Sharon A Tooze

We describe a large consanguineous pedigree from a remote area of Northern Pakistan, with a complex developmental disorder associated with wide-ranging symptoms, including mental retardation, speech and language impairment and other neurological, psychiatric, skeletal and cardiac abnormalities. We initially carried out a genetic study using the HumanCytoSNP-12 v2.1 Illumina gene chip on nine family members and identified a single region of homozygosity shared amongst four affected individuals on chromosome 7p22 (positions 3059377-5478971). We performed whole-exome sequencing on two affected individuals from two separate branches of the extended pedigree and identified a novel nonsynonymous homozygous mutation in exon 9 of the WIPI2 (WD-repeat protein interacting with phosphoinositide 2) gene at position 5265458 (c.G745A;pV249M). WIPI2 plays a critical role in autophagy, an evolutionary conserved cellular pathway implicated in a growing number of medical conditions. The mutation is situated in a highly conserved and critically important region of WIPI2, responsible for binding PI(3)P and PI(3,5)P2, an essential requirement for autophagy to proceed. The mutation is absent in all public databases, is predicted to be damaging and segregates with the disease phenotype. We performed functional studies in vitro to determine the potential effects of the mutation on downstream pathways leading to autophagosome assembly. Binding of the V231M mutant of WIPI2b to ATG16L1 (as well as ATG5-12) is significantly reduced in GFP pull-down experiments, and fibroblasts derived from the patients show reduced WIPI2 puncta, reduced LC3 lipidation and reduced autophagic flux.

Abstract WIPI2 is a member of the human WIPI protein family (seven-bladed b-propeller proteins binding phosphatidylinositols, PROPPINs), which play a pivotal role in autophagy and has been implicated in the pathogenesis of several neurological conditions. The homozygous WIPI2 variant c.745G>A; p.(Val249Met) (NM_015610.4) has recently been associated with a neurodevelopmental disorder in a single family. Using exome sequencing and Sanger segregation analysis, here, two novel homozygous WIPI2 variants [c.551T>G; p.(Val184Gly) and c.724C>T; p.(Arg242Trp) (NM_015610.4)] were identified in four individuals of two consanguineous families. Additionally, follow-up clinical data were sought from the previously reported family. Three non-ambulant affected siblings of the first family harbouring the p.(Val184Gly) missense variant presented with microcephaly, profound global developmental delay/intellectual disability, refractory infantile/childhood-onset epilepsy, progressive tetraplegia with joint contractures and dyskinesia. In contrast, the proband of the second family carrying the p.(Arg242Trp) missense variant, similar to the initially reported WIPI2 cases, presented with a milder phenotype, encompassing moderate intellectual disability, speech and visual impairment, autistic features, and an ataxic gait. Brain MR imaging in five patients showed prominent white matter involvement with a global reduction in volume, posterior corpus callosum hypoplasia, abnormal dentate nuclei and hypoplasia of the inferior cerebellar vermis. To investigate the functional impact of these novel WIPI2 variants, we overexpressed both in WIPI2-knockout HEK293A cells. In comparison to wildtype, expression of the Val166Gly WIPI2b mutant resulted in a deficient rescue of LC3 lipidation whereas Arg224Trp mutant increased LC3 lipidation, in line with the previously reported Val231Met variant. These findings support a dysregulation of the early steps of the autophagy pathway. Collectively, our findings provide evidence that biallelic WIPI2 variants cause a neurodevelopmental disorder of variable severity and disease course. Our report expands the clinical spectrum and establishes WIPI2-related disorder as a congenital disorders of autophagy.

). • Preparation of liposomes to monitor the kinetics of ATG8 lipidation in a real-time manner.

Abstract Disclosure: M. Korbonits: Novo Nordisk, Ipsen, Pfizer, Inc.. X. Wang: None. S. Barry: None. O. Suleyman: None. D. Stefano: None. N. Uddin: None. C.L. Hall: None. P. Laura: None. P. Chapple: None. H. Sian: None. V. Morales: None. K. Bianchi: None. M. Duchen: None. S. Patel: None. E. Aksoy: None. G. Czibik: None. A. Borovikov: None. H. Björnsson: None. H. Van Esch: None. S. Tooze: None. C. Brennan: None. O. Haworth: None. Background: The aryl hydrocarbon receptor interacting protein (AIP) is a highly conserved and ubiquitously expressed chaperone protein. For over a decade, heterozygous loss-of-function mutations in the AIP gene have been linked to childhood-onset pituitary adenomas. Our data here reveal that AIP is vital for metabolic processes, well beyond its role in the pituitary gland. Results - patients Biallelic loss of the Aip gene leads to embryonic (mouse) or larval (fruit file, C. elegans) lethality. Surprisingly, we identified children born with deleterious biallelic variants of AIP. These variants include three distinct homozygous mutations across four kindreds. Two of these variants are absent in the gnomAD variant database, while the third one has a minor allele frequency (MAF) of 0.000003097, identified only in heterozygote state. In the first family, consanguineous, two close relatives were affected. Two of the 4 kindreds (with the same variant) were from the same geographical area. The affected patients have a severe multisystem metabolic disorder characterized by failure to thrive following birth. Two children died before their first birthday ultimately of heart failure. The patients show non-infectious hyperthermia, hypercalcaemia, hypercalciuria with nephrocalcinosis, chronic diarrhoea, tachycardia, hypertension and delayed development. Results -experimental Using patient-derived dermal fibroblasts and Aip knockout mouse embryonic fibroblasts, we have discovered that AIP was required to support proteasome activity, induction of autophagy, lysosome function, maintaining metabolic homeostasis during periods of excessive metabolic demand. aip knockout zebrafish recapitulated the severe phenotype of the children, exhibiting cardiomegaly and death after yolk depletion when autophagy is required for cells to adapt to periods of starvation. Our results demonstrate that AIP plays a crucial role in initiating autophagy to maintain proteostasis in response to nutrient deprivation and during periods of high metabolic demand. In summary, we have identified a novel and complex paediatric disorder linked to biallelic AIP variants. Furthermore, we have in part uncovered the molecular mechanisms contributing to the severe phenotype, highlighting AIP’s critical role in autophagy and metabolic regulation. Presentation: Monday, July 14, 2025