VariantEvidence: Expression of recombinant GMT was performed in E. coli. Firstly, the wild-type protein showed high solubility compared to the mutant of which only a minor fraction was soluble. Furthermore, enzyme activity was measured and the mutant GMNT lacked any activity while the wild-type exibits activity of >200 pmol/min/ug.

Variant: NM_018960.4:c.296G>A (p.Arg99His)

VariantEvidence: Human HEK293 cells were transfected with either wild-type or mutant protein and cloned, with mRNA and protein levels being determined by PCR and western blotting. The mutant protein levels were just on the threshold of being detected, while the wild-type was over-expressed. This further exemplifies the pathogenicity of the gene.

Variant: NM_018960.4:c.296G>A (p.Arg99His)

Case1: female, 2.3y (initial referral), Italian, Family 1

CasePresentingHPOs: HP:0001508, (failure to thrive)

HPOsFreeText: birthweight 3060 g, SGOT 11 mo 150 U/L, SGPT 11 mo 160 U/L, 1.8y elevated plasma lactic acid 2.9 mmol/L. 3.9 y alkaline phosphatase 928 U/L, 4.7 y serum tricglycerides 184 mg/dl, fasting plasma methionine 644 umol/L. increased urinary methionine excretion 40 umol/mmol. increased lactic acid excretiom 107 umol/mmol. mild centrilobular fibrosis in liver. some eosinophils present in liver.

CaseNotHPOs: HP:0001404, HP:0001397, HP:0012465, HP:0001396, (hepatocellular necrosis, hepatic steatosis, elevated hepatic iron concentration, cholestasis)

NotHPOsFreeText: hepatic dysfunction. normal tHcy.

CaseAddInfo: proband, second child, low methionine diet (300 mg/day) at 2.4 y decreased plasma methionine to normal.

Case (Level 2)

Variant1: NM_018960.5(GNMT):c.149T>C (p.Leu50Pro)

ClinVar: 4169

CAID: CA248600

gnomAD: 0.0002319 (East Asian)

Variant2: NM_018960.5(GNMT):c.529C>A (p.His177Asn)

ClinVar: 4170

CAID: CA248601

gnomAD: 0.0006993 (European non-Finnish)

CaseAddInfo: compound heterozygote, Leu50Pro maternal mutation, His177Asn paternal mutation. The amino acid shifts are one # different than those predicted in the paper (Leu49Pro and His176Asn).

Case2: male, 9.6 y (evaluation), Italian, Family 1

CasePresentingHPOs:

HPOsFreeText: birth weight 3850 g, 5y slightly elevated serum transaminases SGOT 60 U/L, SGPT 52 U/L. elevated alkaline phosphatase 1042 U/L). elevated triglycerides (342 mg/dl). fasting plasma methionine elevated to 426 umol/L. elevated tHcy 13 umol/L, elevated urinary methionine 34 umol/mmol. increased pyruvate excretion.

CaseNotHPOs:

NotHPOsFreeText: normal neurological examination and normal somatic growth. normal serum amylase and lipase. hepatic dysfunction.

CaseAddInfo: proband, first child

Case (Level 2)

Variant1: NM_018960.5(GNMT):c.149T>C (p.Leu50Pro)

ClinVar: 4169

CAID: CA248600

gnomAD: 0.0002319 (East Asian)

Variant2: NM_018960.5(GNMT):c.529C>A (p.His177Asn)

ClinVar: 4170

CAID: CA248601

gnomAD: 0.0006993 (European non-Finnish)

CaseAddInfo: compound heterozygote, Leu50Pro maternal mutation, His177Asn paternal mutation. The amino acid shifts are one # different than those predicted in the paper (Leu49Pro and His176Asn).

Case1 (Level 1)

Case1: male, 5y (at time of study, born 1998), Greece, Gypsy (Roma)

CasePresentingHPOs: HP:0002587, HP:0008872, HP:0001662, HP:0003235 (projectile vomiting (at birth), feeding difficulties in infancy, bradycardia (infancy), hypermethioninemia)

HPOsFreeText: 3350 g birthweight, elevated serum aminmotransferases, mildly elevated ALT and AST at 2y, mild ferritin elevation 173 ng/ml, serum triglycerides 155 mg/dl, total cholesterol 248 mg/dl, aspartate transaminase 31 to 154 U/L, alanine transaminase 34-180 U/L, urine methionine 508-627 umol/L, methionine excretion 60 umol/mmol, plasma methionine 1005-1049 umol/L, plasma AdoMet 1643-2222 nmol/L, plasma cystathionine 740-900 nmol/L, plasma phosphatidylcholine 2622-2641 umol/L, plasma betaine 218-382 umol/L, plasma dimethylglycine 11.7-12.1 umol/L

CaseNotHPOs: HP:0002240 (hepatomegaly)

NotHPOsFreeText: normal apgar score, normal blood ammonia, normal lactate, normal tyrosine, , normal plasma N-methylglycine 1.4-1.6 umol/L, normal plasma AdoHcy 32-43 nmol/L, normal plasma tHcy 11.1-12.1 umol/L, normal plasma tCys 206-217 umol/L, normal plasma choline 10.2-14.0 umol/L

CaseAddInfo: Adopted at 9 mo, no family info,

CasePMIDs: n/a

Case1 (Level 2)

CasePreviousTesting: metabolite assays: tHcy- fluorescent polarization method; urinary organic acids- gas chromatography mass spectrometry; plasma methionine, tHcy, cystathionine, tCys, dimethylglycine, sarcosine- capillary gas chromatography

CaseMethod1: PCR

CaseGenotypingMethod: genomic DNA isolated from blood samples (affected patient and 10 healthy American controls). 1.9 kb 5'-fragment of GNMT gene prepared using PCR primers and 1.8 kb 3' fragment prepared using other primers. PCR fragments were purified. Sequencing done by dideoxy sequencing method. Plasmid pGE3 containing wild-type GNMT cDNA was used to prepare mutated human GNMT cDNA. Fragments of all exons and most introns and 300 nucelotides at 5' and 3' flanking ends were sequence using the GNMT sequence specified by GI 8671581 which found a single nucleotide substitution in AAC codon of exon 3:3415A>G.

Variant: NM_018960.5(GNMT):c.422A>G (p.Asn141Ser)

ClinVar: 218931

CAID: CA347847

gnomAD: not in gnomAD

CaseAddInfo: No additional point mutation was found in the coding regions of patients GNMT gene, so the paper concludes that either he is homozygous or he has a deletion on the other allele that overlaps 3415A>G change and prevented PCR amplification in region of question.

Case 1 (Variant Evidence)

VariantEvidence: This variant was expressed in E. coli cells and investigated for protein levels. After the standard incubation time/temperature (15-20 min, 25 deg C) there was no detectable activity. Only after incubation for 60 min at 30 deg C was any measurable amount of sarcosine present, and only then at less than 1% of normal levels.

Variant: NM_018960.5(GNMT):c.422A>G (p.Asn141Ser)

ClinVar: 218931 https://www.ncbi.nlm.nih.gov/clinvar/variation/218931/

gnomAD: not in gnomAD