Case765: Female, 9 years report, Spain

CasePresentingHPOs: HP:0003153, HP:0001256, HP:0007359, HP:0012000 (Cystathioninuria, Mild mental retardation, Partial seizures, EEG with generally recognized spikes)

HPOsFreeText: Elevated urine cystathionine (750 umol/g creatine; reference <46) and Elevated tHcys in urine (61 umol/g creatine; reference <20), Elevated plasma tHcys (19 umol/l; reference <7.5), Elevated plasma cystathionine (10 umol/l; reference =0), Post treatment normal urine cystathionine (34 umol/g creatine)

CaseNotHPOs: HP:0003345 (Glutathione synthetase deficiency)

NotHPOsFreeText: N/A

CaseAddInfo: Patient is homozygous for this variant

CasePreviousTesting: Karyotype, magnetic resonance imaging, specific genetic tests for neurogenetic diseases and screening for inborn errors of metabolism (lactate, pyruvate, ammonium, uric acid, amino acids, organic acids, glycosaminoglycanes, creatine and guanidinoacetate, purines and pyrimidines, and isoelectric focusing pattern of transferrin), as well as folate, pyridoxine and cobalamine deficiencies, neural tumors and genetically determined homocystinurias, were all ruled out.

CaseGenotypingMethod: All twelve exons of the CTH gene and the intronic flanking sequences of the subjects and their parents was PCR amplified and directly sequenced.

Variant: NM_001902.5:c.200C>T (p.Thr67Ile)

ClinVar: 2939 https://www.ncbi.nlm.nih.gov/clinvar/variation/2939/

gnomAD: Ashkenazi Jewish 9.934e-3, Overall 6.308e-3 https://gnomad.broadinstitute.org/variant/1-70881670-C-T

CaseGM01566: Female, 16 years report, Caucasian, Europe

CasePresentingHPOs: HP:0003153 (Cystathioninuria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is a compound heterozygote for this variant, Variants were confirmed in trans by parental testing

CasePreviousTesting: N/A

CaseGenotypingMethod: Primers were developed and the CTH exons 1-12 and exon-intron boundaries were PCR amplified and sequenced. All sequence variations were verified by restriction enzyme analysis and/or repeat sequencing.

Variant1: NM_001902.5:c.200C>T (p.Thr67Ile)

ClinVar: 2939 https://www.ncbi.nlm.nih.gov/clinvar/variation/2939/

gnomAD: Ashkenazi Jewish 9.934e-3, Overall 6.308e-3 https://gnomad.broadinstitute.org/variant/1-70881670-C-T

Variant2: CTH, 1-BP DEL, 1220C AKA: NM_001902.5:c.1064del (p.Thr355Ilefs*19)

ClinVar: 2938 https://www.ncbi.nlm.nih.gov/clinvar/variation/2938/

gnomAD: European (Non-Finnish) 1.551e-4, Overall 8.845e-5 https://gnomad.broadinstitute.org/variant/1-70904381-AC-A

Case2928: Female, 2 years diagnosis, Czech

CasePresentingHPOs: HP:0001270, HP:0001263, HP:0003153, HP:0001250, HP:0002240, HP:0000980, HP:0000613, HP:0001744 (Motor delay, Psychomotor retardation, Cystathioninuria, Seizures, Hepatomegaly, Paleness, Photophobia, Splenomegaly)

HPOsFreeText: Elevated total homocysteine (34.0 μM/l; reference 5.1–13.9), Elevated cystathionine (4.4 μM/l; reference 0.04–0.34), Normal total cysteine (250 μmol/l; reference 203–369), Responsive to B6 treatment, Birth weight was 3000 g

CaseNotHPOs: HP:0000315, HP:0000939, HP:0000818, HP:0001977 (Ocular abnormality, Osteoporosis, Abnormality of the endocrine system, Abnormal thrombosis)

NotHPOsFreeText: N/A

CaseAddInfo: Patient is compound heterozygous for these mutations

CasePreviousTesting: Cystathionine (Cysta), total cysteine (tCys), and total homocysteine (tHcy) panels

CaseGenotypingMethod: PCR amplification of CTH coding regions and exon-intron boundaries was performed. These fragments were sequenced and aligned with a reference to find mutations. Those found were confirmed by restriction digest of 100 controls.

Variant1: NM_001902.5:c.200C>T (p.Thr67Ile)

ClinVar: 2939 https://www.ncbi.nlm.nih.gov/clinvar/variation/2939/

gnomAD: Ashkenazi Jewish 9.934e-3, Overall 6.308e-3 https://gnomad.broadinstitute.org/variant/1-70881670-C-T

Variant Too Long

Variant2: NP_001893.2:p.Gly57_Gln196del

ClinVar:

gnomAD:

Variant1: NM_001902.5:c.200C>T (p.Thr67Ile)

Variant2: NM_001902.5(CTH):c.718C>G (p.Gln240Glu)

VariantEvidence: Mutant proteins were expressed in the Escherichia coli by site-directed mutagenesis, with the codon change confirmed by DNA sequencing. Recombinant CTH was expressed and the proteins were purified and the levels detected. The activity of the T67I mutant was 0.6 μmol min−1 mg−1 protein, 3.5-fold lower than the wild-type, with H2S generation from homocysteine and cysteine showing similar decreases of 4-fold and 2-fold respectively. The Q240E mutant was significantly lower, with only 0.03 μmol min−1 mg−1 protein and no detectable H2S generation. See Purification and Kinetic Properties of the Patient Mutations T67I and Q240E for details.

Furthermore, the variant was tested for PLP responsiveness. While the wild-type alleles exhibited ~80% content, the T67I and Q240E variants only displayed 19% and 0.3-1% saturation respectively. See Figure 3 for details.

Variant1: NM_001902.5:c.200C>T (p.Thr67Ile)

Variant2: NM_001902.5(CTH):c.718C>G (p.Gln240Glu)

VariantEvidence: Mutant proteins were expressed in the Escherichia coli by site-directed mutagenesis, with the codon change confirmed by DNA sequencing. Recombinant CTH was expressed and the proteins were purified and the levels detected. The activity of the T67I mutant was 0.6 μmol min−1 mg−1 protein, 3.5-fold lower than the wild-type, with H2S generation from homocysteine and cysteine showing similar decreases of 4-fold and 2-fold respectively. The Q240E mutant was significantly lower, with only 0.03 μmol min−1 mg−1 protein and no detectable H2S generation. See Purification and Kinetic Properties of the Patient Mutations T67I and Q240E for details.

Furthermore, the variant was tested for PLP responsiveness. While the wild-type alleles exhibited ~80% content, the T67I and Q240E variants only displayed 19% and 0.3-1% saturation respectively. See Figure 3 for details.