Autism Spectrum Disorder (ASD) in a Developing country. A Clinical and Descriptive Study of 75 Infant and Young Patients

Autism spectrum disorder (ASD) involves a complex interplay
of both genetic and environmental risk factors, with immune
alterations and synaptic connection deficiency in early life.


Introduction
Autism spectrum disorder (ASD) involves a complex interplay of both genetic and environmental risk factors, with immune alterations and synaptic connection deficiency in early life. In the past decade, studies of ASD have substantially increased, in both humans and animal models. Immunological imbalance (including autoimmunity) has been proposed as a major etiological component in ASD, taking into account increased levels of pro-inflammatory cytokines observed in postmortem brain from patients, as well as autoantibody production [1]. Evidence indicates an increased offspring vulnerability to ASD through advanced maternal and paternal age, valproate intake, toxic chemical exposure, maternal diabetes, enhanced steroidogenic activity, immune activation, and multifactorial [4]. An associations between early developmental exposure to ambient pesticides and autism spectrum disorder has been recently reported [5,6]. Etiology of ASD likely involves environmental factors triggering physiological abnormalities in genetically sensitive individuals. One of these major physiological abnormalities is mitochondrial dysfunction, which may affect a significant subset of children with ASD. Co-occurring medical disorders and associated physiological abnormalities in individuals with autism spectrum disorder (ASD) show strongest associations with epilepsy, sleep, as well as gastrointestinal and immune functioning [7]. Individuals with autism spectrum disorder (ASD) exhibit severe difficulties in social interaction, motor coordination, behavioral flexibility, and atypical sensory processing, with considerable interindividual variability [8].
Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is clinically defined by abnormalities in reciprocal social and communicative behaviors and an inflexible adherence to routinised patterns of thought and behavior. Laboratory studies repeatedly demonstrate that autistic individuals experience difficulties in recognizing and understanding the emotional expressions of others and naturalistic observations show that they use such expressions infrequently and inappropriately to regulate social exchanges [9]. In a previous work we have reported the maternal stress and the relationship with hyperactivity and attention deficit (ADHD) in ASD patients [10]. In the present study we have carried out a clinical and descriptive study of autism spectrum disorder (ASD), and the involved risk factors and comorbidities in a developing country facing a severe and critical sociopolitical situation. We emphasize the maternal pathological processes during pregnancy, an underestimated pathology thus far as an essential risk factor for ASD, mainly in developing countries facing sociopolitical crisis.

Material and Methods
We have examined 75

Tactile Hypersensitivity and GABA Concentration in the Sensorimotor Cortex
In the present study we have found tactile hypersensitivity in a 10% of patients examined typically characterized by either over-or under-responsiveness to stimulation. Impaired responses to tactile stimulation are a commonly reported symptom among children with autism spectrum disorder (ASD) suggesting a functional deficit in the somatosensory inhibitory system in autism [13]. Puts et al. According to Puts et al. (2017) and Sapey-Triomphe et al. [14].
tactile hypersensitivity is correlated with reduced GABA levels measured by magnetic resonance spectroscopy in brain areas processing touch, such as sensorimotor cortex.

Hyperconnected ASD Children (Asperger syndrome)
In the present study we have found 10% ASD children with higher functioning activity. Asperger's syndrome seems to be considerably more common than "classic" autism. The syndrome is more prevalent in boys than in girls. The clinical characteristics of Asperger's syndrome are probably influenced by many factors, including organic and genetic factors [15] . In addition, impaired integrative cognitive processing such as social cognition or executive function, restricted interest, and compulsive repetition of the same act are observed in savant individuals. All these are significantly relevant to the behavioral characteristics observed in individuals with autistic spectrum disorders (ASD).
A neurocognitive model of savant syndrome should explain these cognitive features and the juxtaposition of outstanding talents with cognitive disabilities [20]. According to these Authors, the disruption of connectivity between the prefrontal cortex and other regions is considered to be a particularly important factor because the prefrontal region shows the most influential inhibitory control on other cortical areas. We propose that these neural mechanisms as the underlying causes for the emergence of savant ability in ASD.

ASD and Psychomotor Delay
We have found psychomotor delay in 5% of ASD patients. In our studied we have found the followings diseases during mother pregnancy: urinary infections, behavioral disturbances like anxiety, fobias, hyperactivity, toxoplasmosis and Zika virus infections, viral hepatitis, viral hepatitis, viral hepatitis, hyperemesis, oligohydramnios and lost of amniotic fluid, twin pregnancy, pre-eclampsia, aging placenta, cesarean surgery, high blood pressure, maternal sepsis, diabetes, hepatic coma, hypothyroidism, obesity, and social parenteral problems, such as excessive work, low economy and poor social conditions, environmental contamination, labor and conjugal stress. Such maternal pathology could be considered as risk and/or etiological factors in ASD children. We should keep in mind that this maternal pathology is frequently observed in developing countries, mainly in those facing sociopolitical crisis.

Language Disorders in ASD and Sensory Processes Alterations
In the present study we have found language disorders in a 5% of patients studied, such as delay in the onset of language or early vocalization, regressive changes of language, mutism, gestural, escatologic and digital language. Sensory processing alterations are highly prevalent in autism spectrum disorder (ASD). Neurobiologically-based theories of ASD propose that abnormalities in the processing of temporal aspects of sensory input could underlie core symptoms of ASD. For example, rapid auditory temporal processing is critical for speech perception, There is significant clinical heterogeneity in language and communication abilities of individuals with autism spectrum disorders (ASD). However, no consistent pathology regarding the relationship of these abilities to brain structure has emerged [25].
Lo et al. [26] hypothesized that the dual stream language network is altered in autism, and that this alteration could be revealed by changes in the relationships between microstructural integrity and functional activation. Microstructural integrity of the left dorsal and left ventral pathways responsible for language processing and the functional activation of the connected brain regions were studied in youths with high-functioning autism. The altered structurefunction relationships in autism suggest possible involvement of the dual pathways in supporting deficient semantic processing.

Sensory and Multisensory Function in Autism Spectrum Disorder
We found in ASD children neurosensory disorders, such as Neurobiological research that has been conducted has pointed toward dysfunction in the excitation/inhibition balance of the central nervous system of those with ASD. Future endeavors into understanding tactile processing differences in ASD will greatly benefit from controlled experiments driven by neurobiological

Learning and Memory Deficit in ASD
We have found 10% learning and memory deficit in the ASD

Aggressivity and Autoagressivity in ASD
In the present study we found 10% of ASD patients with

ASD and Sleep Disorders
We have found sleep disorders in a 5% of ASD patients. Liu et al.
[38] examined sleep patterns, sleep problems, and their correlates in children with autism spectrum disorders, and found ASD patients with bedtime resistance, insomnia, parasomnias, sleep disordered breathing, morning rise problems, and with daytime sleepiness.
Their results showed that both dyssomnias and parasomnias are very prevalent in children with ASD. Allik et al. [39] reported sleep patterns in school-age in children with Asperger syndrome or highfunctioning autism.

ASD and Hyperexcitability and Attention Deficit (ADHD)
We have found 10% ASD patients with hyperactivity and According to these Authors, children with typical BECTS may have an increased risk of suffering from symptoms of ASD and ADHD.
Children with late onset of seizures may be more likely to develop neuropsychological disturbances regarding ASD and ADHD.

ASD and Mood Disorders
In the present study we have reported mood disorders in 3% of ASD patients examined. We have found autistic patients with anxiety, separation anxiety, fobias, impulsivity, and desafiant At the present time there is a substantial uncertainty regarding the extent and nature of autism spectrum disorder (ASD) and bipolar affective disorder (BPAD) co-occurrence, due to disparate findings in previous studies [56,57].

ASD and Epilepsy
We have found partial or focal convulsive syndromes and febrile seizures in 3% of ASD patients. Canitano [60], the association between autism spectrum disorder (ASD) and epilepsy is well-known.

Abnormalities on electroencephalography (EEG) results
have been reported in patients with ASD without a history of seizures. However, little is known about the relationship between abnormalities on EEG results and the core features of ASD. El Achkar and Spence [61]. consider that the established risk factors Milovanovic et al. [62] 2019 conclude that epilepsy, as well as subclinical epileptic discharges, showed small effects on motor skills in patients with autism, and had no effect on adaptive behavior, and communication/socialization/daily living skills. Tuchman [63] analyses the relationship of autism spectrum disorder and epilepsy and considers that the association of epilepsy and autism spectrum disorders (ASD) is best understood by examining the relationship between social cognition, nonsocial cognition, and epilepsy. The relationship between ASD and epilepsy is bidirectional and is strongly linked to intellectual disability (ID). The risk of developing ASD in children with epilepsy is highest in children with early onset seizures, with a high prevalence in children with infantile spasms. The risk of developing epilepsy in children first diagnosed with ASD is highest in those with ID. The prevalence of seizures in ASD increases with age. When epilepsy and ASD coexist, they share common pathophysiological mechanisms A high prevalence of epilepsy and interictal epileptiform discharges (IED) have been recently reported among children with ASD [62,64]. Loussouarn et al. [65] emphasized the high prevalence of EEG abnormalities in children with ASD without epilepsy, and that EEG should only be performed at epilepsy onset, and more precisely when a clinical interview has confirmed that repetitive paroxysmal events could be seizures. Pacheva et al. [66] postulate that in the occurrence of epilepsy among children with ASD we should define the type of epileptic seizures and syndromes, the age of onset of epilepsy, EEG abnormalities, the used antiepileptic drugs and the therapeutic responses for seizures and autistic behavior, as well as to find some correlations between epilepsy and gender, etiology and intellectual disability (ID).

Males and Females
In our study we have reported exhibiting ASD disconnected child prevalent in males than females, but the underlying neurobiology of this sex bias remains unclear. According to Smith et al. [67], given its involvement in ASD, its role in sensorimotor, cognitive, and socio-affective processes, and its developmental sensitivity to sex hormones, and the cerebellum are candidates for understanding this sex difference. These Authors used restingstate functional magnetic resonance imaging (fMRI) to investigate sex-dependent differences in cortico-cerebellar organization in ASD. Post hoc tests revealed a pattern of cortico-cerebellar hyperconnectivity in ASD females and a pattern of hypoconnectivity in ASD males. Furthermore, cortico-cerebellar FC in females more closely resembled that of control males than that of control females. In relationship among gastrointestinal symptoms, problem behaviors, and internalizing symptoms in children and adolescents with autism spectrum disorder, the majority of patients experienced constipation, about half experienced stomachaches or stomach pain, and others experienced nausea or diarrhea. Young children with aggressive problem behaviors were 11.2% more likely to have co-occurring nausea; whereas, older children showed more complex relationships between internalizing symptoms and GI symptoms [69].

Autism and Intelligent Fathers
Some parents of the patients examined in the present study are highly educated and intelligent. Original case descriptions of autism noted that parents of the affected children tended to be highly educated and intelligent. Recent genetic studies indicate that risk for autism spectrum disorders (ASD) is associated with high intelligence. The relationship between paternal IQ and offspring risk of ASD was non-monotonic and varied by the presence of co-occurring disorders, probably reflecting phenotypic diversity among affected individuals [73].

Maternal Infections and Diseases During Pregnancy
We have above described the numerous pathological conditions of mothers during pregnancy considered as risks factor such as urinary infections, behavioral disturbances like anxiety and fobias, hyperactivity and attention deficit, toxoplasma and Zika virus infections, hyperemesis, oligohydramnios and loss of amniotic fluid, twin pregnancy pre-eclampsia, aging placenta, cesarean surgery, high blood pressure, maternal sepsis, diabetes and hepatic coma, hipotiroidism, viral hepatitis, obesity, and social problems, such as excessive work, low economy and poor conditions environmental contamination, labor and conjugal stress. According to Ornoy et al. with anti-β2GP1 IgG antibodies and thyroid disease with antithyroid peroxidase (TPO) antibodies, preeclampsia and some other autoimmune diseases with IgG antibodies that might affect fetal brain development. Other related factors are maternal infections (rubella and CMV with fetal brain injuries, and possibly Influenza with fever, prolonged fever and maternal inflammation, especially with changes in a variety of inflammatory cytokines and antibodies that cross the placenta and affect the fetal brain.
Langrigde et al. [76] included hypertension, asthma, urinary tract infection, some types of ante-partum haemorrhage, any type of preterm birth, elective C-sections, breech presentation, poor fetal growth and need for resuscitation at birth, with all factors showing an increased risk. These findings support most of pathological entities on maternal pregnancy described in the present study.
In the present study we found some mother with Zika virus infection during pregnancy. Vianna et al. [77]. suggest a mechanism encompassing neurological and immunological pathways that

Immune Dysfunction in ASD
We have herein reported allergic diseases, such as bronchial asthma and atopic dermatitis (3%) in ASD patients. Dysregulation in immune responses during pregnancy increases the risk of a having a child with an autism spectrum disorder (ASD). Asthma is one of the most common chronic diseases among pregnant women, and symptoms often worsen during pregnancy [80]. Two main

Finally, an important portion of individuals diagnosed with ASD
has elevated peripheral cytokines and chemokines and associated neuroinflammation [85]. Scientific research studies emerging within the past two decades suggest that immune dysfunction and inflammation have pathogenic influences through different mechanisms, all leading to both a chronic state of low grade inflammation, and alterations in the central nervous system and immune response, respectively [86]. Neuro-inflammation and neuro-immune abnormalities have now been established in ASD as key factors in its development and maintenance [87].
Inflammation in the brain and CNS has been reported by several groups with notable microglia activation and increased cytokine production in postmortem brain specimens of young and old individuals with ASD [88]. Further, it is the heterogeneity within this disorder that has brought to light much of the research emerging within the past two decades suggests that immune dysfunction is a viable risk factor contributing to the neurodevelopmental deficits observed in autism spectrum disorders (ASD). During gestation, prenatal insults including maternal infection and subsequent immunological activation may increase the risk of autism in the child. Similarly, the presence of maternally derived anti-brain autoantibodies found in ~20% of mothers whose children are at risk for developing autism has defined an additional subphenotype of ASD [89].
According to Ahmad et al. [90], dysregulation of T cell According to Gata-Garcia and Diamond [91], these evidences support a contribution of maternal brain-reactive antibodies to neurodevelopmental disorders. An interplay between antibodies, genetics, and other environmental factors is likely to determine the specific neurodevelopmental phenotypes and their severity. These results strongly support the importance of anti-neuronal and glial protein autoantibodies biomarker in screening for ASD children and further confirm the importance of the involvement of the maternal immune system as an index that should be considered in fetal in utero environmental exposures. Mast cell activation by allergic, infectious, environmental and stress-related triggers, especially perinatally, would release pro-inflammatory and neurotoxic molecules. We speculate these could disrupt the gut-blood-brain barriers, thus contributing to brain inflammation and ASD pathogenesis. Increased mast cell responsiveness may define at least a subgroup of ASD subjects, who could benefit from inhibition of mast cell activation [93].
Autoantibodies against brain epitopes in mothers of children with ASD and many such children strongly correlate with allergic symptoms and indicate an aberrant immune response, as well as disruption of the blood-brain barrier (BBB). Recent epidemiological studies have shown a strong statistical correlation between risk for ASD and either maternal or infantile atopic diseases, such as asthma, eczema, food allergies and food intolerance, all of which involve activation of mast cells (MCs). These unique tissue immune cells are located perivascularly in all tissues, including the thalamus and hypothalamus, which regulate emotions. MC-derived inflammatory and vasoactive mediators increase BBB permeability [94][95][96].
Mast cells (MCs) are located perivascularly close to neurons and microglia, primarily in the leptomeninges, thalamus, hypothalamus and especially the median eminence.
Corticotropin-releasing factor (CRF) is secreted from the hypothalamus under stress and, together with neurotensin (NT), can stimulate brain MCs to release inflammatory and neurotoxic mediators that disrupt the blood-brain barrier (BBB), stimulate microglia and cause focal inflammation. CRF and NT synergistically stimulate MCs and increase vascular permeability; these peptides can also induce each other's surface receptors on MCs leading to autocrine and paracrine effects. As a result, brain MCs may be involved in the pathogenesis of "brain fog," headaches, and autism spectrum disorders (ASDs), which worsen with stress. CRF and NT are significantly increased in serum of ASD children compared to normotypic controls further strengthening their role in the pathogenesis of autism [97].

The Genetic Architecture of ASD
The genetic architecture of ASD has become increasingly clear and increasingly complex with estimates of at least 1000 genetic alterations associated with the risk for ASD [4]. In the last 10 years, there have been significant advances in understanding the genetic basis for ASD, critically supported through the establishment of ASD bio-collections and application in research. Collectively, these include mapping ASD candidate genes, assessing the nature and frequency of gene mutations and their association with ASD clinical subgroups, insights into related molecular pathways such as the synapses, chromatin remodelling, transcription and ASD-related brain regions [98]. Multiple lines of evidence from genetic linkage studies to animal models implicate aberrant cortical plasticity and metaplasticity in the pathophysiology of autism spectrum disorder (ASD) and fragile X syndrome (FXS) [99].
Data from whole-genome screens in multiplex families suggest interactions of at least 10 genes in the causation of autism. Thus far, a putative speech and language region at 7q31-q33 seems most strongly linked to autism, with linkages to multiple other loci under investigation. Cytogenetic abnormalities at the 15q11-q13 locus are fairly frequent in people with autism, and a "chromosome 15 phenotype" was described in individuals with chromosome 15 duplications. Among other candidate genes are the FOXP2, RAY1/ ST7, IMMP2L, and RELN genes at 7q22-q33 and the GABA(A) receptor subunit and UBE3A genes on chromosome 15q11-q13 [100].
Prior structural MRI studies demonstrated atypical gray matter characteristics in siblings of individuals with autism spectrum disorder (ASD). However, they did not clarify which aspect of gray matter is related to the endophenotype (i.e. genetic vulnerability) of ASD. This proof-of-concept study suggests that an ASD endophenotype emerges in sulcal depth SD and that neural bases for ASD diagnosis can be discerned from the endophenotype when accounted for the difference between TD siblings [101].

Mitochondrial Dysfunction in Children with ASD
The different clinical symptoms found in ASD patients as observed in the present study suggest the dysfunction of a cell energy organelle as mitochondria. Rose et al. [6]. further, cortical thickness differences were observed to be greater in younger individuals and in those with sex, age and full-scale intelligence (FIQ) lower and to be related to neuroanatomical heterogeneity and overall clinical severity. [104].

Neuropathological Findings and Brain Overgrowth in ASD
Neuropathological studies appear to have identified common abnormalities in the cerebellum and limbic system, marked Purkinje cell loss in the cerebellar hemispheres, together with retained fetal neuronal circuitry in cerebellar nuclei and increased neuronal packing in specific regions of the limbic system, amygdala, and hippocampus [105] The most replicated neuroanatomical finding in autism is the tendency toward brain overgrowth, especially in younger children. Research shows that both gray and white matter are enlarged. Proposed mechanisms underlying brain enlargement include abnormal inflammatory and neurotrophic signals that lead to excessive, aberrant dendritic connectivity via disrupted pruning and cell adhesion, and enlargement of white matter due to excessive gliogenesis and increased myelination. Amyloid-β protein precursor (βAPP) and its metabolites, more commonly associated with Alzheimer's disease (AD), are also dysregulated in autism plasma and brain tissue samples [106].

Synaptopathology Involved in Autism Spectrum Disorder
Multiple studies have revealed that mutations in genes like NRXN,

Some Neurochemical Research in ASD
Pavăl [109] has proposed a dopamine hypothesis of autism spectrum disorder. that autistic behavior arises from dysfunctions in the midbrain dopaminergic system and that a dysfunction of the mesocorticolimbic circuit leads to social deficits, while a dysfunction of the nigrostriatal circuit leads to stereotyped behaviors. This hypothesis is based clinical studies of dopamine antagonists which seem to have improving effects on autistic behavior. The 5-HT transporter (SERT) gene (SLC6A4) has been associated with whole blood 5-HT levels and ASD susceptibility. strong support for the hypothesis that altered 5-HT homeostasis can impact risk for ASD traits and provide a model with construct and face validity that can support further analysis of ASD mechanisms and potentially novel treatments [110].
Elevated whole blood serotonin, or hyperserotonemia, was the first biomarker identified in autism spectrum disorder (ASD) and is present in more than 25% of affected children. The serotonin system is a logical candidate for involvement in ASD due to its pleiotropic role across multiple brain systems both dynamically and across development Emerging data from both neuroimaging and postmortem samples also indicate changes in the brain serotonin system in ASD. Genetic linkage and association studies of both whole blood serotonin levels and of ASD risk point to the chromosomal region containing the serotonin transporter (SERT) gene in males but not in females. In ASD families with evidence of linkage to this region, multiple rare SERT amino acid variants lead to a convergent increase in serotonin uptake in cell models [111].
Children with ASD tend to have higher free radicals than antioxidants compared with their matched controls. This disequilibrium of oxidative stress has been associated with the pathogenesis of this neurocognitive disorder [112]. Gilbert et al. [113] formulate the hypothesis that there may be a link between cholesterol, vitamin D, and steroid hormones which subsequently impacts on the development of at least some of patients with ASD.
Our hypothesis, posits that there may be links between cholesterol metabolism, which we will refer to as "steroid metabolism"