CaseSA6P: Male, 7 months onset, European

CasePresentingHPOs: HP:0001988, HP:0000825, HP:0001250, HP:0001987 (Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia, Seizures, Hyperammonemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Has father with confirmed variant and similar phenotypes

CasePreviousTesting: Leucine load test showed leucine sensitivity; Protein/leucine restriction and dioxazide was effective at reducing symptoms

CaseGenotypingMethod: Genomic DNA was prepared from all patients via peripheral blood leukocytes. The GLUD1 gene exons 1-13 and the flanking intronic regions were PCR-amplified and directly sequenced. All variants were confirmed via restriction enzyme analysis. Additionally, paternity testing was performed by HLA typing and by southern blot analysis of DNA fragments as well as PCR analysis of STRs.

Variant: NM_005271.5(GLUD1):c.965G>A (p.Arg322His)

ClinVar: 16129, https://www.ncbi.nlm.nih.gov/clinvar/variation/16129/

gnomAD: Not in gnomAD

CaseSA3P: Female, 16 months onset, European

CasePresentingHPOs: HP:0001988, HP:0000825, HP:0001987 (Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia, Hyperammonemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is confirmed de novo via parental teting and confirmed paternity

CasePreviousTesting: Protein/leucine restriction was effective at reducing symptoms

CaseGenotypingMethod: Genomic DNA was prepared from all patients via peripheral blood leukocytes. The GLUD1 gene exons 1-13 and the flanking intronic regions were PCR-amplified and directly sequenced. All variants were confirmed via restriction enzyme analysis. Additionally, paternity testing was performed by HLA typing and by southern blot analysis of DNA fragments as well as PCR analysis of STRs.

Variant: NM_005271.5(GLUD1):c.965G>A (p.Arg322His)

ClinVar: 16129, https://www.ncbi.nlm.nih.gov/clinvar/variation/16129/

gnomAD: Not in gnomAD

CaseSA5P: Female, 3 months onset, European

CasePresentingHPOs: HP:0001988, HP:0000825, HP:0001249, HP:0001250 (Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia, Intellectual disability, Seizure)

HPOsFreeText: N/A

CaseNotHPOs: HP:0001987 (Hyperammonemia)

NotHPOsFreeText: N/A

CaseAddInfo: Has father with confirmed variant and similar phenotypes

CasePreviousTesting: Leucine load test showed leucine sensitivity; Protein/leucine restriction and dioxazide was effective at reducing symptoms

CaseGenotypingMethod: Genomic DNA was prepared from all patients via peripheral blood leukocytes. The GLUD1 gene exons 1-13 and the flanking intronic regions were PCR-amplified and directly sequenced. All variants were confirmed via restriction enzyme analysis. Additionally, paternity testing was performed by HLA typing and by southern blot analysis of DNA fragments as well as PCR analysis of STRs.

Variant: NM_005271.5(GLUD1):c.965G>A (p.Arg322His)

ClinVar: 16129, https://www.ncbi.nlm.nih.gov/clinvar/variation/16129/

gnomAD: Not in gnomAD

CaseSA2P: Female, 3 months onset, European

CasePresentingHPOs: HP:0001988, HP:0000825, HP:0001987 (Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia, Hyperammonemia)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is confirmed de novo via parental teting and confirmed paternity

CasePreviousTesting: Leucine load test showed leucine sensitivity in some patients; Protein/leucine restriction was effective at reducing symptoms

CaseGenotypingMethod: Genomic DNA was prepared from all patients via peripheral blood leukocytes. The GLUD1 gene exons 1-13 and the flanking intronic regions were PCR-amplified and directly sequenced. All variants were confirmed via restriction enzyme analysis. Additionally, paternity testing was performed by HLA typing and by southern blot analysis of DNA fragments as well as PCR analysis of STRs.

Variant: NM_005271.5(GLUD1):c.965G>A (p.Arg322His)

ClinVar: 16129, https://www.ncbi.nlm.nih.gov/clinvar/variation/16129/

gnomAD: Not in gnomAD

FamilySA1: European

FamilyPresentingHPOs: HP:0001988, HP:0000825, HP:0001987 (Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia, Hyperammonemia)

HPOsFreeText: N/A

FamilyNotHPOs: N/A

NotHPOsFreeText: N/A

FamilyGenotypingMethod: Genomic DNA was prepared from all patients via peripheral blood leukocytes. The GLUD1 gene exons 1-13 and the flanking intronic regions were PCR-amplified and directly sequenced. All variants were confirmed via restriction enzyme analysis.

FamilyPreviousTesting: Leucine load test showed leucine sensitivity in some patients; Other causes of hypoglycemia were excluded (normal values for plasma cortisol, growth hormone, lactate, ammonia, amino acids, and urine organic acids)

FamilyAddInfo: N/A

Variant: NM_005271.5(GLUD1):c.820C>T (p.Arg274Cys)

ClinVar: 16128, https://www.ncbi.nlm.nih.gov/clinvar/variation/16128/

gnomAD: Not in gnomAD

Affected: 5

Segregations: 4

AddSegregationInfo: Pedigree in Figure 1a

CaseSA4P: Female, 12 months onset, European

CasePresentingHPOs: HP:0001988, HP:0000825, HP:0001987, HP:0002167 (Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia, Hyperammonemia, Neurological speech impairment)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Has mother with confirmed variant and similar phenotypes

CasePreviousTesting: Leucine load test showed leucine sensitivity; Protein/leucine restriction was effective at reducing symptoms

CaseGenotypingMethod: Genomic DNA was prepared from all patients via peripheral blood leukocytes. The GLUD1 gene exons 1-13 and the flanking intronic regions were PCR-amplified and directly sequenced. All variants were confirmed via restriction enzyme analysis. Additionally, paternity testing was performed by HLA typing and by southern blot analysis of DNA fragments as well as PCR analysis of STRs.

Variant: NM_005271.5(GLUD1):c.965G>A (p.Arg322His)

ClinVar: 16129, https://www.ncbi.nlm.nih.gov/clinvar/variation/16129/

gnomAD: Not in gnomAD

NonPatientCell: EFO:0002529 (INS-1), EFO:0000856 (islet of Langerhans), CL:0000182 (Hepatocyte)

GeneFunction: GO:0004353 (glutamate dehydrogenase [NAD(P)+] activity)

GeneAlteration: Recombinant adenoviruses encoding WT human GLUD1 and the S498L variant were generated via an expression system, with the human GLUD1 gene being amplified and sub-cloned into a plasmid with a promoter. Digestion using EcoRV was used to discriminate between the wild-type and the mutant with sequencing verifying each step of the cloning. This adenovirus was amplified in HEK-293 cells and INS-1E β-cells were transduced with it. Mouse and human hepatocytes were isolated and seeded before being transduced with the same adenoviruses. The transfection efficency was confirmed via immunoblotting.

FunctionalEvidence: The enzymatic activity was evaluated in both wild-type and mutant mice, with both showing very low activity without glutamate concentrations and the mutant showing higher activity when glutamate was increased. The reductive direction using α-ketoglutarate as substrate was also tested with similar increased results for the mutant. Sensitivity of both lines to the reaction activator (ADP) and inhibitor (GTP) levels were tested, showing that the mutant was more sensitive to the ADP activator and slightly reduced for the GTP inhibitor (particularly in the presence of ADP). Mitochondiral function in wid-type and mutant cells was assessed, demonstrating that glucose response from mutant and WT cells was preserved but that glutamine stimulation abnormally generates ATP in mutant cells.Calcium stimulation was also activated by both glutamine and glucose in mutant cells while only being affected by glucose in WT. Studies in mutant and WT-transfected mouse and human islets showed increased insulin and ammonia production in mutants from stimulation through this responsive glutamine sensitivity. All of this supports GLUD1's pathogenic mechanism, specifically causing a hyperctive mutant protein that responds to glutamine levels rather than simply glucose.

FAVariants: NM_005271.5(GLUD1):c.1493C>T (p.Ser498Leu)

ClinVar: 16122, https://www.ncbi.nlm.nih.gov/clinvar/variation/16122/

NonHumanModel: Mouse (Mus musculus)

GeneAlteration: Two constructs were created, containing with the WT human GDH sequence or the H507Y variant along with the rat insulin promotoe and human growth hormone genomic DNA sequence to stabilize the construct and drive β-cell-specific transcription of the cDNA sequence. These were microinjected into fertilized eggs of B6SJLF1 mice, transferred into pseudopregnant females, and PCR-amplified/sequenced to confirm the succesful insertion of the transgene. Offspring were crossed with C57BL/6 mice and generated two lines of mutant along with one line of WT transgenic mice: TG-1, TG-2, and huGDH.

ModelPhenotype: MP:0000189, MP:0008770, MP:0002079, MP:0005309 (hypoglycemia, decreased survivor rate, increased circulating insulin level, hyperammonemia)

ModelFreeText: Higher sensitivity to leucine and glutamine ramp stimulation, Increased ATP/ADP ratios

HumanPhenotype: HP:0001987, HP:0001988, HP:0000825 (Hyperammonemia, Recurrent hypoglycemia, Hyperinsulinemic hypoglycemia)

HumanFreeText: N/A

ModelEvidence: The two lines of mice transgnically modified with the H507Y variant were observed compared to the WT mice. Both lines of mice had notable and spontaneous hypoglyemia, specifically affecting both the mouse survivial rate and fertility compared to the control line. Cultured insulin islets were compared and demonstrated significantly increased insulin secretion when exposed to glutamine as well as showing an increased sensitivity to leucine. This also results in elevated ammonia as a byproduct, since the elevated reaction product also yields NH4+. This directly results to the pathogenic mechanism of H507Y, and other pathogenic GLUD1 variants, in humans. These variants reduce the protein's sensitivity to regulation by GTP, causing increased glutamate oxidation and insulin production by raising the ATP/ADP ratio artifically.

ModelVariant: NM_005271.5(GLUD1):c.1519C>T (p.His507Tyr)

ClinVar: 16121, https://www.ncbi.nlm.nih.gov/clinvar/variation/16121/

FamilySA2: Serbia

FamilyPresentingHPOs: HP:0002197, HP:0001988, HP:0001987, HP:0000825, HP:0001263 (Generalized-onset seizure, Recurrent hypoglycemia, Hyperammonemia, Hyperinsulinemic hypoglycemia, Global developmental delay)

HPOsFreeText: N/A

FamilyNotHPOs: N/A

NotHPOsFreeText: N/A

FamilyGenotypingMethod: Genetic analysis was perfomed on the GLUD1 gene.

FamilyPreviousTesting: Diazoxide treatment proved somewhat effective for each affected individual;

FamilyAddInfo:

Variant: NM_005271.5(GLUD1):c.820C>T (p.Arg274Cys)

ClinVar: 16128, https://www.ncbi.nlm.nih.gov/clinvar/variation/16128/

gnomAD: Not in gnomAD

Affected: 2

Segregations: 1

AddSegregationInfo: Not enough segregations

CaseSA1: Male, Serbia

CasePresentingHPOs: HP:0025190, HP:0001987, HP:0000825, HP:0001988, HP:0001252, HP:0025502, HP:0001263 (Generalized tonic-clonic seizure, Hyperammonemia, Hyperinsulinemic hypoglycemia, Recurrent hypoglycemia, Muscular hypotonia, Overweight, Global developmental delay)

HPOsFreeText: Born at term with a birth weight of 3850 g, birth length of 51 cm, and an Apgar score of 10

CaseNotHPOs: HP:0002353 (EEG abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are healthy and non-consanguineous with no family history,

CasePreviousTesting: At 9 weeks blood glucose at 2.0 mmol/L, serum insulin level of 32.4 mIU/L, plasma ammonia level was of 277 μmol/L; No abnormalities in plasma amino acid levels nor in urinary organic acid concentrations

CaseGenotypingMethod: Genetic analysis was perfomed on the GLUD1 gene.

Variant: NM_005271.5(GLUD1):c.1493C>T (p.Ser498Leu)

ClinVar: 16122, https://www.ncbi.nlm.nih.gov/clinvar/variation/16122/

gnomAD: Not in gnomAD