owencqueen/cgbench_data huggingface dataset:
Gene: ACADVL (HGNC:92)
Evidence Code: PVS1 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: PVS1 — Very Strong
Modification Type: Disease-specific
Loss of function is a known mechanism for VLCAD Deficiency. The specifications below are based on published guidance for assigning strength of evidence for PVS1 (Abou Tayoun et al., 2018; PMID: 30192042). There are multiple transcripts for ACADVL. The major isoform, NM_000018.4, encodes a 655 amino acid precursor protein that contains a 40 amino acid N-terminal target sequence that is removed during uptake (Aoyama et al., 1995; PMID: 7668252). In a joint project between NCBI and EMBL-EBI (MANE), NM_000018.4 was designated as the most relevant transcript.
Nonsense or Frameshift:




Use caution when interpreting LOF variants at the 3’ end of the gene.


All nonsense and frameshift variants will meet PVS1 unless the variant is predicted to be missed by nonsense-mediated decay (NMD).


NMD is not predicted if the variant is in the last exon (exon 20) or in the last 50 nucleotides of the penultimate exon (exon 19).
Canonical Splice Site (+1, +2, -1, -2):


All donor/acceptor sites follow the GT/AG rule, except for the donor splice site of intron 8, which begins with GC. PVS1 should not be applied for variants in the splice donor site of intron 8 since the impact of GC donor splice sites is not well understood.


For +1 or +2 GT donor splice site variants, the exon immediately 5’ of the variant is predicted to be skipped. For -1 or -2 AG acceptor splice site variants, the exon immediately 3’ of the variants is predicted to be skipped. For the predicted in frame/out of frame consequences for exon skipping in ACADVL (NM_000018.4), see Appendix 1.
Deletions:


For single or multi-exon deletions that result in an out-of-frame consequence, use PVS1 unless NMD is not predicted to occur. If NMD is not predicted to occur, use PVS1_Moderate.


If a deletion results in an in-frame consequence, the deletion must encompass one or more exons in order to apply PVS1. Consult Appendix 1 and the PVS1 decision tree to assign a strength.
Duplications:


Single and multi-exon duplications have not been reported in ACADVL. Consult the PVS1 decision tree to assign the strength.
Initiation codon:


The next in-frame methionine is at position 6 (on transcript NM_000018). However, the first 40 amino acids comprise the leader sequence in the precursor peptide and are important for proper localization of the protein (Aoyama et al., 1995; PMID: 7668252). Therefore, initiator codon variants will meet PVS1_Strong.
Gene: ACADVL (HGNC:92)
Evidence Code: PS1 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: PS1 — Strong
Modification Type: No change
Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.
Gene: ACADVL (HGNC:92)
Evidence Code: PS2 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: PS3 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: PS3 — Strong
Modification Type: Disease-specific
Functional evidence from non-patient derived material with only a single variant best reflects the variant-level phenotype. Apply patient-derived evidence in PP4.


Apply criteria at the level determined by validation parameters (see flowchart below). VLCAD assays available now do not meet the criteria that is being proposed now regarding the types of controls etc. but the published VLCAD assays are well established with many positive and negative controls being run.


Hesse et al., 2018 (PMID 30194637) reviewed enzymatic testing in lymphocytes as a confirmatory tool in newborns identified by screening. Molecular testing was performed after in most patients.


If an enzyme activity assay has >20% activity it cannot be weighted above PS3_supporting regardless of flowchart results.
Gene: ACADVL (HGNC:92)
Evidence Code: PS4 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: PM1 — Original ACMG Summary
Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.
Gene: ACADVL (HGNC:92)
Evidence Code: PM1 — Moderate
Modification Type: Disease-specific
Examples of mutational hot spots:
It has been cited that the CpG dinucleotides in the codon for arginine326 and arginine429 are mutational hot spots, since CrT (R326C and R429W) and GrA (R326H and R429Q) mutations of the CpG di- nucleotide are present in both codons. Article for reference is Clear Correlation of Genotype with Disease Phenotype in Very–Long-Chain Acyl-CoA Dehydrogenase Deficiency – by Andresen et al., 1999 (PMID 9973285).


Please refer to “Structural Basis for Substrate Fatty Acyl Chain Specificity: Crystal Structure of Human Very-Long-Chain acyl-CoA Dehydrogenase” by McAndrew et al., 2008 (PMID 18227065) (note – numbering in this reference is for the mature peptide without the mitochondrial signal peptide, so amino acid position numbers are lower by 40 than numbering based on NM-000018.4) and “Compared effects of missense mutations in Very-Long-Chain Acyl-CoA Dehydrogenase deficiency: Combined analysis by structural, functional and pharmacological approaches” by Gobin-Limballe et al., 2010 (PMID 20060901) for identifying important structural regions for proper enzyme function such as binding sites and active sites of the enzyme. Based on this and information from UniProt the following regions are designated to be important functional domains:


p.214-223, p.249-251, p.460-466, p.562: Nucleotide and substrate binding.


p.481-516: Membrane binding.


p.1-40: Mitochondrial signal peptide.


Curators may seek approval from the expert panel for identifying new hot spots or critical regions as discovered in literature searches, in uniport, HGMD, etc. for inclusion.
Gene: ACADVL (HGNC:92)
Evidence Code: PM2 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: PM2 — Supporting
Modification Type: Disease-specific
Variants with a highest population minor allele frequency (MAF) <0.001 (0.1%) in any continental population with >2000 alleles in gnomAD will meet PM2_supporting.


The 0.001 cutoff is based on a disease frequency of 1:100,000, genetic heterogeneity of 100%, penetrance of 75%, and maximum allelic contribution of 20%, based on the most common pathogenic ACADVL variant, c.848T>C (p.Val283Ala): gnomAD MAF: 0.002238 (289/129106; 1 homozygote; European Non-Finnish) and overall frequency: 0.001224 (346/282744; 2 homozygotes). This variant would not meet PM2_supporting, but it is assumed that the most common variants have already been identified so a conservative cutoff was chosen.


See Appendix 2 for calculations.


It is acceptable for an ACADVL variant to be present in controls because VLCAD deficiency is a recessive condition. It is also possible for homozygous ACADVL variants to be present in population databases due to later onset of the condition. If homozygous variants are present, the number should be noted and discussed with an expert.


SVI guidance: use PM2 as Supporting (not Moderate) based on absence or rarity not being in the odds range expected for a moderate piece of evidence. This EP will track the impact of not using Moderate for PM2.
Gene: ACADVL (HGNC:92)
Evidence Code: PM3 — Original ACMG Summary
For recessive disorders, detected in trans with a pathogenic variant
Note: This requires testing of parents (or offspring) to determine phase.
Gene: ACADVL (HGNC:92)
Evidence Code: PM3 — Moderate
Modification Type: Disease-specific
Details of the cDNA change must be used to describe any variants used as evidence for PM3. If the variant is described only as an amino acid change, this is not sufficient. Probands must also meet PP4 criteria to be counted.


If more than one case has the same genotype and the variants are not confirmed in trans, then only one case should be used for assigning points to avoid overcounting evidence if the variants are actually in cis and hence inherited together in multiple individuals or potentially counting the same case twice. If the variants are confirmed to be in trans, more than one individual with the same genotype can be counted as long as the reports do not represent the same case.


Following SVI guidance for PM3, use the scoring system below to determine the strength of evidence for PM3.


These variant interpretation guidelines should be used to determine the classification of the “other variant” in order to determine the appropriate number of points to assign.


For a variant to be “confirmed in trans” in a compound heterozygous patient, parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed. Otherwise, the phase of the variants is unknown. Parental testing is not required for homozygous cases.
Gene: ACADVL (HGNC:92)
Evidence Code: PM4 — Original ACMG Summary
Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.
Gene: ACADVL (HGNC:92)
Evidence Code: PM4 — Moderate
Modification Type: No change
Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.
Gene: ACADVL (HGNC:92)
Evidence Code: PM5 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: PM5 — Moderate
Modification Type: Disease-specific
These variant interpretation guidelines should be used to determine the classification of the other missense change in order to determine whether this rule can be applied.


Use PM5_Supporting if the other variant is Likely Pathogenic.
Gene: ACADVL (HGNC:92)
Evidence Code: PM6 — Original ACMG Summary
Assumed de novo, but without confirmation of paternity and maternity.
Gene: ACADVL (HGNC:92)
Evidence Code: PM6 — Moderate
Modification Type: No change
Assumed de novo, but without confirmation of paternity and maternity.
Gene: ACADVL (HGNC:92)
Evidence Code: PP1 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: PP1 — Supporting
Modification Type: Disease-specific
Following SVI guidance, use the scoring system below to determine the strength of evidence for PP1.


For segregation counting, do not count probands as a segregation.
o Affected segregations = # affected individuals in the family with the variants - 1.


Affected segregations are defined as affected family members (typically siblings) who harbor the variant in question and a second variant on the remaining allele.


Unaffected segregations are defined as unaffected family members, typically siblings, who are at risk to inherit the two variants identified in the proband. These individuals should be either wild-type for both variants identified in the proband, or a heterozygous carrier for a single variant.


Unaffected, carrier parents DO NOT count as unaffected segregations.


There may be scenarios where individuals other than siblings could be counted as segregations, such as in families where one parent is affected with the autosomal recessive disorder, in large families with multiple branches, or in consanguineous families.
Gene: ACADVL (HGNC:92)
Evidence Code: PP2 — Original ACMG Summary
Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.
Gene: ACADVL (HGNC:92)
Evidence Code: PP3 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: PP3 — Supporting
Modification Type: Disease-specific
Missense changes with a REVEL score >0.75 will meet PP3.


For in-frame deletions and insertions, use PROVEAN and Mutation Taster. Results must be consistent to count.


For non-canonical splice site variants, use Splice AI, MaxEntScn and NNSplice. Based on data from Jaganathan et al., 2019 (PMID: 30661751), Houdayer et al., 2012 (PMID: 22505045) and Tang et al., 2016 (PMID: 27313609), PP3 can be applied if there is, a SpliceAI “high score” (Δ Score ≥ 0.5 “confidently predicted splice variants”) (exclude any results with Δ Score ≤ 0.2 from consideration of pathogenicity, <0.2 are not “predicted to alter splicing”), >15% reduction using MaxEntScn and >5% reduction using NNSplice. Two out of the three predictors must be consistent to count.


For SpliceAI’s cryptic splice-site rules, the creation of a new splice-site with Δ Score ≥ 0.5 may be enough to produce a large proportion of aberrant transcripts.


If a new splice site is predicted to be generated, this rule can be applied if the newly generated splice site is significantly stronger than the wild type site (Δ Score ≥ 0.5 using SpliceAI; >15% difference using MaxEntScn).


Do not apply this rule for canonical splice site changes meeting PVS1.
Gene: ACADVL (HGNC:92)
Evidence Code: PP4 — Original ACMG Summary
Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.
Gene: ACADVL (HGNC:92)
Evidence Code: PP4 — Moderate
Modification Type: Disease-specific
Abnormal tests that are consistent with VLCAD deficiency include deficient VLCAD enzyme activity in patient cells (leukocytes, fibroblasts, liver, heart, or skeletal muscle, or amniocytes), abnormal C14:1 acylcarnitine values from newborn screening (NBS), and abnormal acylcarnitine values from follow-up plasma analysis.


To award a given tier of evidence, at least one proband harboring the variant must meet at least one of the minimum requirements below. If multiple analyses are completed, only one result needs to be within the allowed ranges to meet this rule as values can fluctuate due to age or diet: 


PP4_moderate (Must meet at least one of the below criteria):


VLCAD enzyme activity (β-Oxidation Flux) ≤20% of normal.


NBS C14:1 Levels ≥ 1.0 μM AND:


VLCAD enzyme activity (β-Oxidation Flux) 21-27% of normal OR Assertion of reduced VLCAD activity without specific levels OR Follow-Up Plasma Acylcarnitine analysis “consistent with VLCADD” without specific levels.
Gene: ACADVL (HGNC:92)
Evidence Code: PP4 — Supporting
Modification Type: Disease-specific
Abnormal tests that are consistent with VLCAD deficiency include deficient VLCAD enzyme activity in patient cells (leukocytes, fibroblasts, liver, heart, or skeletal muscle, or amniocytes), abnormal C14:1 acylcarnitine values from newborn screening (NBS), and abnormal acylcarnitine values from follow-up plasma analysis.


To award a given tier of evidence, at least one proband harboring the variant must meet at least one of the minimum requirements below. If multiple analyses are completed, only one result needs to be within the allowed ranges to meet this rule as values can fluctuate due to age or diet: 


PP4_supporting (Must meet at least one of the below criteria):


VLCAD enzyme activity (β-Oxidation Flux) 21-27% of normal.


Assertion of reduced VLCAD activity without specific levels.


NBS C14:1 Levels from >0.8 μM.


Assertion of abnormal NBS “consistent with VLCADD” without specific levels AND: Follow-Up Plasma Acylcarnitine analysis “consistent with VLCADD” without specific levels.
Gene: ACADVL (HGNC:92)
Evidence Code: PP5 — Original ACMG Summary
Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.
Gene: ACADVL (HGNC:92)
Evidence Code: BA1 — Original ACMG Summary
Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.
Gene: ACADVL (HGNC:92)
Evidence Code: BA1 — Stand Alone
Modification Type: Disease-specific
Variants with a highest population minor allele frequency (MAF) ≥0.007 (0.7%) in any continental population with >2000 alleles in gnomAD will meet BA1.


See Appendix 2 for calculations.
Gene: ACADVL (HGNC:92)
Evidence Code: BS1 — Original ACMG Summary
Allele frequency is greater than expected for disorder.
Gene: ACADVL (HGNC:92)
Evidence Code: BS1 — Strong
Modification Type: Disease-specific
Variants with a highest population minor allele frequency (MAF) ≥0.0035 (0.35%) in any continental population with >2000 alleles in gnomAD will meet BS1.


See Appendix 2 for calculations.
Gene: ACADVL (HGNC:92)
Evidence Code: BS2 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: BS3 — Original ACMG Summary
Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.
Gene: ACADVL (HGNC:92)
Evidence Code: BS3 — Strong
Modification Type: Disease-specific
In vitro expression:






50% enzyme activity
Splicing assays:






For non-canonical splicing variants, use BS3 if there is evidence demonstrating normal splicing with no evidence of abnormal splicing (RT-PCR and/or RNA sequencing).


These studies can be performed using patient-derived cells or heterologous cultured cells.


Any variants meeting the requirement for in vitro expression or splicing assays can meet BS3, but if the variant meets the description for both, BS3 should only be counted once.
Gene: ACADVL (HGNC:92)
Evidence Code: BS4 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: BS4 — Strong
Modification Type: No change
Lack of segregation in affected members of a family.
Gene: ACADVL (HGNC:92)
Evidence Code: BP1 — Original ACMG Summary
Missense variant in a gene for which primarily truncating variants are known to cause disease.
Gene: ACADVL (HGNC:92)
Evidence Code: BP2 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: BP2 — Supporting
Modification Type: No change
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.
Gene: ACADVL (HGNC:92)
Evidence Code: BP3 — Original ACMG Summary
In frame-deletions/insertions in a repetitive region without a known function.
Gene: ACADVL (HGNC:92)
Evidence Code: BP4 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: BP4 — Supporting
Modification Type: Disease-specific
Missense changes with a REVEL score <0.5 will meet BP4.


For in-frame deletions and insertions, use PROVEAN and Mutation Taster. Results must be consistent to count.
For non-canonical splice site variants, use Splice AI, MaxEntScn and NNSplice. Based on data from Jaganathan et al., 2019 (PMID: 30661751), Houdayer et al., 2012 (PMID: 22505045) and Tang et al., 2016 (PMID: 27313609), PP3 can be applied if there is a with Δ Score ≤ 0.2 , <10% reduction using MaxEntScn and <2% reduction using NNSplice. Two out of the three predictors must be consistent to count.


Do not apply this rule if there is evidence for creation of a cryptic splice site.


Can be used with BP7 code.
Gene: ACADVL (HGNC:92)
Evidence Code: BP5 — Original ACMG Summary
Variant found in a case with an alternate molecular basis for disease.
Gene: ACADVL (HGNC:92)
Evidence Code: BP6 — Original ACMG Summary
Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.
Gene: ACADVL (HGNC:92)
Evidence Code: BP7 — Original ACMG Summary
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.
Gene: ACADVL (HGNC:92)
Evidence Code: BP7 — Supporting
Modification Type: No change
Can be used with BP4 code.