| Functional Data | Well-established functional studies show no deleterious effect BS3 | Missense in gene with low rate of benign missense variants and path. missenses common PP2 | Mutational hot spot or well-studied functional domain without benign variation PM1 | Well-established functional studies show a deleterious effect PS3 | |
| Segregation Data | Non-segregation with disease BS4 | Co-segregation with disease in multiple affected family members PP1 | Increased segregation data → | ||
| De novo Data | De novo (without paternity & maternity confirmed) PM6 | De novo (paternity & maternity confirmed) PS2 | |||
| Allelic Data | Observed in trans with a dominant variant BP2 Observed in cis with a pathogenic variant BP2 | For recessive disorders, detected in trans with a pathogenic variant PM3 | |||
| Other Database | Reputable source w/out shared data = benign BP6 | Reputable source = pathogenic PP5 | |||
| Other Data | Found in case with an alternate cause BP5 | Patient's phenotype or FH highly specific for gene PP4 | |||
Table 3 Criteria for Classifying Pathogenic Variants
| Very strong evidence of pathogenicity | |
|---|---|
| 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 | |
| Strong evidence of pathogenicity | |
| 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 |
| 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 | |
| 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 | |
| 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. | |
| Moderate evidence of pathogenicity | |
| 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 |
| PM2 | Absent from controls (or at extremely low frequency if recessive) (see Table 6)in Exome Sequencing Project, 1000 Genomes or ExAC |
| Caveat: Population data for indels may be poorly called by next generation sequencing | |
| PM3 | For recessive disorders, detected in trans with a pathogenic variant |
| Note: This requires testing of parents (or offspring) to determine phase | |
| PM4 | Protein length changes due to in-frame deletions/insertions in a non-repeat 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 |
| PM6 | Assumed de novo, but without confirmation of paternity and maternity |
| Supporting evidence of pathogenicity | |
| 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 | |
| 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 |
| 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. | |
| PP4 | Patient's phenotype or family history is highly specific for a disease with a single genetic etiology |
| PP5 | Reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation |
Table 4 Criteria for Classifying Benign Variants
| Stand-Alone evidence of benign impact | |
|---|---|
| BA1 | Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes, or ExAC |
| Strong evidence of benign impact | |
| BS1 | Allele frequency is greater than expected for disorder (see table 6) |
| 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 |
| BS3 | Well-established in vitro or in vivo functional studies shows no damaging effect on protein function or splicing |
| 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 anautosomal dominant disorder, further confounding an apparent lack of segregation. | |
| Supporting evidence of benign impact | |
| BP1 | Missense variant in a gene for which primarily truncating variants are known to cause disease |
| 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 |
| BP3 | 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. | |
| BP5 | 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 |
| BP7 | 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 |
Table 5 Rules for Combining Criteria to Classify Sequence Variants
Pathogenic
- 1 Very Strong (PVS1) AND
- ≥1 Strong (PS1–PS4) OR
- ≥2 Moderate (PM1–PM6) OR
- 1 Moderate (PM1–PM6) and 1 Supporting (PP1–PP5) OR
- ≥2 Supporting (PP1–PP5)
- ≥2 Strong (PS1–PS4) OR
- 1 Strong (PS1–PS4) AND
- ≥3 Moderate (PM1–PM6) OR
- 2 Moderate (PM1–PM6) AND ≥2 Supporting (PP1–PP5) OR
- 1 Moderate (PM1–PM6) AND ≥4 Supporting (PP1–PP5)
Likely Pathogenic 본문, mean >90% certainty of a variant being disease-causing
- 1 Very Strong (PVS1) AND 1 Moderate (PM1–PM6) OR
- 1 Strong (PS1–PS4) AND 1–2 Moderate (PM1–PM6) OR
- 1 Strong (PS1–PS4) AND ≥2 Supporting (PP1–PP5) OR
- ≥3 Moderate (PM1–PM6) OR
- 2 Moderate (PM1–PM6) AND ≥2 Supporting (PP1–PP5) OR
- 1 Moderate (PM1–PM6) AND ≥4 Supporting (PP1–PP5)
Benign
- 1 Stand-Alone (BA1) OR
- ≥2 Strong (BS1–BS4)
Likely Benign
- 1 Strong (BS1–BS4) and 1 Supporting (BP1–BP7) OR
- ≥2 Supporting (BP1–BP7)
* Variants should be classified as Uncertain Significance if other criteria are unmet or the criteria for benign and pathogenic are contradictory.
Pathway Analysis
Goal
confirm the involvement of x pathways, highlight the role of x and its potential interaction with — the more genes for any given pathway are identified, the greater the confidence that this pathway should be prioritized over others
- — concentrations of proteins in the same complex are less noisy compared with proteins that are not within one complex31
| Gene-disease phenotype association | PATHway analysis | |||||
|---|---|---|---|---|---|---|
| biomarkers | ||||||
| PADD | ||||||
| Pathogenicity of variant | Web tool ve.genecards.org | https://www.genome.jp/kegg/tool/map_pathway2.html | EnrichR | IPA, 으로부터 app을 이미 download 받아놓았으니, https://takeda-… | open source https://s… | |
| Pathway Analysis | … Analytics … | |||||
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Top continuation matrix — De novo Data | De novo (without paternity & maternity confirmed) PM6 / De novo (paternity & maternity confirmed) PS2 | Both halves of the row are visible; the leftmost subcell of the same matrix continuing onto this page is empty. |
| Table 3 PVS1 row | single or multi-exon deletion | The crop edge clips the closing parenthesis after single or multi-exon deletion; verb. text continues with in a gene where loss of function (LOF) is a known mechanism of disease. |
| Table 5 Likely Pathogenic heading | Likely Pathogenic 본문, mean >90% certainty of a variant being disease-causing | Korean prefix 본문, immediately precedes the English clause; both are inline as written. |
| Pathway Analysis row — IPA cell | IPA, 으로부터 app을 이미 download 받아놓았으니, https://takeda-… | The hyperlink is truncated after https://takeda-; the remainder of the URL is below the visible area of the body crop and only partially visible in the status-bar capture. |