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




CCDS VCEP notes:
Loss of function (LOF) of GAMT is a known mechanism of disease for guanidinoacetate methyltransferase deficiency (GAMT-D). There are examples of various LOF variants, including nonsense and frameshift, in GAMT in individuals with GAMT-D (
https://databases.lovd.nl/shared/variants/GAMT?search_var_status=%3D%22Marked%22%7C%3D%22Public%22
 ). The specifications below are based on the PVS1 decision tree (Figure 1, Abou Tayoun et al, 2018, PMID 30192042).


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.




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.


To apply PVS1, splice site variants must have no detectable 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.




Initiator codon variants




To our knowledge, initiator codon variants have not been reported in GAMT (01/2019) but may occur.


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: PVS1 — Strong
Modification Type: Strength
In frame loss of >10% of the protein.




CCDS VCEP notes:
Loss of function (LOF) of GAMT is a known mechanism of disease for guanidinoacetate methyltransferase deficiency (GAMT-D). There are examples of various LOF variants, including nonsense and frameshift, in GAMT in individuals with GAMT-D (
https://databases.lovd.nl/shared/variants/GAMT?search_var_status=%3D%22Marked%22%7C%3D%22Public%22
 ). The specifications below are based on the PVS1 decision tree (Figure 1, Abou Tayoun et al, 2018, PMID 30192042).


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.




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.


To apply PVS1, splice site variants must have no detectable 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.




Initiator codon variants




To our knowledge, initiator codon variants have not been reported in GAMT (01/2019) but may occur.


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: PVS1 — Moderate
Modification Type: Strength
Initiator codon variant.


Single exon or larger deletion resulting in loss of <10% of the protein.




CCDS VCEP notes:
Loss of function (LOF) of GAMT is a known mechanism of disease for guanidinoacetate methyltransferase deficiency (GAMT-D). There are examples of various LOF variants, including nonsense and frameshift, in GAMT in individuals with GAMT-D (
https://databases.lovd.nl/shared/variants/GAMT?search_var_status=%3D%22Marked%22%7C%3D%22Public%22
 ). The specifications below are based on the PVS1 decision tree (Figure 1, Abou Tayoun et al, 2018, PMID 30192042).


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.




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.


To apply PVS1, splice site variants must have no detectable 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.




Initiator codon variants




To our knowledge, initiator codon variants have not been reported in GAMT (01/2019) but may occur.


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: None
CCDS VCEP notes:




This criterion is applicable as described.


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.
Gene: GAMT (HGNC:4136)
Evidence Code: PS3 — Strong
Modification Type: Disease-specific
RT-PCR evidence of mis-splicing for non-canonical +1, +2, -2, -1 intronic variants with no evidence of normal splice products.
GAMT specifications:
Any variant meeting the description for splicing assays below can meet PS3 or PS3_Supporting, and variants meeting the description for in vitro activity assays can meet PS3_Supporting. If a variant meets the description for both e.g., a splice site variant with evidence of abnormal splicing and deficient GAMT activity in vitro, PS3 must only be counted once.
In vitro expression
PS3_Supporting can be assigned if a variant is expressed in GAMT-deficient fibroblasts or 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. See Appendix 2 for further details on GAMT functional assays.
Splicing assays
For non-canonical splicing variants, use PS3 if there is RT-PCR and/or RNA sequencing evidence demonstrating only abnormal splice products, with no evidence of normal splicing.


Evidence of abnormal splicing includes transcripts of alternative length or with specific intron or exon inclusion/exclusion. These studies can be performed on mRNA extracted from patient-derived cells, or by inserting the mutant genomic DNA into plasmid vectors and introducing these into human or other mammalian host cells.


Note that in patients who are compound heterozygotes for a splicing variant and another variants type that does not disrupt splicing, such as a missense variant, evidence of normal splicing is expected. However, the presence of normal splice products could complicate the assessment of the impact of the splice variant. Therefore, if there is any evidence of normal splice products, either when using RNA from patient cells or in an in vitro expression system, use PS3_Supporting.


PP3 may also be used for non-canonical splice variants meeting PS3 or PS3_Supporting.
Gene: GAMT (HGNC:4136)
Evidence Code: PS3 — Supporting
Modification Type: Disease-specific
<15% control activity when variant is expressed in GAMT-deficient fibroblasts or HeLa cells, as reported in PMID 24415674, 26003046, and 26319512.


RT-PCR evidence of mis-splicing for non-canonical intronic variants with evidence of normal splice products.
GAMT specifications:
Any variant meeting the description for splicing assays below can meet PS3 or PS3_Supporting, and variants meeting the description for in vitro activity assays can meet PS3_Supporting. If a variant meets the description for both e.g., a splice site variant with evidence of abnormal splicing and deficient GAMT activity in vitro, PS3 must only be counted once.
In vitro expression
PS3_Supporting can be assigned if a variant is expressed in GAMT-deficient fibroblasts or 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. See Appendix 2 for further details on GAMT functional assays.
Splicing assays
For non-canonical splicing variants, use PS3 if there is RT-PCR and/or RNA sequencing evidence demonstrating only abnormal splice products, with no evidence of normal splicing.


Evidence of abnormal splicing includes transcripts of alternative length or with specific intron or exon inclusion/exclusion. These studies can be performed on mRNA extracted from patient-derived cells, or by inserting the mutant genomic DNA into plasmid vectors and introducing these into human or other mammalian host cells.


Note that in patients who are compound heterozygotes for a splicing variant and another variants type that does not disrupt splicing, such as a missense variant, evidence of normal splicing is expected. However, the presence of normal splice products could complicate the assessment of the impact of the splice variant. Therefore, if there is any evidence of normal splice products, either when using RNA from patient cells or in an in vitro expression system, use PS3_Supporting.


PP3 may also be used for non-canonical splice variants meeting PS3 or PS3_Supporting.
Gene: GAMT (HGNC:4136)
Evidence Code: PM2 — Supporting
Modification Type: Strength,Disease-specific
Allele frequency <0.0004 (<0.04%) in all populations in gnomAD.




CCDS VCEP notes:
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%.


GAMT specifications:




All subpopulations in gnomAD 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 (See Appendix 3).
Note – PM2 will NOT be used at moderate strength; PM2 will only be applied as a Supporting criterion.


If homozygotes are observed, the variant will meet BS2 (assuming 100% penetrance for an individual with 2 pathogenic variants in trans).
Gene: GAMT (HGNC:4136)
Evidence Code: PM3 — Very Strong
Modification Type: Strength
Consult specifications for assigning strength of evidence for PM3.


GAMT specifications:




Following SVI guidance for PM3 (
https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf
), use the scoring system below.


Parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed in order to confirm that the variants are in trans if the patient is compound heterozygous.
Gene: GAMT (HGNC:4136)
Evidence Code: PM3 — Strong
Modification Type: Strength
Consult specifications for assigning strength of evidence for PM3.


GAMT specifications:




Following SVI guidance for PM3 (
https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf
), use the scoring system below.


Parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed in order to confirm that the variants are in trans if the patient is compound heterozygous.
Gene: GAMT (HGNC:4136)
Evidence Code: PM3 — Moderate
Modification Type: None
Consult specifications for assigning strength of evidence for PM3.


GAMT specifications:




Following SVI guidance for PM3 (
https://clinicalgenome.org/site/assets/files/3717/svi_proposal_for_pm3_criterion_-_version_1.pdf
), use the scoring system below.


Parental testing, or another appropriate molecular method (such as cloning each allele separately followed by sequencing), must have been performed in order to confirm that the variants are in trans if the patient is compound heterozygous.
Gene: GAMT (HGNC:4136)
Evidence Code: PM4 — Moderate
Modification Type: None
CCDS VCEP notes:




Stop loss variants in GAMT have not been reported, as far as we are aware.




GAMT specifications:
Use this rule “as is” for 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
GAMT specifications:




If the pathogenicity of another missense change at the same amino acid residue is unknown, determine its pathogenicity using these specifications in order to determine if this criterion can be used. If the variant is pathogenic, use PM5. If the variant is likely pathogenic, use PM5_Supporting.
Gene: GAMT (HGNC:4136)
Evidence Code: PM5 — Supporting
Modification Type: Strength
GAMT specifications:




If the pathogenicity of another missense change at the same amino acid residue is unknown, determine its pathogenicity using these specifications in order to determine if this criterion can be used. If the variant is pathogenic, use PM5. If the variant is likely pathogenic, use PM5_Supporting.
Gene: GAMT (HGNC:4136)
Evidence Code: PP3 — Moderate
Modification Type: Strength
Non-canonical splice site variant predicted to be more deleterious than a previously observed pathogenic variant at the same nucleotide.




GAMT specifications:




For missense changes, those with a REVEL score more than 0.75 will meet PP3.


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


For non-canonical splice site variants (e.g., +3, -3), use SpliceAI ((
https://spliceailookup.broadinstitute.org/
 ) and varSEAK (
https://varseak.bio/
). Results must be consistent to apply this criterion.


For SpliceAI, any donor loss or acceptor loss with a score >0.5. For varSEAK, any variant with splicing class 4 or 5. Evidence for creation of a cryptic splice site should also be assessed.


Do not apply this rule for canonical splice site changes meeting PVS1.


Upgrade to PP3_Moderate if a different non-canonical splice site variant at the same position is known to be pathogenic AND the newly observed variant is at least as deleterious as the previously observed variant, based on in silico prediction (Human Splicing Finder, MaxEntScan) e.g. variant being interpreted is +3C>G, previously reported variant is +3C>T and +3C>G is predicted to be more deleterious than +3C>T.
Gene: GAMT (HGNC:4136)
Evidence Code: PP3 — Supporting
Modification Type: None
REVEL score >0.75 for missense variants.


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


Predicted impact on splicing by SpliceAI and varSEAK.




GAMT specifications:




For missense changes, those with a REVEL score more than 0.75 will meet PP3.


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


For non-canonical splice site variants (e.g., +3, -3), use SpliceAI ((
https://spliceailookup.broadinstitute.org/
 ) and varSEAK (
https://varseak.bio/
). Results must be consistent to apply this criterion.


For SpliceAI, any donor loss or acceptor loss with a score >0.5. For varSEAK, any variant with splicing class 4 or 5. Evidence for creation of a cryptic splice site should also be assessed.


Do not apply this rule for canonical splice site changes meeting PVS1.


Upgrade to PP3_Moderate if a different non-canonical splice site variant at the same position is known to be pathogenic AND the newly observed variant is at least as deleterious as the previously observed variant, based on in silico prediction (Human Splicing Finder, MaxEntScan) e.g. variant being interpreted is +3C>G, previously reported variant is +3C>T and +3C>G is predicted to be more deleterious than +3C>T.
Gene: GAMT (HGNC:4136)
Evidence Code: PP4 — Strong
Modification Type: Disease-specific
Any combination of two or more of the data types listed for PP4_Moderate and PP4 OR significantly decreased creatine peak and visible GAA peak on 1H-MRS (see points scheme in main document)
Note: Do not apply this rule if the variant meets BA1, or otherwise meets criteria for benign or likely benign status.


GAMT specifications:
The following table shows the weight used for PP4 depending upon the data that is available. Assign points for each type of data present, and then add up the points.
PP4 = 1-2 points
PP4_Moderate = 3 points
PP4_Strong = 4 points
Two or more data types are recommended to reach moderate and required to reach strong.




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
Elevated GAA and low or low normal creatine in urine, OR elevated GAA and low creatine in plasma OR


Significantly decreased creatine peak on 1H-MRS (GAA not measured/not reported) OR


GAMT activity <5% normal in fibroblasts.
Note: Do not apply this rule if the variant meets BA1, or otherwise meets criteria for benign or likely benign status.




GAMT specifications:
The following table shows the weight used for PP4 depending upon the data that is available. Assign points for each type of data present, and then add up the points.
PP4 = 1-2 points
PP4_Moderate = 3 points
PP4_Strong = 4 points
Two or more data types are recommended to reach moderate and required to reach strong.




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 — Supporting
Modification Type: Disease-specific
Elevated GAA in plasma or urine (with no creatine measurement)
Note: Do not apply this rule if the variant meets BA1, or otherwise meets criteria for benign or likely benign status.




GAMT specifications:
The following table shows the weight used for PP4 depending upon the data that is available. Assign points for each type of data present, and then add up the points.
PP4 = 1-2 points
PP4_Moderate = 3 points
PP4_Strong = 4 points
Two or more data types are recommended to reach moderate and required to reach strong.




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: BA1 — Stand Alone
Modification Type: Disease-specific
Allele frequency >0.003 (0.3%) in gnomAD in any continental population in gnomAD with >2000 alleles.


GAMT specifications:




Any variant with a frequency >0.003 (based on the estimated prevalence 1 in 114,000, PMID 24071436) in gnomAD (max allelic contribution = 100%; max genetic contribution = 100%)(See Appendix 3).


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 in any continental population in gnomAD with >2000 alleles.
GAMT specifications:




Any variant with a frequency >0.001 (based on the estimated prevalence 1 in 114,000 (PMID: 24071436) in gnomAD (max allelic contribution = 40%; max genetic contribution = 100%)(See Appendix 3).


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).