GlcNAc-Asn is a biomarker for NGLY1 deficiency

Abstract

Substrate-derived biomarkers are necessary in slowly progressing monogenetic diseases caused by single-enzyme deficiencies to identify affected patients and serve as surrogate markers for therapy response. N-glycanase 1 (NGLY1) deficiency is an ultra-rare autosomal recessive disorder characterized by developmental delay, peripheral neuropathy, elevated liver transaminases, hyperkinetic movement disorder and (hypo)-alacrima. We demonstrate that N-acetylglucosamine-asparagine (GlcNAc-Asn; GNA), is the analyte most closely associated with NGLY1 deficiency, showing consistent separation in levels between patients and controls. GNA accumulation is directly linked to the absence of functional NGLY1, presenting strong potential for its use as a biomarker. In agreement, a quantitative liquid chromatography with tandem mass spectrometry assay, developed to assess GNA from 3 to 3000 ng/ml, showed that it is conserved as a marker for loss of NGLY1 function in NGLY1-deficient cell lines, rodents (urine, cerebrospinal fluid, plasma and tissues) and patients (plasma and urine). Elevated GNA levels differentiate patients from controls, are stable over time and correlate with changes in NGLY1 activity. GNA as a biomarker has the potential to identify and validate patients with NGLY1 deficiency, act as a direct pharmacodynamic marker and serve as a potential surrogate endpoint in clinical trials.

Keywords: GNA; GlcNAc-Asn; N-glycanase 1; NGLY1 deficiency; biomarker.

Graphical Abstract

Fig. 1

A model for the generation of GNA in NGLY1 deficiency. (A) Normal degradation of misfolded proteins through the ERAD pathway. (B) NGLY1 deficiency leads to generation and accumulation of GNA. N = Asn; D = Asp

Fig. 2

GNA elevation found to correlate with NGLY1 deficiency in semiquantitative analysis of patient sample metabolites. (A) Targeted semi-quantitative analysis of disease-associated oligosaccharides in NGLY1-deficient patient urine compared to unaffected related control samples. (B) Semi-quantitative asparagine-linked oligosaccharides in NGLY1-deficient patient urine and unaffected siblings and parents. (C) Semiquantitative identification and analysis of metabolites in NGLY1-deficient patient and unaffected control plasma.

Fig. 3

Development of a quantitative method to detect GNA shows accumulation of GNA in NGLY1 KO cells. (A) GNA analysis across concentrations in surrogate matrix (PBS + BSA, standard curve, average 2 replicates). (B) GNA accumulation in various NGLY1-deficient cell lines. (C) WT versus NGLY1 KO HEK293 cell mixing and GNA detection. (D) Expression of NGLY1 reduces GNA in NGLY1 KO HepG2 cells. ^*P < 0.05; ^**P < 0.01; ^***P < 0.0001.

Fig. 4

The rat model of NGLY1 deficiency shows accumulation of GNA in all tissues. (A) Consistently high levels of GNA detected in Ngly1 KO rats compared to HET and WT animals across longitudinal measurements in a 10-week study (PNDs 33–110). (B) Elevated levels of GNA detected in CSF, plasma and urine in Ngly1 KO rats at terminal collection. (C) Freeze–thaw study in rat plasma and urine demonstrates an increase in GNA signal post-thaw in urine and relative stability in plasma. (D) GNA is consistently higher across tissue types in Ngly1 KO rats compared to HET and WT rats at PNS ~110. S.C. = Spinal Cord; ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Fig. 5

GNA accumulates in NGLY1-deficient patient samples. (A) The quantitative assay demonstrates GNA is elevated in the plasma of patients with NGLY1 deficiency compared to unaffected parent and sibling control samples. (B) The quantitative assay demonstrates GNA is elevated in the urine of patients with NGLY1 deficiency compared to unaffected related control samples. (C) GNA level in patient plasma does not significantly correlate with donor age by linear model.