Connective-Related Symptoms and Neurodevelopmental Disorders: An Exploratory Case-Control Comparative Study in Children

1. Introduction

Autistic Spectrum Disorders (ASD), Attention Deficit/Hyperactivity Disorder (ADHD), and Tourette Syndrome (TS) are neurodevelopmental disorders (NDD) that typically emerge during childhood and persist throughout life. These conditions manifest in diverse ways, leading to impairments in personal, social, academic, and occupational functioning [1,2]. The aetiopathogenesis of these disorders is thought to involve a complex interplay of genetic, epigenetic, and environmental factors. Extensive research into the heritability of NDDs suggests that a substantial proportion of phenotypic variance can be attributed to genetic influences [3,4]. Notably, certain genes associated with ASD have also been implicated in ADHD and TS [4].

Although ASD, ADHD, and TS are traditionally viewed as distinct conditions, differences in the timing of abnormal neurodevelopment and, more importantly, the neural circuits involved distinguish these disorders [5]. However, emerging evidence indicates that they may share partially overlapping genetic factors [4,6]. Specific genes and genetic regions have been identified that appear to contribute to a shared genetic predisposition for ASD, ADHD, and TS [4]. This genetic overlap is further supported by the significant symptomatological similarities among these disorders, which include attentional difficulties, impulsivity, repetitive or ritualistic behaviours, deficits in communication and social interaction, obsessive traits, anxiety disorders, and abnormalities in sensory processing [2,4,7].

Moreover, ASD, ADHD, and TS seem to exhibit overlapping neuropathological mechanisms. These disorders share features such as long-range underconnectivity and short-range overconnectivity in brain networks, reflecting similar patterns of neural dysfunction [8]. Consequently, it has been hypothesised that ASD, ADHD, and TS may represent a spectrum of related neurodevelopmental disorders, collectively termed "abnormal connectivity spectrum disorders” [9].

A broader perspective on the central and peripheral connectivity alterations characterising NDDs has been proposed through the "Connectivome Theory" [10]. This theory underscores the role of connective tissue in various organs, highlighting its multifunctional properties, including structural support, connection, nourishment, regulation, and modulation among different cellular elements.

The primary objective of this study is to examine the prevalence of pathologies associated with connective tissue alterations in individuals diagnosed with ASD, ADHD, or TS and to compare these findings with those from a healthy control group. A secondary aim is to evaluate whether these symptoms are observed at a significant rate among the parents of affected individuals.

2. Materials and Methods

The questionnaire (see Appendix), designed to investigate symptoms associated with connective tissue disorders, was administered to the families of 120 individuals diagnosed with NDD, specifically ASD, ADHD, and TS, as well as to a control group attending the Child and Adolescent Neuropsychiatry Outpatient Clinics at the University Hospital of Verona, Italy. The questionnaire gathered sociodemographic data and assessed the presence of 10 symptom categories in both the patients and their parents. These categories included:

Striae rubrae, skin irregularities, or redness;

Excessive sweating of the hands and feet;

Back pain, transient muscle pain in the limbs, or chronic fatigue;

Hip dysplasia, scoliosis, or kyphosis;

Flat feet;

Constipation, diarrhoea, or alternating bowel patterns;

Heartburn, gastroesophageal reflux, or hiatal hernia;

Use of orthodontic appliances;

Tactile, visual, auditory, olfactory, or gustatory hypersensitivity;

Myopia or drooping eyelids, including unilateral or bilateral ptosis.

The same questionnaire was also administered to a control group comprising 44 families with typically developing, healthy children attending the clinic. The data collection period spanned from December 2019 to January 2022. Parents or caregivers actively participated in the study by responding to structured questionnaires, contributing to the data acquisition.

The study was approved by the Ethical Committee of the University Hospital of Verona under the following codes: CESC 2243 (Paediatric Clinic, University Hospital of Verona) and CESC 2242 (Child and Adolescent Neuropsychiatry Outpatient Clinics, University Hospital of Verona). Written informed consent was obtained from each participant's parents.

Statistical Analysis

Continuous Gaussian variables were summarised as mean values with standard deviations (S.D.). Categorical variables were described using counts and percentages.

The presence of symptoms was compared across controls, TS, ASD, and ADHD groups using logistic regression models. In these models, the dependent variable was the presence of the symptom, while case-control status served as the independent variable. Age and sex were included as covariates to adjust the estimates.

To address issues of separation and potential convergence difficulties caused by the low prevalence of certain symptoms, Firth's penalised maximum likelihood method was employed in the logistic regression analyses [11,12].

Statistical analyses were conducted using the STATA 16.0 software package (www.stata.com). A significance threshold of 5% was applied for all statistical tests.

3. Results

A total of 164 participants were included in the study. Of these, 44 (26.8%) were healthy controls, while the remaining 120 (73.2%) were cases: 48 (29.3%) with ASD, 36 (22.0%) with ADHD, and 36 (22.0%) with TS. The mean age of the cases was 10.1 years (S.D.: 3.6), compared to 9.5 years (S.D.: 2.5) for the controls.

The control group was evenly distributed by sex, with 22 males (50.0%) and 22 females (50.0%). In contrast, most cases were male, comprising 110 participants (91.7%).

The analysis presented in Table 2 demonstrates that flat feet are significantly more prevalent among individuals with ASD (52.1%), ADHD (52.8%), and TS (38.9%) compared to the control group (13.6%), indicating a strong association with the NDD under investigation. Heartburn, gastro-oesophageal reflux, and hiatus hernia are notably more frequent in individuals with TS (27.8%) relative to the other groups. Similarly, the use of orthodontic appliances is more common in subjects with TS (41.7%) compared to the other cohorts. Hypersensitivity is markedly more prevalent in individuals with ASD (56.3%), ADHD (50.0%), and TS (58.3%) compared to controls (18.2%), further supporting a strong correlation with the NDD included in the study. Additionally, myopia and ptosis are observed more frequently in individuals with ADHD (30.6%) compared to the other groups (Table 1, Figure 1).

The analysis in Table 2 indicates that individuals with ASD exhibit a lower tendency to experience back pain, transient muscle pain in the limbs, and chronic fatigue (OR 0.25; 95% CI: 0.053–1.16; p = 0.076). The data also reveal a strong association between flat feet and various NDD, including ADHD (OR 6.73; 95% CI: 2.097–21.63; p = 0.001), ASD (OR 7.20; 95% CI: 2.438–21.23; p < 0.001), and TS (OR 3.70; 95% CI: 1.107–12.34; p = 0.034). Additionally, there is a potential trend towards increased use of orthodontic appliances in TS (OR 3.20; 95% CI: 0.887–11.51; p = 0.076).

A significant association between hypersensitivity and NDD was also identified. Individuals with hypersensitivity demonstrate a higher propensity for ADHD (OR 4.11; 95% CI: 1.385–12.19; p = 0.011), ASD (OR 5.90; 95% CI: 2.160–16.12; p = 0.001), and TS (OR 5.35; 95% CI: 1.738–16.47; p = 0.003), confirming a robust association between hypersensitivity and these disorders. Finally, the analysis revealed a significant association between myopia or ptosis and ADHD (OR 13.12; 95% CI: 1.859–92.56; p = 0.01).

Table 3. The table presents a comparative analysis of the percentage distribution of symptoms among all patients, their fathers, and their mothers, alongside the statistical significance of the observed associations.

Symptoms		Overall patients with NDD		Father’s patients		Mother’s patients		Statistical analysis
		Total n. (ASD+ADHD + TS)	%	n.	%	.	%	O.R.	p-value	p-overall
Totals (n.)		120
Striae roubre / Skin irregularities / Reddened skin	absent	109	90.8	15	13.8	7	6.4	2.44	0.206	0.330
	present	11	9.2	5	45.5	0	0.0
Excessive sweating of hands and feet	absent	97	80.8	11	11.3	10	10.3	1.49	0.480	0.299
	present	23	19.2	4	17.4	2	8.7
Mal di schiena / dolori muscolari transitori agli arti / stanchezza cronica	absent	104	86.7	28	26.9	21	20.2	1.7	0.341	0.353
	present	16	13.3	6	100.0	5	31.6
Back pain / transient muscle pain in the limbs / chronic fatigue	absent	110	91.7	21	19.1	19	17.3	2.08	0.283	0.862
	present	10	8.3	3	30.0	3	30.0
Flat foot	absent	62	51.7	5	8.1	4	4.5	8.3	<0.001	<0.001
	present	58	48.3	18	31.0	18	31.0
Constipation/diarrhoea / abdominal discomfort	absent	103	85.8	23	22.3	8	7.8	3.01	0.041	0.302
	present	17	14.2	8	47.1	4	23.5
Heartburn / gastroesophageal reflux / hiatal hernia	absent	101	84.2	33	32.7	25	24.8	1.68	0.344	0.389
	present	19	15.8	9	47.4	6	31.6
Heartburn / gastroesophageal reflux / hiatal hernia	absent	84	70.0	23	27.4	10	11.9	1.48	0.372	0.109
	present	36	30.0	12	33.3	10	27.8
Tactile, visual, auditory, olfactory or gustatory hyper sensoriality	absent	54	45.0	8	14.8	3	5.6	3.31	0.006	<0.001
	present	66	55.0	24	36.4	10	15.2
Myopia or drooping eyelids / unilateral or bilateral eyelid ptosis	absent	100	83.3	28	28.0	26	26.0	1.27	0.635	0.059
	present	20	16,7	9	45,0	6	30,0

Legend: ASD, Autism Spectrum Disorder; ADHD, Attention Deficit Hyperactivity Disorder; NDD, neurodevelopmental disorders; O.R, odds ratio; TS, Tourette Syndrome.

Table 3. The table presents a comparative analysis of the percentage distribution of symptoms among all patients, their fathers, and their mothers, alongside the statistical significance of the observed associations.

Symptoms		Overall patients with NDD		Father’s patients		Mother’s patients		Statistical analysis
		Total n. (ASD+ADHD + TS)	%	n.	%	.	%	O.R.	p-value	p-overall
Totals (n.)		120
Striae roubre / Skin irregularities / Reddened skin	absent	109	90.8	15	13.8	7	6.4	2.44	0.206	0.330
	present	11	9.2	5	45.5	0	0.0
Excessive sweating of hands and feet	absent	97	80.8	11	11.3	10	10.3	1.49	0.480	0.299
	present	23	19.2	4	17.4	2	8.7
Mal di schiena / dolori muscolari transitori agli arti / stanchezza cronica	absent	104	86.7	28	26.9	21	20.2	1.7	0.341	0.353
	present	16	13.3	6	100.0	5	31.6
Back pain / transient muscle pain in the limbs / chronic fatigue	absent	110	91.7	21	19.1	19	17.3	2.08	0.283	0.862
	present	10	8.3	3	30.0	3	30.0
Flat foot	absent	62	51.7	5	8.1	4	4.5	8.3	<0.001	<0.001
	present	58	48.3	18	31.0	18	31.0
Constipation/diarrhoea / abdominal discomfort	absent	103	85.8	23	22.3	8	7.8	3.01	0.041	0.302
	present	17	14.2	8	47.1	4	23.5
Heartburn / gastroesophageal reflux / hiatal hernia	absent	101	84.2	33	32.7	25	24.8	1.68	0.344	0.389
	present	19	15.8	9	47.4	6	31.6
Heartburn / gastroesophageal reflux / hiatal hernia	absent	84	70.0	23	27.4	10	11.9	1.48	0.372	0.109
	present	36	30.0	12	33.3	10	27.8
Tactile, visual, auditory, olfactory or gustatory hyper sensoriality	absent	54	45.0	8	14.8	3	5.6	3.31	0.006	<0.001
	present	66	55.0	24	36.4	10	15.2
Myopia or drooping eyelids / unilateral or bilateral eyelid ptosis	absent	100	83.3	28	28.0	26	26.0	1.27	0.635	0.059
	present	20	16,7	9	45,0	6	30,0

Legend: ASD, Autism Spectrum Disorder; ADHD, Attention Deficit Hyperactivity Disorder; NDD, neurodevelopmental disorders; O.R, odds ratio; TS, Tourette Syndrome.

Given that both flat feet and hypersensitivity were found to be more prevalent in each NDD under investigation, we performed a familial analysis across the entire cohort of parents. Flat feet were common (OR 8.3) among fathers (31.0%), mothers (31.0%), and patients (43.8%; p < 0.001). The symptom of tactile, visual, auditory, olfactory, or gustatory hypersensitivity was more frequent (OR 3.31) among fathers (36.4%) compared to mothers (15.2%), and it was associated with a high prevalence of the symptom in patients (55%; p = 0.006).

4. Discussion

In our study, we identified a strong association between flat feet, hypersensitivity, and the neuropsychiatric conditions of ASD, ADHD, and TS. The use of orthodontic appliances was more frequent in individuals with TS, and myopia or ptosis was notably prevalent in those with ADHD. Additionally, flat feet and hypersensitivity were more common among the parents affected by the NDD under investigation.

Specifically, flat feet were notably more prevalent in individuals with ASD (52.1%), ADHD (52.8%), and TS (38.9%) compared to the control group (13.6%). The aetiology of flat feet can be either congenital or acquired. However, determining the precise cause of paediatric flat feet is complex, as it may occur in isolation or as part of a broader syndrome [13]. For example, certain congenital disorders, such as Ehlers-Danlos syndrome, can present with both flat feet and NDDs due to underlying connective tissue abnormalities [14]. In connective tissues, elastic fibres play a crucial role in the extracellular matrix, contributing to the elasticity and resilience of tissues. These fibres give connective tissues their elasticity and resilience [15]. Although no direct evidence links flat feet to alterations in the extracellular matrix, the structural integrity of the foot is mainly dependent on connective tissues, primarily composed of extracellular matrix components.

In our study, we observed that hypersensitivity was significantly more prevalent in individuals with TS (58.3%; OR 5.35, 95% CI 1.738-16.47), ASD (56.3%; OR 5.9, 95% CI 2.160-16.12), and ADHD (50.0%; OR 4.11, 95% CI 1.385-12.19) compared to controls (18.2%). In the context of ASD, hypersensitivity refers to an increased sensitivity to stimuli such as sound, light, touch, taste, and smell. Research has suggested that this heightened sensitivity is associated with abnormalities in glial cells, which may disrupt the development of the myelin sheath, leading to delays in response times [16,17]. There is a substantial body of literature supporting the association between sensory alterations and ASD [10] [18,19,20]. Tactile and auditory hypersensitivity has been identified as a predictor for an ASD diagnosis [21]. Moreover, connective tissue plays a crucial role in the structure and function of the outer, middle, and inner ear and the central auditory pathways.

In our study, myopia and ptosis were more prevalent in subjects with ADHD (30.6%; OR 13.12, 95% CI 1.859-92.56) compared to those with TS (13.9%), ASD (8.3%), and controls (2.3%). Previous studies have not identified a significant association between myopia and ptosis [22]. However, individuals with ASD often exhibit defects in oculomotor activity and the pupillary sphincter response to the light reflex [23] and frequently display refractive deficits [24]. The most common ocular issues in ASD include difficulties with eye alignment (convergence insufficiency) and refractive errors [20]. The connective tissue in the eyes plays a crucial role in supporting the blood vessels and nerves that comprise the retina [25]. Additionally, the corneal-scleral framework primarily comprises connective tissue [25,26].

The use of orthodontic appliances was more common in individuals with TS (41.7%) compared to other groups and controls (OR 3.20, 95% CI 0.887-11.51). Orthodontic appliances (oral orthotics, occlusal splints) have been investigated as potential treatments for reducing tics in individuals with TS. Several studies have explored using customised oral splints and dental orthodontic devices to manage tics [27]. In recent years, reports have suggested that dental orthodontic devices, typically used for treating temporomandibular joint (TMJ) disorders, may also effectively reduce tics when worn by individuals with TS [28].

Family history is strongly associated with the risk of ASD. The individual risk of ASD increases with closer genetic relationships [3]. A reanalysis of a previous study on the familial risk of ASD estimated that 83% of the risk could be attributed to genetic factors, suggesting that genetics play a pivotal role in the development of ASD [29]. In the familial analysis of our study, which included patients diagnosed with ASD, ADHD, and TS, flat feet were observed frequently in fathers (31%), mothers (31%), and patients (48.3%). Sensory hypersensitivity, which was commonly observed in patients (55%), was more prevalent in fathers (24%) compared to mothers (10%). Myopia and ptosis, which were present in 16.7% of the patients, were more frequent in fathers (45%) than in mothers (30%).

In the context of ASD, ADHD, and TS, abnormalities in connective tissue may represent a shared underlying factor. This hypothesis is supported by emerging research that underscores the interconnectedness of these NDD through physical manifestations [30]. In ASD, ADHD, and TS, abnormalities in connective tissue may represent a shared underlying factor. This hypothesis is supported by emerging research that highlights the interconnectedness of these NDD through physical manifestations [31].

Several limitations may affect the validity and generalisability of the present study. The small sample size for each NDD subgroup and the gender imbalance may introduce bias in the results. Clinic-based recruitment could lead to selection bias, and the control group may not represent the general population. Additionally, reliance on self-reported questionnaires without evaluating their reliability and validity may result in recall bias. The subjective nature of some symptoms also lacks clinical verification. The use of multiple testing increases the risk of false positives. The study does not account for all potential confounders, and its cross-sectional design prevents the establishment of causal inferences. Finally, while the familial analysis suggests a genetic component, it is limited by the absence of genetic testing.

5. Conclusions

In conclusion, the study identified significant associations between certain NDDs and physical symptoms, such as flat feet and sensory hypersensitivity. These symptoms were also frequently observed in parents, suggesting a strong familial component. This finding implies that overlapping aetiological factors may manifest in various ways, with connective tissue abnormalities potentially serving as a common underlying factor. Further research is required to confirm and expand upon these findings.