Because early detection of autism is linked to significantly improved outcomes, the discovery of early predictors could make all the difference in a child’s development.
Dr. Ray Bahado-Singh, a geneticist and Chair of Obstetrics and Gynecology for Beaumont Health and the Oakland University William Beaumont School of Medicine and his research team, identified key biomarkers for predicting autism in newborns.
The preliminary, collaborative study used Artificial Intelligence, a computer-based technology which scans a map of the human genome.
The team’s findings could lead to an accessible, standardized newborn screening tool which uses a simple blood test, Dr. Bahado-Singh said, enabling earlier intervention, reducing disability and improving outcomes.
The project compared DNA from 14 known cases of autism to 10 control cases and featured researchers from the Oakland University William Beaumont School of Medicine, Albion College and the University of Nebraska Medical Center.
Results appeared in the journal Brain Research.
“Compared to what is currently available, these findings provide a more direct method which could be employed earlier on, shortly after birth,” Dr. Bahado-Singh said. “It’s been shown that children who are treated earlier do better in life.”
Symptoms of autism include sensory processing difficulties, anxiety, irritability, sleep dysfunction, seizures and gastrointestinal disorders.
According to Autism Speaks, nearly half of 25-year-olds diagnosed with autism have never held a paying job.
In the United States, the majority of costs associated with autism are for adult services – an estimated $175 to $196 billion a year, compared to $61 to $66 billion a year for children.
The team’s findings could lead to an accessible, standardized newborn screening tool which uses a simple blood test, Dr. Bahado-Singh said, enabling earlier intervention, reducing disability and improving outcomes.
Although the American Academy of Pediatrics recommends all children be screened between 18-24 months, children in large portions of the U.S. do not receive recommended clinical screenings.
Lori Warner, Ph.D., director of the Ted Lindsay Foundation HOPE Center which treats children with autism at Beaumont Children’s called the findings optimistic.
“We are always looking for new ways to make a difference in the lives of our patients,” Dr. Warner said. “Getting them into therapy early on is a proven way to make their path, and that of their families, easier and more meaningful.”
Dr. David Aughton, Genetics Chief for Beaumont Children’s, said he looks forward to additional, larger follow-up studies.
“Although it has been thought for many years that the underlying cause of a significant proportion of autism is likely to be nongenetic in nature, this study takes a very pragmatic and important first step toward investigating the epigenome — the inheritable changes in gene expression — and identifying those underlying nongenetic influences.
The authors call for larger follow-up studies to validate their findings, and I eagerly look forward to learning the outcome of those validation studies.”
Autism spectrum disorder (ASD), which appears in the first years of life, is associated with abnormalities such as buccal sensory sensitivity, taste and texture aversions, speech apraxia and changes in salivary ribonucleic acid expression [1–9].
It has been estimated that one in 59 American children is affected by ASD, and there has been a marked increase in its incidence and prevalence over the last decades [10]. There are a number of co-occurring pathologies in ASD (Table 1).
Early clinical interventions can improve symptom trajectory, but do not completely abrogate ASD symptoms, and pharmacologic interventions are limited.
One novel avenue for diagnostic and therapeutic research is the emerging association between ASD and oral bacteria communities [1,11]. This review will discuss the possible relationship between oral bacteria and the biologic and symptomologic aspects of ASD, focusing particularly on the clinical implications for diagnostic and therapeutic development.
Table 1.
Co-occurring diseases in ASD (from ref [29]).
| Brain-related comorbidities |
|---|
| Altered metabolite profile in urine and blood |
| Fragile X syndrome, Rett syndrome and tuberous sclerosis |
| Mitochondrial dysfunction |
| Gut-related co-morbidities |
| Gastrointestinal symptoms |
| Increased permeability of the intestinal epithelial barrier |
| Decreased expression of brush-border disaccharides in the intestinal epithelium |
| Other co-morbidities |
| Altered expression of tight junction protein in the BBL |
| Increased amounts of activated microglia cells |
Dental problems in ASD
Children with autism can have multiple medical and behavioral problems that make adequate oral hygiene and dental treatment difficult to perform.
In a study of 61 children with ASD, aged 6–16 years (45 males and 16 females), higher caries prevalence, poor oral hygiene and extensive unmet needs for dental treatment compared to controls without autism were reported [12].
This could promote dissipation of oral bacteria to the circulation and potentially the brain [13], initiated by widespread dental plaque-induced diseases such as caries and gingivitis/periodontitis [14–16].
Studies on oral bacteria in ASD
Qiao et al. [11] used high throughput sequencing to compare the oral microbiota in children with ASD to healthy controls (Table 2).
Approximately 1 ml of non-stimulated, naturally outflowed saliva was first collected. Then, supragingival plaques were obtained separately from caries-free molars in four quadrants (upper right, upper left, lower right and lower left) per subject. The 111 samples were divided into four groups: 1) salivary samples from healthy controls (HS; n = 27); 2) dental samples from healthy controls (HP; n = 26); 3) salivary samples from ASD patients (AS; n = 32); 4) dental samples from ASD patients (AP; n = 26).
The transcriptional activity of the salivary and dental microbiota in ASD patients differed markedly from that of healthy children. In children with ASD, a lower bacterial diversity was demonstrated than in controls, consistent with findings from the gut [17,18]. This finding was particularly pronounced in dental plaque samples.
The genera Haemophilus in saliva and Streptococcus in dental plaque were significantly more abundant in ASD whereas Prevotella, Selenomonas, Actinomyces, Porphyromonas and Fusobacterium were reduced. A depletion of the Prevotellaceae family co-occurrence network was also detected in plaque from ASD patients. In saliva, no phylotypes were highly correlated with decayed, missing, filled teeth or surfaces (DMFT/S). In dental plaque, however, six phylotypes including Streptococcus, Actinomyces and Capnocytophaga were positively associated with DMFT/S. Accordingly, presence of dental caries was more related to the microbiota of dental plaque than to that of saliva.
Aggregatibacter segnis (OTU220) was positively associated with bleeding on probing, gingival index and periodontitis. The bacterial patterns observed in individuals with ASD suggested a possible role for microorganisms in this disorder, but did not establish a causal relationship. The results also suggested that aversion of ASD patients to dental hygiene interventions might be one mechanism for oral dysbiosis.
Table 2.
Clinical trials performed on the oral microbiota in children with autism spectrum disorder (ASD).
| Authors/ref | Age (yrs) | Method | Groups | Results and Conclusions |
|---|---|---|---|---|
| Hicks et al. [1] | 2–6 | RNA extraction & Shotgun sequencingGenetic activity of oral microbiota were examined | ASD (n = 180)DD (n = 60)TD (n = 106) | 12 taxa were altered between groups28 taxa distinguished ASD patients with and without GI disturbances5 microbial ratios distinguished ASD from TD3 microbial ratios distinguished ASD from DDGI microbe disruption in ASD extended to pharynxOral microbiome profiling has a potential to evaluate ASD status |
| Qiao et al. [11] | 7–14 | High throughput sequencing | ASD (n = 32)Controls (n = 27) | Salivary and dental microbiota distinct from those of controlsLower bacterial diversity in ASD than in controls, especially in dental samplesHaemophilus (saliva) and Streptococcus (plaques) were significantly higher in ASD, while commensals like Prevotella, Selenomonas, Actinomyces, Porphyromonas and Fusobacterium were reducedDepletion of Prevotellaceae co-occurrence network detected in ASD (dental plaque)Distinguishable bacteria were correlated with clinical indices (disease severity and oral health status; dental caries)Diagnostic models based on key microbes constructed with 96.3% accuracy in salivaThe habitat-specific profile of the oral microbiota in ASD may help diagnosis of ASD |
ASD = autism spectrum disorder; DD = developmental delay; TD = typically developing
In a second study [1], changes in the salivary microbiome of children 2–6 years old were identified across three developmental profiles: ASD (n = 180), non-ASD with development delay (DD; n = 60) and typically developing (TD; n = 106) children (Table 2).
Actively transcribing taxa were quantified and tested for differences between the groups and within ASD endophenotypes. Between the developmental groups, 12 bacterial taxa differed. Of particular note, 28 taxa were distinctly active among ASD patients with gastrointestinal (GI) disturbance.
By group classification, five microbial ratios distinguished ASD from TD children (79.5% accuracy), three separated ASD from DD (76.5% accuracy) and three identified ASD children with GI disturbance from ASD peers without GI comorbidities (85.7% accuracy). There were significant differences in microbial transcription of energy metabolism and lysine degradation pathways across the ASD, TD and DD groups.
The results indicated that GI microbial disruption in ASD likely extends to the oropharynx. Given the largely unidirectional transit of bacteria from the oropharynx to the lower GI tract, this implies that oral dysbiosis may actually serve as a primary source for a portion of the fecal dysbiosis reported in numerous ASD studies [19–21].
Oral microbiota affecting the intestine
Studies in animals and humans have demonstrated that oral bacteria can be transferred to the gut, changing its microbial composition and perhaps even host immune responses [22–24]. Oral bacteria and stool bacteria overlapped in almost half (45%) of the subjects in the Human Microbiome Project [25].
The ectopic transfer of oral bacteria has also been reported in patients with systemic diseases, such as inflammatory bowel disease [26]. Co-occurring GI problems are common in children with ASD [27]. GI symptoms were four times more prevalent in children with ASD than in children with typical development [28].
The GI symptoms seen in individuals with autism can include constipation, diarrhea, bloating, abdominal pain, reflux, vomiting, gaseousness and foul-smelling stools (for a review see [29]). Such symptoms may be related to the lower bacterial diversity reported in children with ASD [17].
Ectopic transfer of oral bacteria can occur in patients with localized ‘chronic’ periodontitis. Porphyromonas gingivalis, which is proposed as a keystone bacterium in this disease [30,31], causes dysbiosis in the periodontal microbiota.
This may lead to microbial dysregulation in the gut since each day 108–1010 of P. gingivalis can be swallowed [32,33]. Changes in the gut microbiota composition could induce permeability of the gut barrier and immune activation leading to systemic inflammation.
Ectopic colonization of oral bacteria in the intestines has been found to drive T-helper (TH)-1 cell induction and inflammation [22]. In one case study, Klebsiella spp. isolated from the saliva of a patient with inflammatory bowel disease were marked inducers of TH-1 cells.
Ongoing colonization by oral bacteria was suggested to perpetuate gut microbiota dysbiosis and chronic inflammation. In this setting, the oral cavity can serve as a reservoir for potential intestinal pathobionts that aggravate intestinal disease. Wang et al. [27] and Ashwood [34] have also reported abnormalities in intestinal immunity in children with ASD.
Dysbiosis of the intestinal microbiota
Dysbiosis of the intestinal microbiota is an emerging etiological factor proposed for ASD [17,29,35–40]. The GI microbiome is thought to influence host behavior and neurodevelopment through the ‘microbial-gut-brain axis’ [41,42].
Imbalance in the intestinal microbiota or its metabolites may affect several complex behaviors (such as emotional and anxiety-like behaviors), and influence brain development or modulate cognition [11,43–45].
A microbiota-gut-brain axis is based on a bidirectional physiologic connection where information between the host microbiome, gut and brain are exchanged [46]. This likely involves cross talk between the central nervous system and microbes within the GI tract through direct neural activation, immune modulation, and hormonal, peptidergic and epigenetic signaling [47–50]. Below, we consider how each of these factors may be translated to an ‘oral-brain axis’
Oral microbiota and the brain
How oral microbiota may reach the brain
There are several plausible pathways for bacteria in the mouth to reach the brain and directly influence neuro-immune activity and inflammation [51] (Figure 1).
Even routine dental procedures can cause bacteremia [52], and a portion of these microbes may traverse the blood–brain barrier (BBB). Altered transcript expression has been described in microglia of ASD individuals, and disrupted microglia function could impair BBB integrity [53].
Increased permeability of the BBB has been described in children with ASD [54]. This could expose the brain to bacterial metabolites, thereby triggering an inflammatory response and altering metabolic activity within the central nervous system [29].
Prolonged disruption of energy metabolism within neurons, oligodendrocytes and glia could lead to structural changes in the cortex, hippocampus, amygdala or cerebellum, which have all been documented in ASD individuals [29].
Figure 1.
Direct and indirect mechanisms of infecting the brain. In the direct mechanism, the oral cavity infects the olfactory tract, and the olfactory nerve transfer the bacteria to the brain. In other mechanisms, bacteria inside the mouth infect the blood and find their way via blood, blood–brain barrier (BBB), perivascular spaces and circumventricular organs to the brain (figure is based on concepts presented in ref [51] and collected from ref [55]).
How oral microbiota may affect the brain: inflammation
Central nervous system inflammation has been a prominent feature in studies of both animal models and post-mortem brains from individuals with ASD. For example, a study by Morgan and colleagues described the up-regulation of microglia in the ASD brain [56].
Cytokines and chemokines are also elevated in the cerebrospinal fluid of ASD patients [57,58]. Moreover, genes associated with immune and inflammatory responses are activated in the ASD cortex [59].
There appears to be a general dysregulation of the immune system towards a pro-inflammatory phenotype in ASD individuals [58,60]. Such inflammation in the developing brain may lead to synapse malfunction [61].
A significant reduction of both synaptic transmission and excitability has been observed when hypoxia and inflammation occur in combination, whereas re-oxygenation leads to neuronal hyper-excitability [62].
Malfunctioning synapses may cause the release of vasopressin, which has been shown to affect social behavior [61]. Interestingly, induction of inflammation early in gestation may promote an ASD-like phenotype through increased synaptic excitation [58] (Figure 2).
In this process, early life exposure to inflammation might prime microglial cells to become hyper-responsive to subsequent insults [63]. Notably, chronic application of periodontal pathogens in mice have resulted in the development of neuropathological changes consistent with Alzheimer’s disease (a condition in which cortical inflammation is a decisive factor) [64].
Oral bacteria reaching the brain could reduce the anti-oxidative capacity and lead to reduction in the ability of mitochondria to produce energy in ASD individuals [65]. Gram-negative, putative periodontal pathogens, are rich in lipopolysaccharide (LPS) which has pro-inflammatory activity. Leakage of LPS through the BBB in ASD individuals could lead to inflammation in the central nervous system. Furthermore, increased levels of LPS in individuals with autism have been found to correlate with high levels of IL-6, a pro-inflammatory cytokine [66].
How oral microbiota may affect the brain: metabolic alterations
Microbial communities have a significant impact on metabolism within the human GI tract [67,68]. Thus, oral dysbiosis in ASD could lead to disruptions in the metabolome – a putative mechanism for ASD pathogenesis [69–71].
There are indications that increases in acetate and propionate, as well as decreases in butyrate (short-chain fatty acids of bacterial origin), can be involved in the development of ASD together with indoles [29] (Figure 3). There are also increased levels of 3-(3-hydroxyphenyl)-3-hydroxypropionic acid, 3-hydroxyphenylacetic acid and 3-hydroxyhippuric acid in children with ASD, which together indicate potential perturbations in the phenylalanine metabolism [72].
These metabolites are related to the abundance of Clostridium spp. and associated with aggravated restricted and repetitive behaviors in children. The high abundance of intestinal Clostridium detected in ASD may reflect a pathogenic role for these particular organisms [73].
Whether metabolic changes result from the oral microbiota composition in children with ASD remains to be determined. However, a study of oral microbe transcription across 346 children (including 180 with ASD) identified ASD-specific changes in pathways involving lysine degradation – a precursor to the neurotransmitter glutamate, that has been implicated in ASD pathogenesis [1].
By using saliva samples from this same cohort, the authors also described ASD-specific alterations in human microRNA expression that were associated with microbial activity, and implicated in cell growth and metabolism pathways [1]. Such findings provide a framework for human–microbial interaction at the biochemical level that may have functional consequences for host behavior.
Proposed mechanisms for development of ASD.
Cause–effect relationship between ASD and microbes?
Although numerous studies have identified microbial disruptions in patients with ASD and linked those disruptions to symptoms and behavior, we still do not fully understand the mechanism by which microbial communities are dysregulated in individuals with ASD. Furthermore, it is unclear if the microbial patterns described in individuals with ASD cause ASD symptoms, or result from behaviors common to the ASD phenotype.
Phenotype
Microbial dysbiosis may be influenced by the ASD phenotype. This could occur through resistance to dental hygiene, lack of a varied diet, and placing objects into the mouth as sensory seeking behavior. Discontinuation of oral hygiene in 29 orally healthy individuals for 4, 7 and 10 days, and assessment 14 days after resumption of oral hygiene, was associated with a significant increase in relative abundance of potential cariogenic Leptotrichia species and a decrease in Streptococcus species [74].
This study demonstrated the importance of regular oral hygiene on the maintenance of oral homeostasis. Furthermore, dental caries can be caused by ecological imbalance of commensal microbiota (mainly due to lack of a varied diet, such as frequent carbohydrate consumption) [75]. Placing foreign objects (e.g. toys, dirt, etc.) in the mouth is yet another source of dysbiosis, because these objects can be contaminated with microorganisms from unwashed hands in contact with other human body fluids [76].
Discerning the importance of ASD phenotype as a modulator of the oral and intestinal microbiota will likely require parallel studies in both humans and animal models. One potential strategy to help elucidate the cause/effect dilemma involving microbial disruption and ASD, is to establish ASD in a gnotobiotic animal and examine the potential of major members of the oral and intestinal microbiota (for example, oral P. gingivalis and Klebsiella spp. [22,77], and intestinal Clostridium spp. [17]) to induce ASD symptoms. Furthermore, promising pilot studies on microbiota transfer therapy (i.e. fecal transfer) should be extended to include double-blinded placebo controlled trials with well-defined a-priori hypotheses for functional outcome measures. Studies carefully examining the interaction between probiotic therapies (e.g. Bifidobacterium) and antibiotic therapies (e.g. vancomycin, minocycline) may also provide useful information about whether microbial modulation can alter ASD behaviors [78,79].
Environmental and genetic factors
Although most cases of ASD are idiopathic [17], both environmental and genetic factors are likely important for ASD development [80,81]. Exposure to environmental risk factors or genetic risk transmission can affect the maternal microbiome [21].
Offspring acquires a large portion of their microbiome from mothers during the birth process. Whether birth occurs via the vaginal canal or by cesarean section significantly affects the infant’s microbiome [82–84]. Thus, delivery mode might play a role in certain neurodevelopmental disorders. To date, studies examining the relationship between ASD and cesarean sections have demonstrated mixed results [85–87].
Changes in the microbiome due to stress might also be transferred to offspring during birth, initiating microbial dysbiosis that lasts into adulthood [88–90]. It has been reported that early life exposures to plastics and other chemicals can affect the infant microbiota [21].
Disentangling the relationship between these exposures, microbiome profiles and developmental trajectories is a difficult task that will require careful, comprehensive data collection, and powerful statistical models that can account for the interplay of many different environmental factors.
Source:
Beaumont Health
Media Contacts:
Maryanne MacLeod – Beaumont Health
