CaseAD: Male, 5 yrs onset

CasePresentingHPOs: HP:0000750, HP:0002572, HP:0012537, HP:0001944, HP:0001649, HP:0002789, HP:0001252, HP:0001942, HP:0001987, HP:0002919, HP:0012120 (Delayed speech and language development, Episodic vomiting, Food intolerance, Dehydration, Tachycardia, Tachypnea, Muscular hypotonia, Metabolic acidosis, Hyperammonemia, Ketonuria, Methylmalonic aciduria)

HPOsFreeText: Frequent catabolism episodes with vomiting were noticed without decompensation

CaseNotHPOs: HP:0001270, HP:0001919, HP:0011015 (Motor delay, Acute kidney injury, Abnormal blood glucose concentration)

NotHPOsFreeText: Propionyl-CoA carboxylase activity was within normal ranges in fibroblasts

CaseAddInfo: Parents are non-consanguineous with no family history, Dietary restriction and supplementation showed amelioration of symptoms

CasePreviousTesting: Normal ECG and MRI results; Metabolic acidosis (pH = 7.08, bicarbonates = 7.3 mmol/L, normal lactic acid, PCO2 = 25 mmHg) and hyperammmoniemia (100 µmol/L) present; Large increase in plasmatic propionylcarnitine (17.5 µmol/L; N < 0.75), undetectable methylmalonylcarnitine, and high urinary excretion of 3-hydroxypropionate and methylcitrate

CaseGenotypingMethod: Next-generation sequencing in the proband revealed a homozygous nonsense founder variant in the MCEE gene.

Variant: NM_032601.4(MCEE):c.139C>T (p.Arg47Ter)

ClinVar: 2343, http://www.ncbi.nlm.nih.gov/clinvar/variation/2343

gnomAD: 0.0004884, https://gnomad.broadinstitute.org/variant/2-71351575-G-A

CaseHE9: 6 month onset

CasePresentingHPOs: HP:0001250, HP:0001943, HP:0001942, HP:0012120 (Seizure, Hypoglycemia, Metabolic acidosis, Methylmalonic aciduria)

HPOsFreeText: Elevated proionyl-carnitine and methyl-citrate in urine

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is homozygous for this variant,

CasePreviousTesting: Fibroblast cell lines taken from 150 patients with mild but clear MMAuria were studied; Urinary MMA at 143–184 mmol/mol creat. (ref 0.3-1.1); Low propionate incorporation in fibroblasts both +/- OHCbl of 3.0 nmol/16 h/mg (ref 3.5–24.4 −OHCbl, 4.3–28.9 +OHCbl); Not responsive to Vitamin B12 treatment

CaseGenotypingMethod: Genomic DNA and RNA extraction was performed and the MCEE gene was sequenced to identify any likely pathogenic variants.

Variant: NM_032601.3:c.158T>G (p.Ile53Arg)

CAID: CA347189175, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA347189175

gnomAD: Not in gnomAD

CaseWG2797: N/A

CasePresentingHPOs: HP:0012120 (Methylmalonic aciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is asymptomatic and has an affected sibling with similar aciduria

CasePreviousTesting: Propionate Incorporation w/o hydroxycobalamin at 3.9 nmols/mg protein/18hr; Propionate Incorporation with hydroxycobalamin at 4.0 nmols/mg protein/18hr; Adenosylcobalamin synthesis percentage at 12.4%; Elevated methylmalonic values of 1400 µmol/mmol creatinine (reference 0.8-5.2 µmol/mmol creatinine)

CaseGenotypingMethod: Genomic DNA was extracted from patient fibroblasts, sequenced, and the three MCEE/thirteen MUT exons were analyzed and amplified. Any reported mutations were resequenced from fresh PCR product.

Variant: NM_032601.4(MCEE):c.139C>T (p.Arg47Ter)

ClinVar: 2343, https://www.ncbi.nlm.nih.gov/clinvar/variation/2343/

gnomAD: 0.0004884, https://gnomad.broadinstitute.org/variant/2-71351575-G-A

CaseWG2098: 3 yrs onset

CasePresentingHPOs: HP:0001251, HP:0004302, HP:0001260, HP:0002313, HP:0012120 (Ataxia, Functional motor deficit, Dysarthria, Spastic paraparesis, Methylmalonic aciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband has normal 14C-propionate incorporation, suggesting normal MCEE protein activity level; The high gnomAD frequency and homozygotes suggests this is likely a benign polymorphism

CasePreviousTesting: Normal MRI; Propionate Incorporation w/o hydroxycobalamin at 7.0 nmols/mg protein/18hr; Propionate Incorporation with hydroxycobalamin at 8.2 nmols/mg protein/18hr; Adenosylcobalamin synthesis percentage at 12.7%; Elevated methylmalonic values of 621 µmol/mmol creatinine (reference 0.8-5.2 µmol/mmol creatinine)

CaseGenotypingMethod: Genomic DNA was extracted from patient fibroblasts, sequenced, and the three MCEE/thirteen MUT exons were analyzed and amplified. Any reported mutations were resequenced from fresh PCR product.

Variant: NM_032601.4(MCEE):c.178A>C (p.Lys60Gln)

ClinVar: 203812, https://www.ncbi.nlm.nih.gov/clinvar/variation/203812/

gnomAD: 0.01463 (10 homozygotes), https://gnomad.broadinstitute.org/variant/2-71351536-T-G

CaseWA: Female, 5 yrs onset, French-Canadian

CasePresentingHPOs: HP:0002013, HP:0001944, HP:0001289, HP:0002367, HP:0001942, HP:0031962, HP:0001987, HP:0003236, HP:0001946, HP:0002919, HP:0000651, HP:0012120 (Vomiting, Dehydration, Confusion, Visual hallucinations, Metabolic acidosis, Elevated serum anion gap, Hyperammonemia, Elevated serum creatine kinase, Ketosis, Ketonuria, Diplopia, Methylmalonic aciduria)

HPOsFreeText: Low incorporation of 14C-propionate (4.1 nmol propionate/mg protein/18 h; control mean 10.8, SD 3.7) with no response to hydroxycobalamin

CaseNotHPOs: HP:0002151 (Increased serum lactate)

NotHPOsFreeText: N/A

CaseAddInfo: Parents are non-consanguineous with unremarkable family history, Treatment with carnitine and restricted protein intake normalized symptoms, Sequencing of the mother revealed Variant 2 without Variant 1 confirming compound heterozygosity

CasePreviousTesting: Metabolic acidosis values w/ elevated anion gap (pH = 6.96, bicarbonate 3.5 mmol/L and pCO2 15.9 mm Hg, anion gap 25.5 mmol/L); Mild hyperammonemia (107 μmol/L; ref. < 50); Elevated creatine kinase (from 498 IU/L initially up to 3062 IU/L, ref. < 150); Organic acid profiles were analyzed via gas chromatography–mass spectrometry with Table 1 showing relevant organic acid values; Proband was negative for propionyl-coA carboxylase deficency (811 pmol/min/mg protein with control mean 1123, PCCA and PCCB variants were negative) and ruled out MUT/cobalamin issues as well; Fusion experiments showed negative results for mut, cblA, and cblB complementation but no increase in activity when fused with MCEE confirming the pathway blockage

CaseGenotypingMethod: Extracted DNA from blood was amplified and three coding exons/splice site junctions of MCEE were Sanger sequenced. A deletion/duplicaiton analysis yeilded normal results. To search for an intronic sequence variant, total RNA was extracted and reverse-transcribed to confirm a 92-bp insertion between Exons 2 and 3. Resulting sequencing of this genomic area revealed a deep intronic variant that created a new consensus donor splice site that leads to a premature stop codon.

Variant1: NM_032601.4(MCEE):c.139C>T (p.Arg47Ter)

ClinVar: 2343, http://www.ncbi.nlm.nih.gov/clinvar/variation/2343

gnomAD: 0.0004884, https://gnomad.broadinstitute.org/variant/2-71351575-G-A

Variant2: NM_032601.3:c.379-644A>G

CAID: CA892631472, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA892631472

gnomAD: Not in gnomAD

CaseAN: Male, 78 yrs report, Germany, White

CasePresentingHPOs: HP:0003774, HP:0001638, HP:0000822, HP:0002099, HP:0012125, HP:0001297, HP:0002134, HP:0002549, HP:0000738, HP:0000739, HP:0000746, HP:0002322, HP:0002172, HP:0012120 (Stage 5 chronic kidney disease, Cardiomyopathy, Hypertension, Asthma, Prostate cancer, Stroke, Abnormality of the basal ganglia, Deficit in phonologic short-term memory, Hallucinations, Anxiety, Delusions, Resting tremor, Postural instability, Methylmalonic aciduria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Mother had dementia, Proband is diagnosed with Parkinson's disease, Sanger sequencing confirmed compound heterozygous as unaffected sons are heterozygous for one variant each

CasePreviousTesting: Montreal Cognitive Assessment (MoCA) score of 17/30 in 2013; Mild MMA-uria, together with increased plasma levels of propionyl-carnitine, was demonstrated in 2016 via metabolic investigation (values in Table 1)

CaseGenotypingMethod: Whole genome sequencing data was analyzed using a pipeline restricted to 681 genes known to cause IEM. Heterozygous mutations in MCEE were identified.

Variant1: NM_032601.4(MCEE):c.139C>T (p.Arg47Ter)

ClinVar: 2343, http://www.ncbi.nlm.nih.gov/clinvar/variation/2343

gnomAD: 0.0004884, https://gnomad.broadinstitute.org/variant/2-71351575-G-A

Variant2: NM_032601.3:c.419del (p.Lys140Argfs*6)

CAID: CA1702578, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA1702578

gnomAD: 0.00007076, https://gnomad.broadinstitute.org/variant/2-71337211-CT-C

CaseDOIII-3: Female, 12 yrs report, White

CasePresentingHPOs: HP:0001531, HP:0002020, HP:0001942, HP:0001944, HP:0002789, HP:0002013, HP:0002014, HP:0002919, HP:0012120 (Failure to thrive in infancy, Gastroesophageal reflux, Metabolic acidosis, Dehydration, Tachypnea, Vomiting, Diarrhea, Ketonuria, Methylmalonic aciduria)

HPOsFreeText: Elevated methylcitric acid

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Parents are first-cousin consanguineous, Son is healthy but second daughter shares phenotypes with the proband and shows clear methylmalonic aciduria, Proband and her sister are confirmed homozygous for this variant via parental testing

CasePreviousTesting: Serum methylmalonic acid was 1314 ng/ml (normal < 32); Vitamin B12 levels were normal (927 pg/ml, normal 200–1100); Vitamin B12 injections improved patient condition; At 12.5 yrs carnitine, plasma AAs, blood counts, and basic metabolic panels are normal but methylmalonic acid remains high at 180–1456 mmol/mol creatinine; Fibroblasts showed normal AdoCbl synthesis, contrary to expected from MMAA or MMAB variants; MUT and MMAB were sequenced with no variation found

CaseGenotypingMethod: Genomic DNA was isolated and PCR-amplified before directly sequencing all exons and flanking intronic sequences of the MCEE gene. Restriction enzyme digest confirmed the phase of the variant in the proband and the parents.

Variant: NM_032601.4(MCEE):c.139C>T (p.Arg47Ter)

ClinVar: 2343, https://www.ncbi.nlm.nih.gov/clinvar/variation/2343/

gnomAD: 0.0004884, https://gnomad.broadinstitute.org/variant/2-71351575-G-A