A male infant was spontaneously delivered at 37 + 3 weeks of gestation in good general condition (Apgar 9/10/10), with a weight of 2,820 g (20th percentile), and a length of 52 cm (73rd percentile). Delivery occurred seven days after rupture of membranes, with clear amniotic fluid. Pregnancy (gravida 3, para 2) was uncomplicated, except for borderline abnormal fetal and umbilical Doppler findings two weeks before delivery. On examination, macrocephaly (head circumference 38 cm, >99th percentile), hypospadias, and right foot hexadactyly were noted. There was no family history of genetic or neurological diseases.

At 12 h of life, the infant was admitted to the NICU with suspected sepsis. He presented with poor general condition, hypothermia, hypoglycemia, mildly elevated IL-6 [81 pg/ml, (0–50)], and normal initial CRP concentration [0.82 mg/dl, (0–1.0)] (Figures 1A–C). Serial blood cultures at diagnosis were negative, while stool PCR was positive for rotavirus. A sharp rise in the inflammatory markers (IL-6 284 pg/ml and CRP 10.41 mg/dl) prompted broad-spectrum antibiotics (Figure 1D). Respiratory adaptation disorder initially required CPAP, followed by invasive ventilation after colonic perforations on days 8 and 11 (Figure 1A). Surgery revealed an extensive gangrenous perforation involving the transverse and descending colon, requiring partial resection of the transverse colon and creation of an ostomy. Three days later, a second perforation in the transverse colon was treated with a protective terminal ileostomy. Histopathology showed patchy ulcerative necrotizing colitis with a mixed capillary-lymphatic vascular malformation. During hospitalization, the infant required prolonged parenteral nutrition, which caused transient severe cholestasis (Figure 1A). Initial neurological evaluation (EEG, cranial ultrasound, MRI) was unremarkable (Figure 2A).

Ultra-rapid whole genome sequencing (urWGS) identified a de novo postzygotic point variant, c.1030G>A, in exon 5 of the PIK3CA gene (NM_006218.3). This variant results in a p.(Val344Met) amino acid substitution in the helical domain of the PI3K p110-α subunit and has been previously associated with PROS (6, 9–11). The same variant was also detected by panel sequencing of the resected intestine at a similar allele frequency (48.67% and 47.12%) as in the blood.

The infant exhibited persistent lymphopenia (Figure 3A) with markedly reduced B (CD19⁺) and T (CD3⁺) cells compared to neonates with sepsis (Figure 3B). Despite the overall reduction in the T cell pool, the proportions of CD8⁺, CD4⁺, and γδ T cells within the CD3⁺ population were preserved, with a discrete increase in Treg cells (Figures 3C,D). Lymphocyte frequencies normalized after the initial hospitalization (Figure 3B), suggesting a transient alteration of the immune response to acute infection-related stress.

On day 10 of hospitalization, during colonic perforation, both pro-inflammatory (IL-6, CXCL-10, IFN-γ) and anti-inflammatory cytokines were elevated (Figure 4A). Meanwhile, CD4⁺ and CD8⁺ T cell functionality remained preserved, with expression of activation markers (CD69 and CD25) and TNF-α production following anti-CD3/anti-CD28 stimulation comparable to controls (Figure 4B). Monocyte frequency (CD14+) increased (Figure 4C) with a shift from classical inflammatory (CD14⁺ CD16⁻) to non-classical anti-inflammatory (CD14⁺ CD16⁺) phenotypes during hospitalization (Figure 4D).

At five months of age, the EEG showed intermittent frontal delta activity and isolated sharp waves, without seizures or infantile spasms. While growth was age-appropriate (Figure 5A), head circumference remained percentile-volatile (Figure 5B). By six months, cranial ultrasound revealed progressive hydrocephalus with enlargement of ventricles I-III, which was confirmed by MRI (Figure 2B). The MRI included a high-resolution, flow-sensitive, thin-slice T2 SPACE sequence, showing an anatomically patent aqueduct and visible CSF flow phenomena. A VP shunt was placed at seven months; however, by 12 months, the patient developed refractory ascites. Serial cerebrospinal fluid (CSF) removal and abdominal punctures were required due to ongoing CSF overproduction (Figure 2C). CSF analysis showed markedly elevated albumin levels [2,520 mg/L, (120–240)], indicating pronounced barrier dysfunction. As an incidental finding during follow-up, a 6 mm diameter benign intraspinal tumor of the nerve roots at the L3/L4 level was detected, with stable size on subsequent evaluations (Figure 2D). From 13 months of age, an off-label therapy trial with acetazolamide significantly reduced CSF and ascites volumes, facilitating motor development. By 15 months, the child achieved independent walking with only mild motor delay. He feeds orally, vocalizes, and performs small tasks. Long-term development outcomes remain to be determined.

EDTA blood was processed using the Illumina DNA PCR-Free Library Preparation Tagmentation Kit, followed by whole genome sequencing (WGS) on an Illumina NovaSeq 6000 sequencer. Sequencing reads were aligned to the human reference genome GRCh38. TruSight™ Software (Illumina, Suite v2.6) and center-specific bioinformatics pipeline were used for alignment, variant calling, variant annotation, filtering, and curation. In addition to variant allele frequency data, prediction tools including phyloP, SIFT, PolyPhen-2, FATHMM, CADD, and REVEL were used. Furthermore, LOVD (https://databases.lovd.nl/shared/), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar), and gnomAD (https://gnomad.broadinstitute.org/) were screened for reported entries of the identified variant. Variant interpretation followed the standards and guidelines of the American College of Medical Genetics and Genomics (18).

Inclusion and sample collection were conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the Hannover Medical School (No. 10856_BO_K_2023). Written informed consent was obtained from all donors, parents, or guardians in the case of children.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density gradient centrifugation from EDTA blood samples collected at different time points after birth from the studied patient, as well as from nine uninfected healthy neonates (−3 days of age) and two neonates diagnosed with bacterial sepsis (2–3 days of age) at the AUF DER BULT Children's and Youth Hospital, Hannover, Germany. Additionally, PBMCs were isolated from an EDTA blood sample of a healthy adult for in vitro assays.

Freshly isolated PBMCs were incubated for 20 min at room temperature with the following antibodies: anti-CD45 BUV395 (HI30; BD Bioscience), anti-CD3 BUV661 (UCHT1; BD Bioscience), anti-CD8 BUV805 (SK1; BD Bioscience), anti-CD4 BV570 (RPA-T4; BioLegend), anti-CD127 BV650 (A019D5; BioLegend), anti-CD25 PE-Fire700 (M-A250; BioLegend), anti-γδ TCR PE (REA591; Miltenyi Biotec), anti-Vδ2 PerCPVio700 (REA771; Miltenyi Biotec), anti-Vγ9 FITC (REA470; Miltenyi Biotec), anti-Vδ1 VioGreen (REA173; Miltenyi Biotec), anti-CD45RA BV605 (HI100; BioLegend), anti-CD14 BB700 (MφP9; BD Bioscience), anti-CD16 BUV496 (3G8; BD Bioscience), anti-CD56 BUV564 (NCAM16.2; BD Bioscience), anti-CCR7 BV785 (G043H7; BioLegend), anti-CD19 Pe-Cy7 (HIB19; BD Bioscience), anti-IgD BV510 (IA6-2; BD Bioscience), and anti-CD27 Alexa Fluor (O323; BioLegend). Dead cells were detected using Zombi-NIR staining. After washing off excess antibodies, cells were acquired on an Aurora spectral flow cytometer (Cytek) using SpectroFlo v2.2.0 (Cytek). Flow cytometry data were analyzed in Flowjo 10.0 software.

Plasma cytokines were measured using the LEGENDplex™ Human Essential Immune Response Panel (BioLegend) according to the manufacturer's instructions.

Plates were coated overnight at 4°C with anti-CD3 antibody (BioLegend, #300438) at a final concentration of 4 µg/ml. Freshly isolated PBMCs (0.5  ×  10⁶ cells/ml) were cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS (Sigma), 1% GlutaMAX, 50 µM β-mercaptoethanol, 1% penicillin-streptomycin (all Gibco), and 100 U/ml IL-2. Anti-CD28 antibody was added to the coated wells at a final concentration of 1 µg/ml. Cells were incubated in a humidified CO₂-incubator at 37°C for 48 h. After incubation cells were stained for 20 min at room temperature with fixable viability dye (Zombie NIR, BioLegend) and the following antibodies: anti-CD45 BUV395 (HI30; BD Bioscience), anti-CD14 BB700 (MφP9; BD Bioscience), anti-CD19 APC-Fire810 (HIB19; Biolegend), anti-CD3 BUV661 (UCHT1; BD Bioscience), anti-TCRγδ PE (REA591; Miltenyi Biotec), anti-CD4 APC (SK3; BD Bioscience), anti-CD8 BV510 (SK1; Biolegend), anti-CD69 BUV737 (FN50; FN50), anti-CD25 PE Fire 700 (M-A251; Biolegend), intracellular anti-TNF-α Alexa Fluor 700 (MAb11; BioLegend), intracellular anti-IFN-γ (B27; BioLegend). Acquisition was performed on an Aurora spectral flow cytometer (Cytek).