owencqueen/cgbench_data huggingface dataset:
Gene: GAMT (HGNC:4136)
Evidence Code: PVS1 — Very Strong
Modification Type: None
Nonsense-mediated decay predicted


Nonsense and frameshift variants




All nonsense and frameshift variants will meet PVS1 unless a premature termination codon is predicted to be missed by nonsense-mediated decay (NMD) because it is located in the last exon (exon 6) or the last 50 bases of the penultimate exon of the gene (exon 5, c.520). In that case, PVS1_Strong or PVS1_Moderate will be applied, depending on whether >10% or <10% of the protein is lost.




Splice site variants (+1, +2, -1, -2)




All canonical splice site pairs in GAMT are GT-AG.


For any canonical splice site variant (+1, +2, -1, -2), the exon immediately adjacent to the variant is predicted to be skipped i.e. upstream exon skipped for canonical donor splice site variants and downstream exon skipped for canonical acceptor splice site variants.


For the predicted in frame/out of frame consequences of exon skipping and assigned strength of PVS1, see Appendix 1.


If this criterion is applied, PP3 (in silico splice site prediction tools) should not be used.


Use SpliceAI to look for nearby (+/- 20 nucleotides) strong consensus splice sequence that may reconstitute in-frame splicing. Otherwise, the PVS1 strength should be reduced accordingly.


Non-canonical splice variants, such as +3 or -3, will not meet PVS1, but could meet PS3 and/or PP3 criteria.




Deletions (single or multi exon)




If a single or multi-exon deletion results in an out of frame consequence, use PVS1 unless not predicted to undergo NMD. 


If not predicted to undergo NMD, use PVS1_Strong if >10% of the protein is predicted to be removed, and use PVS1_Moderate if <10% of the protein is predicted to be removed.


If the consequence is in frame, the deletion must encompass one or more exons for PVS1 to apply. Use PVS1_Strong if more than 10% of the protein is removed and PVS1_Moderate if <10% of the protein is removed.


If the in frame deletion is smaller than one exon, PVS1 does not apply; consider using PM4.
Appendix 1 can be used to predict the consequences of single exon deletions.




Duplications




Use the PVS1 decision tree (Figure 1, Abou Tayoun et al, 2018, PMID 30192042) to assess the impact of duplications.
Gene: GAMT (HGNC:4136)
Evidence Code: PVS1 — Strong
Modification Type: Strength
Single exon or larger deletion resulting in loss of >10% of the protein.
 


Nonsense and frameshift variants




All nonsense and frameshift variants will meet PVS1 unless a premature termination codon is predicted to be missed by nonsense-mediated decay (NMD) because it is located in the last exon (exon 6) or the last 50 bases of the penultimate exon of the gene (exon 5, c.520). In that case, PVS1_Strong will be applied if >10% of the protein is lost.




Splice site variants (+1, +2, -1, -2)




All canonical splice site pairs in GAMT are GT-AG.


For any canonical splice site variant (+1, +2, -1, -2), the exon immediately adjacent to the variant is predicted to be skipped i.e. upstream exon skipped for canonical donor splice site variants and downstream exon skipped for canonical acceptor splice site variants.


Use SpliceAI to look for nearby (+/- 20 nucleotides) strong consensus splice sequence that may reconstitute in-frame splicing. 


For considerations for strength at which PVS1 may be applied see Appendix 1.


If this criterion is applied, PP3 (in silico splice site prediction tools) should not be used.


Non-canonical splice variants, such as +3 or -3, will not meet PVS1, but could meet PS3 and/or PP3 criteria.




Deletions (single or multi exon)




If a single or multi-exon deletion results in an out of frame consequence, use PVS1 unless not predicted to undergo NMD.


 If not predicted to undergo NMD, use PVS1_Strong if >10% of the protein is predicted to be removed, and use PVS1_Moderate if <10% of the protein is predicted to be removed.


If the consequence is in frame, the deletion must encompass one or more exons for PVS1 to apply. Use PVS1_Strong if more than 10% of the protein is removed and PVS1_Moderate if <10% of the protein is removed.


If the in frame deletion is smaller than one exon, PVS1 does not apply; consider using PM4.
Appendix 1 can be used to predict the consequences of single exon deletions.




Duplications




Use the PVS1 decision tree (Figure 1, Abou Tayoun et al, 2018, PMID 30192042) to assess the impact of duplications.
Gene: GAMT (HGNC:4136)
Evidence Code: PVS1 — Moderate
Modification Type: Strength
Single exon or larger deletion resulting in loss of <10% of the protein.
   


Nonsense and frameshift variants




All nonsense and frameshift variants will meet PVS1 unless a premature termination codon is predicted to be missed by nonsense-mediated decay (NMD) because it is located in the last exon (exon 6) or the last 50 bases of the penultimate exon of the gene (exon 5, c.520). In that case, PVS1_Moderate will be applied if <10% of the protein is lost.




Splice site variants (+1, +2, -1, -2)




All canonical splice site pairs in GAMT are GT-AG.


Use SpliceAI to look for nearby (+/- 20 nucleotides) strong consensus splice sequence that may reconstitute in-frame splicing. 


For any canonical splice site variant (+1, +2, -1, -2), if RT-PCR data predicts in-frame loss of <10% of the protein, apply PVS1_Moderate.


If this criterion is applied, PP3 (in silico splice site prediction tools) should not be used.


Non-canonical splice variants, such as +3 or -3, will not meet PVS1, but could meet PS3 and/or PP3 criteria.




Initiator codon variants




All initiator codon variants will meet PVS1_Moderate. The next in-frame methionine is at amino acid position 42 (based on NP_000147.1).




Deletions (single or multi exon)




If a single or multi-exon deletion results in an out of frame consequence, use PVS1 unless not predicted to undergo NMD. 


If not predicted to undergo NMD, use PVS1_Strong if >10% of the protein is predicted to be removed, and use PVS1_Moderate if <10% of the protein is predicted to be removed.


If the consequence is in frame, the deletion must encompass one or more exons for PVS1 to apply. Use PVS1_Strong if more than 10% of the protein is removed and PVS1_Moderate if <10% of the protein is removed.


If the in frame deletion is smaller than one exon, PVS1 does not apply; consider using PM4.
Appendix 1 can be used to predict the consequences of single exon deletions.




Duplications




Use the PVS1 decision tree (Figure 1, Abou Tayoun et al, 2018, PMID 30192042) to assess the impact of duplications.
Gene: GAMT (HGNC:4136)
Evidence Code: PS1 — Strong
Modification Type: General recommendation
This criterion is applicable for any variant resulting in the same amino acid change as a variant that has been previously established as pathogenic by the CCDS VCEP, by assessment using these criteria, regardless of nucleotide change.


If the variant is in the last 3 nucleotides of an exon, further analysis using splicing site prediction algorithms (see PP3) and data from the literature (if available) is required to investigate the impact on splicing.


PS1 may also be applied for splicing variants under specific circumstances (see Table 3 in PMID: 37352859).
Gene: GAMT (HGNC:4136)
Evidence Code: PS1 — Moderate
Modification Type: General recommendation,Strength
This criterion is applicable for any variant resulting in the same amino acid change as a variant that has been previously established as likely pathogenic by the CCDS VCEP, by assessment using these criteria, regardless of nucleotide change.


If the variant is in the last 3 nucleotides of an exon, further analysis using splicing site prediction algorithms (see PP3) and data from the literature (if available) is required to investigate the impact on splicing.


PS1_Moderate may also be applied for splicing variants under specific circumstances (see Table 3 in PMID: 37352859).
Gene: GAMT (HGNC:4136)
Evidence Code: PS1 — Supporting
Modification Type: General recommendation,Strength
PS1_Supporting may be applied for splicing variants under specific circumstances (see Table 3 in PMID: 37352859).
Gene: GAMT (HGNC:4136)
Evidence Code: PS3 — Supporting
Modification Type: Disease-specific,Strength
PS3_Supporting can be assigned if a variant is expressed in GAMT-deficient fibroblasts, HeLa cells and has <15% of the control value, for values published in Mercimek-Mahmutoglu et al, 2014, PMID 24415674; Mercimek-Mahmutoglu et al, 2016, PMID 26319512; or DesRoches et al, 2016, PMID 26003046.
Gene: GAMT (HGNC:4136)
Evidence Code: PM2 — Supporting
Modification Type: Disease-specific,Strength
Allele frequency <0.0004 (<0.04%) in all populations in gnomAD.


It is acceptable for a GAMT variant to be present in controls, if heterozygous, because GAMT-D is a recessive disorder. Homozygotes should not be seen in a population database, such as gnomAD, because the penetrance of this condition in individuals with biallelic pathogenic variants is expected to be 100% and the condition is expected to present with severe symptoms early in life.


GAMT specifications:




All subpopulations in gnomAD v4.0 must have a maximum allele frequency less than 0.0004 (the highest population minor allele frequency of the most common pathogenic GAMT variant, c.327G>A, in gnomAD). Any variant with a frequency below this cutoff will meet PM2_Supporting.


If homozygotes are observed, or variant is confirmed in trans with a known pathogenic variant, the variant will meet BS2 (assuming 100% penetrance for an individual with 2 pathogenic variants in trans).
Gene: GAMT (HGNC:4136)
Evidence Code: PM4 — Moderate
Modification Type: None
GAMT specifications: Use this rule “as is” for stop loss variant, and in frame deletions and insertions of 2 or more amino acids, but downgrade to PM4_Supporting for single amino acid deletions and insertions.
Gene: GAMT (HGNC:4136)
Evidence Code: PM5 — Moderate
Modification Type: None
This criterion is applicable for any variant resulting in a different amino acid change, at the same amino acid position, as a variant that has been previously established as pathogenic by the CCDS VCEP, by assessment using these criteria, regardless of nucleotide change.


If the variant is in the last 3 nucleotides of an exon, further analysis using splicing site prediction algorithms (see PP3) and data from the literature (if available) is required to investigate the impact on splicing. 


If the variant is likely pathogenic, use PM5_Supporting.
Gene: GAMT (HGNC:4136)
Evidence Code: PM5 — Supporting
Modification Type: Strength
This criterion is applicable for any variant resulting in a different amino acid change, at the same amino acid position, as a variant that has been previously established as likely pathogenic by the CCDS VCEP, by assessment using these criteria, regardless of nucleotide change.


If the variant is in the last 3 nucleotides of an exon, further analysis using splicing site prediction algorithms (see PP3) and data from the literature (if available) is required to investigate the impact on splicing.  


If the variant is likely pathogenic, use PM5.
Gene: GAMT (HGNC:4136)
Evidence Code: PP3 — Strong
Modification Type: General recommendation,Strength
Missense variant with a REVEL score equal to or >0.932 (based on guidance from Pejaver et al, 2022, PMID: 36413997).
Gene: GAMT (HGNC:4136)
Evidence Code: PP3 — Moderate
Modification Type: General recommendation,Strength
Missense variant with a REVEL score 0.773-0.932 (based on guidance from Pejaver et al, 2022, PMID: 36413997).
Gene: GAMT (HGNC:4136)
Evidence Code: PP3 — Supporting
Modification Type: General recommendation,None
Missense variant with a REVEL score 0.644-0.773 (based on guidance from Pejaver et al, 2022, PMID: 36413997).


In frame deletion or insertion predicted deleterious by PROVEAN and MutationTaster. Results must be consistent to count.


For non-canonical splice site variants (e.g., +3, -3), predict the impact on splicing by using SpliceAI, 
https://spliceailookup.broadinstitute.org/
 , and apply PP3 for a score equal to or >0.2 (as indicated in PMID: 37352859, Table 1 and Figure 4). Assess the possibility of activation of cryptic splice sites.
Gene: GAMT (HGNC:4136)
Evidence Code: PP4 — Strong
Modification Type: Disease-specific
4 points based on any combination of the following. Two or more data types are required to apply PP4_Strong:




Elevated urine guanidinoacetate with or without low or low normal creatine (1 point).


Elevated plasma guanidinoacetate with or without low or low normal creatine (2 points).


Significantly decreased creatine peak in brain magnetic resonance spectroscopy with or without visible guanidinoacetate peak (3 points).


GAMT enzyme activity <5% of normal (3 points).


Variant must meet PM2_Supporting for PP4 to apply at any strength.


For PP4 to be applied at strong, full GAMT gene sequencing, including all coding exons and intron/exon boundaries, must have been carried out. If not, consider downgrading.
Gene: GAMT (HGNC:4136)
Evidence Code: PP4 — Moderate
Modification Type: Strength
3 points based on any combination of the following. Two or more data types are recommended to apply PP4_Moderate:




Elevated urine guanidinoacetate with or without low or low normal creatine (1 point).


Elevated plasma guanidinoacetate with or without low or low normal creatine (2 points).


Significantly decreased creatine peak in brain magnetic resonance spectroscopy with or without visible guanidinoacetate peak (3 points).


GAMT enzyme activity <5% of normal (3 points).


Variant must meet PM2_Supporting for PP4 to apply at any strength.
Gene: GAMT (HGNC:4136)
Evidence Code: PP4 — Supporting
Modification Type: Disease-specific
1-2 points based on: 




Elevated urine guanidinoacetate with or without low or low normal creatine (1 point).


Elevated plasma guanidinoacetate with or without low or low normal creatine (2 points).


Variant must meet PM2_Supporting for PP4 to apply at any strength.
Gene: GAMT (HGNC:4136)
Evidence Code: BA1 — Stand Alone
Modification Type: Disease-specific
Allele frequency >0.003 (0.3%) in gnomAD v4.0 in any continental population with >2000 alleles (based on the estimated prevalence 1 in 114,000, PMID 24071436) in gnomAD v4.0 (max allelic contribution = 100%; max genetic contribution = 100%).


Use the highest population minor allele frequency (MAF) in any given continental population with >2,000 alleles (European non-Finnish, African, East Asian, South Asian, Latino) (PMID 30311383).
Gene: GAMT (HGNC:4136)
Evidence Code: BS1 — Strong
Modification Type: Disease-specific
Allele frequency >0.001 (0.1%) in gnomAD v4.0 in any continental population with >2000 alleles (based on the estimated prevalence 1 in 114,000 (PMID: 24071436) in gnomAD v4.0 (max allelic contribution = 40%; max genetic contribution = 100%).


Use the highest population minor allele frequency (MAF) in any given continental population with >2,000 alleles (European non-Finnish, African, East Asian, South Asian, Latino) (PMID 30311383).
Gene: GAMT (HGNC:4136)
Evidence Code: BS2 — Strong
Modification Type: None
Observed in the homozygous state in a healthy adult, or confirmed in trans with a variant that has been classified as pathogenic by the CCDS VCEP using these criteria.
Gene: GAMT (HGNC:4136)
Evidence Code: BS3 — Supporting
Modification Type: Disease-specific,Strength
In vitro assays in which a variant is expressed in GAMT-deficient cultured cells (e.g. GAMT-deficient fibroblasts) or in-fusion High-Fidelity cloning of GAMT transcript and site directed mutagenesis to generate missense variant overexpressed in HeLa cells and measurement of GAMT activity in cells for wild-type and missense variant. Any variant with enzyme activity at or above 30% of normal in the following publications meets BS3_Supporting (Mercimek-Mahmutoglu et al, 2014; PMID 24415674; Mercimek-Mahmutoglu et al, 2016, PMID 26319512; DesRoches et al, 2016, PMID 26003046).
Gene: GAMT (HGNC:4136)
Evidence Code: BP4 — Supporting
Modification Type: General recommendation,None
REVEL score <0.29 for missense variants (based on guidance from Pejaver et al, 2022, PMID: 36413997).


In frame deletion or insertion predicted benign by PROVEAN and MutationTaster.


No predicted impact on splicing by SpliceAI, 
https://spliceailookup.broadinstitute.org/
 , based on a score <0.1 (as indicated in PMID: 37352859, Table 1 and Figure 4).
Gene: GAMT (HGNC:4136)
Evidence Code: BP7 — Strong
Modification Type: General recommendation,Strength
Experimental evidence, such as RT-PCR, shows no impact on splicing. Follow the decision tree outlined in Figure 5, Walker et al, 2023, PMID: 37352859. Note that splicing may appear normal in compound heterozygous patients if the splicing defect generates a transcript that is degraded by nonsense-mediated decay. Therefore, caution must be used when assessing the data prior to applying this code.
Gene: GAMT (HGNC:4136)
Evidence Code: BP7 — Supporting
Modification Type: None
A synonymous (silent) variant OR an intronic variant at or beyond positions +7 and -21, for which SpliceAI, 
https://spliceailookup.broadinstitute.org/
, predicts no impact on splicing (score <0.1).