Variant1: NM_000257.4(MYH7):c.1633G>A (p.Asp545Asn)

ClinVar: 264607 https://www.ncbi.nlm.nih.gov/clinvar/variation/264607/

gnomAD: 0.00001759, https://gnomad.broadinstitute.org/variant/14-23897049-C-T

Variant2: NM_000257.4(MYH7):c.2678C>T (p.Ala893Val)

ClinVar: 177763 https://www.ncbi.nlm.nih.gov/clinvar/variation/177763/

gnomAD: Not in gnomAD

Variant3: NM_000257.3:c.2713T>C (p.Cys905Arg)

CAID: CA389047302 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000257.3%3Ac.2713T%3EC

gnomAD: Not in gnomAD

Variant4: NM_000257.4(MYH7):c.2863G>A (p.Asp955Asn)

ClinVar: 264608 https://www.ncbi.nlm.nih.gov/clinvar/variation/264608/

gnomAD: Not in gnomAD

Variant5: NM_000257.3:c.415G>T (p.Val139Leu)

CAID: CA389052864 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000257.3%3Ac.415G%3ET

gnomAD: Not in gnomAD

Variant6: NM_000257.4(MYH7):c.495G>A (p.Met165Ile)

ClinVar: 524948 https://www.ncbi.nlm.nih.gov/clinvar/variation/524948/

gnomAD: Not in gnomAD

Variant7: NM_000257.3:c.547G>A (p.Gly183Arg)

CAID: CA389052574 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000257.3%3Ac.547G%3EA

gnomAD: Not in gnomAD

Variant8: NM_000257.3:c.5754C>G (p.Asn1918Lys)

CAID: CA389034544 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000257.3%3Ac.5754C%3EG

gnomAD: Not in gnomAD

Variant9: NM_000257.3:c.5773C>G (p.Arg1925Gly)

CAID: CA257807342 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000257.3%3Ac.5773C%3EG

gnomAD: Not in gnomAD

Variant10: NM_000257.4(MYH7):c.689T>C (p.Phe230Ser)

ClinVar: 263543 https://www.ncbi.nlm.nih.gov/clinvar/variation/263543/

gnomAD: Not in gnomAD

Variant11: NM_000257.3:c.715_717del (p.Asp239del)

CAID: CA913184869 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000257.3%3Ac.715_717del

gnomAD: Not in gnomAD

Variant12: NM_000257.4(MYH7):c.732+1G>A

ClinVar: 14127 https://www.ncbi.nlm.nih.gov/clinvar/variation/14127/

gnomAD: 0.00005437, https://gnomad.broadinstitute.org/variant/14-23900793-C-T

Variant13: NM_000257.3:c.798T>A (p.Tyr266Ter)

CAID: CA389052023 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000257.3%3Ac.798T%3EA

gnomAD: Not in gnomAD

Variant14: NM_000257.4(MYH7):c.847T>G (p.Tyr283Asp)

ClinVar: 66081 https://www.ncbi.nlm.nih.gov/clinvar/variation/66081/

gnomAD: Not in gnomAD

Variant15: NM_000257.3:c.902T>A (p.Leu301Gln)

CAID: CA389051788 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_000257.3%3Ac.902T%3EA

gnomAD: Not in gnomAD

Variant 1: NM_002225.3:c.1207T>A

Variant 2: NM_002225.4(IVD):c.863C>T

VariantEvidence: This variant is located in the turn region next to the alpha helix where hydrogen bonding is favorable, this location along with the presentation of severe phenotypes and biochemical profiles in the patients harboring these variants suggests a genotype-phenotype correlation (fig. 4)

Variant: NM_002225.3:c.749G>C

VariantEvidence: This variant is located in the coil region of the protein where hydrogen bonding is limited, thus the variant would not have significant effects on bonding. This along with the mild phenotype in Patient 5 suggests a mild genotype-phenotype correlation for this variant

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)

Families 1 and 2

Same single variant (NKX6-2: c.121A>T; p.Lys41∗ (NM_177400.2)) segregated in both Families 1 and 2. No relation between the families.

Haplotype analysis performed on each individual in both families which located four areas of homozygosity between each individual.

These shared regions include 1.4M region on chromosome 10 and a haplotype block around NKX6-2 (founder effect support).

Family 3

Variant, (NM_177400.2; c.487C>G; p.Leu163Val), replaces conserved leucine within the first helix of the homeodomain, therefore destabilizing the protein.

Families 1 and 2

Variant, NKX6-2: c.121A>T; p.Lys41∗ (NM_177400.2), results in stop codon at amino acid 41 resulting in mRNA decay which causes the loss of the functional homeobox.

NM_000152.4(GAA):c.-32-13T>G

NM_000152.4(GAA):c.-32-13T>G

NM_000152.4(GAA):c.-32-13T>G

PMID: 21930696

Gene: DLD

Disease: Pyruvate dehydrogenase E3 deficiency

MonDO: MONDO:0009529

InheritancePattern: Autosomal recessive

Case1

Variant: NM_013391.3:c.326A>G (p.His109Arg)

VariantEvidence: The above mutation was introduced into hDMGDH cDNA and both the wild-type and mutant sequences were expressed in E. coli, Saccharomyces cerevisiae, and other mammalian tissue culture samples. In the mutant E coli, no active enzyme could be detected. Similarly, no antigen could be detected in the other cells. The wild-type cells had detectable activity and much higher levels of mRNA than the mutants. Western blot of the cellular extracts showed that the levels of wild-type and mutant protein were similar, but the activity was undetectable.

Likely Variant Evidence for Variant: c.466C>T (p.Arg156Ter) ClinVar: 39743 and Variant:c.222del (p.Met74IlefsTer15) CAID: CA891862642

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

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.

Variant 1: NM_000257.4(MYH7):c.2167C>G (p.Arg723Gly)

ClinVar: 42885, https://www.ncbi.nlm.nih.gov/clinvar/variation/42885/

gnomAD: n/a

Variant 2: NM_000257.4:c.599C>T (p.Ala200Val)

CAID: CA389052449, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA389052449

gnomAD: n/a

Variant: NM_013391.3:c.326A>G (p.His109Arg)

VariantEvidence: The above variant was expressed in E coli through an expression vector and DMGDH activity was measured and purified for both the mutant and the wild-type. The flavination in the mutant protein showed a 2-fold decrease compared to the wild-type and the under-flavinated mutant revealed a 27-fold decrease in enzyme activity compared to the flavinated wild-type. Experiments with trypsin digestion also confirmed that the under-flavinated variants may be less stable than the wild-type enzyme.

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.