Table 2.

Summary table of OGT-CDG variant characterization

Variant	L254F	A259T	R284P	A319T	E339G
Domain	TPR	TPR	TPR	TPR	TPR
Polyphen Score	0.999	1.000	0.967	0.999	1.000
In vitro Glycosyltransferase Activity	•Normal activity (CKIIα) •↓ activity (de-O-GlcNAcylated HEK293 cell lysate) •Normal activity (HEK293 overexpression system)	Normal activity (CKIIα)	•Normal activity (CKIIα) •↓ activity (de-O-GlcNAcylated HEK293 cell lysate)	Normal activity (CKIIα)	Normal activity (CKIIα)
In vitro HCFC1 Glycosyltransferase Activity	Normal activity	Normal activity	Reduced activity	Normal activity	Normal activity
In vitro HCFC1 Protease Activity	Normal activity	Normal activity	Reduced activity	Normal activity	Normal activity
Kinetic Analysis	•Km 1.86-fold change, kcat 0.78-fold change versus WT (CKIIα protein substrate) •Km and Vmax unchanged (peptide substrate)	Km 1.29-fold change, kcat 0.78-fold change versus WT (CKIIα protein substrate)	Km 1.29-fold change, kcat 0.78-fold change versus WT (CKIIα protein substrate)	Km 2.07-fold change, kcat 0.73-fold change versus WT (CKIIα protein substrate)	Km 1.71-fold change, kcat 0.89-fold change versus WT (CKIIα protein substrate)
Dimerization	Normal dimerization of recombinant TPR domain	Normal dimerization of recombinant TPR domain	Normal dimerization of recombinant TPR domain	Normal dimerization of recombinant TPR domain	Normal dimerization of recombinant TPR domain
Thermal Stability	↓	↓	↓	↓	↓
Global O-GlcNAc Levels	Unchanged (hESCs)	Unchanged (hESCs)	Unchanged (hESCs)	N/A	Unchanged (hESCs)
Global OGT Levels	Unchanged (hESCs)	Unchanged (hESCs)	Unchanged (hESCs)	N/A	Unchanged (hESCs)
Global OGA Levels	Unchanged (hESCs)	Unchanged (hESCs)	Unchanged (hESCs)	N/A	Unchanged (hESCs)
Gene Expression	•Altered (patient derived lymphoblastoids) •Altered (hESCs)	Altered (hESCs)	Altered (hESCs)	N/A	Altered (hESCs)
Pluripotency Markers	Unchanged POU5F1 (Oct4), SOX2, NANOG (hESCs)	Unchanged POU5F1 (Oct4), SOX2, NANOG (hESCs)	Unchanged POU5F1 (Oct4), SOX2, NANOG (hESCs)	N/A	Unchanged POU5F1 (Oct4), SOX2, NANOG (hESCs)
Animal and Cell Models	Drosophila: •Non-performers in habituation learning •↑ synaptic bouton number, ↑ NMJ length (not significantly), ↑ number of NMJ branches	N/A	Drosophila: •↓ climbing speed •Non-performers in habituation learning (homozygous) •Deficits in habituation learning (heterozygous) •↑ synaptic bouton number, ↑ length of NJM, ↑ perimeter of NMJ, ↑ number of NMJ branches	Drosophila: •Deficits in habituation learning (homozygous) •↑ synaptic bouton number, ↑ NMJ length (not significantly), ↑ NMJ length	N/A
References	(31, 32, 40, 49)	(32)	(30, 32, 49)	(32, 49)	(32)

Variant	Δ155–177	N567K	N648Y	C921Y
Domain	TPR	Catalytic	Catalytic	Catalytic
Polyphen Score	N/A	0.998	1.000	1.000
In vitro Glycosyltransferase Activity	N/A	↓ (TAB1)	↓ (TAB1)	↓ (TAB1)
In vitro HCFC1 Glycosyltransferase Activity	N/A	↓ activity	N/A	↓ activity
In vitro HCFC1 Protease Activity	N/A	↓ activity	N/A	Normal activity
Kinetic Analysis	N/A	Km 1.45-fold change, kcat 0.17-fold change, kcat/Km 0.08-fold change versus WT (peptide substrate)	Kd 6.88-fold change versus WT (fluorescent probe)	Vmax 0.25-fold change, Km 0.18-fold change, kcat/Km unchanged versus WT (peptide substrate)
Dimerization	N/A	N/A	N/A	N/A
Thermal Stability	↓ thermal stability	N/A	Normal thermal stability	Normal thermal stability
Global O-GlcNAc Levels	•Unchanged (patient derived fibroblasts) •Unchanged (iPSCs) •↓ (differentiated iPSCs) •↓ (NPCs)	Unchanged (pluripotent mESCs)	↓ (mESCs)	•↓ (undifferentiated, mESCs) Unchanged (mESCs, clonogenic conditions)
Global OGT Levels	•↓ (patient derived fibroblasts) •↓ (iPSCs) •↓ (differentiated iPSCs) •↓ (NPCs)	Unchanged (pluripotent mESCs)	Unchanged (mESCs)	•↑ (undifferentiated mESCs) ↑(mESCs, clonogenic conditions)
Global OGA Levels	•↓ (patient derived fibroblasts) •↓ (iPSCs) •↓ (differentiated iPSCs) •↓ (NPCs)	•↓ (pluripotent mESCs) •Unchanged (differentiated mESCs)	↓ (mESCs)	•Unchanged (undifferentiated mESCs) Unchanged (mESCs, clonogenic conditions)
Gene Expression	•Unchanged OGT, OGA mRNA (patient derived fibroblasts) •↑ OGT, ↓ OGA mRNA (iPSCs) •Unchanged OGT, ↓ OGA mRNA (differentiated iPSCs) •↑ NEFH, all other ectoderm markers unchanged (differentiated iPSCs) •Unchanged OGT, OGA mRNA (NPCs)	↑ GABPA mRNA (mESCs)	N/A	Unchanged Ogt, ↓ Oga mRNA (undifferentiated mESCs)
Pluripotency Markers	•Unchanged POU5F1 (Oct4), SOX2, NANOG mRNA, Unchanged NANOG, OCT4, SOX2 protein (iPSCs)	Unchanged Sox2, Oct4 during first 6 days of differentiation (mESCs)	N/A	•Unchanged Oct4 and Sox2 mRNA, Oct4 and Sox2 protein (undifferentiated mESCs) ↓ Oct4, Sox2 protein (mESCs, clonogenic conditions)
Animal and Cell Models	•Unchanged pluripotency, differentiation, and morphology (iPSCs) •Unchanged neural progenitor markers (NPCs) •Fewer and larger rosettes, larger apical lumens (NPCs)	Drosophila: •↓ O-GlcNAc (adult heads) mESCs: •↓ neurite length •↓ proteolytic cleavage of HCFC1 Mice: •Altered inheritance distribution •↓ Global GlcNAc, ↓ OGA protein, ↓ OGT protein (brain) •↓ Oga, ↓ Ogt mRNA, ↓ DIs, ↓ PEs (brain) •↓ body weight, ↓ nose-to-tail length, ↑ pancreas weight, slim appearance •↓ brain weight	Mice: •↓ Global GlcNAc, ↓ OGA protein, ↓ OGT protein (brain) •↓ Oga, ↓ Ogt mRNA, ↓ DIs, ↓ PEs (brain) •↓ body weight, ↓ nose-to-tail length, ↑ pancreas weight, slim appearance •↓ brain weight	Drosophila: •↓ O-GlcNAc, ↑ OGT protein (adult heads) •↓ O-GlcNAc, ↑ OGT protein (embryos) •↓ O-GlcNAc, unchanged OGT protein (larvae) •↑ ectopic bristle penetrance •↓ NMJ area, ↓ NJM length, ↓ NMJ bouton numbers •↓ total sleep, ↓ duration and more frequent sleep bouts mESCs: ↓alkaline phosphatase staining (clonogenic conditions) Mice: •↓ Global GlcNAc, ↓ OGA protein, ↓ OGT protein (brain) •↓ Oga, ↓ Ogt mRNA, ↓ DIs, ↓ PEs (brain) •↓ body weight, ↓ nose-to-tail length, ↓ body fat mass, ↑ lean body mass, ↓ glycemia, ↑ pancreas weight, slim appearance •↓ skull length, ↓ skull size, ↓ absolute brain weight, rounder and smaller skull Hypothesized anxiety phenotype, compulsive behavior, altered spatial working memory
References	(30, 44)	(34, 50)	(33, 50)	(35, 50, 51)

Summarized are characteristics of the six Tetratricopeptide repeat (TPR) domain variants and three catalytic domain variants of OGT discussed in this review. This table shows the results of characterizations of variants in vitro, in cellulo, and in vivo alongside phenotypes observed in vivo. Abbreviations used within include WT (wild-type), TPR (Tetratricopeptide repeat), CKIIα (casein kinase II alpha), TAB1 (TAK-1 binding protein), hESCs (human embryonic stem cells), iPSCs (induced pluripotent stem cells), mESCs (mouse embryonic stem cells), NPCs (neural progenitor cells), NMJ (neuromuscular junction), DI (detained introns), PE (decoy exon), ↑ (upregulated or increased), ↓ (downregulated or decreased), N/A (information unavailable or not determined).