Exome Study of Single Nucleotide Variations in Patients with Syndromic and Non-Syndromic Autism Reveals Potential Candidate Genes for Diagnostics and Novel Single Nucleotide Variants

Abstract

Autism spectrum disorder (ASD) is a neurodevelopmental impairment that occurs due to mutations related to the formation of the nervous system, combined with the impact of various epigenetic and environmental factors. This necessitates the identification of the genetic variations involved in ASD pathogenesis. We performed whole exome sequencing (WES) in a cohort of 22 Bulgarian male and female individuals showing ASD features alongside segregation analyses of their families. A targeted panel of genes was chosen and analyzed for each case, based on a detailed examination of clinical data. Gene analyses revealed that specific variants concern key neurobiological processes involving neuronal architecture, development, and function. These variants occur in a number of genes, including SHANK3, DLG3, NALCN, and PACS2 which are critical for synaptic signaling imbalance, CEP120 and BBS5 for ciliopathies, SPTAN1 for spectrins structure, SPATA5, TRAK1, and VPS13B for neuronal organelles trafficking and integrity, TAF6, SMARCB1, DDX3X, MECP2, and SETD1A for gene expression, CDK13 for cell cycle control, ALDH5A1, DPYD, FH, and PDHX for mitochondrial function, and PQBP1, HUWE1, and WDR45 for neuron homeostasis. Novel single nucleotide variants in the SPATA5, CEP120, BBS5, SETD1A, TRAK1, VPS13B, and DDX3X genes have been identified and proposed for use in ASD diagnostics. Our data contribute to a better understanding of the complex neurobiological features of autism and are applicable in the diagnosis and development of personalized therapeutic approaches.

1. Introduction

Autism spectrum disorder (ASD) is a neurodevelopmental impairment with early onset, characterized by social difficulties, deterioration of communication, repetitive behaviors, stereotyped and restricted interests, and atypical sensory stimuli perception [1,2,3,4]. Comorbid psychiatric and behavioral disorders are common in individuals with ASD, including ADHD, mood disorders, anxiety, obsessive compulsive disorder, irritability, aggression, substance use disorders, self-injurious behaviors, gender dysphoria, suicidality, psychosis, catatonia, and schizophrenia spectrum disorders [4]. Currently, the diagnostics of ASD are symptomatic and according to the Diagnostic and Statistical Manual of Mental Disorders-5th edition (DSM-5), it belongs to the group of neurodevelopmental disorders (NDDs) [5]. In the present medical nomenclature, ASD covers several NDDs: autism, atypical autism, Asperger’s syndrome, and a pervasive developmental disorder not otherwise specified.

The DSM-5 redefines ASD as a single continuum rather than a collection of distinct categories, which triggers a significant evolvement of the diagnostic criteria for ASD. The heterogenous nature of the spectra could be grouped into two critical domains: social communication deterioration and restricted, repetitive behaviors, that must present early in development and significantly affect daily functioning [6]. The Classification of Functioning, Disability, and Health for Children and Youth (ICF-CY) provides basic indicators which can be applied to different forms of ASD diagnosis (https://icd.who.int/en/ (accessed on 5 May 2025)). ASD has a large etiological heterogeneity due to variability in its phenotype observed in symptoms, onset, and severity. In these complex conditions, multiple genetic, epigenetic, and environmental factors additively contribute to symptom expression [7]. About 85% of the cases of ASD are estimated to have idiopathic ASD (with undefined causes for its pathogenesis), and just about 15% are considered to be secondary ASD (where a specific cause can be identified) [7]. No biomarkers are currently available for diagnosis or monitoring of ASD progression [8].

Molecular research has defined multigene dysfunction as an important causative factor of ASD, whereas its heterogeneity could be explained by the multigene model influenced by metabolomic factors [8]. Genetic studies have confirmed that ASD has a strong genetic basis and genetic heterogeneity [9]. It can be a distinct clinical phenotype or syndromic, related to the most common genetic syndromes [10]. Non-syndromic ASD could be polygenic and multifactorial, determined by specific combinations of environmental and genetic factors, or a single gene mutation can result in developing the disease in the relatively benign form of sporadic non-syndromic ASD [11]. In syndromic cases, ASD is a symptom of a more profound developmental disorder that includes multiple phenotypes, such as dysmorphic features, intellectual disability, and epilepsy [12]. The most common related with autism genetic syndromes comprise: Down, Fragile X, tuberous sclerosis complex, neurofibromatosis 1, Cornelia de Lange, Angelman, Coffin–Lowry, Cohen Laurence–Moon–Biedel, Marinesco-Sjogren, Moebius, Prader–Willi Rett syndrome, phenylketonuria, mitochondrial disorders, and many others [10,13]. The prevalence of ASD within these syndromes varies but can reach or exceed 60% [13]. The specific factors that determine the characteristics of subgroups of autism patients in each individual syndrome remain unknown [10].

Recent large-scale genomic studies using whole exome sequencing (WES) and chromosomal microarray analysis have significantly advanced our understanding of genetic contributions to ASD and related neurodevelopmental disorders [14]. These studies have identified novel genes with potential diagnostic value and suggest that clustering missense mutations indicates that normal development may be disrupted through activating or dominant-negative mechanisms [14]. Various genetic changes have been found in individuals with autism spectrum disorder (ASD), including missense and nonsense mutations, which alter protein function, and copy number variations, where segments of DNA are either duplicated or deleted, affecting gene expression [15]. Three major categories of genetic risk are implicated in ASD: common polygenic variations, rare inherited, and de novo mutations. Single nucleotide variants (SNVs), defined as rare nucleotide changes with an allele frequency < 1%, are of particular interest due to their potential to affect protein structure and function [16]. Pathogenicity of SNVsis evaluated according to guidelines from the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP), which classify variants as pathogenic, likely pathogenic, of uncertain significance (VUS), likely benign, or benign [17]. Dynamically growing data points to ASD association with a variety of genes and their single nucleotide variants (SNVs) [18]. A study based on the Australian database for ASD patients reports that single nucleotide variants (SNVs) are considered to account for 40–50% of ASD cases [19]. Lots of ASD candidate genes have been highlighted using genomic analyses for determining allelic diversity, mode of inheritance, and phenotypic impact of inherited and de novo variants of ASD and NDD genes [20,21,22,23,24,25,26]. According to the Simons Foundation Autism Research Initiative (SFARI) gene database, over 1000 gene candidates have been associated with ASD (About the Gene Scoring Module, https://gene.sfari.org/about-gene-scoring/ (accessed on 8 June 2025)). Mutations associated with autism often affect genes, related to a variety of metabolic disorders in the patients. Specific metabolites and metabolic pathways significantly differ in children with ASD compared to normally developing individuals [27].

SNVs may be either de novo arising during gametogenesis or embryonic development, or inherited from one or both parents [18]. It has been reported that postzygotic mutations account for numerous de novo harmful mutations resulting in mosaicism [28].

In this study, we performed whole exome sequencing on a cohort of 22 Bulgarian male and female individuals showing ASD features alongside segregation analyses of their families. We applied a targeted gene panel approach, selecting genes based on their clinical relevance to ASD and other neurodevelopmental conditions. This work aims to contribute to ASD gene discovery, support variant classification efforts, and improve understanding of the genetic architecture underlying ASD. Our goal was to identify rare de novo and inherited SNVs and assess their potential phenotypic impact, especially in relation to neurodevelopmental comorbidities.

2. Materials and Methods

In total, 22 Bulgarian patients (13 males and 9 females) with syndromic and non-syndromic autism spectrum disorder were selected from the medical records of Genetic Medico-Diagnostic Laboratory Genica, based on the joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology [17]. Each patient was clinically assessed by neurological and psychiatric examinations in children’s neurological and psychiatric clinics in Bulgaria. To assess structural and functional impairments in the brain, electroencephalography (EEG), computed tomography (CT), and magnetic resonance imaging (MRI) were used. Electromyography (EMG) was performed to evaluate muscle tone. The psychometric tests used for diagnosing ASD were Autism Diagnostic Interview Revised (ADI-R) [29], Autism Diagnostic Observation Schedule Generic (ADOS) [30], and Wechsler intelligence scale for children—5th edition. Some patients were diagnosed on the basis of fifth edition of Diagnostic and Statistical Manual of Mental Disorders (DSM-5) [5]. Written informed consent was obtained from the patients’ guardians, as well as from all family members tested.

High molecular weight DNA was extracted from EDTA-venous blood by standard salting-out method. The quality of the extracted DNA was assessed by direct spectrophotometry.

Whole exome sequencing (WES) was conducted in the partner laboratories “Admera Health, LLC, NJ, USA”, and Clinical Institute of Medical Genetics, UMC Ljubljana, Ljubljana, Slovenia. Clinically relevant genes were analyzed via a specialized software, GensearchNGS (PhenoSystems SA, Basel, Switzerland).

To verify the variants detected by WES, the target regions of the human genome, were multiplied by polymerase chain reaction (PCR). The amplified fragments were sequenced by Sanger’s method with BigDye^® Terminator v.3.1 sequencing kit (Applied Biosystems, Foster City, CA, USA). Electrophoretic separation of sequence products was performed via an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The obtained data were processed automatically by the program ABI3130 Data Collection Software and received in the form of an electrophoregram with Sequencing Analysis software v.5.1.1. Additionally, segregation analysis in the family was performed via Sanger sequencing using parental DNA samples, extracted from venous blood. For the targeting sequencing, the list of utilized primers used to validate selected var-iants is provided in a Supplementary File (see Supplementary Table S1).

The interpretation of the detected genetic variants was performed according to the classification criteria of the guidelines of the American College of Medical Genetics and Genomics/Association of Molecular Pathology (ACMG/AMP), taking into account the clinical manifestations and the results from the segregation analyses performed in the families [17].

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Sofia University “St. Kliment Ohridski”, Protocol No 93-M-412/1.10.2024 for studies involving humans (Approval date: 1 October 2024).

3. Results

The genetic variants which were detected by WES in our cohort of 22 Bulgarian patients (13 boys and 9 girls) having syndromic and non-syndromic phenotype are presented in

Table 1. Genetic variants detected by whole exome sequencing in a cohort of 22 Bulgarian patients. VUS—Variant of uncertain significance. * The classification is in accordance with the ACMG/AMP guidelines, Richards et al., 2015 [20].

Case	Gene ID	Sex	Age of Testing (Years)	Syndromic/Non-Syndromic Phenotype	Variant (GRCh37)	Variant Type	Zygosity	Inheritance	Pathogenicity *
1	MECP2	Male	2	Syndromic	chrX:g.153296071dup, NM_004992.3: c.1208dup, p.(Glu404Ter)	Nonsense	Hemizygous	Maternal	Likely pathogenic
2	TAF6	Female	15	Syndromic	chr7: g.99711522A>G, NM_001190415.1: c.323T>C, p.(Ile108Thr)	Missense	Homozygous	Biparental	Likely pathogenic
3	SMARCB1	Male	6	Syndromic	chr22: g.24145549C>T, NM_003073.3: c.568C>T, p.(Arg190Trp)	Missense	Heterozygous	de novo	Likely pathogenic
4	PACS2	Male	5	Syndromic	chr14: g.105834449G>A, NM_001100913.3: c.625G>A, p.(Glu209Lys)	Missense	Heterozygous	de novo	Pathogenic
5	WDR45	Female	4	Syndromic	chrX:g.48933330del, NM_007075.3:c.601_602del, p.(Leu201LysfsTer21)	Frameshift	Heterozygous	de novo	Likely pathogenic
6	PQBP1	Male	34	Syndromic	chrX:g.48760017C>T, NM_001032381.1:c.586C>T, p.(Arg196Ter)	Nonsense	Hemizygous	Maternal	Pathogenic
7	SPATA5	Male	13	Syndromic	chr4: g.123855300G>A, NM_145207.2: c. 554G>A, p.(Gly185Glu) p.	Missense	Heterozygous	Maternal	VUS
					chr4: g.123900503C>T, NM_145207.2: c.1831C>T, (Pro611Ser)	Missense	Heterozygous	Paternal	VUS
8	NALCN	Female	7	Syndromic	chr13: g.101944423A>G, NM_052867.2: c.965T>C, p.(Ile322Thr)	Missense	Heterozygous	de novo	Likely pathogenic
9	FH	Female	8	Syndromic	chr1: g.241667402G>A, NM_000143.4: c.1048C>T, p.(Arg350Trp)	Missense	Homozygous	Biparental	Likely pathogenic
10	CEP120	Male	3	Syndromic	chr5: g.122758670A>C, NM_153223.3: c.23T>G, p.(Leu8Trp)	Missense	Heterozygous	Paternal	VUS
					chr5: g.122700222G>C NM_153223.3: c.2548C>G, p.(Arg850Gly)	Missense	Heterozygous	Maternal	VUS
11	BBS5	Male	12	Syndromic	chr2:g.170343603G>A, NM_152384.3:c.167G>A, p.(Arg56Lys)	Missense	Heterozygous	Maternal	Likely pathogenic
					chr2:170354136G>C, NM_152384.3: c.619-1G>C	Splice site	Heterozygous	Paternal	Pathogenic
12	SPTAN1	Male	6	Syndromic	chr9: g.131394565C>T, NM_001130438.3: c.6922C>T, p.(Arg2308Cys)	Missense	Heterozygous	de novo	Likely pathogenic
13	VPS13B	Female	10	Syndromic	chr8:g.100844840_100844849delinsAC, NM_152564.5: c.9574_9583delinsAC, p.(Val3192ThrfsTer33)	Frameshift	Heterozygous	Paternal	Likely pathogenic
					chr8: g.100733139C>T, NM_152564.5: c.6914C>T, p.(Thr2305Ile)	Missense	Heterozygous	Maternal	VUS
14	SHANK3	Male	17	Non-syndromic	chr22: g.51153476G>A, NM_001372044.2: c.2490+1G>A	Splice site	Heterozygous	de novo	Pathogenic
	DLG3				chrX: g.69712394G>A, NM_021120.4:c.1721G>A, p.(Arg574Gln)	Missense	Hemizygous	Maternal	VUS
15	CDK13	Male	10	Syndromic	chr7: g.40085606A>T NM_003718.5: , c.2525A>T, p.(Asn842Ile)	Missense	Heterozygous	de novo	Pathogenic
16	PDHX	Female	3	Syndromic	chr11: g.35016549C>T, NM_003477.3: c.1336C>T, p.(Arg446Ter)	Nonsence	Homozygous	Biparental	Pathogenic
17	SETD1A	Male	15	Syndromic	chr16: g.30995020delG, NM_014712.3: c.4879del, p.(Val1627TrpfsTer41)	Frameshift	Heterozygous	de novo	Pathogenic
18	TRAK1	Female	5	Non-syndromic	chr3:g.42240742T>A, NM_001042646.3:c.1187T>A, p.(Ile396Asn)	Missense	Heterozygous	de novo	VUS
19	ALDH5A1	Female	3	Syndromic	chr6:g.24515433dup NM_170740.1:c.804dup, p.(Val269fsTer19)	Frameshift	Heterozygous	Paternal	Pathogenic
					chr6:g.24528277G>A NM_170740.1:c.1265G>A, p.(Gly422Asp)	Missense	Heterozygous	Maternal	Likely pathogenic
20	DPYD	Male	6	Syndromic	chr1:g.97915614C>T, NM_000110.3: c.1905+1G>A	Splice site	Homozygous	Biparental	Likely pathogenic
21	DDX3X	Female	2	Syndromic	chrX:g.41203374C>A, NM_001356.3:c.857C>A, p.(Ala286Asp)	Missense	Heterozygous	de novo	VUS
22	HUWE1	Male	8	Syndromic	chrX:g.53578038C>T, NM_031407.7:c.9209G>A, p.(Arg3070His)	Missense	Hemizygous	de novo	Pathogenic

. All patients share autistic features, neuropsychological delay, and intellectual disability. The detected genetic variants are classified as pathogenic, likely pathogenic, or VUS (variants of uncertain significance) according to the ACMG/AMP guidelines [17]. In total, 16 cases were concluded to carry pathogenic or likely pathogenic variants. In one of the cases, likely pathogenic and VUS were detected in the autosomal recessive VPS13B gene, which segregate in the family (one is maternally inherited and the other one is paternally inherited). Another patient carries a pathogenic de novo variant in the SHANK3 gene and maternally inherited VUS in the DLG3 gene. The last 4 cases carry variants classified as VUS in different genes (see Table 1). The performed segregation analysis in the affected families revealed 10 de novo cases, 11 cases with variants segregating in the family, and a case with one de novo and one maternally inherited variant. We identified novel single nucleotide variants in the SPATA5, CEP120, BBS5, SETD1A, TRAK1, VPS13B, and DDX3X genes, according to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar (accessed on 5 May 2025)) and LOVD (https://www.lovd.nl/ (accessed on 5 May 2025)).

The cohort of 22 patients had been examined clinically and neuropsychologically in children’s neurological and psychiatric clinics in Bulgaria prior to genetic testing. All the patients share autistic features of poor social interaction, language use, speech deterioration, and repetitive behavior, combined with neuropsychological delay and intellectual disability. Their specific additional neurological and psychiatric profiles, as well as other clinical characteristics, accordingly with the findings from the WES analyses, are represented in

Table 2. Neurological and psychiatric profiles, and the corresponding genetic analysis findings of the cohort of 22 Bulgarian patients.

Case	Gene ID	Sex	Age of Testing (Years)	Clinical and Neuropsychological Profile of the Patient
1	MECP2	Male	2	West syndrome: abnormal EEG, chaotic brain waves (hypsarrhythmia), specific infantile spasms with twitching of the head, arms, body tremors, stereotyped movements, and epileptic seizures, combined with axial muscular hypotony and motor developmental delay, mild fascial dysmorphism and smaller left auricle, clinodactyly of second left toe, and normal metabolic screening; communication deficits, lack of speech, and responding to commands, stereotyped movements.
2	TAF6	Female	15	Congenital cerebellar hypoplasia, hypotrophy (underrepresented subcutaneous fat tissue), ataxic gait, mild muscular hypotony, discretely impaired fine motor skills, mild mental retardation, defects in sound pronunciation, speech delay, mood disorders, insufficient concentration, and anxiety.
3	SMARCB1	Male	6	Epileptic seizures, focal epileptiform changes, and generalized paroxysmal manifestations of myocytic type, facial dysmorphism, delay in speech and neuropsychiatric development, deterioration in communication, infrequent eye contact, and stereotyped movements.
4	PACS2	Male	5	Microcephaly, facial dysmorphism, discrete facial symmetry, antimongoloid eye slits, epicanthus, hypertelorism, ocular coloboma, facial dysmorphism, backward rotated dysplastic auricles, muscular hypotony, epileptic seizures, and delay in speech and psychomotor development.
5	WDR45	Female	4	Epileptic encephalopathy with late epileptic spasms and focal seizures, moderate mental retardation, communication deficits.
6	PQBP1	Male	34	Confined atrophy of the brain, mental retardation since early childhood, behavioral stereotypes, tics, communication deficits.
7	SPATA5	Male	13	EEG abnormality, delayed onset of psychomotor development, speech delay, stereotypic movements.
8	NALCN	Female	7	Delay in speech, neuropsychiatric and psychomotor development, generalized muscular hypotension and hyporeflexia in the neonatal period, ulnar deviation of the fingers and hip dysplasia, speech and communication deterioration; lack of organic pathological changes in the examined intracranial anatomical components according to MRT of CNS and MR spectroscopy.
9	FH	Female	8	Microcephaly and unspecified encephalopathy; transfontanel ultrasound analyses shows mild diffuse dilatation of subarachnoid space and of the lateral ventricles; seizures, generalized muscular hypotension, delay in speech, and neuropsychiatric development.
10	CEP120	Male	3	Delay in speech and neuropsychiatric development, epileptic seizures, dolichocephaly, and hyperprolinemia type I.
11	BBS5	Male	12	Polydactyly, undeveloped expressive speech, communication deterioration, anxiety, psychomotor and sensory deterioration, and repetitive behavioral patterns.
12	SPTAN1	Male	6	Febrile seizures, behavior deterioration, and communication difficulties.
13	VPS13B	Female	10	Abnormal EEG, paralysis cerebralis, divergent strabismus, hydrocephaly, large fontanelle in infancy, planovagus deformity of the feet, pronounced scoliosis, severe neurodevelopmental disorder, speech delay, and stereotyped movements.
14	SHANK3	Male	17	Loss of previous skills over the years, loss of speech, and deterioration in communication.
	DLG3
15	CDK13	Male	10	Moderate mental retardation, significant behavioral disorder requiring care and treatment; mental and physical developmental delay, lack of speech, stereotyped movements and behavior, lack of attention, and communication deficits.
16	PDHX	Female	3	Cerebral cortex atrophy, demyelination, mental retardation, significant behavioral disorder, alalia, central quadriplegia, microcephaly, blindness, and leukodystrophy.
17	SETD1A	Male	15	Neurobehavioral retardation, facial dysmorphism, muscular hypotony, enterocolitis, hepatosplenomegaly, and hemangioma parietis thoracis since birth.
18	TRAK1	Female	5	Speech delay and communication and behavioral deteriorations.
19	ALDH5A1	Female	3	Global developmental delay, including speech and behavioral disorders, hypotonia, coordination problems, hyporeflexia, movement disorders, and epilepsy.
20	DPYD	Male	6	Microcephaly, severe developmental delay, hypotonia, seizures, speech delay, and communication difficulties.
21	DDX3X	Female	2	Facial dysmorphism, delay in speech, and psychomotor development.
22	HUWE1	Male	8	Microcephaly, epilepsy, severe mental retardation, significant behavioral deterioration, lack of speech, and delay in motor development.

.

4. Discussion

The underlying mechanisms of autism spectrum disorder (ASD) remain incompletely understood, with both inherited and de novo variants contributing to its complex etiology. Although ASD presents with a consistent behavioral profile, its causes are heterogeneous, involving genetic, chromosomal, and environmental components. Disruptions in genes regulating neuronal structure and function, even when not associated with well-defined syndromes, can lead to non-syndromic ASD.

We conducted whole exome sequencing (WES) and segregation analysis in 22 Bulgarian families with ASD probands and additional neurodevelopmental comorbidities. The cohort consisted of 13 males and 9 females (male-to-female ratio: 1.44), with a mean age at testing of 6.8 years (range: 2–34; SD: 7.01). The age of the patient varies due to the time when they were sent for WES. ASD appears more frequently in males than in females [31]. According to the statistics described by numerous authors, the male–female ratio is 4–5:1 [23]. Although ASD is more frequently diagnosed in males, the higher proportion of females in our cohort likely reflects the limited sample size and the tendency for genetic testing to be performed in cases with more severe phenotypes.

This study identified a range of potentially damaging de novo and inherited single nucleotide variants (SNVs), highlighting allelic diversity and helping to clarify inheritance patterns and genotype–phenotype correlations.

Table 3. Single nucleotide variants (SNVs) identified by whole exome sequencing in 22 Bulgarian patients. The table includes the affected gene and protein, a summary of each gene’s role in neuronal structure or function, symptoms observed in each of the 22 patients, and its classification according to the SFARI Gene database in relation to autism spectrum disorder (ASD).

Gene (SNV)	Protein Name	Role in Neuronal Structure or Function	SFARI Classification
NALCN	Sodium Leak Channel, Non-Selective	Regulates resting membrane potential and excitability [32,33,34]	Strong candidate
PACS2	Phosphofurin Acidic Cluster Sorting Protein 2	Synaptic signaling, organelle communication, calcium signaling, and mitochondrial function [35,36,37]	Syndromic
SHANK3	ProSAP SH3 and multiple ankyrin repeat domain protein 3	Synapse formation and maintenance [38,39,40,41,42]	High confidence
DLG3	disks large membrane-associated guanylate kinase scaffold protein 3, synapse-associated protein 102 (SAP-102)	Synaptic signaling involved in N-methyl-D-aspartate receptor clustering at excitatory synapses; synaptic plasticity [43,44]	Not listed
ALDH5A1	Aldehyde Dehydrogenase 5 Family Member A1	GABA metabolism, mitochondrial function [45,46,47,48,49]	High confidence, syndromic
DPYD	Dihydropyrimidine Dehydrogenase	Mitochondrial enzyme [50] and pyrimidine degradation [50,51,52,53]	Strong candidate
FH	Fumarate hydratase	Mitochondrial function, Krebs cycle enzyme; maintaining levels of neurotransmitters like glutamate, aspartate, and GABA [54]	Not listed
PDHX	pyruvate dehydrogenase X	Mitochondrial function; links glycolysis to tricarboxy acid cycle; neurotransmitter balance conversion of pyruvate to acetyl-CoA, maintaining levels of neurotransmitters like glutamate, aspartate, and GABA [42,55,56,57]	Not listed
TAF6	TATA-Box Binding Protein Associated Factor 6	Transcription initiation complex component [58,59]	Strong candidate
MECP2	Methyl-CpG Binding Protein 2	Transcription regulation [60,61,62]	High confidence, syndromic
SMARCB1	SWI/SNF-related, matrix- associated, actin-dependent regulator of chromatin subfamily B member 1	Chromatin remodeling complex subunit [63,64,65,66]	Not listed
DDX3X	DEAD-Box Helicase 3 X-Linked	RNA metabolism, translation initiation [67,68,69]	High confidence, syndromic
SETD1A	SET domain containing protein 1A or histone methyltransferase	Histone methylation and transcription regulation [70,71,72,73]	High confidence, syndromic
CDK13	cyclin-dependent kinase 13	Cell cycle control factors; transcriptional regulation and RNA splicing [74,75,76,77]	Syndromic
CEP120	centrosomal protein 120	Ciloigenesis, axonal growth, and cerebellar development [78,79]	Not listed
BBS5	BBSome	Cilia function and intracellular transport [80,81,82,83]	Not listed
SPTAN1	αII spectrin subunit	Membrane structure; synaptic support [84,85]	Not listed
SPATA5	spermatogenesis-associated protein 5	Mitochondrial dynamics; ATP production in neurons [86,87,88,89]	Not listed
TRAK1	trafficking kinesin binding protein 1	Mitochondrial transport in neurons; [90]	Not listed
VPS13B	vacuolar sorting protein 13	Golgi integrity; vesicle trafficking in neurons [91]	High confidence, strong candidate
PQBP1	Polyglutamine binding protein-1	Neuron homeostasis; clearance of neurotoxic proteins [92]	Not listed
WDR45	WD repeat-containing protein 45	Macroautophagy; removal of damaged organelles [93]	Not listed
HUWE1	HECT, UBA, and WWE Domain Containing E3 Ubiquitin Protein Ligase 1	Protein degradation and cortical development [94]	Syndromic

summarizes all detected SNVs, their functional roles in neurons, and SFARI gene classification where applicable.

Exome sequencing of 22 Bulgarian patients revealed SNVs affecting key intraneuronal mechanisms that align with each individual’s clinical presentation. These findings support the potential for personalized diagnostic and therapeutic approaches in autism and related neurodevelopmental disorders.

4.1. SNVs Associated with Synaptic Structure, Function, and Signaling Imbalance of Neurons in ASD

In our exome sequencing study of individuals with ASD, we identified rare variants in genes implicated in synaptic regulation and neuronal excitability, including NALCN, PACS2, SHANK3, and DLG3. These findings support the role of disrupted excitatory-inhibitory signaling in ASD pathogenesis. A de novo heterozygous missense variant in NALCN was detected in a female patient presenting with hypotonia, joint deformities, microcephaly, developmental delay, and ASD. The variant likely impairs sodium leak currents, contributing to neuromuscular dysfunction. A male patient with epilepsy, hypotonia, and ASD carried a de novo PACS2 variant, potentially contributing to the ASD phenotype and the early-onset epileptic encephalopathy through impaired ion channel regulation. Another male patient with ASD had a de novo heterozygous splice-site variant in SHANK3. The alteration likely affects alternative splicing, leading to production of variable variants of SHANK3 molecules and potentially disrupting synaptic function. Notably, this patient also carries a hemizygous maternally inherited DLG3 variant, which is not currently listed in the SFARI database but is classified in ClinVar as likely pathogenic/variant of uncertain significance (https://www.ncbi.nlm.nih.gov/clinvar/variation/224095/ (accessed on 5 May 2025)), associated with X-linked intellectual disability 90. The combined presence of SHANK3 and DLG3 variants coincide with progressive loss of communication skills and worsening ASD symptoms. These cases underscore the genetic complexity of ASD and suggest that multiple rare SNVs presented in a single person may contribute to more severe phenotypes.

4.2. SNVs Implicated in Mitochondrial Dysfunction and ASD

Variants in mitochondrial metabolism in ALDH5A1, DPYD, FH, PDHX, and SPATA5 were identified implicating mitochondrial dysfunction in a subset of ASD cases. Compound heterozygous variants in ALDH5A1 were found in a female patient with ASD, epilepsy, hypotonia, developmental delay, and motor coordination deficits. Given its role in GABA metabolism, ALDH5A1 deficiency likely disrupts neurotransmitter balance, contributing to the clinical presentation. A homozygous splice-site variant in DPYD was identified in a male patient with microcephaly, severe developmental delay, hypotonia, seizures, and ASD, potentially leading to toxic metabolite accumulation. A female patient with microcephaly, developmental delay, and seizures carried a homozygous missense variant in FH. As FH encodes fumarate hydratase, essential for mitochondrial energy production, the variant likely impairs neuronal energy balance. A homozygous PDHX variant was found in a female patient with cerebral atrophy, severe intellectual disability, leukodystrophy, and ASD phenotype. The variant is expected to impair glucose metabolism in the brain, resulting in neurodegeneration. Although FH and PDHX are not currently listed in the SFARI Gene database, their involvement in mitochondrial energy pathways and the presence of ASD-related features suggest they merit further investigation as ASD candidate genes. Additionally, compound heterozygous variants in SPATA5 were identified in a male patient with EEG abnormalities, delayed psychomotor development, speech delay, and stereotypies. SPATA5 is essential for mitochondrial dynamics and ATP production in neurons. Disruption of its function may impair axonal growth and synaptic signaling, contributing to the observed neurodevelopmental phenotype. This case is described in detail in a separate study [89].

4.3. SNVs Associated with ASD and Defects in Gene Expression: Transcription Factors, Chromatin Remodeling, and Histone Methylation

We identified rare variants in genes involved in transcription regulation, chromatin remodeling, and histone methylation, including MECP2, TAF6, SMARCB1, DDX3X, and SETD1A. These variants are likely to contribute to ASD phenotypes through disrupted gene expression and neurodevelopmental processes. A male patient with a hemizygous nonsense variant in MECP2 showed symptoms overlapping with West syndrome and ASD features. MECP2 is a key transcription regulator in the brain [62], where both overexpression and underexpression can cause neurodevelopmental disorders [60]. Mutations in MECP2 are the primary cause of Rett syndrome (RTT), a condition mostly affecting females, although affected males often experience more severe symptoms and reduced survival [95]. Besides Rett Syndrome, MECP2 mutations have been identified in individuals with classic autism suggesting that loss-of-function mutations of MECP2 may contribute to ASD [96]. Our findings emphasize the need for further investigation into the diverse roles of MECP2 variants in neuronal dysfunction and neurodevelopment. While MECP2 mutations are primarily linked to Rett syndrome and classic autism, this case highlights the phenotypic variability associated with partial loss-of-function.

A homozygous TAF6 variant was identified in a female patient with intellectual disability, ASD, muscular hypotonia, and cerebellar hypoplasia likely impairing neuronal gene expression. A de novo missense variant in SMARCB1 was found in a male with ASD, seizures, and facial dysmorphism. Though not in the SFARI database, it may disrupt synaptic development. A de novo heterozygous missense variant in DDX3X was detected in a female patient with facial dysmorphism and developmental delay. Given its established role in RNA metabolism and high-confidence association with ASD, the variant likely impairs neuronal proliferation and differentiation. Another male patient presented with muscular hypotonia, developmental delay, and atypical autism, and carried a de novo heterozygous frameshift variant in SETD1A. The gene encodes a component of a histone methyltransferase complex, and the variant likely disrupts transcriptional regulation during neurodevelopment.

4.4. SNVs Associated with ASD and Cell Cycle Regulation, Ciliopathies, and Spectrin Function

In our ASD cohort, we identified a de novo heterozygous missense variant in CDK13 in a male patient with cognitive impairment, moderate intellectual disability, and ASD. Given CDK13’s role in transcriptional regulation and RNA splicing, the variant may impair neuronal differentiation and synapse formation, supporting its potential involvement in ASD-related neurodevelopment.

Variants associated with ciliopathies were also observed. A male patient with compound heterozygous missense variants in CEP120 presented with ASD, speech delay, epilepsy, dolichocephaly, and hyperprolinemia type I. CEP120 plays a role in centriole biogenesis and cerebellar development; its disruption may underlie the neurological phenotype. Another male patient carried biallelic variants in BBS5, presenting with polydactyly, developmental delay, and ASD. BBS5 encodes a subunit of the BBSome, essential for cilia function and intracellular transport. Although CEP120 and BBS5 are not currently listed in the SFARI Gene database, their roles in neurodevelopment suggest they may be relevant to ASD pathogenesis.

We also identified a de novo heterozygous missense variant in SPTAN1 in a male patient with seizures and ASD traits. SPTAN1 encodes a neuronal spectrin that supports membrane stability and synaptic signaling. The variant may disrupt the spectrin-actin cytoskeleton, affecting ion channel organization and contributing to the observed phenotype.

4.5. SNVs Associated with ASD and Affecting Neuronal Organelle Trafficking and Homeostasis

We identified variants in TRAK1 and VPS13B genes involved in intracellular trafficking and organelle maintenance—processes critical for neuronal function. A female patient carried a de novo heterozygous missense variant in TRAK1, a gene involved in mitochondrial transport along axons and dendrites. Although previously associated with neurodevelopmental disorders, TRAK1 is not currently listed in the SFARI database. Given the ASD features in our patient, its role in ASD warrants further investigation. Another female patient presented with compound heterozygous variants (frameshift and missense) in VPS13B. She exhibited severe neurodevelopmental delay, speech impairment, stereotypies, cerebral palsy, and skeletal malformations. VPS13B is essential for Golgi integrity and vesicle trafficking, and its disruption likely contributed to the complex phenotype observed.

We also identified variants in genes regulating neuronal homeostasis. A male patient carried a maternally inherited hemizygous variant in PQBP1, presenting with moderate intellectual disability, behavioral stereotypies, and progressive neuropsychiatric decline. PQBP1 is involved in immune responses and proteostasis, and the variant likely impairs synaptic maintenance over time. A de novo heterozygous frameshift variant in WDR45 was found in a female patient with epileptic encephalopathy, developmental delay, and ASD features. WDR45 regulates autophagy, and the variant likely disrupted neuronal clearance of damaged components. Another male patient carried a de novo hemizygous missense variant in HUWE1, presenting with microcephaly, epilepsy, severe intellectual disability, and marked ASD traits. HUWE1 encodes a ubiquitin ligase essential for cortical development and protein degradation, and its disruption may explain the severity of the phenotype. These findings highlight the role of proteostasis and organelle maintenance in ASD pathogenesis.

The genes identified in our cohort are involved in critical neurobiological processes, including gene expression, mitochondrial function, synaptic signaling, organelle trafficking, neuronal homeostasis, cell cycle regulation, and the structural integrity of cilia and spectrins (Figure 1).

Among all the mutations mentioned, novel single nucleotide variants in the SPATA5, CEP120, BBS5, SETD1A, TRAK1, VPS13B, and DDX3X genes were identified in our cohort. These variants were not previously reported in the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar (accessed on 5 May 2025)) and LOVD (https://www.lovd.nl/ (accessed on 5 May 2025)) databases. While associations with ASD have not been firmly established for genes like SPATA5, CEP120, and TRAK1, our findings suggest a potential role in ASD pathogenesis that warrants further investigation. Although FH and PDHX are not listed in SFARI, we detected homozygous variants in these genes in ASD patients, supporting their inclusion in future ASD-related metabolic gene panels.

There are several limitations and challenges to our study. ASD is highly heterogeneous, both genetically and clinically, necessitating large sample sizes for the discovery of rare genetic variations. Moreover, the demographic profile of the ASD patients in Bulgaria, together with the relatively small number of the Bulgarian population (about 6,718,470 people) and its economic status, results in the fact that only a small subset of the ASD patients have the opportunity to participate in genetic investigations. Genetic testing is primarily pursued by individuals presenting with severe psychosomatic and neurodevelopmental disorders, whereas those exhibiting milder or more limited ASD symptoms are often underrepresented or excluded from such diagnostic evaluations. Moreover, the lack of alignment of our data with findings reported in SFARI most probably is due to the fact that this database comprises results obtained by already performed investigations, but ASD is still not fully studied and unriddled.

5. Conclusions

Our findings support the view that autism spectrum disorders (ASDs) are genetically and clinically heterogeneous. Mutations implicated in ASD affect diverse neuronal functions, including synaptic architecture, excitatory-inhibitory balance, mitochondrial metabolism, and cellular processes such as chromatin remodeling, transcription regulation, organelle trafficking, and protein degradation. Several of these single nucleotide variants (SNVs) disrupt the epigenomic landscape, potentially contributing to the broad phenotypic variability observed in ASD.

Notably, SNVs in SHANK3, DLG3, and PQBP1 may have prognostic value for neurodegenerative conditions with autistic features in later life. In our cohort of 22 patients, we identified novel SNVs in SPATA5, CEP120, BBS5, SETD1A, TRAK1, VPS13B, and DDX3X genes not currently listed in ClinVar or the SFARI database. These genes encode proteins essential for neuronal function and, given their presence in patients with ASD traits, merit further investigation as potential contributors to neurodevelopmental disorders.

In particular, SNVs in SPATA5, CEP120, and TRAK1 may be linked to ASD-related symptoms in our patients.

Our study adds new data to public mutation databases and contributes to a more comprehensive understanding of autism’s molecular underpinnings, with implications for diagnosis and the development of targeted therapies.