Glycerophosphoinositol is Elevated in Blood Samples From CLN3^Δex7-8 pigs, Cln3^Δex7-8 Mice, and CLN3-Affected Individuals

Abstract

Introduction: CLN3 Batten disease is a rare pediatric neurodegenerative lysosomal disorder caused by biallelic disease-associated variants in CLN3. Despite decades of intense research, specific biofluid biomarkers of disease status have not been reported, hindering clinical development of therapies. Thus, we sought to determine whether individuals with CLN3 Batten disease have elevated levels of specific metabolites in blood. Methods: We performed an exhaustive metabolomic screen using serum samples from a novel minipig model of CLN3 Batten disease and validated findings in CLN3 pig serum and CSF and Cln3 mouse serum. We further validate biomarker candidates with a retrospective analysis of plasma and CSF samples collected from participants in a natural history study. Plasma samples were evaluated from 22 phenotyped individuals with CLN3 disease, 15 heterozygous carriers, and 6 non-affected non-carriers (NANC). Results: CLN3 pig serum samples from 4 ages exhibited large elevations in 4 glycerophosphodiester species: glycerophosphoinositol (GPI), glycerophosphoethanolamine (GPE), glycerophosphocholine (GPC), and glycerophosphoserine (GPS). GPI and GPE exhibited the largest elevations, with similar elevations found in CLN3 pig CSF and Cln3 mouse serum. In plasma samples from individuals with CLN3 disease, glycerophosphoethanolamine and glycerophosphoinositol were significantly elevated with glycerophosphoinositol exhibiting the clearest separation (mean 0.1338 vs 0.04401 nmol/mL for non-affected non-carriers). Glycerophosphoinositol demonstrated excellent sensitivity and specificity as a biomarker, with a receiver operating characteristic area under the curve of 0.9848 ( P = .0003). Conclusions: GPE and GPI could have utility as biomarkers of CLN3 disease status. GPI, in particular, shows consistent elevations across a diverse cohort of individuals with CLN3. This raises the potential to use these biomarkers as a blood-based diagnostic test or as an efficacy measure for disease-modifying therapies.

CLN3 deficiency leads to neurological and metabolic perturbations during early development

“…We could not detect a decrease of cln3 mRNA levels in MUT1, reducing the chance that transcriptional adaptation mechanisms (triggered by mutant mRNA decay) are compensating cln3 loss-of-function phenotypes in our zebrafish model (El-Brolosy et al 2019). Finally, the very distinct accumulation of GPDs and decrease of BMP species in MUT1 larvae confirm that we have indeed successfully knocked out Cln3 function, as the exact same metabolic changes were measured in samples derived from patients with CLN3 disease (Hobert and Dawson 2007; Brudvig et al 2022; Laqtom et al 2022). The association between BMPs and CLN3 has been known for many years, with metabolic labeling studies showing early on that CLN3 may be involved in BMP synthesis (Hobert and Dawson 2007).…”

“…5B) , whereas glycerophosphoethanolamine (GPE) was not significantly changed and glycerophosphoserine (GPS) was not detected in the extracts. While this work was ongoing, accumulation of GPDs has also been found in yeast and mammalian models of CLN3 deficiency (Laqtom et al 2022; Brudvig et al 2022), suggesting that CLN3 plays a highly conserved role in maintaining normal GPD levels across species. Phospholipid precursors such as phosphocholine and phosphoethanolamine were also detected at significantly higher levels in the mutant compared to WT larvae (Fig.…”

“…The association between BMPs and CLN3 has been known for many years, with metabolic labeling studies showing early on that CLN3 may be involved in BMP synthesis (Hobert and Dawson 2007). By contrast, the accumulation of GPDs in CLN3 deficient mammalian cells and tissues as well as in extracellular fluids (CSF and plasma) of CLN3 patients was only shown very recently in two independent studies (Laqtom et al 2022;Brudvig et al 2022), while the work presented here was ongoing in the zebrafish and human cerebral organoids. It is remarkable that we were able to detect GPD accumulation by untargeted metabolomics in an unbiased way (before results from the other groups were published) and in extracts prepared from whole 5 dpf larvae.…”

High-resolution promoter interaction analysis in Type 3 Innate Lymphoid Cells implicates Batten Disease geneCLN3in Crohn’s Disease aetiology

“…Structurally, CLN3 encodes a transmembrane lysosome protein whose C- and N-ends localise to the cytoplasm 106 . While CLN3 function remains not fully elucidated, recent studies have demonstrated that it is required for glycerophospholipid catabolism 107,108 . Glycerophospholipids are key structural components of cell membranes, but also play a variety of regulatory roles, including in innate immunity 109 .…”

Cerebrospinal Fluid Protein Biomarker Discovery in CLN3

“…CLN3 codes for a 468-amino acid predicted transmembrane protein that is implicated in many cellular pathways but awaiting a clearly defined function . Recent studies identified elevated levels of glycerophosphodiester species in CLN3 mouse and pig models and CLN3-affected individuals, , with the work from Laqtom et al suggesting an essential role of CLN3 protein in the clearance of these compounds from the lysosome. The most common disease-associated variant, a 966-base pair deletion present in ∼80% of chromosomes in affected individuals, is predicted to remove exons 7 and 8 and trigger either nonsense-mediated mRNA decay , or production of a truncated product with novel function. − …”