevant roles played by 5-HT during neurodevelopment , and because they help us interpret contributions to autism by serotoninergic genes. TPH2 and the 5-HT transporter (5-HTT) play pivotal roles in central serotoninergic neurotransmission. TPH2 determines the rate of 5-HT biosynthesis in the CNS [9–11], whereas the 5-HTT terminates the action of extracellular 5-HT on its receptors through reuptake . During neurodevelopment, 5-HT exerts impressive neurotrophic effects on cell proliferation, differentiation, and migration, programmed cell death, cell-cell coupling, synaptogenesis and cytoskeletal plasticity . Many of these processes appear altered in neuropathological studies of autistic brains [2, 3]. Furthermore, a recent quantitative brain imaging study has revealed increased cortical gray matter volumes in autistic patients carrying 5-HTT gene variants yielding slower extracellular 5-HT clearance rates, resulting in higher extracellular 5-HT levels . This finding parallels results obtained in 5-HTT knockout mice backcrossed into C57BL/6J, displaying increased neuronal cell density and thickness of supragranular and infragranular neocortical layers compared to wild-type mice . In contrast to these neurobiological data, strongly supporting the trophic effects exerted by elevated extracellular 5-HT levels in autism, results of genetic studies on serotoninergic gene variants in autism are mostly negative or mixed at best. Indeed, the present and prior studies jointly indicate that (a) in the vast majority of cases, functional serotoninergic gene variants play a modulatory, but not a causal role in autism; (b) 5-HT synthesis does not contribute to produce increased levels and/or prolonged persistence of extracellular 5-HT, which largely stems from reduced 5-HT uptake and could possibly play a role in eccessive neurotrophism and macrocephaly in autism; (c) 5-HT could modulate not only morphological features such as head circumference, but also repetitive and stereotypic behaviours in autism, as well as in OCD [7, 13, 14, 38]. Unfortunately the approved Italian translation of the Autism Diagnostic Interview-Revised (ADI-R) has become available only recently , and this instrument was not available when the vast majority of our sample was recruited. However, the presence/absence of relevant verbal and motor stereotypic behaviors that was assessed here by an experienced clinician at intake largely corresponds to the current presence of "stereotyped utterances and delayed echolalia", "hand and finger mannerisms", "other complex mannerisms or stereotyped body movements", and "midline hand movements", as assessed by ADI-R items n. 33, 77, 78, and 79, respectively . Therefore, despite representing a crude, single time-point measure, it allows us to exclude prominent roles for TPH2 gene variants in these behavioural symptoms. Searches for modulatory effects on repetitive and stereotypic behaviors will require finer, possibly longitudinal measures.
On the other hand, GLO1 allele A419 seemingly exerts a protective effect among unaffected siblings, whose allelic transmission patterns differ significantly from those of their autistic siblings, both using TDT and FBAT. The discrepancy between initial findings by Junaid and Colleagues , proposing A419 as an autism vulnerability allele, and our findings may stem from at least two potential sources. First, it is important to notice that the A419 allele frequencies found by Junaid and Colleagues  in autistic patients are very similar to those reported here (0.6056 vs 0.5785 and 0.5693 in our Italian and Caucasian-American patients, respectively), whereas their control allele frequencies are much lower than those found in our controls (0.4400 vs 0.5439). It is likely that a small sample size including only 50 controls, encompassing both normal subjects and patients with Batten disease or fragile-X syndrome instead of randomly chosen unaffected individuals, may have diminished the reliability of allele frequency estimates in the general population, skewing case-control statistics . The definition of A419 as a risk allele for autism would thus not seem fully justified already on the basis of the genetic data set presented in the initial study . Secondly, the present study is the first one including also unaffected siblings, which were not assessed previously . The existence of significant differences in allelic transmission rates between our autistic patients and unaffected siblings does point towards the existence of functional GLO1 gene variants, deserving further characterization in order to understand their protective role in families with an autistic proband. These variants may be represented by the A419 allele itself, but could also consist in polymorphisms located in the transcriptional regulatory regions of the GLO1 gene and in linkage disequilibrium with the C419A SNP. This possibility is consistent with the decreased glyoxalase I enzymatic activity found in post-mortem neocortical tissues of autistic patients, as compared to controls .