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. Author manuscript; available in PMC: 2026 Feb 20.
Published in final edited form as: Epilepsy Res. 2025 Jan 31;210:107514. doi: 10.1016/j.eplepsyres.2025.107514

4-Phenylbutyrate restored GABA uptake, mitigated seizures in SLC6A1 and SLC6A11 Microdeletions/ 3p- Syndrome: from Cellular Models to Human Patients

Melissa B DeLeeuw 1,2,3, Wangzhen Shen 3, Xiaojuan Tian 4, Changhong Ding 4, Karishma Randhave 3, Jing-Qiong Kang 1,2,3,5,6,§
PMCID: PMC12919845  NIHMSID: NIHMS2141431  PMID: 39923323

Abstract

Background:

Haploinsufficient deletions of GABA transporter 1 (GAT-1)- encoding SLC6A1, and GABA transporter 3 (GAT-3)-encoding SLC6A11 are implicated in epileptic syndromes. Despite their significance, the impact of these deletions has not been characterized. Our previous work on SLC6A1 missense mutations prompted a clinical trial for Ravicti (NCT04937062), a glycerol formulation of 4-phenylbutyrate (PBA), for treatment-resistant epilepsy. We observed phenotypic overlap between trial-eligible SLC6A1 mutation patients and 3p- syndrome patients carrying deletions of SLC6A1 and SLC6A11. This study characterizes the functional impact of these deletions and assesses the urgent question of whether 3p- syndrome patients could benefit from this treatment.

Methods:

Chromosomal microarray analysis identified a deletion affecting one allele of both SLC6A1 and SLC6A11 in two pediatric patients with 3p- syndrome. Clinical phenotyping included electroencephalogram (EEG) recordings and neurodevelopmental assessments. Functional characterization was conducted using 3H-labeled GABA uptake assays and Western blotting in HEK293T cells, comparing haploinsufficient and missense variant models.

Results:

The haploinsufficient GAT-1 and GAT-3 conditions demonstrated reduced GABA uptake and protein expression, comparable to known SLC6A1 missense variants. Post-treatment EEGs showed a moderate reduction in epileptiform discharges following PBA administration, and patients exhibited improved motor function. However, varying degrees of cognitive impairments persisted.

Conclusions:

Haploinsufficiency of SLC6A1 and SLC6A11 contributes to the epileptic phenotypes observed in 3p- syndrome, marking this as the first study to biochemically characterize the functional impact of these deletions. Treatment with PBA may provide therapeutic benefits, particularly for addressing seizures and motor deficits, though further exploration of PBA’s long-term effects in patients with 3p- syndrome is warranted.

Keywords: 3p- Syndrome, GABA transporter 1, GABA Transporter 3, genetic epilepsy

Introduction

3p- syndrome, also known as 3p- deletion syndrome or partial monosomy 3p, is a rare autosomal genetic disorder caused by distal deletions on the short arm (p-arm) of the 3rd chromosome.1,2 These deletions result in haploinsufficiency of genes located on chromosome 3p, leading to a variety of clinical manifestations. Patients with 3p- syndrome often present with a spectrum of symptoms, including seizures, neurodevelopmental delay, motor delay, intellectual disability, hypotonia, craniofacial abnormalities, and, in rarer cases, gastrointestinal (GI) issues, polydactyly, and other organ malformations.2,3,4 Understanding the genetic and molecular foundations of this syndrome is crucial for developing targeted therapeutic interventions.

The identification of genetic variants underlying rare neurological disorders is crucial for developing targeted therapies. Previous studies have documented clinical cases of 3p- syndrome; however, the specific genes involved and their roles in the clinical phenotype are not fully understood, as the deleted region can vary case-by-case.3,4 A significant finding by a 2014 study highlighted a rare but recurring proximal microdeletion in the 3p25.3 region, encompassing the genes SLC6A1 and SLC6A11, which encode the gamma-aminobutyric acid (GABA) transporters GAT-1 and GAT-3, respectively.5 This deletion is associated with consistent phenotypes, suggesting these genes play a pivotal role in the pathogenesis of 3p- syndrome. The 3p25.3 region is thus considered critical for the primary features of 3p- syndrome.1,5

GABA is the primary inhibitory neurotransmitter in the mammalian central nervous system (CNS), and its homeostatic function is crucial for proper neurodevelopment and neurotransmission.6,7 GAT-1, encoded by SLC6A1, is a principal GABA transporter in the central nervous system, localized in GABAergic neurons and astrocytes.8 It is responsible for the reuptake of GABA from the synaptic cleft, thereby rapidly terminating neurotransmission and modulating neuronal excitation and inhibition.9 Pathogenic variants in SLC6A1 have been associated with a spectrum of neurodevelopmental disorders, including epilepsy syndromes, intellectual disabilities, motor delays, hypotonia, and autism spectrum disorder (ASD).8,10–15 There is a notable and recurring phenotypic overlap between certain cases of 3p- syndrome and disorders caused by pathogenic SLC6A1 variants, suggesting a shared pathogenic mechanism.5,16–17 GAT-3, encoded by SLC6A11, is another major GABA transporter, primarily expressed in thalamic astrocytes and, like GAT-1, is involved in the reuptake of GABA from the synaptic cleft to maintain homeostasis of GABA in the brain and modulating tonic inhibition, particularly in non-synaptic regions.18–22 In contrast to SLC6A1, the role of SLC6A11 (encoding GAT-3) in disease is less clear. Although no specific variants in SLC6A11 have been reported, deletions affecting this gene are recurrent in 3p- syndrome. Recent reports suggest that the loss of GAT-3 function may contribute to the neurological manifestations observed in these patients, including seizures, ASD, and developmental delays.5,18 Despite the possible contribution of GAT-3 in various neurological conditions, the function of GAT-3 has never been evaluated in disease models.

This study aims to elucidate the functional consequences of the microdeletion of SLC6A1 and SLC6A11 in 3p- syndrome and to investigate the potential of 4-phenylbutyrate (PBA) as a novel therapeutic approach for this condition as the compound has been trialed on SLC6A1 variants mediated disorders with promising results.34 Ravicti, which contains glycerol phenylbutyrate (GPB) as its active ingredient, is an FDA-approved medication primarily used to treat urea cycle disorders. GPB is metabolized in the body to release 4-phenylbutyrate (PBA), a chemical chaperone with potential therapeutic benefits in diseases involving protein misfolding and trafficking defects. Our lab previously reported PBA’s efficacy as a rescue-of-function treatment for pathogenic SLC6A1 variants, which are typically treatment-resistant, like 3p- syndrome.10,18,23–25 We have found PBA to restore GABAergic function by improving the trafficking of wild-type GAT-1 to the cell membrane.23 This improvement in protein trafficking is crucial as it helps restore the function of GAT-1, thus potentially reducing seizure frequency and other related symptoms in patients with SLC6A1 variants. Here, we tested PBA for its effects on SLC6A1 and SLC6A11 deletion conditions to evaluate its effectiveness as a therapy for 3p- syndrome patients.

In this study, we engineered a functional hGAT-3-YFP plasmid for in vitro experiments, performed 3H radioactive GABA uptake assays to evaluate transporter functionality, and conducted western blot analyses to assess protein expression in heterologous cells. Additionally, we report two novel cases of deletions encompassing both SLC6A1 and SLC6A11, contributing to the understanding of their roles in 3p- syndrome and potential therapeutic strategies. This comprehensive approach provides insights into the molecular mechanisms underlying 3p- syndrome and highlights the potential of pharmacochaperoning as a viable therapeutic strategy.

Patient Background and Genetic Data

In this study, we report two novel cases of 3p- syndrome involving haploinsufficient microdeletions encompassing both SLC6A1 and SLC6A11, which encode the GABA transporters GAT-1 and GAT-3, respectively. These deletions result in the loss of one functional copy of each gene, leading to haploinsufficiency—a condition where the single remaining functional copy of the gene is insufficient to maintain normal physiological function. These deletions represent a rare but recurring genetic abnormality within the 3p25.3 region of chromosome 3, a critical locus implicated in a range of neurodevelopmental and neuropsychiatric disorders.

This rare genetic alteration was found de novo in both cases, with no similar deletions detected in the parents, suggesting that these are spontaneous events. Genotyping, karyotyping, and electroencephalograms (EEGs) were provided by the parents of the affected children. All patient data were anonymized, and PHI (Protected Health Information) was redacted to protect the identity of the patients, ensuring compliance with ethical standards.

Patient 1

Patient 1, a male child, aged 2 years and 9 months (33 months), has been under clinical observation since the age of 8 months due to significant developmental delays and neurological concerns. Despite being within normal ranges for physical growth parameters, including head circumference, height, and weight, the child has exhibited considerable delays in reaching developmental milestones. These include difficulties with sitting up independently, standing, and walking, as well as limited engagement with toys, recognizing sounds, and responding to voices. Behavioral assessments revealed that the child struggles with social interactions, including making eye contact and recognizing familiar people, and demonstrates limited verbal communication, raising concerns about intellectual disability.

Neurological evaluations, including electroencephalogram (EEG) testing, indicated abnormal brain activity consistent with epilepsy or seizure disorders. This diagnosis aligns with the child’s observed motor and cognitive delays. Additionally, global developmental delay (GDD) has been identified, indicating that the child is behind in multiple areas of development, encompassing motor skills, speech, and cognitive functions. Family history appears non-contributory, with no reported cases of neurological or developmental disorders.

A chromosomal microarray analysis (CMA) was performed on Patient 1, revealing a significant genetic alteration on chromosome 3p25.3. Specifically, the analysis identified a 466 kilobase pairs (Kbp) deletion in the region spanning from 10,709,750 to 11,175,970 base pairs, according to the human genome reference sequence GRh37. This deletion is notable because it encompasses several critical genes, including SLC6A1 and SLC6A11, both of which are involved in important neurological functions.

Variants or deletions in SLC6A1 are associated with a spectrum of neurological disorders, including myoclonic-atonic epilepsy (MAE), a condition characterized by a combination of seizures and developmental delay.8,17,24,29,30 The presence of a deletion in this gene suggests a significant disruption of GABAergic signaling, which likely contributes to the clinical manifestations observed in the patient, such as seizures, developmental delays, and potentially other neurodevelopmental disorders.17,31 Additionally, although the exact clinical implications of SLC6A11 deletions are less well-documented compared to SLC6A1, the loss of this gene in conjunction with SLC6A1 could exacerbate the neurological dysfunction due to a broader impairment of GABAergic neurotransmission.

Ultimately, the 466 Kbp deletion on chromosome 3p25.3 encompassing SLC6A1 and SLC6A11 is highly likely to be pathogenic, contributing to the patient’s epilepsy and neurodevelopmental challenges. This genetic alteration provides a critical piece of the diagnostic puzzle, highlighting the importance of GABAergic dysfunction in the patient’s clinical presentation. The identification of this deletion also opens potential avenues for targeted therapeutic interventions, such as the use of GABA-modulating drugs, to mitigate the neurological symptoms associated with this deletion.

The pre-treatment electroencephalogram (EEG) recordings for Patient 1 recorded at 30 months old provide critical insights into the neurological basis of his developmental delays and seizure activity. The EEG, conducted over an 11-hour period, revealed a background rhythm predominantly in the 6 Hz range, with voltage amplitudes between 20–80 μV. For a child of this age, a background rhythm in the theta range (6–7 Hz) is developmentally appropriate; however, the persistent slowing and irregularities observed in Patient 1’s EEG are indicative of cortical dysfunction and correlate with the child’s global developmental delay (GDD), which includes delays in motor, cognitive, and speech development. The EEG further demonstrated frequent epileptiform discharges, particularly during sleep, characterized by high-amplitude slow waves and sharp waves in the temporal regions. These findings are consistent with a diagnosis of epilepsy, aligning with the observed clinical symptoms of seizure activity. Although no specific seizure events were recorded during the EEG session, the presence of these interictal discharges suggests a high susceptibility to seizures, aligning with the parents’ report of the Patient 1’s history of seizures, reinforcing the need for anti-seizure medication management.

Patient 2

Patient 2, a male child aged 3 years and 5 months (41 months), has been diagnosed with a deletion involving both the SLC6A1 and SLC6A11 genes. His medical history reveals a series of developmental challenges, including significant delays in motor and cognitive milestones. Although physically within normal growth parameters, Patient 2 has exhibited delays in reaching critical developmental milestones such as walking, speaking, and social engagement. Observational reports indicate that he struggles with making eye contact, responding to verbal cues, and interacting with his environment, raising concerns about potential intellectual disability and autism spectrum disorder (ASD).

Genetic testing of Patient 2 revealed a significant deletion involving the SLC6A1 and SLC6A11 genes on chromosome 3p25.3. This chromosomal deletion is crucial as it disrupts the function of GABA transporters GAT-1 and GAT-3, which play a vital role in inhibitory neurotransmission in the brain. The loss of SLC6A1, which encodes GAT-1, is strongly associated with myoclonic-atonic epilepsy (MAE) and a spectrum of neurodevelopmental disorders. This disruption in GABAergic signaling likely contributes to the patient’s seizures, cognitive delays, and other neurodevelopmental challenges.

The additional deletion of SLC6A11, which encodes GAT-3, may further exacerbate the neurological dysfunction, given GAT-3’s role in GABA reuptake in glial cells. The combined deletion of these two genes suggests a broader impairment in the GABAergic system, which is critical for maintaining the balance between excitatory and inhibitory signals in the brain. The genetic findings, confirmed through a comprehensive SNP array analysis, provide a clear genetic basis for Patient 2’s clinical symptoms and underscore the importance of targeted therapeutic interventions that can address the underlying GABAergic dysfunction. These genetic insights also suggest that Patient 2’s condition could serve as a model for understanding similar cases, where multiple GABA transporter genes are involved.

At the age of 3 years, 2 months (38 months), prior to starting treatment with Ravicti, Patient 2’s EEG revealed severe epileptic activity, providing a clear indication of his neurological dysfunction. The baseline EEG, conducted 1–2 months before treatment initiation, showed extensive epileptiform discharges and abnormal brain activity, which were concerning for the progression of his condition. This severe epileptic activity aligns with the genetic findings of deletions in the SLC6A1 and SLC6A11 genes, both of which are implicated in GABAergic signaling and epilepsy.

Following baseline evaluations, both patients were treated with Ravicti under the supervision of their medical teams to target the identified GABAergic dysfunction. Post-treatment outcomes were assessed after three months, providing insights into the potential therapeutic effects of Ravicti in managing the neurological deficits associated with these deletions.

Methods

Patients with confirmed SLC6A1, SLC6A11 microdeletions

Two pediatric patients with confirmed microdeletions encompassing both the SLC6A1 and SLC6A11 genes were evaluated for this study. For both patients, informed consent was obtained from their parents or legal guardians, in compliance with ethical standards for research involving minors. Both patients were clinically assessed at specialized pediatric neurology and epilepsy centers. Clinical data collected included detailed developmental histories, seizure types and frequency, response to anti-seizure medications (ASMs), motor and cognitive development, and family history. General and neurological examinations were performed by pediatric neurologists, and video electroencephalograms (EEGs) were reviewed by qualified electroencephalographers.

The cDNAs for coding GABA transporter 1

The plasmid cDNA encoding enhanced yellow fluorescent protein (EYFP)-tagged rat GAT-1 was sub-cloned into the expression vector pCMV. QuikChange Site-directed Mutagenesis kit was utilized to introduce the GAT-1(Ser295Leu) and GAT-1(Ala288Val) variants into wild-type GAT-1 coding sequence. The product from polymerase chain reaction was transformed using DHα competent cells and finally plated. A clone was chosen and grown overnight, replicating the cDNA. The GAT-1(Ser295Leu) and GAT-1(Ala288Val) variants were confirmed by DNA sequencing. Both the wild-type and the variant cDNAs of GAT-1 were prepared with Qiagen Maxiprep kit.

The cDNAs for coding GABA transporter 3

The plasmid cDNA encoding enhanced yellow fluorescent protein (EYFP)-tagged human GAT-3 was sub-cloned into the expression vector pCMV. QuikChange Site-directed Mutagenesis kit and overlap extension polymerase chain reaction were utilized to introduce the GAT-3 coding sequence in the selected vector. The product from polymerase chain reaction was first ligated with the vector and then was transformed using DHα competent cells and finally plated. A clone was chosen and grown overnight, replicating the cDNA. The complete GAT-3 plasmid was confirmed by DNA sequencing. The wild-type cDNA of GAT-3 was prepared with Qiagen Maxiprep kit.

Polyethylenimine (PEI) transfection

Standard transfection protocols were performed using human embryonic kidney 293T (HEK293T) cells.26 24 hours before transfection HEK293T cells were split and seeded with an equal number into plates. For radiolabeling GABA uptake, 0.5μg of the cDNAs with PEI at a ratio of 1:2.5μl was transfected in 35mm dish. The cDNAs were combined with Dulbecco modified Eagle medium (DMEM) and a PEI/DMEM mixture. For total protein expression, 3μg cDNAs also with 1:2.5μl PEI ratio were used and transfected in 60mm dish. Transfected HEK293T cells incubated for 48 hours. After incubation, proteins were harvested as described below.

Western blot analysis of total GAT-1 and GAT-3 protein

Live transfected HEK293T cells were washed with phosphate buffered saline (1×PBS, pH 7.4) 3 times and then cells were lysed in RIPA buffer (20 mM Tris, 20 mM EGTA, 1 mM DTT, 1 mM benzamidine), supplemented with 0.01 mM PMSF, 0.005 μg/mL leupeptin, and 0.005 μg/mL pepstatin for 30 min at 4° C. The samples were then sonicated and subject to protein concentration determination and followed by SDS-PAGE and immunoblotted. Primary antibodies used were anti-GAT-1 1:500 (Alomone Labs, AGT-001 or Synaptic System, 274102), anti-GAT-3 1:500 (Synaptic System, 274302), and anti- β-actin 1:1000 (Abclonal, AC004). Secondary antibodies used were LI-COR IRDye 680LT Goat anti-Mouse IgG Secondary Antibody (926–68020) and IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926–32211), both 1:10,000.

Radioactive 3H-labeled GABA uptake assay

The radioactive 3H-labeled GABA uptake assay in HEK293T cell model. Cells were cultured in 5 mm2 dishes 4 days before the GABA uptake experiment in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were then transfected with equal amounts of the wild-type or the deletion-mimicking cDNAs (0.5 μg) with PEI at a ratio of 1 μg cDNA:2.5 μl of PEI for each condition at 48 hrs after cell seeding. GABA uptake assay was carried out 72 hrs after transfection. The cells were incubated with preincubation solution for 30 min and then incubated with preincubation solution containing 1μci/ml 3H-labeled GABA and 10 μM unlabeled GABA for 30 min at room temperature. After washing, the cells were lysed with 0.25 N NaOH for 1 hr. Acetic acid glacial was added and lysates were then determined on a liquid scintillator with QuantaSmart. The flux of GABA (pmol/μg/min) was averaged with at least triplets for each condition at each transfection. The average counting was taken as n = 1. The untransfected condition was taken as a baseline that was subtracted from both the wild-type and the deletion conditions. The pmol/μg/min in the deletion conditions was then normalized to the wild-type from each experiment, which was arbitrarily taken as 100%.

Data analysis

Numerical data were expressed as mean ± SEM. Proteins were quantified by Odyssey software and data were normalized to loading controls and then to wild-type transporter proteins, which was arbitrarily taken as 1 in each experiment. The radioactivity of GABA uptake was measured in a liquid scintillator with QuantaSmart. The flux of GABA (pmol/μg/min) in the wild-type GAT-1 samples was arbitrarily taken as 100% each experiment. For statistical significance, we used one-way analysis of variance (ANOVA) with Newman-Keuls test or Student’s unpaired t-test. In some cases, one sample t-test was performed (GraphPad Prism, La Jolla, CA), and statistical significance was taken as p < 0.05.

Results

1. Genetic and Phenotypic Features in 3p- Deletion Syndrome and Overlap with SLC6A1 Variant-Mediated Disorders

The chromosomal microdeletion affecting the 3p25.3 region, which encompasses both the SLC6A1 and SLC6A11 genes, has been identified as a region of interest in 3p- syndrome. The SLC6A1 and SLC6A11 genes encode GABA transporter 1 (GAT-1) and GABA transporter 3 (GAT-3), respectively. Normal function of these genes is necessary for maintaining homeostatic GABAergic neurotransmission (Fig. 1A). The deletion of one or both of these genes can lead to a disruption of this balance, resulting in the neurological manifestations seen in patients with 3p- syndrome. Of particular interest, a haploinsufficient double microdeletion of both SLC6A1 and SLC6A11 has been documented as a rare, but recurring, genetic alteration in this syndrome, contributing significantly to the observed phenotypes.

Figure 1. Comparative Analysis of 3p- Deletion Syndrome and SLC6A1 Variant-Mediated Disorders.

Figure 1.

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A) Chromosomal location of 3p25.3 deletions highlighting the involvement of SLC6A1 and SLC6A11. This panel illustrates the specific region on chromosome 3p that is deleted in 3p- syndrome and corresponding table pinpointing the critical genes affected. B) Comparative phenotypic manifestations of 3p- syndrome versus disorders mediated by mutations in SLC6A1. This diagram delineates the clinical features associated with 3p- syndrome alongside those found in conditions stemming from SLC6A1 mutations, underlining the overlapping and distinct symptoms. C) Diagram of 2D GAT-1 and GAT-3 protein structures. GAT-1 shows the positions of the A288V and S295L mutations. Each mutation is annotated with its associated clinical phenotypes, providing a molecular-genetic link to observed clinical outcomes in patients with these specific SLC6A1 mutations.

In the small subset of patients with this haploinsufficient double microdeletion, including the two novel patients presented in this study, overlapping phenotypic features are noted when compared to patients having certain SLC6A1 missense variants.5,17,18 The overlap includes, but is not limited to: hypotonia, neurodevelopmental delay (NDD), autism spectrum disorder (ASD), motor delays, and seizures (Fig. 1B). This phenotypic overlap is clinically significant, as these clinical presentations are hallmarks of SLC6A1 missense variants. Furthermore, these missense variants serve as an inclusion criterion for the Ravicti clinical trial, which targets GABA transporter dysfunction.27,34 The shared features between these two patient groups suggest that the therapeutic potential of Ravicti could be expanded to treat 3p- syndrome patients with the SLC6A1 and SLC6A11 double microdeletion, highlighting the importance of further exploring this pharmacological avenue.

Specifically, two SLC6A1 missense variants that we have previously identified and characterized—Ser295Leu (S295L) and Ala288Val (A288V)—exhibit phenotypes closely aligned with those of the 3p- syndrome patients (Fig. 1C). These variants show partial to complete loss of GABA uptake function, accompanied by significantly reduced GAT-1 protein expression.12,28 Our previous research demonstrated that PBA provides a robust rescue of function for these variants by alleviating endoplasmic reticulum (ER) stress induced by impaired protein trafficking.24,28 These findings also provide a strong foundation for the hypothesis that PBA could be expanded as a therapeutic option for 3p- syndrome patients with the SLC6A1 and SLC6A11 double microdeletion, given the shared phenotypic and mechanistic overlap.

2. Reduced GABA Uptake Function and GAT-1 and GAT-3 protein expression in Single Gene 3p- Syndrome Deletion Conditions.

In our study, we initially established a baseline functionality for the GABA transporters GAT-1 and GAT-3 in wild-type conditions by transfecting HEK293T cells with both cDNAs to ensure both plasmids were reliable and robust and measuring GABA uptake function by high-throughput 3H GABA uptake assay. To verify correct expression, the wild-type GAT-1 and GAT-3 conditions were treated with their specific inhibitors, CI-966 [50μM] and SNAP5114 [30μM], respectively, by including the inhibitors in the 3H-labeled GABA flux solution. Both of which exhibited significant, almost complete, reduction in uptake; reduced to ~6% in GAT-1 with CI-966 treatment and ~3% in GAT-1 with SNAP5114 treatment compared with untreated GAT-1 (Fig. 2A) (****P < 0.0001). In order to mimic the haploinsufficiency observed in 3p- syndrome, we co-transfected HEK293T cells with a mixture of wild-type cDNAs and empty vector pcDNA at a 1:1 ratio to generate a hemizygous condition with untransfected ‘U’ HEK293T cells, full dose of empty-vector pcDNA, and known GAT-1(S295L) and GAT-1(A288V) heterozygous missense variants included as controls. Compared with cells transfected only with wild-type cDNAs, both GAT-1 and GAT-3 hemizygous conditions had reduced GABA uptake function (Fig. 2B) (****P < 0.0001). To investigate the protein expression deficits in haploinsufficient conditions mimicking 3p- syndrome, we transfected HEK293T cells with total cDNAs of 3μg, adjusting for hemizygous and double microdeletion conditions by co-transfecting equal parts of wild-type cDNA and empty vector pcDNA. This method was designed to simulate the reduced gene dosage observed in 3p- syndrome. Protein levels were assessed by Western blot, using specific antibodies against GAT-1 and GAT-3, and normalization was done relative to β-actin to ensure accuracy in quantification. Both GAT-1 and GAT-3 single hemizygous conditions showed a significant reduction in expression (−0.5031±0.03341 for GAT-1; −0.2749±0.04100 for GAT-3) (Fig. 2C and Fig. 2D). Faint high molecular weight bands observed (Fig. 2D) represent the YFP tag on the GAT-3 plasmid. This tag confirms the integrity and successful expression of the transfected cDNA construct in HEK293T cells. Importantly, this high molecular mass band corresponding to GAT-3 is faint compared to that of GAT-1, likely reflecting differences in the abundance or expression dynamics of the two constructs. This observation highlights the distinct properties of the GAT-3 construct and underscores its successful incorporation into the experimental system. As this is the first report characterizing this specific cDNA construct in a Western blot, the detection of the YFP tag provides an additional layer of validation for its use in simulating GAT-3 expression under experimental conditions. The protein expression deficits observed in the haploinsufficient conditions mimicking 3p- syndrome are comparable to the protein expression impairments previously characterized in SLC6A1 missense variants exhibiting similar phenotypes.24

Figure 2: Reduced GABA Uptake Function and GAT-1 and GAT-3 protein expression in Single Deletion (SLC6A1 or SLC6A11) Conditions on par with SLC6A1 Missense Mutations Exhibiting Similar Phenotypes.

Figure 2:

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A) GABA uptake under baseline conditions in HEK293T cells expressing wild-type GAT-1 and GAT-3. The cells were incubated with preincubation solutions containing 3H GABA without or with Cl-966 [50 μM] or SNAP5114 [30 μM] for 30 min before being counted on a liquid scintillator with QuantaSmart. Demonstrates robust functionality of both transporters with a significant decrease in uptake upon treatment with specific inhibitors (****P < 0.0001), confirming the effective expression of the intended transporters. B) GABA uptake in HEK293T cells under haploinsufficient conditions mimicking 3p- syndrome deletions, showing a significant reduction in GABA uptake compared to wild-type with untransfected ‘U’ HEK293T cells, empty-vector pcDNA, and known GAT-1(S295L) and GAT-1(A288V) heterozygous missense variants included as controls (****P < 0.0001). Values were expressed as mean ± SEM. One-way analysis of variance with Sidak’s post hoc tests for multiple comparisons for A and B. C) Western blot showing protein expression levels of GAT-1 in wild-type (WT) and hemizygous conditions mimicking haploinsufficient deletion of GAT-1. The intensity of bands was quantified, and data normalized to internal loading controls. Expression levels in the hemizygous condition showed a significant reduction compared to WT, as analyzed by unpaired t-test (****P<0.0001), indicating the impact of haploinsufficiency on protein levels. Error bars represent SEM (−0.5031±0.03341). D) Western blot analysis of GAT-3 in WT and hemizygous conditions, mimicking haploinsufficient deletion of GAT-3. Normalized integrated density values (IDVs) are shown, with a significant decrease in protein expression in the hemizygous condition versus WT, as analyzed by unpaired t-test (****P<0.0001, SEM −0.2749±0.04100), demonstrating the effects of gene deletion on GAT-3 protein levels. Beta-actin was used as loading control (LC) for C and D.

3. Reduced GABA Uptake Function in Double Gene Deletion Condition and Moderate Rescue by 4-Phenylbutyrate (PBA) Treatment.

To explore the deficits associated with the double SLC6A1 and SLC6A11 microdeletion as reported in this study, we co-transfected HEK293T cells with a mixture of GAT-1 and GAT-3 wild-type cDNAs at a 1:1 ratio to generate a double hemizygous condition. A chromosomal schematic illustrates the wild-type condition, containing two functional copies of GAT-1 and GAT-3, and the deletion condition, where only one functional copy of either GAT-1, GAT-3, or both is present (Fig. 3A). We then treated the double microdeletion condition with CI-966 [50μM] and SNAP5114 [30μM] inhibitors to verify expression and determine differential uptake contribution between GABA transporters. When compared to both GAT-1 and GAT-3 wild-type conditions, the double GAT-1/GAT-3 microdeletion condition showed significant reduction in GABA uptake function (Fig. 3B) (****P < 0.0001). In the context of our investigations into the efficacy of PBA as a therapeutic intervention for 3p- syndrome, Figure 4 offers crucial insights into its mechanism and effectiveness under deletion conditions. Notably, PBA treatment [2mM] concentration for 24 hours resulted in a significant yet moderate increase in GABA uptake function across all conditions (Fig. 3C). This improvement, while statistically significant, did not restore GABA uptake to wild-type levels, particularly in the deletion conditions. Such findings indicate moderate rescue capability of PBA in deletion conditions, suggesting that while PBA enhances functional activity, it does not completely compensate for the lost function due to gene deletions

Figure 3: Reduced GABA Uptake Function in Double (SLC6A1 and SLC6A11) Deletion Condition and Moderate Rescue by 4-Phenylbutyrate (PBA) Treatment.

Figure 3:

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A) Chromosomal schematic of deletion conditions reported in patient cases. Wild-type conditions contain two functional copies of GAT-1 and GAT-3, whereas deletion conditions involve the loss of one functional copy of either GAT-1, GAT-3, or both, as characterized and reported in this study. B) GABA uptake in the double deletion model, mimicking the recurring deletion of both SLC6A1 and SLC6A11. This condition shows a significant drop in uptake functionality compared to single transporter expressions (****P < 0.0001), assessed before and after treatment with inhibitors, underscoring the combined effect of these deletions. Values were expressed as mean ± SEM. One-way analysis of variance with Sidak’s post hoc tests for multiple comparisons for B. C) Functional 3-H radiobinding assay data comparing untreated with 24-hour PBA [2mM] treatment. Shows an increase in GABA uptake across all conditions, analyzed using multiple unpaired t-tests with false discovery rate (FDR) adjustments to account for multiple comparisons. This suggests a functional rescue despite the absence of increased protein expression. Error bars represent SEM.

Figure 4: 4-Phenylbutyrate (PBA) Has a Moderate Rescue of GAT-1 and GAT-3 Protein Expression in 3p- Syndrome Deletion Conditions.

Figure 4:

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A) Combined Western blot analysis of WT, hemizygous conditions for GAT-1, GAT-3, and co-expression in double deletion mimicking the 3p- syndrome with both SLC6A1 and SLC6A11 deletions. Each protein was probed with specific antibodies against GAT-1 and GAT-3. Beta-actin was used as loading control (LC). The normalized IDVs for GAT-1 and GAT-3, respectively, were analyzed with ordinary one-way ANOVA with post hoc Dunnett’s test for multiple comparisons. Both GAT-1 and GAT-3 show significant decreases in protein expression in hemizygous and double deletion conditions compared to WT (****P<0.0001, ***P<0.001, **P<0.01). B) Combined Western blot analysis comparing untreated with 24-hour PBA [2mM] treatment of WT, hemizygous conditions for GAT-1 and GAT-3, and co-expression GAT-1/GAT-3 hemizygous double deletion mimicking the 3p- syndrome with both SLC6A1 and SLC6A11 deletions. Each protein was probed with specific antibodies against GAT-1 and GAT-3. Beta-actin was used as loading control (LC). C) Western Blot analysis measuring protein expression in hemizygous and double deletion conditions relative to wild-type GAT-1. Displays a moderate increase in protein expression with PBA treatment, indicative of a partial rescue effect using multiple unpaired t-tests with Welch’s correction, suggesting some variability in the effectiveness of PBA on protein stability. D) Western Blot analysis measuring protein expression in hemizygous and double deletion conditions relative to wild-type GAT-3. No increase in protein expression following PBA treatment is observed; in fact, the double deletion condition shows a decrease in expression. These findings were assessed using multiple unpaired t-tests with Welch’s correction.

4. 4-Phenylbutyrate (PBA) Has a Moderate Rescue of GAT-1 and GAT-3 Protein Expression in 3p- Syndrome Deletion Conditions.

To assess the potential impact of PBA on protein expression in 3p- syndrome deletion conditions, we examined its effects on GAT-1 and GAT-3 levels in both hemizygous and double deletion contexts. Here we provide a detailed analysis of Western blot results, comparing untreated and PBA-treated samples to evaluate whether PBA can partially rescue the expression of these critical GABA transporters under deletion conditions observed in 3p- syndrome patients. The double GAT-1/GAT-3 hemizygous microdeletion condition showed reduced expression compared to both GAT-1 and GAT-3 wild-type expression (Fig. 4A). Notably, in the ‘Co-Exp.’ condition, the GAT-1 band appeared at a lower molecular weight compared to the WT and Hemi groups in the SDS-PAGE. This shift likely results from the co-transfection of 1.5 μg GAT-1 and 1.5 μg GAT-3 cDNA, which reduced the total cDNA dose for GAT-1 compared to the WT condition (3 μg). Additionally, the co-expression of GAT-1 and GAT-3 may introduce competition for cellular transcriptional and translational machinery, potentially affecting GAT-1’s post-translational modifications and stability. Such factors could lead to altered electrophoretic mobility and the observed shift of protein mass. The protein expression deficits observed in the haploinsufficient conditions mimicking 3p- syndrome are comparable to the protein expression impairments previously characterized in SLC6A1 missense variants exhibiting similar phenotypes.24,28

Interestingly, the analysis of protein expression for GAT-1 and GAT-3 post-PBA treatment did not show a significant enhancement in the levels of these proteins (Fig. 4B, 4C, and Fig. 4D). However, the ratio of the band with the higher molecular mass over the band with the lower molecular mass was increased in PBA treated cells, suggesting enhanced protein folding and maturation. This observation is pivotal, as it implies that the rescue effect of PBA in deletion conditions might not operate primarily through increasing the total protein amount but enhancing protein refolding, which is also observed in missense mutation scenarios. By aiding the folding and trafficking of misfolded proteins, PBA can alleviate ER stress and make the cellular environment more conducive for protein folding and trafficking. This is consistent with the functional assay., The increase in GABA uptake without a corresponding increase in protein expression suggests more efficient protein trafficking and more functional transporters are at play. Additionally, other possible mechanisms for PBA’s action in deletion conditions could include allosteric modulation of existing GABA transporters, enhancing their activity without altering protein quantity. Alternatively, PBA may activate compensatory pathways that support GABA uptake or improve the membrane stability and local environment of GABA transporters, thus enhancing their functional efficacy. These mechanisms would not necessarily involve changes in protein expression levels but would instead optimize the function of existing protein molecules or the cellular environment.

This disconnection between functional rescue and total protein expression in deletion conditions highlights a nuanced role of PBA, highlighting functional state, instead of the total amount of the transporter in PBA rescue. This is consistent with our hypothesis that that PBA enhance protein clearance and promote trafficking as observed in the GABAA receptor mutation mouse model Gabrg2+/Q390X of Dravet syndrome.28,35 We have previously demonstrated that PBA reduced ER stress by enhancing the mutant protein clearance.28 Understanding these alternative pathways is important for developing tailored therapeutic approaches that harness PBA’s full potential under varying genetic contexts such as those presented by 3p- syndrome and SLC6A1 missense mutations mediated disorders.

While PBA demonstrates a capacity to moderately enhance GABA uptake in 3p- syndrome deletion conditions, it does so without significantly increasing GAT-1 or GAT-3 protein levels. This points to a potential role for PBA in modulating transporter activity through mechanisms other than protein expression enhancement, which warrants further investigation to fully elucidate and optimize its therapeutic application in such complex genetic disorders.

5. Identification and Characterization of 3p- Syndrome

In this study, we present a detailed genetic and neurological characterization of two patients diagnosed with 3p- syndrome, focusing on a significant 466 kilobase pairs (Kbp) deletion on chromosome 3p25.3 and its correlation with the patient’s clinical and electroencephalographic (EEG) findings. This deletion encompasses critical genes involved in GABAergic neurotransmission, specifically SLC6A1 and SLC6A11, which encode the GABA transporters GAT-1 and GAT-3, respectively.

The patients presented with a spectrum of neurological symptoms, including epilepsy, neurodevelopmental delay, and hypotonia, which are phenotypes that overlap significantly with those seen in disorders associated with SLC6A1 variants. This overlap highlights the critical role of GABAergic signaling in maintaining neurological health and highlights the potential impact of these deletions on GABA transporter function.

The identification of these haploinsufficient microdeletions in SLC6A1 and SLC6A11 provides new insights into the genetic basis of 3p- syndrome and offers a compelling case for targeted therapeutic intervention. Given the established efficacy of 4-phenylbutyrate (PBA) in rescuing GAT-1 function in missense variant conditions24, this study seeks to explore whether PBA could also be effective in treating patients with these specific deletions. Such findings would not only extend the applicability of PBA (Ravicti) as a therapeutic agent but could also propose the first targeted drug therapy for this subset of 3p- syndrome patients, offering a novel approach to managing this debilitating condition. The recurring nature of this deletion suggests that while rare, it is a significant contributor to the clinical variability seen in 3p- syndrome, warranting further investigation and consideration in therapeutic development.

5.1. Patient 1

After three months of treatment with Ravicti, Patient 1 demonstrated moderate improvement in several clinical aspects, although progress was gradual. The child showed increased cognitive engagement, with a higher level of alertness and improved response to social cues. Behavioral assessments post-Ravicti treatment indicated that the child was more willing to interact with certain toys, such as playing on a slide and engaging in limited social play. However, challenges in understanding verbal commands and unnatural hand movements persisted. Treatment recommendations for the child include a comprehensive rehabilitation program comprising physical therapy (PT), occupational therapy (OT), and speech therapy, aimed at improving motor function, language development, and cognitive abilities. Sensory integration therapy and interactive play therapy were also suggested to help enhance the child’s environmental engagement and behavioral responses. Parental guidance has been emphasized as a critical component of the child’s care, with parents advised on activities and strategies to support developmental progress at home. While some challenges remain, these moderate improvements suggest a potential benefit of continued Ravicti treatment in addressing the neurological deficits linked to the SLC6A1 and SLC6A11 deletions.

Following treatment with Ravicti, a notable improvement in the EEG pattern of Patient 1 was observed (Fig. 5A). The post-treatment EEG, similarly recorded over an extended period, showed a remarkable reduction in epileptiform discharges and an increase in the background rhythm frequency to a near-normal range of 7–8 Hz. The amplitude also became more consistent, ranging from 30–50 μV, suggesting improved cortical function. These changes are indicative of a significant positive response to the treatment, demonstrating an enhancement in the overall stability of neuronal activity. These EEG improvements align with clinical gains observed post-treatment, including moderate improvements in motor stability and sociability (Fig. 5B).

Figure 5: Clinical Outcomes and EEG Analysis of a 3p- Syndrome Patient Treated with Ravicti (Metabolized Form of PBA).

Figure 5:

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A) EEG comparisons for Patient 1 before and after three months of treatment with Ravicti. The pre-treatment EEG for Patient 1 shows frequent, severe seizure activities with abnormal background discharges, indicating significant neurological disruption. The post-treatment EEG demonstrates a remarkable reduction in epileptic activity, clearer background, and improved EEG patterns, suggesting enhanced neurological stability and reduced seizure frequency due to the treatment. B) Summary of clinical observations and EEG results for Patients 1 and 2 before and after treatment with Ravicti. The table reflects various clinical categories such as seizures, motor development, cognitive/communication skills, hypotonia, and other observations, highlighting the changes observed pre- and post-treatment. For Patient 1, notable improvements include better motor skills and social interaction. Patient 2 shows significant advancements in motor skills and social responsiveness. Both patients exhibit decreased seizure activity and improved motor and cognitive functions post-treatment, underscoring the efficacy of 4-Phenylbutyrate in enhancing the quality of life and clinical outcomes for individuals with 3p- syndrome.

5.2. Patient 2

Neurological assessments, including EEG recordings, have shown severe epileptic activity, further complicating his clinical picture. This case highlights the multifaceted challenges in managing a child with significant neurodevelopmental delays and epilepsy, particularly in the context of a rare genetic deletion affecting key neurotransmitter transporters. Despite the challenges in managing his condition, a follow-up EEG conducted 6–8 weeks after starting Ravicti showed a remarkable improvement. The reduced epileptic activity on the follow-up EEG was described as “remarkable” by a leading pediatric neurologist, suggesting that Ravicti had a significant positive impact on the child’s neurological function. However, the EEG findings must be balanced with the clinical observations of developmental regression in speech, as the child, who had started to babble and say “dada,” lost these abilities after beginning Ravicti, although the parents also noted improvements in other areas such as attention span, eye contact, and interaction with toys. Despite setbacks in speech development, improvements in mood, sociability, and seizure control were evident (Fig. 5B). The pre-treatment EEG serves as a critical baseline for assessing the effectiveness of ongoing and future treatments, highlighting the need for careful monitoring and potential adjustments in therapy to maximize benefits while minimizing adverse effects.

The EEG abnormalities observed in both patients, particularly the slowed background activity and the presence of epileptiform discharges, provide a physiological basis for the presenting neurological and developmental challenges. These findings, when viewed alongside the comprehensive clinical assessment and chromosomal microarray analysis (CMA) revealing a deletion involving the SLC6A1 and SLC6A11 genes, underscore the impact of disrupted GABAergic signaling in this patient. The pre-treatment EEG results establish a critical baseline for evaluating the effectiveness of subsequent therapeutic interventions, including the use of anti-seizure medications and targeted rehabilitation therapies designed to address the developmental delays and improve overall neurological function. The post-treatment EEG findings further reinforce the potential therapeutic efficacy of Ravicti in mitigating some of the neurological impacts associated with the deletion affecting the GABA transporter genes, providing a foundation for ongoing clinical management and potentially guiding future therapeutic strategies for similar cases.

The findings from both patients underscore the potential therapeutic impact of Ravicti in addressing neurological deficits associated with 3p- syndrome. Improvements were observed across several clinical domains, including seizure frequency, EEG abnormalities, motor development, and sociability (Fig. 5B). While some challenges, such as persistent delays and developmental regression in specific areas, remain, the overall results suggest that Ravicti offers a promising avenue for mitigating some of the effects of haploinsufficient deletions in SLC6A1 and SLC6A11. These outcomes warrant further investigation to optimize therapeutic strategies and address individual variability in treatment response.

Discussion

3p- syndrome, characterized by deletions on the short arm of chromosome 3, is a rare disorder with diverse clinical manifestations, including neurodevelopmental delay, epilepsy, motor dysfunction, and intellectual disability. The deletions affecting the SLC6A1 and SLC6A11 genes, which encode GABA transporters 1 (GAT-1) and 3 (GAT-3), respectively, have emerged as critical genetic factors contributing to the neurological features of this syndrome. GABA is the brain’s primary inhibitory neurotransmitter, and the regulation of its levels is essential for maintaining homeostatic excitation and inhibition balance in neural circuits.32 In this study, we investigated the functional and molecular consequences of these deletions, compared them to SLC6A1 missense variants, and explored the therapeutic potential of 4-phenylbutyrate (PBA/Ravicti) in alleviating GABAergic deficits in 3p-syndrome.

Our study begins by establishing that SLC6A1 and SLC6A11 deletions disrupt GABAergic signaling in ways that are comparable to SLC6A1 missense variants. In 3p- syndrome patients harboring a double microdeletion of both genes, we observed overlapping phenotypic features with patients carrying pathogenic SLC6A1 missense variants, such as epilepsy, neurodevelopmental delay (NDD), autism spectrum disorder (ASD), and motor delays.5,17,24 These phenotypes are hallmarks of SLC6A1-related disorders, emphasizing the critical role of SLC6A1 and SLC6A11 in maintaining proper GABAergic function. The significant overlap between the clinical manifestations of these two patient groups raises the intriguing possibility that the therapeutic approaches currently being explored for SLC6A1 variants, namely PBA/Ravicti, could be applicable to 3p- syndrome patients as well.

In our functional assays, we confirmed that deletions in SLC6A1 and SLC6A11 result in significantly impaired GABA uptake, mimicking the loss of function seen in SLC6A1 missense variants like S295L and A288V. Both transporters, GAT-1 and GAT-3, are responsible for the reuptake of GABA from the synaptic cleft, which modulates synaptic activity and prevents hyperexcitation. Consistently, absence seizure activity was detected in both Slc6a1+/S295L and Slc6a1+/A288V mouse models.11,24 Using HEK293T cells expressing wild-type GAT-1 and GAT-3, we demonstrated robust GABA uptake in the cells expressing the GAT-1 and GAT-3 transporters, with significant reduction following inhibition by the GAT-1-specific inhibitor CI-966 and the GAT-3-specific inhibitor SNAP5114. When we mimicked haploinsufficiency by co-transfecting wild-type cDNAs with empty vectors, we observed a marked decrease in GABA uptake (****P < 0.0001), which was further compounded when both SLC6A1 and SLC6A11 were deleted (Fig. 2).

These deficits were strikingly similar to those observed in the heterozygous S295L and A288V missense variants of SLC6A1, both of which lead to impaired GABA transporter function.24 This similarity suggests that the loss of one copy of these genes (haploinsufficiency) is sufficient to cause significant disruption in GABAergic signaling, thus contributing to the overlapping phenotypes in these patient populations. The functional deficits observed in 3p- syndrome deletion conditions mirror the deficits in GABA uptake seen in missense variants, supporting our hypothesis that these genetic alterations result in comparable disturbances in inhibitory neurotransmission.

We extended our investigation to assess the protein expression of GAT-1 and GAT-3 in hemizygous and double deletion conditions. Western blot analysis revealed that the hemizygous conditions for both SLC6A1 and SLC6A11 showed significant reductions in protein expression levels, which were further exacerbated in the double deletion condition (Fig. 3). These results are consistent with the reduced functionality observed in the GABA uptake assays and underscore the critical role that gene dosage plays in regulating transporter expression and function. The loss of one functional allele in 3p- syndrome patients result in insufficient protein production to maintain normal GABAergic signaling, contributing to the severe neurological phenotypes observed. Interestingly, the protein expression deficits in the deletion conditions were comparable to those previously reported for S295L and A288V missense variants of SLC6A1, further reinforcing the mechanistic similarities between these genetic alterations. In both cases, the reduced expression and function of GAT-1 leads to impaired GABA uptake and, consequently, to the development of epilepsy and other neurological symptoms.

The therapeutic potential of 4-phenylbutyrate (PBA/Ravicti) in addressing these functional deficits is of particular interest. In SLC6A1 missense variants like S295L and A288V, the pathomechanism involves impaired protein trafficking caused by endoplasmic reticulum (ER) stress due to the accumulation of misfolded variant GAT-1 protein.28 PBA acts as a pharmacochaperone, facilitating the proper folding and trafficking of GAT-1 to the cell membrane, thereby restoring GABA uptake function.24,33 Our previous studies have shown that PBA provides a robust rescue of function in these missense variants, effectively reducing ER stress and improving GABAergic neurotransmission.

However, in the deletion conditions seen in 3p- syndrome, the therapeutic effect of PBA was moderate compared to its strong efficacy in missense variants. We hypothesize that this moderate effect is due to the absence of ER stress, which is present in the cells expressing the mutant GAT-1 but absent in the deletion conditions. This notion is evidenced in the GAT-1 and GAT-3 expression in the cells with a higher cDNA amount in which condition, the endogenous chaperones are overwhelmed by unfolded or misfolded proteins. This is the case in missense variants. Instead, in haploinsufficient conditions, PBA may exert its effect through alternative mechanisms, such as stabilizing the remaining wild-type protein or modulating compensatory pathways involved in GABAergic signaling. Although the exact mechanisms by which PBA exerts its moderate rescue in deletion conditions remain unclear, our results suggest that it still holds therapeutic potential for 3p- syndrome patients.

We report a detailed analysis of two pediatric patients, both presenting with deletions encompassing the SLC6A1 and SLC6A11 genes, highlights significant phenotypic overlap between 3p- syndrome and SLC6A1-related disorders. Both patients exhibited hallmark symptoms that are commonly associated with SLC6A1-related disorders, including treatment-resistant seizures, profound neurodevelopmental delay, and hypotonia. These clinical features, consistent across both cases, underscore the shared pathogenic mechanisms likely driven by disruptions in GABAergic neurotransmission due to the loss of SLC6A1 and SLC6A11 function. The identification of overlapping phenotypes between 3p- syndrome and SLC6A1 missense variants opens new avenues for therapeutic intervention. The moderate improvement observed in Patient 1 following treatment with Ravicti, as evidenced by a reduction in epileptiform discharges, an increase in background rhythm frequency to 7–8 Hz, and consistent amplitude levels (30–50 μV), aligning with near-normal EEG patterns, provides a promising indication that this pharmacochaperone could be beneficial for 3p- syndrome patients. While the effect of PBA in deletion conditions may not be as pronounced as in missense variants, the observed improvements in seizure control, cognitive engagement, and social interactions suggest that continued treatment could provide long-term benefits.

Future studies should focus on elucidating the specific mechanisms by which PBA operates in deletion conditions, as well as exploring combination therapies that could enhance its efficacy. Additionally, expanding the inclusion criteria of the PBA/Ravicti clinical trial to include 3p- syndrome patients with SLC6A1 and SLC6A11 deletions would provide valuable insights into the broader applicability of this therapeutic approach. Given the shared mechanistic underpinnings between 3p- syndrome and SLC6A1-mediated disorders, targeted interventions like PBA hold great promise for improving the quality of life for patients suffering from these debilitating conditions.

In conclusion, our study provides the first and a comprehensive analysis of the functional and molecular deficits associated with SLC6A1 and SLC6A11 deletions in 3p- syndrome. By drawing parallels with SLC6A1 missense variants and exploring the therapeutic potential of PBA, we have laid the groundwork for future research aimed at developing targeted treatments for this rare genetic disorder. The moderate efficacy of PBA in deletion conditions highlights the complexity of the underlying mechanisms and underscores the need for continued investigation into pharmacochaperone therapy for 3p- syndrome patients.

Highlights.

  • Identified two novel cases of 3p- syndrome involving haploinsufficient microdeletions encompassing both SLC6A1 and SLC6A11.

  • Highlighted phenotypic overlap between 3p- syndrome and SLC6A1-related disorders, including seizures, neurodevelopmental delay, and hypotonia.

  • The haploinsufficient GAT-1 and GAT-3 conditions had reduced activity with 3H GABA uptake assay.

  • The haploinsufficient GAT-1 and GAT-3 conditions exhibited reduced protein expression.

  • Data suggest the loss of GAT-1 and GAT-3 function contributes to the neurological manifestations of 3p- syndrome.

  • Investigated 4-phenylbutyrate (PBA) as a potential therapy, demonstrating moderate effect in 3p- syndrome deletion conditions.

Acknowledgements

We would like to thank the patients and their family who participated in this study for their cooperation.

Funding

T32AI007281, T32GM14492, T32HL00773, and NS82635; Cure GABAA, Taysha Gene Therapies, and NIH R01 NS121718.

List of abbreviations

GAT-1

GABA transporter 1

GAT-3

GABA transporter 3

ER

endoplasmic reticulum

PBA

4-phenylbutyrate

Footnotes

Competing interests

The authors declare that they are no competing interests.

Availability of supporting data

Any raw data of functional assay can be made available upon request.

Any clinical information can be made available upon request subject to approval by the appropriate ethical board.

References

  • 1.Narahara K, Kikkawa K, Murakami M, Hiramoto K, Namba H, Tsuji K, … & Kimoto H (1990). Loss of the 3p25. 3 band is critical in the manifestation of del (3p) syndrome: Karyotype‐phenotype correlation in cases with deficiency of the distal portion of the short arm of chromosome 3. American journal of medical genetics, 35(2), 269–273. [DOI] [PubMed] [Google Scholar]
  • 2.Verjaal M, & De Nef MB (1978). A patient with a partial deletion of the short arm of chromosome [DOI] [PubMed] [Google Scholar]
  • 3.Fu J, Wang T, Fu Z, Li T, Zhang X, Zhao J, & Yang G (2021). Case report: a case report and literature review of 3p deletion syndrome. Frontiers in Pediatrics, 9, 618059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Fernandez TV, García‐González IJ, Mason CE, Hernández‐Zaragoza G, Ledezma‐Rodríguez VC, Anguiano‐Alvarez VM, … & Dávalos NO (2008). Molecular characterization of a patient with 3p deletion syndrome and a review of the literature. American Journal of Medical Genetics Part A, 146(21), 2746–2752. [DOI] [PubMed] [Google Scholar]
  • 5.Dikow N, Maas B, Karch S, Granzow M, Janssen JW, Jauch A, … & Moog U (2014). 3p25. 3 microdeletion of GABA transporters SLC6A1 and SLC6A11 results in intellectual disability, epilepsy and stereotypic behavior. American Journal of Medical Genetics Part A, 164(12), 3061–3068. [DOI] [PubMed] [Google Scholar]
  • 6.Fischer FP, Kasture AS, Hummel T, & Sucic S (2022). Molecular and clinical repercussions of GABA transporter 1 variants gone amiss: links to epilepsy and developmental spectrum disorders. Frontiers in molecular biosciences, 9, 834498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Ben-Ari Y Excitatory actions of gaba during development: the nature of the nurture. Nat Rev Neurosci. 2002;3(9):728–739. [DOI] [PubMed] [Google Scholar]
  • 8.Kang JQ.Defects at the crossroads of GABAergic signalling in generalized genetic epilepsies. Epilepsy Res. 2017;137:9–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Beghi E (2020). The epidemiology of epilepsy. Neuroepidemiology, 54(2), 185–191. [DOI] [PubMed] [Google Scholar]
  • 10.Wang J, Poliquin S, Mermer F, et al. Endoplasmic reticulum retention and degradation of a mutation in SLC6A1 associated with epilepsy and autism. Mol Brain. 2020;13(1):76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Mermer F, Poliquin S, Zhou S, Wang X, Ding Y, Yin F, … & Kang JQ (2022). Astrocytic GABA transporter 1 deficit in novel SLC6A1 variants mediated epilepsy: connected from protein destabilization to seizures in mice and humans. Neurobiology of disease, 172, 105810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Conti F, Melone M, de Biasi S, Minelli A, Brecha NC, & Ducati A (1998). Neuronal and glial localization of GAT‐1, a high‐affinity γ‐aminobutyric acid plasma membrane transporter, in human cerebral cortex: With a note on its distribution in monkey cortex. Journal of Comparative Neurology, 396(1), 51–63. [DOI] [PubMed] [Google Scholar]
  • 13.Jensen K, Chiu CS, Sokolova I, & Lester HA (2003). GABA transporter-1 (GAT1)-deficient mice: differential tonic activation of GABAA versus GABAB receptors in the hippocampus. Journal of Neurophysiology, 90, 2690–2701. [DOI] [PubMed] [Google Scholar]
  • 14.Chiu CS, Brickley S, Jensen K, Southwell A, Mckinney S, Cull-Candy S, … & Lester HA (2005). GABA transporter deficiency causes tremor, ataxia, nervousness, and increased GABA-induced tonic conductance in cerebellum. Journal of Neuroscience, 25(12), 3234–3245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Cellot G, & Cherubini E (2014). GABAergic signaling as therapeutic target for autism spectrum disorders. Frontiers in pediatrics, 2, 70. 10.3389/fped.2014.00070 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Johannesen KM, Gardella E, Linnankivi T, Courage C, de Saint Martin A, Lehesjoki AE, … & Møller RS (2018). Defining the phenotypic spectrum of SLC6A1 mutations. Epilepsia, 59(2), 389–402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Mermer F, Poliquin S, Rigsby K, Rastogi A, Shen W, Romero-Morales A, … & Kang JQ (2021). Common molecular mechanisms of SLC6A1 variant-mediated neurodevelopmental disorders in astrocytes and neurons. Brain, 144(8), 2499–2512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Shen W, Nwosu G, Honer M, Clasadonte J, Schmalzbauer S, Biven M, … & Kang JQ (2024). γ-Aminobutyric acid transporter and GABAA receptor mechanisms in Slc6a1+/A288V and Slc6a1+/S295L mice associated with developmental and epileptic encephalopathies. Brain Communications, 6(2), fcae110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Lee TS, Bjørnsen LP, Paz C, Kim JH, Spencer SS, Spencer DD, Eid T, & de Lanerolle NC (2006). GAT1 and GAT3 expression are differently localized in the human epileptogenic hippocampus. Acta neuropathologica, 111(4), 351–363. [DOI] [PubMed] [Google Scholar]
  • 20.Ribak CE, Tong WM, & Brecha NC (1996). GABA plasma membrane transporters, GAT‐1 and GAT‐3, display different distributions in the rat hippocampus. Journal of Comparative Neurology, 367(4), 595–606. [DOI] [PubMed] [Google Scholar]
  • 21.De BS, Vitellaro-Zuccarello L, Brecha NC.. Immunoreactivity for the GABA transporter-1 and GABA transporter-3 is restricted to astrocytes in the rat thalamus. A light and electron-microscopic immunolocalization. Neuroscience. 1998;83(3):815–828. [DOI] [PubMed] [Google Scholar]
  • 22.Pow DV, Sullivan RK, Williams SM, Scott HL, Dodd PR, & Finkelstein D (2005). Differential expression of the GABA transporters GAT-1 and GAT-3 in brains of rats, cats, monkeys and humans. Cell and tissue research, 320(3), 379–392. [DOI] [PubMed] [Google Scholar]
  • 23.Poliquin S, Hughes I, Shen W, Mermer F, Wang J, Mack T, … & Kang JQ (2021). Genetic mosaicism, intrafamilial phenotypic heterogeneity, and molecular defects of a novel missense SLC6A1 mutation associated with epilepsy and ADHD. Experimental neurology, 342, 113723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Nwosu G, Mermer F, Flamm C, Poliquin S, Shen W, Rigsby K, & Kang JQ (2022). 4-Phenylbutyrate restored γ-aminobutyric acid uptake and reduced seizures in SLC6A1 patient variant-bearing cell and mouse models. Brain communications, 4(3), fcac144. 10.1093/braincomms/fcac144 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Laxer KD, Trinka E, Hirsch LJ, Cendes F, Langfitt J, Delanty N, … & Benbadis SR (2014). The consequences of refractory epilepsy and its treatment. Epilepsy & behavior, 37, 59–70. [DOI] [PubMed] [Google Scholar]
  • 26.Cai K, Wang J, Eissman J, Wang J, Nwosu G, Shen W, … & Kang JQ (2019). A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function. Experimental neurology, 320, 112973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Goodspeed K, Pérez-Palma E, Iqbal S, Cooper D, Scimemi A, Johannesen KM, … & Lal D (2020). Current knowledge of SLC6A1-related neurodevelopmental disorders. Brain communications, 2(2), fcaa170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Shen W, Flamm C, Delahanty AJ, Casteel E, Biven M, DeLeeuw MB, Poliquin S, Nwosu G, Randhave K, & Kang JQ (2024). 4-Phenylbutyrate promoted wild-type γ-aminobutyric acid type A receptor trafficking, reduced endoplasmic reticulum stress, and mitigated seizures in Gabrg2+/Q390X mice associated with Dravet syndrome. Epilepsia, 65(1), 204–217. 10.1111/epi.17779 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Mattison KA, Butler KM, Inglis GAS, Dayan O, Boussidan H, Bhambhani V, … & Escayg A (2018). SLC6A1 variants identified in epilepsy patients reduce γ‐aminobutyric acid transport. Epilepsia, 59(9), e135–e141. [DOI] [PubMed] [Google Scholar]
  • 30.Kang JQ, Barnes G (2013). A common susceptibility factor of both autism and epilepsy: functional deficiency of GABA a receptors. Journal of Autism Developmental Disorders, 43, 68–79. [DOI] [PubMed] [Google Scholar]
  • 31.Carvill GL, McMahon JM, Schneider A, Zemel M, Myers CT, Saykally J, … & Mefford HC (2015). Mutations in the GABA transporter SLC6A1 cause epilepsy with myoclonic-atonic seizures. The American Journal of Human Genetics, 96(5), 808–815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Sears SM, & Hewett SJ (2021). Influence of glutamate and GABA transport on brain excitatory/inhibitory balance. Experimental Biology and Medicine, 246(9), 1069–1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Asjad HMM, Kasture A, El-Kasaby A, et al. Pharmacochaperoning in a Drosophila model system rescues human dopamine transporter variants associated with infantile/juvenile parkinsonism. J Biol Chem. 2017;292(47):19250–19265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Grinspan ZM, Burre J, Cross J, Ross ME, Stone A, Basma N, … & Demarest S (2024). 4-Phenylbutyrate for STXBP1 and SLC6A1. Safety, tolerability, seizure, and EEG outcomes. A case series at 2 centers. medRxiv, 2024–11. [Google Scholar]
  • 35.Poliquin S, Nwosu G, Randhave K, Shen W, Flamm C, & Kang JQ (2024). Modulating Endoplasmic Reticulum Chaperones and Mutant Protein Degradation in GABRG2(Q390X) Associated with Genetic Epilepsy with Febrile Seizures Plus and Dravet Syndrome. International journal of molecular sciences, 25(9), 4601. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Any raw data of functional assay can be made available upon request.

Any clinical information can be made available upon request subject to approval by the appropriate ethical board.

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