The proband was a 4-year-old male who presented to the Neurology Clinic of Fujian Provincial Children's Hospital at 2 years and 1 month of age with global developmental delay (GDD), including deficits in fine/gross motor skills and speech. Clinical assessment included neurological examination, developmental evaluation with China Developmental Scale for Children (CDSC) (0–6 years), and neuroimaging with cranial MRI. Electroencephalography (EEG) was deferred due to no clinical seizure history. The patient received intermittent neurodevelopmental therapy (focused on motor coordination and speech habilitation) through the hospital's rehabilitation program.

Trio whole-exome sequencing was performed by Chigene Translational Medicine Research Center Co., Ltd. (Beijing, China) on peripheral blood-derived DNA (proband and parents) using the IDT xGen Exome Research Panel v2.0 on an Illumina NovaSeq 6,000 platform (150 bp paired-end reads, average 100 × coverage). Raw reads were processed (fastq), aligned to GRCh37/hg19 (BWA-MEM v0.7.17), and variants called following GATK (v4.2.6.1) best practices, including duplicate marking, base quality recalibration, and local realignment. Variants were annotated (ANNOVAR) and filtered against population databases (gnomAD/ExAC/1,000 Genomes), then prioritized based on predicted impact using multiple algorithms (SIFT/PolyPhen-2/CADD/REVEL for missense; SpliceAI/dbscSNV for splicing; GERP++/phyloP for conservation). Variants were classified per ACMG/AMP guidelines with ClinGen specifications, integrating population frequency, computational predictions, inheritance patterns, and phenotype correlation (HGMD/ClinVar/OMIM). The MAST4 variant (c.4142G>T) was validated by Sanger sequencing (ABI 3730xl).

The patient, a 4-year-old boy, presented to our institution at 2 years and 1 month of age with global developmental delay affecting fine motor, gross motor, and speech domains. His clinical features included independent ambulation with gait instability, impaired manual dexterity (manifested by slow reaching/grasping and weak grip strength), and significant language delay (limited to single-word utterances with poor articulation). On physical examination, he was alert and responsive with stable vital signs. His head circumference measured 50.5 cm (within normal range), and his anterior fontanelle was closed. Skin examination revealed no jaundice, pallor, edema, or petechiae, with the absence of transverse palmar creases. Facial features included normal ocular motility without hypertelorism or ptosis, along with a mildly flattened nasal bridge. Oropharyngeal structures were intact, without a high-arched palate or cyanosis. Cardiopulmonary and abdominal assessments were unremarkable. Neurological evaluation demonstrated preserved visual and auditory responses, absent startle reflex, mild hypotonia, and symmetrically normal deep tendon reflexes (knee and Achilles ++). Plantar responses (Babinski sign), ankle clonus, and meningeal signs (Kernig's and Brudzinski's signs) were all negative.

The child was born at term with a normal birth weight of 3.1 kg. During the neonatal period, he exhibited heightened startle reflexes and frequent, unexplained crying spells. His motor developmental milestones were as follows: head lifting in the prone position by 2–3 months, stable head control by 4 months, independent sitting by 10 months, crawling by 17 months, and unaided walking by 19 months. At 2 years and 1 month of age, he was referred to our outpatient clinic for evaluation of developmental delays in fine motor skills, gross motor function, and language abilities. A cranial MRI at that time revealed inadequate myelination (Figure 1).

Developmental assessment using the CDSC (0–6 years) for Children at 2 years 1 month and 3 years 6 months revealed persistent delays across all domains (Table 1). This assessment evaluates gross motor skills, fine motor skills, adaptive behavior, language competence, and personal-social behavior. The developmental quotient (DQ) serves as an indicator of overall developmental status, with classifications as follows: DQ ≥ 85 (normal development), 76–84 (borderline delay), 55–75 (mild delay), 40–54 (moderate delay), 25–39 (severe delay), and ≤24 (profound delay). In this case, the patient demonstrated persistent developmental delays, with both DQ scores falling below 70, consistent with mild developmental delay. This sustained subthreshold performance underscores the need for targeted clinical interventions to address deficits across multiple functional domains.

Whole-exome sequencing identified a heterozygous MAST4 missense variant (NM_001164664.2: c.4142G>T, p.Arg1381Leu) in the proband. Trio analysis confirmed its de novo origin (PS2), with Sanger sequencing demonstrating the variant in the proband (heterozygous G/T peaks) and wild-type genotypes (G/G) in both parents (Figure 2). Pedigree analysis revealed an autosomal dominant inheritance pattern with no family history of neurodevelopmental disorders (Figure 3). The variant was absent in major population databases [gnomAD v4.0 (807,162 alleles; 62,784 East Asian), ExAC, and ESP6500], with an allele frequency <0.0001% (PM2_Supporting). Computational tools unanimously predicted pathogenicity: SIFT (deleterious, 0.02), PolyPhen-2 (probably damaging, 0.998), REVEL (pathogenic, 0.88), and CADD (Phred 28.5) (PP3). The variant meets ACMG-AMP criteria for Likely Pathogenic classification (PS2 + PM2_Supporting + PP3). No other pathogenic variants or CNVs were detected that could explain the phenotype.

Multiple sequence alignment revealed exceptional evolutionary conservation of Arg1381 across vertebrate species, suggesting critical functional importance (Figure 4). Structural modeling localized the variant to a disordered loop region (residue 1,381) (Figure 5).

AlphaFold2-based modeling (Google DeepMind) revealed that wild-type MAST4 Arg1381 forms stabilizing hydrogen bonds with Ala1380 and Thr1382 in the kinase domain (Figure 6A). While the p.Arg1381Leu variant maintained similar hydrogen bond numbers, the substitution of a positively charged arginine with hydrophobic leucine likely impairs function through (Figure 6B): (1) disruption of local electrostatic balance, potentially affecting substrate binding; and (2) steric interference with kinase domain accessibility. This charge and structural alteration at a highly conserved residue may compromise protein-protein interactions and catalytic activity, suggesting a plausible mechanism by which this variant contributes to neurodevelopmental pathology through structure-function perturbation. Together, these findings provide strong evidence for the pathogenic potential of this de novo MAST4 variant in the patient's neurodevelopmental disorder.

MAST4, a member of the MAST kinase family, is crucial for neurodevelopment as it regulates axonal guidance and synaptic plasticity via its kinase domain. This regulation is part of the complex molecular mechanisms that ensure proper neural network formation and function. Its predominant expression in cerebellar Purkinje cells, hippocampal regions, and white matter-enriched areas (20), along with embryonic enrichment in the dorsal midline thalamic nuclei and medial prefrontal cortex (8, 21), underscores its importance in early neural circuit formation. Animal studies reveal seizure-induced upregulation of MAST4 in the murine hippocampus (22), suggesting involvement in epileptogenic neuroplasticity. Functionally, MAST4 modulates neuronal survival via FOXO1 phosphorylation and regulates ciliary dynamics through Tctex-1/Cdc42 interactions (8, 10), establishing it as a multifunctional orchestrator of neural development and homeostasis. All of these suggest that MAST4 variants disrupt cellular self-renewal, proliferation, and differentiation (12).

The pathogenicity of MAST4 variants in NDDs remains poorly characterized, as no MAST4-related disorders are currently cataloged in OMIM. Although the Human Gene Mutation Database (HGMD) lists 23 MAST4 variants classified as disease-causing (DM), their clinical significance lacks validation in large cohorts or functional studies. The de novo MAST4 missense variant (c.4142G>T, p.Arg1381Leu) identified in our patient offers several lines of evidence supporting its potential pathogenicity: (1) The variant is absent in gnomAD v4.0, ExAC, and ESP6500, suggesting strong negative selection; (2) Arg1381 is highly conserved across vertebrates (Figure 4), implying functional indispensability in the MAST4 kinase domain; (3) Concordant deleterious predictions from SIFT (deleterious), PolyPhen-2 (probabaly damaging), REVEL (0.885), and CADD (28.5) further bolster its disease association. Structural modeling localizes p.Arg1381Leu to a critical loop region within the kinase domain (Figure 5). The substitution of a charged arginine with a hydrophobic leucine likely destabilizes local protein conformation, impairing MAST4's interaction with downstream substrates (Figure 6). Unlike MAST1 variants, which are associated with megalencephaly and corpus callosum anomalies, our patient's MAST4 variant correlates with delayed myelination and developmental delay, without structural malformations. This divergence may reflect differential substrate specificity among MAST kinases, with MAST4 preferentially influencing white matter integrity via distinct signaling cascades.

Our patient presents a distinct clinical manifestation compared to previously reported MAST4 cases, characterized by a unique combination of significant global developmental delay (DQ < 70) without epileptic manifestations and predominant delayed myelination on MRI (Figure 1), contrasting with the typical structural abnormalities seen in other cases. This phenotypic divergence likely results from the interplay between variant-specific effects and epigenetic modulation. The p.Arg1381Leu variant may disrupt distinct protein interaction networks and substrate recognition, creating a unique perturbation pattern. Importantly, epigenetic mechanisms appear to play a protective role against seizures while potentially exacerbating myelination defects. The adenosine-mediated DNA hypomethylation pathway (23) and histone modification cascades may maintain an anti-epileptic state, while disproportionate disruption of Wnt/β-catenin signaling (24) and mTOR-mediated OPC differentiation (25) likely contribute to the myelination delay. The involvement of SWI/SNF and CHD chromatin remodelers (26) in oligodendrocyte differentiation, along with the known neurodevelopmental roles of H3K4 trimethylation and H3K27 demethylation (27), suggests complex epigenetic modulation of myelination defects. Although direct evidence linking MAST4 to these pathways requires further investigation, the kinase domain's potential to phosphorylate chromatin modifiers and its known regulation of cytoskeletal dynamics suggest plausible mechanistic connections. These findings highlight three key aspects of MAST4 pathobiology: domain-specific effects, epigenetic buffering capacity, and developmental stage-dependent neural vulnerability, underscoring the need for integrated genetic-epigenetic analyses.

Our findings highlight three critical avenues for further investigation: First, constructing MAST4 p.Arg1381Leu mutant mouse models to systematically delineate its spatiotemporal effects on myelination and synaptic plasticity, with a focus on oligodendrocyte differentiation and axon-glia interactions. Second, integrating whole-genome sequencing and chromatin conformation capture (Hi-C) to identify MAST4-interacting epigenetic regulators (e.g., HDAC4, MECP2) and uncover molecular drivers of phenotypic heterogeneity. Third, developing targeted therapies, including allosteric small-molecule modulators or AAV-based gene editing strategies, to restore MAST4 kinase functionality. These efforts will deepen mechanistic insights and accelerate translational applications for MAST4-related disorders.