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Phosphofurin acidic cluster sorting protein 2 (PACS2) is a multifunctional sorting protein that regulates intracellular cargo trafficking and calcium homeostasis. Heterozygous missense variants in PACS2 underlie Developmental and Epileptic Encephalopathy-66, characterized by early-onset seizures, intellectual disability, cerebellar dysgenesis, facial dysmorphism, and coloboma. The most recurrent variant, c.625G>A (p.Glu209Lys), has been identified in multiple unrelated individuals, implicating PACS2 dysfunction in epileptogenesis and neurodevelopmental anomalies. The disorder follows an autosomal dominant inheritance pattern with de novo occurrence of pathogenic variants. Coloboma has also been observed as a shared ocular feature in the PACS1/PACS2/WDR37 axis, suggesting a role in ocular development. Here, we summarize the clinical validity, genetic evidence, and functional data supporting the PACS2–DEE66 association to guide diagnostic decision-making and future research.
To date, four independent probands have been reported with de novo heterozygous PACS2 missense variants, including three individuals harboring c.625G>A (p.Glu209Lys) and one with c.631G>A (p.Glu211Lys), all presenting with DEE66 (n=4 probands) (PMID:33369122; PMID:30684285; PMID:38545008). The recurrent p.Glu209Lys variant has been detected in two unrelated de novo cases and segregates with disease in a familial Saudi pedigree. Each variant is absent from population databases, supporting pathogenicity. No truncating or splice‐site variants have been associated with DEE66. These recurrent de novo missense alleles underscore a gain-of-function or dominant-negative mechanism. This aggregation of probands meets ClinGen case‐level evidence for a strong gene–disease association.
Inheritance of DEE66 is autosomal dominant with de novo emergence of pathogenic PACS2 variants. Extended pedigree data are limited and segregation beyond probands is not well documented, precluding lod score estimation. The variant spectrum is restricted to missense changes affecting acidic cluster residues, with p.Glu209Lys recurrent in three probands and p.Glu211Lys in one individual. No loss-of-function or structural variants have been reported, suggesting that missense alterations disrupt specific protein interactions. There is no evidence for founder alleles. Carrier frequencies remain undefined due to the rarity of DEE66 and absence of PACS2 variants in control cohorts.
Functional studies have elucidated the impact of DEE66‐associated PACS2 variants on protein function. In mammalian cell assays, the p.Glu209Lys and p.Glu211Lys mutations decrease PACS2 phosphorylation, prolong protein half-life, and alter protein–protein interactions, disrupting ER–mitochondrial Ca2+ flux and reducing resistance to apoptosis (PMID:39738582). In Caenorhabditis elegans models, editing orthologous PACS2 variants leads to aberrant localization of PACS proteins in multiple cell types, including neurons, and disrupts WDR37 ortholog expression, indicating evolutionary conservation of the PACS2–WDR37 axis (PMID:38712144; PMID:39031646). These perturbations recapitulate key cellular phenotypes consistent with DEE66 neuropathology. Rescue experiments with human PACS1 indicate potential network redundancy but require further validation. These data support a dominant-negative or gain-of-function mechanism in PACS2‐related encephalopathy.
To date, no studies have robustly disputed the PACS2–DEE66 association or identified loss-of-function variants in unaffected individuals. Phenotypic heterogeneity beyond seizures, intellectual disability, cerebellar malformations, facial dysmorphism, and coloboma has not been described. Control cohort analyses have not uncovered PACS2 missense variants at analogous residues, and in silico predictions uniformly indicate deleterious effects. No alternative disease assignments have emerged. Current evidence remains consistent and supportive of PACS2’s role in DEE66.
Integration of genetic and functional data yields a coherent model in which recurrent de novo PACS2 missense variants dysregulate acidic cluster–mediated protein sorting, leading to disturbed calcium homeostasis, neuronal dysfunction, and DEE66 clinical manifestations. The convergence of multiple independent case reports and mechanistic studies satisfies ClinGen criteria for a strong gene–disease association. While additional family segregation data could augment evidence strength further, current data suffice for diagnostic utility and variant interpretation. Clinically, PACS2 sequencing should be considered in individuals presenting with early-onset epileptic encephalopathy, cerebellar dysgenesis, and facial dysmorphism, particularly when coloboma is observed. Application of these findings will improve molecular diagnosis, enable genetic counseling, and guide future therapeutic research.
Key Take-home: De novo missense mutations in PACS2 cause a distinct autosomal dominant encephalopathy with consistent clinical and cellular phenotypes.
Gene–Disease AssociationStrongFour probands with de novo PACS2 missense variants (three with c.625G>A, one with c.631G>A) segregating with DEE66 (PMID:33369122; PMID:30684285; PMID:38545008); functional data concordant Genetic EvidenceStrongFour independent de novo missense variants in four probands, recurrent p.Glu209Lys (c.625G>A) observed Functional EvidenceModerateCellular assays and C. elegans models demonstrate disrupted protein localization, altered Ca2+ flux, and deleterious phosphorylation changes |