Criteria Specification Registry

Please note this is a beta version of the Criteria Specification Registry. This resource is intended to provide access to Criteria Specifications used and applied by ClinGen Variant Curation Expert Panels and biocurators in the classification of variants.

For general information about ClinGen Expert Panels and Variant Curation please visit: Clinical Domain Working Groups. For specific inquiries regarding content correction or adding a new criteria specification refer to the Help page.

This resource is undergoing updates and testing. Should you encounter any issues regarding the data displayed, lack of functionality or other problems, please let us know by contacting us via email so we can rectify these accordingly. Your help in this regard is greatly appreciated.

Refer to the specification PDF document on the ClinGen website for tables, images, flow charts, etc.

Coming Soon! Future releases of CSpec Registry will include the following features:

Additional attributes to the specification such as inline citations, references, specification type, comments, superscripts, italics, etc.
File attachments to support tables, flow charts and images.

ClinGen Mitochondrial Disease Nuclear and Mitochondrial Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines Version 1_ntDNA

Description: This document contains one of the two sets of ACMG/AMP specifications from the ClinGen Mitochondrial Disease Nuclear and Mitochondrial Variant Curation Expert Panel

Expert Panel: Mitochondrial Diseases VCEP

Approval Status: Approved - Step 4

Last Updated: 04-30-2020

Version: 1.0.0

PDF Document:

JSON-LD Document:

Rules for SLC19A3

Gene: SLC19A3 (HGNC:16266) HGNC Name: solute carrier family 19 member 3 Preferred Transcript: NM_025243.4 Disease: Biotin-responsive basal ganglia disease (MONDO:0011841)

Criteria	Original ACMG Summary	Strength Specifications
PVS1	Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Caveats: • Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7). • Use caution interpreting LOF variants at the extreme 3’ end of a gene. • Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact. • Use caution in the presence of multiple transcripts.	Very Strong Applied per PVS1 flowsheet of Abou Toyoun et al.
PS1	Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.	Strong Same amino acid change as a previously established pathogenic variant regardless of nucleotide change
PS2	De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity.	Strong De novo in a patient with the disease and no family history
PS3	Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.	Supporting Transporter assay showing loss of function
PS4	The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance. Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence.	Not Applicable
PM1	Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.	Not Applicable
PM2	Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. Caveat: Population data for indels may be poorly called by next generation sequencing.	Moderate <0.00005 (<0.0050%)
PM3	For recessive disorders, detected in trans with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase.	Moderate Use per SVI guidance
PM4	Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.	Moderate Protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants
PM5	Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.	Moderate Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before
PM6	Assumed de novo, but without confirmation of paternity and maternity.	Strong De novo in a patient with the disease and no family history Moderate Assumed de novo, but without confirmation of paternity and maternity
PP1	Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. Note: May be used as stronger evidence with increasing segregation data.	Supporting For segregation, an affected is defined as an individual who 1) has brainstem or basal ganglia lesions compatible with SLC19A3-related Biotin-responsive basal ganglia disease OR 2) a person with neurodevelopmental regression or MRI lesions compatible with SLC19A3-related Biotin-responsive basal ganglia disease who had significant clinical improvement in either symptoms or MRI lesions from treatment with biotin and thiamine.
PP2	Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.	Not Applicable
PP3	Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.	Supporting No gene-specific predictors; agree to utilize REVEL, with thresholds of >0.75 and <0.15 for PP3 and BP4, respectively
PP4	Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.	Supporting Patient has/had MRI features of Leigh syndrome with clinical response to biotin/thiamine
PP5	Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.	Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BA1	Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.	Stand Alone >0.001 (>0.1%)
BS1	Allele frequency is greater than expected for disorder.	Strong >0.0005 (>0.050%)
BS2	Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.	Strong Observed in a healthy, untreated, adult individual in the homozygous state
BS3	Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.	Supporting Transporter assay showing no effect on the gene or gene product
BS4	Lack of segregation in affected members of a family. Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation.	Strong Lack of segregation in affected and/or treated members of a family.
BP1	Missense variant in a gene for which primarily truncating variants are known to cause disease.	Not Applicable
BP2	Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.	Supporting Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder; or observed in cis with a pathogenic variant in any inheritance pattern
BP3	In frame-deletions/insertions in a repetitive region without a known function.	Supporting In-frame deletions/insertions in a repetitive region without a known function
BP4	Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.	Supporting No gene-specific predictors; agree to utilize REVEL, with thresholds of >0.75 and <0.15 for PP3 and BP4, respectively
BP5	Variant found in a case with an alternate molecular basis for disease.	Supporting Variant found in a case with an alternate molecular basis for disease
BP6	Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.	Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BP7	A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.	Supporting A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved

Rules for PDHA1

Gene: PDHA1 (HGNC:8806) HGNC Name: pyruvate dehydrogenase E1 subunit alpha 1 Preferred Transcript: NM_000284.4 Disease: Pyruvate dehydrogenase deficiency (MONDO:0019169)

Criteria	Original ACMG Summary	Strength Specifications
PVS1	Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Caveats: • Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7). • Use caution interpreting LOF variants at the extreme 3’ end of a gene. • Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact. • Use caution in the presence of multiple transcripts.	Very Strong Applied per PVS1 flowsheet of Abou Toyoun et al.
PS1	Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.	Strong Same amino acid change as a previously established pathogenic variant regardless of nucleotide change
PS2	De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity.	Strong De novo in a patient with the disease and no family history
PS3	Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.	Not Applicable
PS4	The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance. Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence.	Not Applicable
PM1	Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.	Moderate Located in one of the following functional domains: • thiamine pyrophosphate (TPP) binding site (aa positions 118Y, 119R, 165G, 167V, 195G, 196D, 197G, 198A, 225N, 227Y, 292H). • α β heterodimer interface (aa positions 160F, 162G, 164N, 169A, 172P, 173L, 176G, 177I, 179L, 180A,183Y, 202G,203Q, 209N, 210M, 213L). • α2 β2 heterotetramer interface (aa positions 88R, 140G, 165G, 166I, 197G, 199A, 200N, 201Q, 202G, 205F, 209N, 213L, 228G, 229M, 230G, 231T, 245R, 296D, 300S). • Phosphorylation loop region (aa positions 287Y, 288R, 289Y, 290H, 291G, 292H, 293S, 295S, 296D, 297P, 298G, 299V, 300S, 301Y, 302R, 303T, 304R, 305E, 314S, 315D, 316P).
PM2	Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. Caveat: Population data for indels may be poorly called by next generation sequencing.	Moderate 0.0000092 (<0.00092%)
PM3	For recessive disorders, detected in trans with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase.	Not Applicable
PM4	Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.	Moderate Protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants
PM5	Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.	Moderate Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before
PM6	Assumed de novo, but without confirmation of paternity and maternity.	Strong De novo in a patient with the disease and no family history Moderate Assumed de novo, but without confirmation of paternity and maternity
PP1	Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. Note: May be used as stronger evidence with increasing segregation data.	Supporting Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease
PP2	Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.	Not Applicable
PP3	Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.	Supporting No gene-specific predictors; agree to utilize REVEL, with thresholds of >0.75 and <0.15 for PP3 and BP4, respectively
PP4	Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.	Supporting One of the following criteria are met: (1) Pyruvate radioactive enzyme assay showing decreased (as defined as <3rd percentile of controls) for PDC, activated and decreased ratios (PDC/E3 and/or PDC/CS) in fibroblasts, muscle, and/or lymphocytes; (2) other assays showing decrease in PDC activity (ie: western blot, immunocapture, and activity; commercial kits for research); (3) abnormally high pyruvate and/or pyruvate/lactate ratio
PP5	Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.	Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BA1	Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.	Stand Alone >0.00092 (>0.092%)
BS1	Allele frequency is greater than expected for disorder.	Strong >0.000092 (>0.0092%)
BS2	Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.	Strong Observed in at least two healthy male adults. Note: Individual’s phenotype is well-characterized (not just seen in database of presumed healthy individuals) AND/OR ≥16 hemizygotes in gnomAD Supporting Observed in 4-15 hemizygotes in gnomAD AND/OR Pyruvate radioactive enzyme assay showing normal (defined as >3rd percentile of controls) for PDC, activated and normal ratios (PDC/E3 and/or PDC/CS) in fibroblasts with no evidence of skewed X-inactivation in fibroblasts.
BS3	Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.	Not Applicable
BS4	Lack of segregation in affected members of a family. Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation.	Strong Lack of segregation in affected and/or treated members of a family.
BP1	Missense variant in a gene for which primarily truncating variants are known to cause disease.	Not Applicable
BP2	Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.	Not Applicable
BP3	In frame-deletions/insertions in a repetitive region without a known function.	Supporting In-frame deletions/insertions in a repetitive region without a known function
BP4	Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.	Supporting No gene-specific predictors; agree to utilize REVEL, with thresholds of >0.75 and <0.15 for PP3 and BP4, respectively
BP5	Variant found in a case with an alternate molecular basis for disease.	Supporting Variant found in a case with an alternate molecular basis for disease
BP6	Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.	Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BP7	A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.	Supporting A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved

Rules for POLG

Gene: POLG (HGNC:9179) HGNC Name: DNA polymerase gamma, catalytic subunit Preferred Transcript: NM_002693.2 Disease: Mitochondrial disease (MONDO:0044970)

Criteria	Original ACMG Summary	Strength Specifications
PVS1	Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Caveats: • Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7). • Use caution interpreting LOF variants at the extreme 3’ end of a gene. • Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact. • Use caution in the presence of multiple transcripts.	Very Strong Applied per PVS1 flowsheet of Abou Toyoun et al.
PS1	Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.	Strong Same amino acid change as a previously established pathogenic variant regardless of nucleotide change
PS2	De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity.	Strong De novo in a patient with the disease and no family history
PS3	Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.	Not Applicable
PS4	The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance. Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence.	Strong Rarely, pathogenic variants cause disease in an AD manner. For these variants only, presence in: 2 unrelated probands will be considered supporting evidence, 4 unrelated probands will be considered moderate evidence, 16 unrelated probands will be strong evidence. • Note: This will only be utilized when there is segregation evidence supporting autosomal dominant inheritance
PM1	Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.	Not Applicable
PM2	Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. Caveat: Population data for indels may be poorly called by next generation sequencing.	Moderate <0.0005 (<0.05% )
PM3	For recessive disorders, detected in trans with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase.	Moderate Use per SVI guidance. Note: T251I and P587L are almost always in cis
PM4	Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.	Moderate Protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants
PM5	Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.	Moderate Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before
PM6	Assumed de novo, but without confirmation of paternity and maternity.	Strong De novo in a patient with the disease and no family history Moderate Assumed de novo, but without confirmation of paternity and maternity
PP1	Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. Note: May be used as stronger evidence with increasing segregation data.	Supporting Further define “affected” as an individual in whom there is objective evidence of manifestations consistent with POLG-related disorders spectrum: Alpers-Huttenlocher syndrome (AHS), childhood myocerebrohepatopathy spectrum (MCHS), myoclonic epilepsy myopathy sensory ataxia (MEMSA), ataxia neuropathy spectrum (ANS), and/or progressive external ophthalmoplegia (PEO)
PP2	Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.	Not Applicable
PP3	Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.	Supporting Agree to utilize REVEL, with thresholds of >0.75 and <0.15 for PP3 and BP4, respectively • Will also utilize POLG pathogenicity prediction server if/when live again (PMID: 28480171); both tools (REVEL and server) will have to be in agreement to score
PP4	Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.	Moderate 1. Mitochondrial DNA depletion showing ≤ 20% of controls, OR 2. Multiple mitochondrial DNA deletions (NOTE:depletion and/or deletion analysis must be performed in muscle and/or liver; other tissues such as blood, fibroblast, and buccal are not acceptable; Must be performed in child, as defined as <18 years old) Note: For both scenarios 1 and 2, will only apply if other mtDNA maintenance disorders have been excluded (exome sequencing or comprehensive panel-based testing) Supporting 1. Mitochondrial DNA depletion showing 20-50% of controls in children (< 18 years of age), AND/OR 2. COX negative fibers in muscle in children and/or adults Note: Will only apply if other mtDNA maintenance disorders have been excluded (exome sequencing or comprehensive panel-based testing)
PP5	Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.	Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BA1	Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.	Stand Alone >0.01 (>1%)
BS1	Allele frequency is greater than expected for disorder.	Strong >0.005 (>0.5% - AR)
BS2	Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.	Strong Observed in a healthy adult individual in the homozygous state AND/OR Normal mtDNA content (1. Must be performed in muscle and/or liver; blood, fibroblast, and buccal not acceptable; 2. Must be performed in children only - defined as <18 years old; 3. A normal level is defined as >50%.) Supporting Lack of COX negative fibers in muscle (children and adults)
BS3	Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.	Not Applicable
BS4	Lack of segregation in affected members of a family. Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation.	Strong Lack of segregation in affected and/or treated members of a family.
BP1	Missense variant in a gene for which primarily truncating variants are known to cause disease.	Not Applicable
BP2	Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.	Supporting Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern
BP3	In frame-deletions/insertions in a repetitive region without a known function.	Supporting In-frame deletions/insertions in a repetitive region without a known function
BP4	Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.	Supporting Agree to utilize REVEL, with thresholds of >0.75 and <0.15 for PP3 and BP4, respectively. Will also utilize POLG pathogenicity prediction server if/when live again (PMID: 28480171); both tools (REVEL and server) will have to be in agreement to score
BP5	Variant found in a case with an alternate molecular basis for disease.	Supporting Variant found in a case with an alternate molecular basis for disease
BP6	Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.	Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BP7	A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.	Supporting A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved

Rules for ETHE1

Gene: ETHE1 (HGNC:23287) HGNC Name: ETHE1 persulfide dioxygenase Preferred Transcript: NM_014297.5 Disease: Ethylmalonic encephalopathy (MONDO:0011229)

Criteria	Original ACMG Summary	Strength Specifications
PVS1	Null variant (nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease. Caveats: • Beware of genes where LOF is not a known disease mechanism (e.g. GFAP, MYH7). • Use caution interpreting LOF variants at the extreme 3’ end of a gene. • Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact. • Use caution in the presence of multiple transcripts.	Very Strong Applied per PVS1 flowsheet of Abou Toyoun et al.
PS1	Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. Example: Val->Leu caused by either G>C or G>T in the same codon. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.	Strong Same amino acid change as a previously established pathogenic variant regardless of nucleotide change
PS2	De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity.	Strong De novo in a patient with the disease and no family history
PS3	Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.	Supporting Reduced ETHE1 persulfide dioxygenase
PS4	The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0. See manuscript for detailed guidance. Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence.	Not Applicable
PM1	Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.	Not Applicable
PM2	Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium. Caveat: Population data for indels may be poorly called by next generation sequencing.	Moderate <0.00002 (<0.0020%)
PM3	For recessive disorders, detected in trans with a pathogenic variant Note: This requires testing of parents (or offspring) to determine phase.	Moderate Use per SVI guidance
PM4	Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.	Moderate Protein length changes as a result of in-frame deletions/insertions in a nonrepeat region or stop-loss variants
PM5	Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. Example: Arg156His is pathogenic; now you observe Arg156Cys. Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level.	Moderate Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before
PM6	Assumed de novo, but without confirmation of paternity and maternity.	Strong De novo in a patient with the disease and no family history Moderate Assumed de novo, but without confirmation of paternity and maternity
PP1	Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease. Note: May be used as stronger evidence with increasing segregation data.	Supporting Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease
PP2	Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.	Not Applicable
PP3	Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion. PP3 can be used only once in any evaluation of a variant.	Supporting No gene-specific predictors; agree to utilize REVEL, with thresholds of >0.75 and <0.15 f or PP3 and BP4, respectively
PP4	Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.	Moderate Individual has abnormally high urinary ethylmalonic acid AND one of the following: (1) All of the following symptoms present: -Acrocyanosis -Petechiae -Chronic diarrhea -Developmental delay (2) ≥3 or more of the following biochemical studies: -Abnormally high blood C4-Acylcarnitine esters -Abnormally high blood C5-acylcarnitine -Abnormally high plasma thiosulphate -Abnormally low cytochrome oxidase activity in skeletal muscle (without evidence of other complexes decreased) Supporting Individual has abnoramlly high urinary ethylmalonic acid AND one of the following: (1) 3 of the following features present: -Acrocyanosis -Petechiae -Chronic diarrhea -Developmental delay (2) abnormal laboratory studies in 2 of the following biochemical studies: -Abnormally high blood C4-Acylcarnitine esters -Abnormally high blood C5-acylcarnitine -Abnormally high plasma thiosulphate -Abnormally low cytochrome oxidase activity in skeletal muscle, without evidence of other complexes decreased
PP5	Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.	Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BA1	Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.	Stand Alone >0.001 (>0.1%)
BS1	Allele frequency is greater than expected for disorder.	Strong >0.0002 (>0.020%)
BS2	Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.	Strong Observed in a healthy adult individual in the homozygous state Supporting Normal laboratory values (specific labs outlined in PP4)
BS3	Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.	Not Applicable
BS4	Lack of segregation in affected members of a family. Caveat: The presence of phenocopies for common phenotypes (i.e. cancer, epilepsy) can mimic lack of segregation among affected individuals. Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation.	Strong Lack of segregation in affected and/or treated members of a family.
BP1	Missense variant in a gene for which primarily truncating variants are known to cause disease.	Not Applicable
BP2	Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.	Supporting Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern
BP3	In frame-deletions/insertions in a repetitive region without a known function.	Supporting In-frame deletions/insertions in a repetitive region without a known function
BP4	Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion. BP4 can be used only once in any evaluation of a variant.	Supporting No gene-specific predictors; agree to utilize REVEL, with thresholds of >0.75 and <0.15 f or PP3 and BP4, respectively
BP5	Variant found in a case with an alternate molecular basis for disease.	Supporting Variant found in a case with an alternate molecular basis for disease
BP6	Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.	Not Applicable This criterion is not for use as recommended by the ClinGen Sequence Variant Interpretation VCEP Review Committee. PubMed : 29543229
BP7	A synonymous variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved.	Supporting A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved