Case817: Female, 7 years report, Spain

CasePresentingHPOs: HP:0003153, HP:0002345, HP:0002174 (Cystathioninuria, Action tremor, Postural tremor)

HPOsFreeText: Elevated urine cystathionine (1916 umol/g creatine; reference <46), Normal plasma tHcys (3.5 umol/l; reference <7.5), Post treatment normal urine cystathionine (24 umol/g creatine)

CaseNotHPOs: HP:0003345, HP:0001249 (Glutathione synthetase deficiency, Mental retardation)

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

Case764: Female, 4 years report, Spain

CasePresentingHPOs: HP:0011344, HP:0003153, HP:0001257, HP:0003698, HP:0002474 (Severe psychomotor retardation, Cystathioninuria, Spasticity, Difficulty standing, Communication delay)

HPOsFreeText: Elevated urine cystathionine (1611 umol/g creatine; reference <46) and Elevated tHcys in urine (54.3 umol/g creatine; reference <20), Elevated plasma tHcys (20.5 umol/l; reference <7.5), Post treatment normal urine cystathionine (42 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

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

Case2927: Female, 2 years diagnosis, Czech

CasePresentingHPOs: HP:0001270, HP:0001263, HP:0003153, HP:0012443, HP:0000486, HP:0012795, HP:0000639, HP:0000545, HP:0000646, HP:0002280, HP:0030890, HP:0000488, HP:0001744 (Motor delay, Global developmental delay, Cystathioninuria, Abnormality of brain morphology, Strabismus, Abnormality of the optic disc, Nystagmus, Myopia, Amblyopia, Enlarged cisterna magna, White matter hyperintensity, Retinopathy, Splenomegaly)

HPOsFreeText: Normal total homocysteine (7.1 μM/l; reference 5.1–13.9), Elevated cystathionine (6 μM/l; reference 0.04–0.34), Normal total cysteine (306 μmol/l; reference 203–369), Birth weight was 2400 g, Responsive to B6 treatment

CaseNotHPOs: HP:0002240, HP:0001977 (Hepatomegaly, Abnormal thrombosis)

NotHPOsFreeText: N/A

CaseAddInfo: Patient is homozygous for this mutation

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.

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

Case2929: Male, 4 months diagnosis, Czech

CasePresentingHPOs: HP:0001270, HP:0011344, HP:0003153, HP:0003698, HP:0002300, HP:0004484, HP:0008454 (Motor delay, Severe psychomotor retardation, Cystathioninuria, Difficulty standing, Mutism, Craniofacial asymmetry, Lumbar kyphosis)

HPOsFreeText: Unknown total homocysteine (N/A μM/l; reference 5.1–13.9), Elevated cystathionine (5.4 μM/l; reference 0.04–0.34), Unknown total cysteine (N/A μmol/l; reference 203–369), Response to B6 treatment unknown

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CasePMIDs: 615480

CaseAddInfo: Patient is homozygous for this mutation, Proband is born of father-daughter incest and is therefore consanguineous

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.

Variant: NM_001902.5:c.589C>T (p.Arg197Cys)

CAID: CA906222 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_001902.5%3Ac.589C%3ET

gnomAD: South Asian 1.312e-4, Overall 6.378e-5 https://gnomad.broadinstitute.org/variant/1-70895477-C-T

Case2930: Female, 3 weeks diagnosis, United States

CasePresentingHPOs: HP:0003153 (Cystathioninuria)

HPOsFreeText: Elevated total homocysteine (18.2 μM/l; reference 5.1–13.9), Elevated cystathionine (11.8 μM/l; reference 0.04–0.34), Normal total cysteine (246 μmol/l; reference 203–369)

CaseNotHPOs: HP:0001270, HP:0001263, HP:0100543, HP:0030972 (Motor delay, Global developmental delay, Cognitive impairment, Abnormal systemic blood pressure)

NotHPOsFreeText: Unresponsive to B6 treatment

CaseAddInfo: Patient is compound heterozygous for these mutations, Parental testing has confirmed these variants are in trans

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.932C>T (p.Thr311Ile)

CAID: CA340839077 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvs=NM_001902.5%3Ac.932C%3ET

gnomAD: Not in gnomAD

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:c.589C>T (p.Arg197Cys)

Variant3: NM_001902.5:c.932C>T (p.Thr311Ile)

VariantEvidence: Wildtype for CTH, 3 separate mutations, and an empty vector were all expressed in E. coli vectors using recombinant proteins. This showed a marked decrease in activity in Variants 1 and 2, but no significant change in Variant 3.

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:

CaseGM01781: Female, 18 years report, Caucasian, Europe

CasePresentingHPOs: HP:0003153 (Cystathioninuria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is a compound heterozygote for these variants, 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: NM_001902.5:c.718C>G (p.Gln240Glu)

ClinVar: 2940 https://www.ncbi.nlm.nih.gov/clinvar/variation/2940/

gnomAD: European (Non-Finnish) 1.759e-5, Overall 7.957e-6 https://gnomad.broadinstitute.org/variant/1-70896071-C-G

CaseGM01565: Female, 16 years report, Caucasian, Europe

CasePresentingHPOs: HP:0003153 (Cystathioninuria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is a homozygote for this variant.

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.

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

CaseGM01454: Male, 7 years report, Asian, China

CasePresentingHPOs: HP:0003153 (Cystathioninuria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband is a homozygote for this variant.

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.

Variant: CTH, 2-BP DEL, 940CT AKA: NM_001902.5:c.784_785del (p.Leu262Thrfs*21)

ClinVar: 2937 https://www.ncbi.nlm.nih.gov/clinvar/variation/2937/

gnomAD: East Asian 1.088e-4, Overall 7.960e-6 https://gnomad.broadinstitute.org/variant/1-70897822-ACT-A

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

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.