Synergistic use of glycomics and single-molecule molecular inversion probes for identification of congenital disorders of glycosylation type-1

Abstract

Congenital disorders of glycosylation type 1 (CDG-I) comprise a group of 27 genetic defects with heterogeneous multisystem phenotype, mostly presenting with nonspecific neurological symptoms. The biochemical hallmark of CDG-I is a partial absence of complete N-glycans on transferrin. However, recent findings of a diagnostic N-tetrasaccharide for ALG1-CDG and increased high-mannose N-glycans for a few other CDG suggested the potential of glycan structural analysis for CDG-I gene discovery. We analyzed the relative abundance of total plasma N-glycans by high resolution quadrupole time-of-flight mass spectrometry in a large cohort of 111 CDG-I patients with known (n = 75) or unsolved (n = 36) genetic cause. We designed single-molecule molecular inversion probes (smMIPs) for sequencing of CDG-I candidate genes on the basis of specific N-glycan signatures. Glycomics profiling in patients with known defects revealed novel features such as the N-tetrasaccharide in ALG2-CDG patients and a novel fucosylated N-pentasaccharide as specific glycomarker for ALG1-CDG. Moreover, group-specific high-mannose N-glycan signatures were found in ALG3-, ALG9-, ALG11-, ALG12-, RFT1-, SRD5A3-, DOLK-, DPM1-, DPM3-, MPDU1-, ALG13-CDG, and hereditary fructose intolerance. Further differential analysis revealed high-mannose profiles, characteristic for ALG12- and ALG9-CDG. Prediction of candidate genes by glycomics profiling in 36 patients with thus far unsolved CDG-I and subsequent smMIPs sequencing led to a yield of solved cases of 78% (28/36). Combined plasma glycomics profiling and targeted smMIPs sequencing of candidate genes is a powerful approach to identify causative mutations in CDG-I patient cohorts.

Keywords: CDG type 1 (CDG-I); congenital disorders of glycosylation (CDG); diagnostics by mass spectrometry; glycomics; multi-omics; smMIPs.

FIGURE 1

Total plasma glycoprofiling of N‐tetrasaccharide glycan for subtyping of Group 1 (PMM2‐, MPI‐, ALG1‐, and ALG2‐congenital disorders of glycosylation [CDG]) and N‐pentasaccharide glycan for direct diagnosis of ALG1‐CDG. (A) Relative abundance of the N‐tetrasaccharide in Control, Group 1, and other CDG type 1 (CDG‐I) defects (Others). High abundance of this glycomarker was proposed to screen for Group 1; (B) further differential analysis of Group 1 using the ratio of the Man3/N‐tetrasaccharide glycans distinguished between Group 1.1 (PMM2‐CDG and MPI‐CDG) and Group 1.2 (ALG1‐CDG and ALG2‐CDG); (C) relative abundance of the N‐pentasaccharide in Control, Group 1, and Others. This novel glycomarker is specific for direct diagnosis of ALG1‐CDG; and (D) fragmentation of the N‐pentasaccharide glycan by quadrupole time‐of‐flight (QTOF) MS/MS analysis

FIGURE 2

Total plasma glycoprofiling of several high‐mannose N‐glycans for subtyping of Group 2 (ALG3‐, MPDU1‐, DPM1‐, DPM3‐, SRD5A3‐, DOLK‐, RFT1‐, ALG11‐, ALG13‐, PMM2‐, MPI‐congenital disorders of glycosylation [CDG], and hereditary fructose intolerance [HFI]), Group 3 (ALG12‐ and ALG9‐CDG), and Group 4 (CDG‐I defects other than Groups 1–3). High relative abundance of Man3 glycan (A) and Man4 glycan (B) in CDG‐I with mannosylation defects including Groups 2 and 3, as compared with Control and Group 4; (C) further ratio analysis of relative abundances of Man3/Man4 glycans allow discrimination of Groups 2 and 3; (D) analysis of the Man3/Man5 ratio could be used to differentiate Group 2.1 (ALG3‐, MPDU1‐, and DPM1‐CDG) and Group 2.2 (DPM3‐, SRD5A3‐, DOLK, RFT1‐, ALG11‐, ALG13‐, PMM2‐, MPI‐CDG, and HFI); and (E) the relative abundance of Man7/Man8 glycans can be used as diagnostic glycoprofile for ALG12‐ and ALG9‐CDG (Group 3)

FIGURE 3

Diagnostic flowchart by combining glycomics, genomics, and clinical signatures to unravel the gene defects in 36 congenital disorders of glycosylation (CDG)‐Ix patients

FIGURE 4

N‐glycosylation pathway with indicated glycan abnormalities in congenital disorders of glycosylation type 1 (CDG‐I) defects. Four groups of gene defects (Groups 1–4) were determined on the basis of their total plasma glycoprofiling. A blue font color represents affected genes while a red font color represents a type of CDG on basis of standard CDG nomenclature