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Scalable generation and functional classification of genetic variants in inborn errors of immunity to accelerate clinical diagnosis and treatment

ABSTRACT: Next-generation sequencing is pivotal for diagnosing inborn errors of immunity (IEI) but predominantly yields variants of uncertain significance (VUS), creating clinical ambiguity. Activated-PI3Kδ syndrome (APDS) is caused by gain-of-function (GOF) variants in PIK3CD or PIK3R1, which encode the PI3Kδ heterodimer. We performed massively parallel base-editing of PIK3CD/PIK3R1 in human T-cells and mapped thousands of variants to a clinically important readout (phospho-AKT/S6), nominating >100 VUS and unannotated variants for functional classification, and validated 27 hits. Leniolisib, an FDA-approved PI3Kδ-inhibitor, rescued aberrant signaling and dysfunction in GOF-harboring T-cells, and revealed partially drug-resistant PIK3R1 hotspots that responded to novel combination therapies of Leniolisib with mTORC1/2-inhibition. We confirmed these findings in T-cells from APDS patients spanning the functional spectrum discovered in the screen. Integrating our screens with population-level genomic studies revealed that APDS may be more prevalent than previously estimated. This work exemplifies a broadly applicable framework for removing ambiguity from sequencing in IEI.

ORGANISM(S): Homo sapiens

PROVIDER: GSE298853 | GEO | 2025/06/05

REPOSITORIES: GEO

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Project description:Objective:mRNA sequencing of sorted naive CD4+ T cells from the spleens of PIK3CD GOF mice and WT littermate mice. Methods:Naive CD4+ T cells were negatively isolated ex vivo(Stemcell Technologies).Total RNA from isolated naive CD4+ T cells was extracted using RNeasy Plus Mini Kit (Qiagen, GmBH, Germany) according to the manufacture’s instruction and checked for RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, US). Qualified total RNA was further purified by RNeasy micro kit (QIAGEN) and RNase-Free DNase Set (QIAGEN). Paired-end libraries were constructed using the TruSeq® RNA Sample Preparation Kit (Illumina, USA) according to the manufacturer’s instructions. The products are purified, enriched, quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer to confirm insert size and calculate the mole concentration. Cluster was generated using cBot with the library diluted to 10 pM and then were sequenced on the Illumina HiSeq 2500 (Illumina, USA). The library construction and sequencing was performed at Shanghai Biotechnology Corporation (Shanghai, China). Results:Compared to the expression profiles for the WT cells, those for the mutant TNaive showed 2,307 genes with differential expression, of which 1,749 were upregulated and 558 were downregulated. Gene ontology analysis showed that PIK3CD GOF TNaive upregulated the expression of genes encoding molecules involved in the cell cycle and mitosis, including E2f1, E2f2, cyclin A2, cyclin B2, Cdk1, Hells and Nuf2, all of which might synergistically promote entry into the cycling phase. In addition, the genes encoding T cell activation, cytokines and cytokines receptors, chemokines and chemokine receptors, transcription factors, and metabolic regulators were differentially regulated in PIK3CD GOF mice compared to WT mice. Kyoto Encyclopedia of Genes and Genomes analysis showed that the altered genes in TNaive from PIK3CD GOF mice displayed significant enrichment of several sets associated with infection, inflammation and autoimmune disease Conlusion:PIK3CD GOF results in a loss of quiescence-associated gene expression patterns in naive T cells by collectively coupling the cell cycle, nutrient metabolism, cell trafficking and signal transduction.

Project description:Glutamate is involved in a variety of metabolic pathways. We reviewed the literature on genetic defects of enzymes that directly metabolise glutamate, leading to inborn errors of glutamate metabolism. Seventeen genetic defects of glutamate metabolising enzymes have been reported, of which three were only recently identified. These 17 defects affect the inter-conversion of glutamine and glutamate, amino acid metabolism, ammonia detoxification, and glutathione metabolism. We provide an overview of the clinical and biochemical phenotypes of these rare defects in an effort to ease their recognition. By categorising these by biochemical pathway, we aim to create insight into the contributing role of deviant glutamate and glutamine levels to the pathophysiology. For those disorders involving the inter-conversion of glutamine and glutamate, these deviant levels are postulated to play a pivotal pathophysiologic role. For the other IEM however-with the exception of urea cycle defects-abnormal glutamate and glutamine concentrations were rarely reported. To create insight into the clinical consequences of disturbed glutamate metabolism-rather than individual glutamate and glutamine levels-the prevalence of phenotypic abnormalities within the 17 IEM was compared to their prevalence within all Mendelian disorders and subsequently all disorders with metabolic abnormalities notated in the Human Phenotype Ontology (HPO) database. For this, a hierarchical database of all phenotypic abnormalities of the 17 defects in glutamate metabolism based on HPO was created. A neurologic phenotypic spectrum of developmental delay, ataxia, seizures, and hypotonia are common in the inborn errors of enzymes in glutamate metabolism. Additionally, ophthalmologic and skin abnormalities are often present, suggesting that disturbed glutamate homeostasis affects tissues of ectodermal origin: brain, eye, and skin. Reporting glutamate and glutamine concentrations in patients with inborn errors of glutamate metabolism would provide additional insight into the pathophysiology.

Project description:Glutathione is a tripeptide composed of glutamate, cysteine and glycine. Glutathione is present in millimolar concentrations in most mammalian cells and it is involved in several fundamental biological functions, including free radical scavenging, detoxification of xenobiotics and carcinogens, redox reactions, biosynthesis of DNA, proteins and leukotrienes, as well as neurotransmission/neuromodulation. Glutathione is metabolised via the gamma-glutamyl cycle, which is catalyzed by six enzymes. In man, hereditary deficiencies have been found in five of the six enzymes. Glutathione synthetase deficiency is the most frequently recognized disorder and, in its severe form, it is associated with hemolytic anemia, metabolic acidosis, 5-oxoprolinuria, central nervous system (CNS) damage and recurrent bacterial infections. Gamma-glutamylcysteine synthetase deficiency is also associated with hemolytic anemia, and some patients with this disorder show defects of neuromuscular function and generalized aminoaciduria. Gamma-glutamyl transpeptidase deficiency has been found in patients with CNS involvement and glutathionuria. 5-Oxoprolinase deficiency is associated with 5-oxoprolinuria but without a clear association with other symptoms. Dipeptidase deficiency has been described in one patient. All disorders are very rare and inherited in an autosomal recessive manner. Most of the mutations are leaky so that many patients have residual enzyme activity. Diagnosis is made by measuring the concentration of different metabolites in the gamma-glutamyl cycle, enzyme activity and in glutathione synthetase and gamma-glutamylcysteine synthetase deficiency, also by mutation analysis. Prenatal diagnosis has been preformed in glutathione synthetase deficiency. The prognosis is difficult to predict, as few patients are known, but seems to vary significantly between different patients. The aims of the treatment of glutathione synthesis defects are to avoid hemolytic crises and to increase the defense against reactive oxygen species. No treatment has been recommended for gamma-glutamyl transpeptidase, 5-oxoprolinase and dipeptidase deficiency.

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Scalable generation and functional classification of genetic variants in inborn errors of immunity to accelerate clinical diagnosis and treatment

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