Toward a better understanding of PHTS heterogeneity: commentary on ‘Cell-type specific deficits in PTEN-mutant cortical organoids converge on abnormal circuit activity’

PTEN hamartoma tumor syndrome (PHTS) is caused by mutations in PTEN or related genes and results in variable clinical presentation with combinations of symptoms including macrocephaly, benign tumors including hamartomas and lipomas, increased lifetime risk of cancer, intellectual disability (ID) and autism spectrum disorder (ASD) (1). The cell biological impact of PTEN loss has been studied extensively in cell-lines and animal models to elucidate mechanisms of cellular proliferation and growth that are the foundation of these symptoms. However, it remains elusive why the clinical manifestations of PHTS exhibit such diversity, even among individuals carrying the same PTEN mutation. Of all physical findings in PHTS patients, the basic biological basis for ID and ASD is the least understood. Changes in stem cell and glial proliferation alter the cellular makeup of the brain. Neuronal hypertrophy, increased excitatory synapse formation and hyperactivity also alter information flow through the brain. Altered oligodendrocyte and microglial function contribute to changes in myelination and the sculpting of synaptic circuits. However, the precise impact of heterozygous PTEN mutations on various cell types during the development of human brain remains unclear. Furthermore, we do not know which or to what degree these cellular changes contribute to ID and ASD. In homozygous loss-of-function models, these changes are robust and easily studied. In heterozygous systems, these changes are subtle and likely subject to gene–gene or gene–environment interactions. Recently, compelling evidence has emerged suggesting that genetic backgrounds play a significant role in the penetrance of ASD associated genes including PTEN (2,3), possibly by influencing the developmental timing of specific cell types in the brain. Nevertheless, it remains uncertain whether there is a convergence of shared functional phenotypes in later stage of development across different genetic backgrounds.

In this issue of Human Molecular Genetics, Pigoni, Uzquiano, Paulsen and Kedaigle et al. model the effect of a heterozygous PTEN mutation using human induced pluripotent stem-cell (iPSC) cortical organoids. The authors investigated the functions of PTEN mutant on human brain development, specifically focusing on cell-type-specific developmental events and their variations across two different genetic backgrounds: Mito210 and PGP1 (independently derived by the laboratories of Bruce Cohen and George Church, respectively). By employing various techniques including single-cell RNA sequencing (scRNA-seq), proteomics and spatial transcriptomics, the authors found that at an early stage of culture (1 month) heterozygous PTEN mutations had no effects on the proportion of any cortical cell types. However, at a later stage of culture (3 months), heterozygous PTEN mutations resulted in an increase in the proportion of outer radial glia (oRG) progenitor cells independent of genetic backgrounds. Interestingly, by comparing the gene expression profiles, the authors found heterozygous PTEN mutations caused divergent changes in the developmental timing of certain cortical projection neurons in the two genetic backgrounds, with accelerated development in Mito210 and delayed development in PGP1. Despite the variations in developmental timing, calcium imaging showed increased activity of local circuits, indicating a convergence on disrupted neuronal activity. This study uncovered some common features associated with PTEN mutations, such as organ overgrowth and increased neuronal excitability, seemingly independent of genetic backgrounds in cultured human brain organoids.

The utilization of human brain organoids as a cutting-edge research model has witnessed a remarkable rise in recent years. These miniature 3D structures offer a unique platform to investigate neurodevelopmental disorders, explore the mechanisms of neurological diseases and evaluate potential therapeutic interventions. In this study, the researchers found at later stages (but not during the early stages), heterozygous PTEN organoids consistently showed an increased proportion of oRG progenitors, irrespective of the genetic background. This time-dependent overgrowth of oRG progenitor cells is intriguing, considering their association with cortical expansion. It could potentially provide insights into the mechanisms underlying ventriculomegaly and macrocephaly, a condition often associated with PTEN mutations. Reportedly, 94% of individuals with PHTS exhibit macrocephaly regardless of whether ASD or ID is exhibited (4). Unfortunately, the study does not provide information about the size of the human brain organoids used. Further research is needed to establish a link between PTEN mutation, macrocephaly and the disrupted regulation of oRG cells during brain development.

Through calcium imaging of intact organoids, the study further revealed that both accelerated and delayed neuronal development phenotypes resulted in increased activity within local circuit, regardless of the genetic background. This finding aligns with previous research that indicates Pten knockout in neurons typically leads to heightened neuronal excitability (5). Additionally, it appears that the basal neuronal activity in PGP1 organoids is relatively higher than that observed in Mito210 organoids, regardless of PTEN mutation. This discrepancy may reflect the asynchronous development of organoids from these two genetic backgrounds. In general, immature neurons, such as those found during early stages of development, tend to exhibit more spontaneous firing activity compared with mature neurons (6). Meanwhile, it is important to note that although calcium imaging can faithfully capture local circuit activity, it lacks the necessary resolution to uncover underlying mechanisms. Conducting comprehensive electrophysiological recordings to examine synaptic transmission, action potentials and general electrophysiological properties of the cells would greatly contribute to obtaining a comprehensive overview of the cellular and circuitry changes induced by PTEN mutants in different genetic backgrounds.

In PHTS patients, only 20–50% meet the criteria for ASD and ID. Of these, the vast majority have de novo heterozygous missense mutations. There have been case reports of inherited mutations with probands displaying ASD and ID and carriers displaying normal intelligence and no ASD (7,8). These observations have led to an interest in how changes in genetic background may influence those cellular phenotypes that ultimately give rise to ASD and ID. The human iPSC system is particularly attractive to examine how genetic background could influence the phenotypic presentation of ID or ASD in different people bearing the same mutation in PTEN. The results of the present paper and a recent paper examining mutant heterozygous PTEN mutation both identify effects of genetic background on cellular phenotype expression (2). These results hold promise for determining which cellular phenotypes covary with specific patient genetic backgrounds displaying ID and ASD. This could lead us to a better understanding of the basic biological basis for the complicated brain abnormalities.

Existing evidence, including this study, suggests a direct correlation between PTEN mutations and specific phenotypes, such as organ overgrowth and increased neuronal excitability. However, the genetic background, on the other hand, can either mitigate or exacerbate the PTEN mutation-related developmental timing phenotypes, thereby contributing to the diverse clinical manifestations observed in PHTS patients. It is crucial to comprehend the intricate interplay between PTEN mutations and genetic backgrounds to accurately predict and interpret the phenotypic consequences of these mutations. Through the utilization of high-throughput multi-omics analysis, the authors have successfully identified significant differential gene expression induced by PTEN mutations. These findings emphasize the necessity for further investigation to unveil unconventional PTEN signaling pathways or parallel signaling cascades that may contribute to the variations in developmental timing across different genetic backgrounds. Additional analyses, such as comparing the differentially expressed genes between Mito210 and PGP1 with and without PTEN mutations, could shed light on the disparities between these two genetic backgrounds and identify the specific genes that underlie the observed differences in developmental timing following PTEN mutation. By delving deeper into these pathways, researchers can attain a more comprehensive understanding of the underlying mechanisms and identify novel factors that influence the diverse phenotypic outcomes associated with PTEN mutations.

With the promise of human-derived cellular models come a variety of questions. Have we detected all of the cellular changes resulting from PTEN loss-of-function? High throughput techniques like scRNA-seq, proteomics and calcium imaging offer unbiased approaches to phenotype detection, but it is unknown whether we can detect changes in neuronal arborization, synapse number and action-potential kinetics in iPSC models. The major challenge for this is that the development of neuronal arborization, synapse number and mature action potential kinetics occurs later in postnatal development and iPSC-derived neurons have not yet been cultivated to reach a fully mature state. Can we understand why some PTEN mutations tend to result in a greater disposition toward cognitive changes versus cancer? While the present study indicates that background influences phenotypes after PTEN mutations, we do not know which phenotypes correlate to the presentation of ASD and ID. It will be important to compare cellular phenotype from iPSCs derived from PHTS patients that vary in the presentation of ASD or ID. Can iPSC models provide a platform for therapeutic translation? In animal models, mTorC1 inhibition has become the gold standard to prevent the onset of cellular phenotypes. We have yet to see if this can be recapitulated in iPSC models. Ultimately, this is an exciting time for both basic and translational sciences because we have at our disposal high-throughput genetic sequencing, the ability to reproduce these genetic changes in model organisms, and the ability to model brain development from iPSCs derived from the population that we seek to both understand and treat. All approaches are necessary and provide valuable clues enabling us to untangle the intricacies of development and developmental disorders.

Conflict of interest statement. The authors have no conflict of interest to declare.