etiopathogenesis of autism spectrum disorders: fitting the pieces of the puzzle together

Medical Hypotheses xxx (2013) xxx–xxx

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Medic al Hypo theses

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Etiopathogenesis of autism spectrum disorders: Fitting the pieces of the puzzle together

Ivan Gentile a,⇑, Emanuela Zappulo a, Roberto Militerni b, Antonio Pascotto b, Guglielmo Borgia a,Carmela Bravaccio c

a Department of Clinical Medicine and Surgery, University of Naples ‘‘Federico II’’, Naples, Italy b Department of Mental and Physical Health and Preventive Medicine, Second University of Naples, Naples, Italy c Department of Medical Translational Science, University of Naples ‘‘Federico II’’, Naples, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 19 February 2013 Accepted 1 April 2013 Available online xxxx

0306-9877/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.mehy.2013.04.002

⇑ Corresponding author. Address: Department of C(Ed. 18), University of Naples ‘‘Federico II’’, via S. PanTel.: +39 0817463178; fax: +39 0817463190.

E-mail address: [email protected] (I. Gentile).

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Autism spectrum disorders (ASD) are disorders of the central nervous system characterized by impairments in communication and social reciprocity. Despite thousands of studies on this topic, the etiopathogenesis of these disorders remains unclear, apart from a general belief that they derive from aninteraction between seve ral genes and the environment. Given the mystery surrounding the etiopathogenesis of ASD it is impossible to plan effective preventive and treatment measures. This is of particular concern due to the progressive increase in the prevalence of ASD, which has reached a figure as high as1:88 children in the USA. Here we present data corroboratin g a novel unifying hypothesi s of the etiopathogenesis of ASD. We suggest that ASD are disorders of the immune system that occur in a very early phase of embryonic development. In a background of genetic predisposition and environmental predisposition (probably vitamin D deficiency), an infection (notably a viral infection) could trigger a deranged immune response which, in turn, results in damage to specific areas of the central nervous system. If proven, this hypothesis would have dramatic consequences for strategies aimed at prevent ing and treating ASD. To confirm or refute this hypothesis, we need a novel research approach, which unlike former approaches in this field, examine the major factors implicated in ASD (genetic, infecti ons, vitamin D defi-ciency, immune system deregulation) not separately, but collectively and simultaneously.

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Introduc tion Notwithstandi ng the large body of studies on the subject, the

Autism spectrum disorders (ASD) are behaviorally defined asdevelopmen tal disorders of the central nervous system characterized by impairment s in communication and social reciprocity,complemen ted by limited, repetitive interests and behaviors [1].The ASD category includes various disorders namely: Autistic disorder (also called ‘‘classic’’ autism), Asperger Syndrome, Pervasive Developmen tal Disorder Not Otherwise Specified (or atypical autism), Childhood Disintegrati ve Disorder and Rett Syndrome. Autis- tic disorder (AD) is the most severe form of ASD. Usually abnormalities appear before 3 years of age. In some cases, AD onset is delayed (the so-called ‘‘regressi ve form’’). One of the most puz- zling aspects of ASD is the increase in the number of diagnoses in recent decades [2–6]. In fact, before the 80s, the prevalence was estimated at 1:2,000 children [5], whereas a recent survey indicates dramatically higher figures (1:88 USA children and 1:54 for males) [6], although this increase might merely reflect im- proved diagnost ic means [7].

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linical Medicine and Surgery sini, 5 I-80131 Naples, Italy.

Etiopatho genesis of autis m spe4.002

etiology of ASD is still unknown . Until the etiology of ASD isknown, it will be difficult to identify effective preventive and treatment measures. This challenge has become dramatic given the progressive increase in the prevalence of ASD and their economic and social impact. Current belief holds that they derive generically from a complex interactio n between several genes and the environment [8–10].

Epidemiologica l and neuroanatomic al investigatio ns suggest that the functional alterations typical of ASD have a prenatal orvery early postnatal origin. In particular, it has been suggested that autism arises from a very early embryonic developmen t defect (approximately day 20–24 of gestation), even in cases of the regressiv e form [11–13]. In this context, it is supposed that the complex genes/en vironmental interplay , thought to underlie ASD,is triggered at this early time in life. Exposure of factors such astoxicants , viruses, hormones, and other environmental pollutant sduring pregnancy could cause or contribute to autism onset (seeTable 1).

Here we present a novel unifying hypothesis of ASD that links the data available on this topic. This novel infective-autoim mune hypothes is could open new approaches to the prevention and treatment of this mysterio us disorder.

c trum disorder s: Fittin g the pieces of the puzzle together. Med Hypothe ses

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Table 1Risk factors of ASD.

Advancing parental age Reichenberg A et al., 2006 [156]Croen LA et al., 2007 [157]Reichenberg A et al., 2010 [158]Hultman CM et al., 2011 [159]Alter MD et al., 2011 [160]Sandin S et al., 2012 [161]van Balkom et al., 2012 [162]Puleo CM et al., 2012 [163]

Maternal infections Stagno S et al., 1985 [95]Gregg NM, 1991 [68]Patterson PH, 2002 [63]Shi L et al., 2003 [64]Patterson PH, 2011 [66]Garbett KA et al., 2012 [60]Malkova NV et al., 2012 [65]

Gestational diabetes Gardener H et al., 2009 [164]Krakowiak P et al., 2012 [149]

Teratogen agents Miyazaki K et al., 2005 [165]Arndt TL et al., 2005 [166]Ornoy A, 2009 [167]Rout UK et al., 2009 [168]Dufour-Rainfray D et al., 2011 [169]

Pesticide exposure Sullivan KM, 2008 [170]Shelton JF et al., 2012 [171]

Thyroid functional alterations Román GC, 2007 [172]Sullivan KM, 2009 [173]Hoshiko S et al., 2011 [174]

Folic acid deregulation Beard CM et al., 2011 [175]Al-Farsi YM et al., 2012 [176]Schmidt RJ et al., 2012 [177]

High levels of prenatal testosterone Knickmeyer RC et al., 2006 [178]Auyeung B et al., 2009 [179]Whitehouse AJ et al., 2010 [180]

Prenatal ultrasound exposure CDC, 2007 [144]Olson CD, 2009 [181]Williams EL et al., 2010 [182]Grether JK et al., 2010 [183]

Fever during pregnancy Zerbo O et al., 2013 [110]

Months of conception/birth Gillberg C, 1990 [105]Barak Y et al., 1995 [184]Stevens MC et al., 2000 [185]Zerbo O et al., 2011 [145]

Mercury exposure Garrecht M et al., 2011 [186]Blanchard KS et al., 2011 [187]Kern JK et al., 2012 [188]

Autoimmune diseases Comi et al., 1999 [52]Licinio J et al., 2002 [56]Sweeten et al., 2003 [53]Croen LA et al., 2005 [57]Molloy CA et al., 2006 [54]Mouridsen SE et al., 2007 [59]Atladottir HO et al., 2009 [55]Keil A et al., 2010 [58]

LatitudeHumble MB, 2010 [189]Hoffman K et al., 2012 [190]

Hygiene hypothesis

Becker KG, 2007 [191]

Oxidative stress Ghanizadeh A et al., 2011 [192]Frustaci A et al., 2012 [193]Heberling CA et al., 2012 [194]Rose S et al., 2012 [195]Ghanizadeh A et al., 2012 [196]Chauhan A et al., 2012 [197]

Urban versus rural living Zachor D et al., 2011 [198]Rai D et al., 2012 [199]Zaroff CM et al., 2012 [200]

Living near a highway Volk HE et al., 2011 [201]

EnvironmentHerbert MR, 2010 [202]Becerra TA et al., 2012 [203]

Vitamine D deficiencyCannell JJ, 2008 [143]Grant WB et al., 2009 [204]Meguid NA et al., 2010 [151]Molloy CA et al., 2010 [154]Mostafa GA et al., 2012 [152]Eyles DW et al., 2012 [141]Kocovska E et al., 2012 [142]

Leaky gut syndrome/ gastrointestinal disturbance D’Eufemia P et al., 1996 [49]de Theije A et al., 2011 [51]Louis P, 2012 [205]Benach JL et al., 2012 [206]

Paracetamol (acetaminophen)Torres AR, 2003 [207]Schultz ST et al., 2008 [208]Schultz ST, 2010 [112]Becker KG et al., 2010 [113]Ghanizadeh A, 2012 [111]

Lyme disease Bransfield RC et al., 2008 [209]Kuhn M et al., 2012 [210]Bransfield RC, 2012 [211]

2 I. Gentile et al. / Medical Hypotheses xxx (2013) xxx–xxx

Please cite this article in press as: Gentil e I et al. Etiopat hogenesis of autis m spe(2013), http ://dx.doi.org/10.1016/ j.mehy.20 13.04.002

Factors associate d with ASD

Genetic factors

Epidemiolog ic and family studies strongly suggest that genetic risk factors are involved in ASD [14].Twin studies revealed a heritability of 38–90% [8,15], which indicates a strong genetic component.However , except for Rett syndrome (attributable in most affected individua ls to mutations of the methyl-CpG-bi nding protein 2(MeCP2) gene) the other forms of ASD are not linked to any particular gene [16]. Genome-wid e association studies, genome-wide copy number variation studies, linkage analyses, low-scale genetic association studies, expression profiling and other low-scale exper- imental studies have associate d ASD with as many as 2,193 genes,2,806 single nucleotide polymorphi sms/Variable Number Tandem Repeats, 4,544 copy number variations and 158 linkage regions [14]. However, genetic syndromes, specific mutations, and metabolic diseases account for less than 20% of autistic patients [17].Monogeni c causes are identifiable in a minority of cases [18]. The remaining subjects have other genetic or multigenic causes and/ or epigenetic influences, i.e. environmental factors that alter gene expression without changing the DNA sequence [3,8,19–21].

Non-genetic factors

Several non-genetic factors have been associate d with ASD (see Table 1). The factors listed in Table 1 can be considered



I. Gentile et al. / Medical Hypotheses xxx (2013) xxx–xxx 3

non-mutuall y exclusive agents that, occurring in a similar, very early period of fetal developmen t can cause alteration s of neurons in specific encephali c areas that are associated with the symptoms of patients with ASD.

It is noteworthy that all these factors are organic and not psychological. In fact, studies carried out using magnetic resonance on autistic brains have revealed amygdala–hippocampal complex alterations [22,23] and abnormalities in cortical gray and white matter volume [24–26]. Moreove r most post-mortem studies revealed alteration in the limbic system (increased cell packing den- sity and reduced neuronal size), in the cerebellum (decreasednumber of Purkinje cells) and in the cerebral cortex (microglia den- sity, cortical dysgenesis) of subjects with ASD [27,28]. Finally, the evidence of increased activated microglia cells implicates the immune system in ASD pathogenes is [29].

Immune system and ASD

Immune abnormalities have been reported to play a pathophy s-iological relevant role in ASD [30,31]. Levels of proinflammatory cytokines have been reported to be elevated in patients with ASD [32]. For example, blood levels of IL-6, INF- c, and TNF- a were higher in autistic individuals than in controls [33,34]. Similar results came from the study of brain cytokines IL- 6, TNF- a, INF- c, GM- CSF and IL-8, which points to an active neuroinflammatory process in ASD patients due to activation of microglia and astroglia [35].

It has also been shown that the inflammatory-associate d nuclear factor kappa-light -chain-enhanc er of activated B cells (NF-jB) isupregulated in both blood [36] and brain tissue [37] of autistic individuals.

El-Ansary et al. reported that the plasma levels of HSP70, TGF- b2,Caspase 7 and INF- c levels, which are biochemical parameters ofinflammation, were high in 20 Saudi autistic male patients than in19 age- and gender-matched control samples. This result confirmsthat neuroinflammation and apoptosis mechanism s are involved in the etiology of autism [38]. Siniscalco et al. investigated the activation of caspases in peripher al blood mononuclea r cells (PBMCs) from 15 ASD children compare d to age-matched healthy controls [39]. They found that mRNA levels of caspase-1, -2, -4, -5were significantly higher in ASD children. Given that caspases are proteases involved both in apoptosis and in mediating innate and adaptive immune responses [40,41], these findings suggest that the immune system is involved in ASD. However, as the authors indicate, it is feasible that the activation of caspases could be aprotective response to ASD-related inflammatory stimuli [39].

Also autoantibod ies to myelin basic protein have been identified in serum of patients with ASD [42]. In addition, patients with ASD have been found to have an increase in autoantib odies against the following brain antigens: nerve growth factor [43], brain endo- thelium [44], cerebellar proteins [45], serotonin receptors [46] andtransglutam inase-2, which is crucial for synaptic stabilization [47].One research group [48] observed that monocyte counts and neopterin levels were higher in autistic children than in gender- and age-matche d healthy controls, which again suggests over-activation of the immune system in ASD patients.

Even the ‘‘leaky gut’’ theory of ASD [49,50], according to which the increased gut permeabilit y observed in some ASD patients could allow substances to enter the blood stream and create or exacerbate damage at central nervous system (CNS) level, as well as the gastrointestinal symptoms observed in a subset of patients with ASD may be read as manifestations of a deregulated immune system. In fact, Tand B lymphocyte gut infiltration is reported in ASD [51].

Moreove r, a large study showed that the frequenc y of autoimmune diseases (such as type 1 diabetes, Hashimo to’s thyroidit isand various immunologica l syndromes) was higher in members of families with autistic children, and especially in mothers [52],

Please cite this article in press as: Gentil e I et al. Etiopatho genesis of autis m spe(2013), http ://dx.doi.org/10.1016/ j.mehy.20 13.04.002

than in control subjects. These findings were confirmed by various other groups [53–59]. In particular, Croen et al. [57] showed that maternal psoriasis diagnose d around the time of pregnancy is significantly associated with a subsequent diagnosis of autism in the child. They also reported a 2-fold increase in the risk of having achild with ASD if the mother was diagnosed with asthma or allergies during pregnancy . An association between a family history oftype 1 diabetes mellitus (a typical autoimm une disease) and infantile autism as well as a significant association between maternal histories of either rheumatoid arthritis or celiac disease and ASD has also been reported [55].

All these findings summarized in this section provide evidence of an etiopathologi cal link or a common substrate between autoimmune disorders and ASD.

Infections and ASD

The above data leave no doubt that ASD are organic diseases ofthe CNS in which the immune system plays a central role. The next question is: what causes this immune disturbance? Also infections might play a role in ASD as happens in several typical autoimmune disease.

Studies conducted in animal models

Prenatal infection induces neuronal damage in animal models of ASD [60–66]. In particular , Willette et al. [61] showed that the administ ration of lipopolys accharide (LPS) (a component of some bacterial membranes) to 9 pregnant female rhesus monkeys caused behavior disturbance s and grey/whi te matter distribution anomalie s in several brain areas in offspring compared to controls.Moreove r, LPS-treated monkeys exhibited a higher cellular reactivity to phytohem agglutinin (PHA) in terms of Interleukin 6 production, during the first months of age. Similarly, Nouel et al. [62]showed a reduction of some specific neurons in the hippocamp usof rat offspring when treated with LPS.

Parallel findings were obtained in mice by Garbett et al. who demonst rated that the maternal immune activation triggered byinfluenza virus mimicked the neural damage mechanism s involved in the genesis of ASD or schizophren ia [60]. Similarly, it has been shown that the colonization of the respiratory tract by the human influenza virus in mid-gestation results in behavioral and pharmacological evidence of impairment of fetal brain developmen tcaused by the maternal antiviral immune response [63,64]. Typical autistic behavior was also found in animal models secondary toinfections ; the authors associated result with infection, which could either directly affect the immune system, or trigger an immune reaction [65,66].

Clinical data

Most studies of the role of infections in ASD have examined viral exposure in affected children [67]. Firstly, Gregg [68] associatedcongenit al infection by rubella virus with specific learning disabilities in babies; this is in line with Desmond et al. [69] who suggested that congenit al infection could contribute to the genesis of autism. Stubbs [70] observed that children with autism have an impaired immune response to rubella vaccination . This might indicate a previous congenital infection with this virus. Chess documented that children with congenital rubella syndrome have anincreased risk of autism [71], prospecti ng, more generally, that insome children, viral congenital infections of the CNS could produce the complex typical symptoms of this disorder [72]. Several isolated reports have linked viral infections to ASD, mainly by herpes- viridae, (herpes simplex virus (HSV), cytomegalovir us (CMV),




varicella zoster virus), mumps virus [73], influenza virus, lymphocytic choriomeni ngitis virus [74], or polyomavir uses [75]. In particular, HSV-related encephalitis has been associated with autism.DeLong et al. [76] reported three children who develope d autism after an encephalopath y of unknown etiology and, in one case, secondary to HSV encephalitis. It should be emphasized that these children had symptom s of autism at an older age compared with the typical onset of the disease, and that resolution of the acute infection coincided with regression of autistic symptoms. Other cases of autism as a result of encephalitis have been reported inadolescents [77,78]. Ghaziuddin et al. reported two patients who had probably been infected in utero or shortly after birth with HSV and presented herpetic encephalitis [79]; both had received a diagnosis of autism according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (Third Edition-Revise d),before the age of 3 years. Indeed herpes viruses can trigger the production of a variety of proinflammatory cytokine s during the acute phase [80–82], as demonstrat ed by high levels of interfero n inbrain tissue during HSV encephali tis [83]. The herpes encephalitis/autism association is also supported by the observation that autism is associated with neuropatho logical alteration s of the temporal lobes [84], which are typical targets of HSV encephalitis [77–79]. A significantly higher prevalence of recent infections, witnessed by enhanced Immunoglobuli n M against HSV2 has been reported in autistic children compare d with healthy controls (65% versus 17.5%) [85]. Moreover, anti-brain antibodie s were significantly more prevalen t in autistic individuals infected with HSV- 2 than in autistic subjects without HSV-2 infection (96% vs. 43%)[85]. This finding suggests a link between HSV-2 infection and autoimmuni ty in subjects with ASD. Several case reports have linked congenital CMV infection to ASD [86–96]. Ivarsson et al. reported two cases of maternal infection with CMV that was followed by congenital infections in two offspring who later received a diagnosis of autism [88]. Kitajima and co-worke rs described the appearance of AD following congenit al CMV infection,witnessed by the occurrence of symptom atic infection at birth,high viremia (CMV–DNA = 6.0 � 106 copies/mL) and diffuse involvement of the placenta, with virus isolation at decidua villi and amnion level [97]. Similar results were obtained by Dogan et al. who reported intracranial brain anomalie s, identified byultrasound examination, and subsequent autism onset, in children with prenatal CMV infection confirmed by the identification of viral genome in amniotic fluid by polymerase chain reaction (PCR)[98]. Hence, several members of the family of herpes viruses are implicated in the pathogenes is of ASD. Interestingl y, in this context, an Italian study assessed the prevalen ce of neurotropic viruses in post-mortem brains of autistic patients compared to controls. The authors found a significant association between the presence of polyomavir us genome (by nested- PCR followed by DNA sequence analysis) and ASD [75].

In contrast with the above-rep orted studies, some cohort studies involving subjects with ASD did not find a significant association between specific infections and pervasive developmental disorders [99–102]. In particular , Anlar et al. [100] found no relation between intrauterine infection by human parvovirus and autism. Similarly , Jorgensen et al. did not observe a link between serum levels of anti-HSV antibodies and AD [99]. Furthermore, nei- ther murine leukemia virus nor xenotropic murine leukemia virus- related virus genomes was found in autopsy examination or inpaternal gametes of patients with ASD [101,102]. One of the features linking ASD and infections is seasonality. Several studies have documented the occurrence of seasonal variations in births of children with autism [103–107]. This suggested that events that occur with a seasonal pattern such as pandemic virus infections may play a role in the etiology of autism. Epidemiolog ical studies conducted in Israel revealed a positive correlation between the rate of viral


meningit is and encephalitis [108] and measles in the general population and the risk of autism [109].

In addition, as some viruses can induce one or more reactiva- tions after the primary infection (notable components of the herpesvirus family), one may speculate that pregnancy-me diated immunosup pression can be associated with a reactivation of avirus that can directly or indirectly trigger one or several mechanisms involved in ASD (see later).

A large robust epidemiolog ical study revealed a significant link between fever during pregnancy and an enhanced risk of autism in the offspring [110]. Also the association between paracetam oluse and autistic disorders may be interpreted as a link between infections occurring in pregnancy and ASD, given that paracetam olis largely used as an antipyreti c drug [111–113]. Moreover, it has been shown that mothers of autistic children have a higher rate ofatopy compared to a control population [57]. This finding may itself be associated with a viral reactivation as this phenomeno n has been described for the herpes virus HHV-6 [114]. Alternatively , the association between the mother’s allergy and ASD may be viewed in the light of a general deregulation of the immune system.

The specific mechanism s by which acute or chronic viral infections may lead to autism are still matter of much debate and spec- ulation. It has been suggested that specific viral infections that occur in a susceptible host at an early stage of pregnancy [67] couldmodify the normal developmen t and differentiation of neural net- works and systems that control behavioral , cognitive, and learning processes (which are specifically impaired in ASD) thereby inducing an altered immune response against neural cells [67,115]. However,how can an infection determine such a complex disorder?

Possible mechani sms linking infections and ASD

As shown in the previous sections, the appearan ce of ASD may be associate d with early exposure to a specific virus. The resulting infection, even subclinical, may interfere with key neurogen etic processes and determine functional neurologi cal deficits. Specifi-cally, viral infection could (i) directly exert a neurotoxic effect;(ii) its neurotoxic effect could be mediated by the immune system [80–82,116].

Direct neurotoxic effect of viral infections

Pregnancy is a well-known immunosuppre ssive condition that prevents rejection of the fetus. However this physiological condition renders women more vulnerable to infections [117,118 ]. Inaddition, the CNS is not fully developed at birth [119] and therefore it is more susceptible to infection-in duced damage [120]. In fact,resistance to viral infections of the CNS is age-relat ed as shown by the progressive reduction of the occurrence of viral encephalitis with age in animal models [121]. It has been suggested that viral encephali tis may directly destroy brain cells and thus determine the onset of ASD. This is supported by reports of cases of autism associate d with post-nata l varicella encephalitis [122] and Stealth Virus Encephalopath y [123].

Based on the foregoing, CNS infections by specific viruses during prenatal developmen t or early postnatal age could induce the developmen t of an ASD in genetically susceptible individuals.

Immune mediated effects of viral infections

As mentioned previously, a viral infection could induce neurological damage by eliciting deregulation of the immune system,thereby leading to autoimm une disturbances. But what is the mechanis m of action of viral infection? Two possible hypothes esare ‘‘molecular mimicry’’ and ‘‘bystand er activation’’ [124].




Molecular mimicry occurs when an immune cell recognizes a viral peptide that resembles a self-pept ide. This determines an immune response which is directed both at the virus and to human tissues that express the cross-rea ctive antigen. Bystander activation is the expansion of an immune response directed at tissues altered byinflammation induced by a viral infection [125,126]. In detail, avirus-induc ed autoimmune phenomenon could affect the developing brain thus generating anatomic abnormalities of neural con- nections [127,128], through the creation of ‘‘nicks’’ or subtle alterations in the myelin sheath [128–130]. Such events may lead to lifelong impairments of higher brain functions such as speech,language, communi cation, and social interaction.

Alternativel y, a viral infection may determine a transient sys- temic increase in the levels of pro-inflammatory cytokine s without viral persistence (‘‘hit-and-go’’ mechanism) or result in the production of chronically elevated levels of several inflammatory cytokines due to viral persisten ce [67]. These cytokines may beproduced directly in the brain or gain access to the CNS through an immature blood–brain barrier [67]. Abnormal levels of cytokines can alter the developmen t of the CNS [116]. In this light,many patients with ASD manifest elements of immune system deregulation , which could indicate an unresolved viral infection contracted before birth [131,132 ]. In other words, infections may trigger a pathological autoimm une response [133–135].

It is noteworthy that these events may occur also in the mother’s immune system. It has been suggested that the mother’s immune system can attack the fetus and cause abnormal neurological development [67]. In support of this theory, Warren et al. [136]found that mothers of children with autism have an increased antibody reactivity against their children’s lymphocytes. Dalton et al.[137] identified antibodies capable of opsonizing the Purkinje cells and other rodent neurons in the serum of a mother of an autistic child. When the serum was injected into pregnant mice, the offspring showed impaired motor coordination and cerebellar abnormalities detected by magnetic resonance spectroscopy compared to controls [137].

However , the next question is: what are the intimate mechanisms that link an infection to these immune system deregula tions in genetically-p redisposed children? And more importantly, how can we explain the increased incidence of ASD that has occurred in recent decades considering that infections are decreasing worldwide and that genomic variabilit y cannot occur in so short atime?

Vitamin D deficiency and ASD

The previous question may be formulated differently: what medical conditions have increased tremendous ly during the last few decades that could link infections, immune deregulation and ASD? One possible answer is vitamin D deficiency [138]. Vitamin D is poorly present in food and it is mainly synthesized byultraviolet B radiation exposure which is able to convert 7-dehydro-c holesterol present in the skin, to pre-vitamin D which,via a spontaneou s isomerizatio n process, turns into vitamin D.Vitamin D is rapidly hydroxylated to 25(OH)D , the circulating form of vitamin D. The kidney further hydroxylates 25(OH)D into 1,25(OH)2 D. This compound is a key factor in maintaining serum calcium levels at physiologica l levels.

The prevalen ce of vitamin D deficiency has increased greatly during recent decades due to life style modifications (workingand living in closed rooms and not in open spaces) and to the increased use of solar filters that reduce vitamin D production bythe skin. This phenomeno n is even more pronounced in pregnant women, to whom a strict avoidance of sun is often recomme nded [138].


However, how can vitamin D be the link between infections and immune deregulation? Indeed, the effects of vitamin D (and its active forms) are not limited to the classic regulatory pathway of calcium metabolism. In fact, vitamin D is also a molecular switch that activates more than 200 target genes, and it is also involved in con- trolling the immune response [124]. In detail, vitamin D is thought to be capable of modulating immune cells, such as monocytes,macroph ages, T-lymphocy tes and B-lymphocy tes [139]. In a typical autoimm une disease such as type 1 diabetes, a study carried out in Finland showed that children who regularly receive adequate supplementati on of vitamin D have a significantly lower risk of diabetes mellitus compared to those who did not take adequate supplem entation [140]. Diabetes is classically associated with viral infection and with genetic predispos ition. Vitamin D deficiencyand an infectious agent (probably a herpesvirus ) have been implicated even in multiple sclerosis [124]. In fact, patients with multiple sclerosis have low serum levels of vitamin D [124]. Both the diabetes and multiple sclerosis are more frequent at higher latitudes, which is in agreement with a lower ultraviolet exposure and therefore a lower vitamin D concentratio n of these populations.

The mechanism proposed in both diseases is that vitamin Ddeficiency causes an exaggerated immune response to an infectious agent. In other words, vitamin D deficiency in a genetically predispos ed person is associated with deregulation of the immune system, creating what is called, to borrow a military term, ‘‘friendly fire’’, i.e. inadvertent firing on one’s own forces while attempti ng tofight enemies. In fact, vitamin D is known to be a modulator of the immune system, and a key factor in establishing and maintain ing self-toler ance [124,139]. Moreover, it has been suggested that vitamin D deficiency alone could be associated with a number of psychiatric and neurodegen erative condition s [141]. In fact, vitamin Ddeficiency in early life affects neuronal differentiation , axonal connectivity , and therefore brain structure and function [141,142]. But apart from these speculative considerations, do we have data indi- cating that vitamin D deficiency is involved in ASD?

Several epidemio logical studies hint at a link between vitamin D and ASD [143,144]. As indicated above, various studies have revealed a positive correlation between latitude and ASD prevalence.In other words, countries at northern latitudes (which are associated with lower ultraviolet exposure and therefore lower vitamin D production) have a higher rate of ASD compared to southern countries . Moreove r, there is an association between the month of conception and the risk of ASD [143]. According to a recent large robust study, children conceived in summer (when vitamin D levels are classically higher) have a lower risk of developing autism than those conceived in winter [145]. These findings may be linked to the higher levels of vitamin D produced during the warm sea- sons. Other studies linked location with the risk of autism. They found a significantly higher rate of ASD in urban populations versus rural populations [146]. This fact, together with the findingof a higher rate of ASD related to air pollution [147] again implicates vitamin D deficiency in ASD. In fact, air pollution, which ismore frequent in urban areas, dramatically reduces ultraviolet penetrati on and finally vitamin D production [148].

Another clue to a link between vitamin D deficiency and ASD isthe well-establi shed association between the mother’s metabolic status and autism [149]; indeed, obese individuals have an increased risk of vitamin D deficiency [138]. Finally, also dark skin has been associated with autism [143,150]. This is again in agreement with a deficiency of vitamin D which is more frequent indark-skin ned people due to the presence of an ever-present sun- screen (melanin) in the skin of these subjects [138].

However, are vitamin D levels lower in autistic children than inhealthy children?

A study conducte d in Egypt showed that patients with autism have significantly lower levels of vitamin D compared to a group



Fig. 1. Novel unifying ethiopathogenetic hypothesis of ASD. ___: indicates a key factor; —: indicates a less important factor.


of healthy age-matche d controls [151]. Similarly, Mostafa et al. reported significantly lower serum levels of 25-OH vitamin D in 50autistic children than in 30 age- and sex-matche d apparently healthy children [152]. Serum 25-hydroxy vitamin D level was negatively correlated with the Childhood Autism Rating Scale [153]. Increased levels of serum anti-MAG auto-antibodies were found in 70% of autistic patients. Interestingl y, serum 25-hydroxy vitamin D levels were negatively correlated with serum levels ofanti-MAG auto-antibo dies, i.e. subjects with lower vitamin D levels had higher titers of anti-MAG autoantibodies [152]. However,other researchers found similar low levels of vitamin D in subjects with ASD and healthy age-matche d controls [154].

Finally, an intriguing aspect of ASD is the prevalen ce of male gender over female gender, the ratio between men and women being 4.7:1 [6]. Again, one may evoke vitamin D deficiency in this phenomeno n. In fact, estrogen, which is high in women, is able toincrease neural 1,25(OH)2 D and therefore it acts as a shield to vitamin D deficiency, unlike testosterone, which makes male brains susceptible to vitamin D deficiency [143,155].

Unifying hypothe sis and conclusi ons

Autism spectrum disorder can be considered a disorder of immune deregulation which, in a very early stage of brain development, causes neurological damage in one or more areas of the brain. From the data described above, ASD probably results from a complex interplay between genetic predisposition (several genes may increase the risk) and environmental predisposition (notablyvitamin D deficiency). An infection or a reactivation (notably a viral infection) probably triggers this deranged immune response. Inother words, the model we propose is that in embryon ic development, an infection or reactivation of the mother (e.g. due to allergy-mediated reactivation or to pregnancy-induce d immuno- deficiency) triggers an autoimm une disorder. Of course, only aminority of infections trigger the immunolog ical and inflammatorycascade of events leading to neurologi cal damage. This may occur ifa genetic predisposition is present and/or if a ‘‘environmenta l’’


predispos ition is present. Several studies identify this ‘‘environmental’’ predisposition as vitamin D deficiency. In fact, vitamin Dplays a role in modulating the immune response to prevent anexaggerated response that could damage host tissues. However,ASD are complex disorders and it is likely that other factors (suchas toxins) may play a role as secondary factors in modulating the severity of the disorder. Autism spectrum disorders should beviewed as a continuum of disorders that differ in severity rather than distinct diseases. The major or minor involvement of one ormore brain areas is probably responsible for the huge symptom variabilit y observed in affected individuals.

Finally, it is conceiva ble that, among these 3 factors (genetic,infections , and vitamin D deficiency), just one alone may beresponsib le for few cases of ASD but it seems that the role of each is potentiated when they act simultaneously in a single person (seeFig. 1). This hypothesis, if proven, would have dramatic prophyla c-tic and therapeutic consequences. For example, the early diagnosis of an infection would lead to the implementati on of preventive (e.g. vaccine) or therapeutic measures (e.g. antivirals); the diagnosis of vitamin D deficiency in pregnancy may be corrected through adequate supplementation, etc. Finally and importantl y, studies aimed at clarifying the etiology of ASD should take into account four major aspects (genetic, infections, vitamin D deficiency, immune system deregulation) together and simultaneously , and not examine them separately, in order to obtain robust data that can confirm or refute the proposed hypothesis.

Conflicts of interest

Each author certifies that he or she has no commercial associations that might pose a conflict of interest in connection with the submitte d article.

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etiopathogenesis of autism spectrum disorders: fitting the pieces of the puzzle together

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