owencqueen/cgbench_data huggingface dataset:
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: PVS1 — Very Strong
Modification Type: Strength
Use 
HNF4A
 PVS1 decision tree.




Variants generating PTCs in exon 10 and last 55 nucleotides of exon 9 (c.1162-1216) are not expected to cause NMD
1


The most 3’ nonsense or frameshift variant is c.1256C>G, p.S419X in the last exon. This variant has been classified as Pathogenic by the MDEP.  There are six other nonsense and frameshift variants in exon 10, none of which have case information and are all currently classified as VUS. The collective evidence supports applying PVS1 for variants at codon 419 (c.1257) and 5’ and PVS1_Supporting for variants at c.1258 (G)/p.Gly420 and 3’.






“Exon skipping or use of a cryptic splice site that preserves reading frame” and “Single to multi-exon deletion that preserves reading frame”


Exons 1, 2 (LRG 4), 3 (LRG 5), 4 (LRG 6), 6 (LRG 8): deletion or skipping causes frameshift: PVS1 


Exons 5 (LRG 7), 7 (LRG 9), 8 (LRG 10), 9 (LRG 11) - deletion or skipping causes in-frame deletion, 52/52/79/51-79 AA deleted, that is >10 % of the protein in each case - PVS1_Strong  


Exon 10 (LRG 12) - 46 AA, contains the transactivation domain, includes stop loss - PVS1_Strong
Gene: HNF4A (HGNC:5024)
Evidence Code: PVS1 — Strong
Modification Type: Strength
Use 
HNF4A
 PVS1 decision tree.




“Exon skipping or use of a cryptic splice site that preserves reading frame” and “Single to multi-exon deletion that preserves reading frame” 


Exons 5 (LRG 7), 7 (LRG 9), 8 (LRG 10), 9 (LRG 11) - deletion or skipping causes in-frame deletion, 52/52/79/51-79 AA deleted, that is >10 % of the protein in each case - PVS1_Strong  


Exon 10 (LRG 12) - 46 AA, contains the transactivation domain, includes stop loss - PVS1_Strong
Gene: HNF4A (HGNC:5024)
Evidence Code: PVS1 — Moderate
Modification Type: Strength
Use 
HNF4A
 PVS1 decision tree.


Apply PVS1_Moderate to initiation codon variants.  MDEP has classified two start codon variants as likely pathogenic (c.3G>A: PM2_Supporting + PP4_Moderate + PP1_Strong + PVS1_Moderate (c.1delA); c.1delA: PM2_Supporting + PP1 + PP4_Moderate + PVS1_Moderate) and there are multiple P/LP variants before the next methionine, p.Met71.
Gene: HNF4A (HGNC:5024)
Evidence Code: PVS1 — Supporting
Modification Type: Strength
Use 
HNF4A
 PVS1 decision tree.


Apply PVS1_Supporting to nonsense or frameshift variants at c.1258 (G)/p.Gly420 and 3’.
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: PS1 — Strong
Modification Type: No change
No change
Gene: HNF4A (HGNC:5024)
Evidence Code: PS1 — Supporting
Modification Type: Strength
PS1 may also be used at a supporting level for canonical and non-canonical splicing variants when a different variant at the same nucleotide has been previously classified as pathogenic and the variant being assessed is predicted by SpliceAI to have a similar (SpliceAI score within 10% of the original variant) or greater deleterious impact.
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: PS2 — Very Strong
Modification Type: Gene-specific,Strength
Use SVI recommended point-based system with specifications for “Phenotype Consistency” per instructions.
Gene: HNF4A (HGNC:5024)
Evidence Code: PS2 — Strong
Modification Type: Gene-specific,Strength
Use SVI recommended point-based system with specifications for “Phenotype Consistency” per instructions.
Gene: HNF4A (HGNC:5024)
Evidence Code: PS2 — Moderate
Modification Type: Gene-specific,Strength
Use SVI recommended point-based system with specifications for “Phenotype Consistency” per instructions.
Gene: HNF4A (HGNC:5024)
Evidence Code: PS2 — Supporting
Modification Type: Gene-specific,Strength
Use SVI recommended point-based system with specifications for “Phenotype Consistency” per instructions.
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: PS3 — Strong
Modification Type: Gene-specific,Strength
Applicable to non-canonical splice site variants that have RNA and in silico evidence of aberrant splicing.
Gene: HNF4A (HGNC:5024)
Evidence Code: PS3 — Supporting
Modification Type: Gene-specific,Strength
List of approved functional studies and guidelines for interpretation:




EMSA for DNA binding


“Decreased function” is defined as activity less than 60% of wildtype 


Note: the effect of the variant on DNA binding will be highly dependent on whether the variant is located within the DNA binding domain.






Luciferase assays for transactivation 


“Decreased function” is defined as activity less than 60% of wildtype  


Note: this threshold is not 100% specific for transactivation (TA) activity and is complicated by the fact that TA activity will vary depend on many factors, for instance cell line that is used (HeLa, INS, MIN6 etc).






Western blotting and indirect immunofluorescence for protein expression (specifically levels and nuclear and cytoplasmic localization, respectively).


Determining appropriate thresholds for protein expression is more difficult due to variability in results due to the complexity of the technique.  Sample preparations, gel loading, transfer efficiency, specificity of the antibody, choice of internal control and inaccurate detection and quantification are some of the factors that can contribute to varying and inconsistent results. If a reduction in protein expression is seen by immunoblotting, then further testing by quantitative PCR (qPCR) is recommended in order to measure the mRNA level and assess whether a reduction in amount of protein is due to a reduced mRNA level.






To use PS3_Supporting, functional study must have been performed on a transfected variant.  If a study was performed on a cell line generated from a patient sample (and therefore contains the variant plus wild-type allele) does not count as PS3_Supporting.
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: PS4 — Strong
Modification Type: Gene-specific,Strength
7 (seven) or more unrelated occurrences = Strong. Variant should meet PM2_Supporting in order to use PS4 at any level (careful review of gnomAD QC data may be necessary to assess whether variant is real or an artifact, especially if variant is in a polyC region).  Phenotype of affected individuals must include diabetes, without clear evidence of an autoimmune etiology.




One or more positive diabetes autoantibodies (IA-2A, ZnT8A+, GAD)
7
,
8
,
9
,
10
 


Very low or negative C-peptide, defined as either fasting or non-fasting random C-peptide (<200pmol/L or 0.6ng/mL)
11
,
12
 or urinary C-peptide/creatinine ratio <0.2 nmol/mmol
8
,
9
Gene: HNF4A (HGNC:5024)
Evidence Code: PS4 — Moderate
Modification Type: Gene-specific,Strength
4-6 unrelated occurrences = Moderate. Variant should meet PM2_Supporting in order to use PS4 at any level. Phenotype of affected individuals must include diabetes, without clear evidence of an autoimmune etiology.




One or more positive diabetes autoantibodies (IA-2A, ZnT8A+, GAD)
7
,
8
,
9
,
10


Very low or negative C-peptide, defined as either fasting or non-fasting random C-peptide (<200pmol/L or 0.6ng/mL)
11
,
12
 or urinary C-peptide/creatinine ratio <0.2 nmol/mmol
8
,
9
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: PM1 — Moderate
Modification Type: Gene-specific,Strength
Applicable to amino acids that directly bind DNA and are necessary for Zinc-finger or homodimer formation
2
 




Directly bind DNA: Asp43, His49, Tyr 50, Gly51, Asp56, Gly57, Lys59, Arg63, Arg64, Arg67, His70, Tyr72, Arg87, Asn88, Arg91, Arg94, Gln109, Arg112   


Homodimer: Arg75, Gln89, Glu111, Asp113 


Zinc finger: Cys38, Cys41, Cys55, Cys58, Cys74, Cys80, Cys90, Cys93
Gene: HNF4A (HGNC:5024)
Evidence Code: PM1 — Supporting
Modification Type: Gene-specific,Strength
This criterion can be used for missense variants in well-conserved regions within the DNA and ligand-binding domains. It can also be used for variants within certain transcription factor binding sites in the promoter (see below for details).




Promoter region: 


c.-132 to c.-151 (HNF6/OC2 and IPF1 binding sites) 


c.-169 to c.-181 (HNF1A/HNF1B binding sites)






DNA binding: 


codons 37-113 (NM_175914.4:c.175C-339C p.Leu37-Asp113) (While the paper describing the crystal structure of 
HNF4A
2
 shows the sequence as amino acids 33-113, amino acids 33-36 do not bind DNA and the conserved sequence starts as Leu37.)






Ligand binding: 


codons 180-220 and 300-350  


(NM_175914.4:c.538G-658G p.Ala180-Val220) 


(NM_175914.4:c.898T-1048G p.Tyr300-Glu350)
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: PM2 — Supporting
Modification Type: General recommendation,Gene-specific
gnomAD 2.1.1 Popmax FAF ≤ 1:333,000 (≤ 0.000003 or 0.0003%) in gnomAD European Non-Finnish population AND ≤ 2 copies observed in ENF AND ≤ 1 copy in any other founder or non-founder population.
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: PM4 — Moderate
Modification Type: Strength
For single amino acid deletions, use as supporting level of evidence.
Gene: HNF4A (HGNC:5024)
Evidence Code: PM4 — Supporting
Modification Type: Strength
For single amino acid deletions/insertions, use as supporting level of evidence
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: PM5 — Strong
Modification Type: Strength
Applicable once two amino acid changes have been classified as pathogenic at the same amino acid residue.
Gene: HNF4A (HGNC:5024)
Evidence Code: PM5 — Moderate
Modification Type: Strength
The novel amino acid change must have a Grantham distance greater than or equal to the previously classified pathogenic variant.
Gene: HNF4A (HGNC:5024)
Evidence Code: PM5 — Supporting
Modification Type: Strength
Apply if the previously classified amino acid change is likely pathogenic (rather than pathogenic) or if the previously classified variant is pathogenic but has a greater Grantham distance than the novel variant.
Gene: HNF4A (HGNC:5024)
Evidence Code: PM6 — Original ACMG Summary
Assumed de novo, but without confirmation of paternity and maternity.
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: PP1 — Strong
Modification Type: General recommendation,Gene-specific
Use thresholds suggested by Jarvik and Browning
5




Single Family : ≤ 1/32 (5 meioses)


> 1 Family : ≤ 1/16 (4 meioses)
Gene: HNF4A (HGNC:5024)
Evidence Code: PP1 — Moderate
Modification Type: General recommendation,Gene-specific
Use thresholds suggested by Jarvik and Browning
5




Single Family : ≤ 1/16 (4 meioses)


> 1 Family : ≤ 1/8 (3 meioses)
Gene: HNF4A (HGNC:5024)
Evidence Code: PP1 — Supporting
Modification Type: General recommendation,Gene-specific
Use thresholds suggested by Jarvik and Browning
5




Single Family : ≤ 1/8 (3 meioses)


> 1 Family : ≤ ¼ (2 meioses)
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: PP3 — Supporting
Modification Type: General recommendation
Use REVEL score of ≥0.70 as supportive evidence of pathogenicity. We also support using SpliceAI to assess the predicted impact of non-canonical splicing variants and synonymous variants: apply PP3 when the predicted change is at least 0.2 
4
,
3
.
Gene: HNF4A (HGNC:5024)
Evidence Code: PP4 — Original ACMG Summary
Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.
Gene: HNF4A (HGNC:5024)
Evidence Code: PP4 — Moderate
Modification Type: Gene-specific
Phenotype: MODY Probability Calculator result ≥50% chance of testing positive
6
 AND negative 
HNF1A
 testing AND presence of at least one additional feature characteristic of _
HNF4A
_-MODY:   




Antibody negative and/or persistent C-peptide after five years following T1DM diagnosis  


Personal or family history of persistent neonatal hypoglycemia  


Personal or family history of large for gestational age (LGA) infants or macrosomia in the absence of sufficient maternal hyperglycemia 


Response to low-dose SU (extreme response- hypoglycemia) 


Biochemical/Molecular phenotypic evidence from patient cell lines 


Fanconi phenotype in conjunction with c.187C>T p.R63W
Gene: HNF4A (HGNC:5024)
Evidence Code: PP4 — Supporting
Modification Type: Gene-specific
MODY Probability Calculator (MPC)
6
 result ≥50% chance of testing positive AND negative 
HNF1A
 testing
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: BA1 — Original ACMG Summary
Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.
Gene: HNF4A (HGNC:5024)
Evidence Code: BA1 — Stand Alone
Modification Type: Gene-specific
gnomAD 2.1.1 Popmax Filtering AF ≥ 1:10,000 (≥ 0.01% or 0.0001).
Gene: HNF4A (HGNC:5024)
Evidence Code: BS1 — Original ACMG Summary
Allele frequency is greater than expected for disorder.
Gene: HNF4A (HGNC:5024)
Evidence Code: BS1 — Strong
Modification Type: Gene-specific
gnomAD 2.1.1 Popmax Filtering AF  ≥ 1:30,000 (≥0.0033% or 0.000033).
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: BS2 — Strong
Modification Type: Gene-specific
Apply to normoglycemic individuals age 70 or older (i.e., genotype positive, phenotype negative)
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: BS3 — Strong
Modification Type: Gene-specific
Applicable to non-canonical splice site variants that have RNA and in silico evidence of normal splicing.
Gene: HNF4A (HGNC:5024)
Evidence Code: BS3 — Supporting
Modification Type: Gene-specific
List of approved functional studies and guidelines for interpretation of data.




EMSA for DNA binding 


“No functional impact” is defined as ≥75% activity of wildtype 


Note: the effect of the variant on DNA binding will be highly dependent on whether the variant is located within the DNA binding domain.






Luciferase assays for transactivation 


“No functional impact” is defined as ≥75% activity of wildtype 


Note: this threshold is not 100% specific for transactivation (TA) activity and is complicated by the fact that TA activity will vary depend on many factors, for instance cell line that is used (HeLa, INS, MIN6 etc). Assays should include controls for WT, T2DM and known MODY variants.






Western blotting and indirect immunofluorescence for protein expression (specifically levels and nuclear and cytoplasmic localization, respectively).   


Determining appropriate thresholds for protein expression is more difficult due to variability in results due to the complexity of the technique.  Sample preparations, gel loading, transfer efficiency, specificity of the antibody, choice of internal control and inaccurate detection and quantification are some of the factors that can contribute to varying and inconsistent results. If a difference in protein expression compared to WT is seen by immunoblotting, then further testing by quantitative PCR (qPCR) is recommended in order to measure the mRNA level and assess whether the difference in amount of protein is due to a reduced mRNA level.
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: BS4 — Strong
Modification Type: General recommendation,Gene-specific
Applicable to family members without variant who have MPC
6
 score ≥50% (i.e., genotype negative, phenotype positive).
Gene: HNF4A (HGNC:5024)
Evidence Code: BP1 — Original ACMG Summary
Missense variant in a gene for which primarily truncating variants are known to cause disease.
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: BP2 — Supporting
Modification Type: General recommendation
Also applicable when in cis or trans with a likely pathogenic variant.
Gene: HNF4A (HGNC:5024)
Evidence Code: BP3 — Original ACMG Summary
In frame-deletions/insertions in a repetitive region without a known function.
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: BP4 — Supporting
Modification Type: General recommendation
Use a REVEL score of ≤0.15 as supportive evidence of no predicted impact on the gene or gene product. We also support using SpliceAI to assess the predicted impact of non-canonical splicing variants and synonymous variants: apply BP4 when the predicted change is below 0.2
3
,
4
.
Gene: HNF4A (HGNC:5024)
Evidence Code: BP5 — Original ACMG Summary
Variant found in a case with an alternate molecular basis for disease.
Gene: HNF4A (HGNC:5024)
Evidence Code: BP5 — Supporting
Modification Type: General recommendation
A variant in another monogenic diabetes gene is Pathogenic/Likely Pathogenic.
Gene: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
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: HNF4A (HGNC:5024)
Evidence Code: BP7 — Supporting
Modification Type: Gene-specific
Apply BP7 when the predicted change from SpliceAI is below 0.2 AND phyloP100 way < 2.0.