Table 2:
Summary of ACMG-AMP Criteria for Rett/Angelman-like Syndromes
| PATHOGENIC CRITERIA | ||
| Criteria | Criteria Description | Specification |
| VERY STRONG CRITERIA | ||
| PVS1 | Null variant in a gene where loss of function is a known mechanism of disease. • Use as defined by ClinGen SVI working group (PMID: 30192042) • FOXG1: PVS1 is applicable up to p.S468. • MECP2: PVS1 is applicable up to p.E472, for any frameshift variant that results in a read-through of the stop codon, for canonical splice site variants predicted to result in an out-of-frame product, and for canonical splice site variants or single in-frame deletions predicted to preserve the reading frame (exon 3). PVS1 is not applicable for initiation codons. • UBE3A: PVS1 is applicable up to p.K841, for any frameshift variant that results in a read-through of the stop codon, for initiation codon variants, and for canonical splice site variants predicted to result in an out-of-frame product. • TCF4: PVS1 is applicable up to p.E643, for any frameshift variant that results in a read-through of the stop codon, for canonical splice site variants predicted to result in an out-of-frame product, and for canonical splice site variants or single in-frame deletions predicted to preserve the reading frame (exon 15). • SLC9A6: PVS1 is applicable up to p.A563, for canonical splice site variants predicted to result in an out-of-frame product, and for canonical splice site variants or single in-frame deletions predicted to preserve the reading frame (exon 10). • CDKL5: Do not use PVS1 for truncating variants in CDKL5 C-terminus (exons 19–21, or after p.P904). The major brain isoform has an alternative C-terminus (NM_001323289.2), PVS1 is applicable up to p.R948, for canonical splice site variants predicted to result in an out-of-frame product, for canonical splice site variants or single in-frame deletions predicted to preserve the reading frame (exons 7, 10, 13), and for the non-coding CDKL5 exon (exon 1). |
Disease-Specific |
| PS2_Very Strong |
De novo (paternity confirmed) in a patient with the disease and no family history. • ≥2 independent occurrences of PS2 • ≥2 independent occurrences of PM6 and one occurrence of PS2. |
Strength |
| PM6_VeryStrong | Confirmed de novo without confirmation of paternity and maternity. • ≥4 independent occurrences of PM6. Evidence from literature must be fully evaluated to support independent events. |
Strength |
| STRONG CRITERIA | ||
| PS1 | Same amino acid change as a previously established pathogenic variant regardless of nucleotide change. | None |
| PS2 | De novo (maternity and paternity confirmed) in a patient with the disease and no family history. | None |
| PS3 | Well-established in vitro or in vivo functional studies supportive of a damaging effect • RNA studies that demonstrate abnormal splicing and an out-of-frame transcript • Do not use for canonical splice site variants and when PVS1 is used |
Disease-Specific |
| PS4 | The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls. • 5+ observations |
Strength |
| PVS1_Strong | Null variant in a gene where loss of function is a known mechanism of disease. • FOXG1: PVS1_Strong is applicable for any truncating variant from p.Gly469 to p.Q480. • UBE3A: PVS1_Strong is applicable for any truncating variant from p.Ala842 to p.G850 and for canonical splice site variants that flank exons 7, 8 (in-frame exons). • SLC9A6: PVS1_Strong is applicable for any truncating variant from p.Cys564 to p.Thr601 and for canonical splice site variants that flank exon 3 (in-frame exon). |
Disease-Specific |
| PM4_Strong | Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. • PM4_Strong is applicable to stop-loss variants in MECP2 and UBE3A. |
Disease-Specific |
| PM5_Strong | Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. • ≥2 different missense changes affecting the amino acid residue. • Do not apply PM1 in these situations. |
Strength |
| PM6_Strong | Confirmed de novo without confirmation of paternity and maternity. • ≥2 independent occurrences of PM6. • Evidence from literature must be fully evaluated to support independent events. |
Strength |
| PP1_Strong | Co-segregation with disease in multiple affected family members • ≥5 informative meiosis • Note: individuals must have disease consistent with reported phenotype (even if on the mild end of spectrum of the disease) |
Strength |
| MODERATE CRITERIA | ||
| PM1 | Located in a mutational hot spot and/or critical and well-established functional domain. • FOXG1: (Forkhead: aa 181–275) • TCF4: (basic Helix-Loop-Helix domain (bHLH): aa 564–617) • CDKL5: (ATP binding region: aa 19–43; TEY phosphorylation site: aa 169–171) • MECP2: (Methyl-DNA binding (MDB): aa 90–162; Transcriptional repression domain (TRD): aa 302–306 • UBE3A: 3’ cysteine binding site: aa 820 • Not to be used for SLC9A6 |
Disease-Specific |
| PM2 | Absent/rare from controls in an ethnically-matched cohort population sample. • Do not use • See PM2_Supporting |
None |
| PM3 | For recessive disorders, detected in trans with a pathogenic variant. • Do not use |
NA |
| PM4 | Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. • Use for in-frame events that are ≥3 amino acids. • CDKL5: Do not use for in-frame deletions/insertions in CDKL5 C-terminus (exons 19–21, or after p.904). • MECP2: Do not use PM4 for in-frame deletions/insertions in the Proline-rich region of gene p.381–p.405) • FOXG1: Do not use PM4 for in-frame deletions/insertions in the Histidine-rich region (p.37–p.57), Proline and Glutamine-rich region (p.58–p.86) and Proline-rich region (p.105–p.112). |
Disease-Specific |
| PM5 | Missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before. • Applicable to all genes as written • A Grantham or BLOSUM score comparison can be used to determine if the variant is predicted to be as or more damaging than the established pathogenic variant. |
None |
| PM6 | Confirmed de novo without confirmation of paternity and maternity. | None |
| PVS1_Moderate | Null variant in a gene where loss of function is a known mechanism of disease. • FOXG1: PVS1_Moderate is applicable for any truncating variant distal of p.Q480. • MECP2: PVS1_Moderate is applicable for any truncating variant distal of p.E472. • UBE3A: PVS1_Moderate is applicable for any truncating variant distal of p.G850. • TCF4: PVS1_Moderate is applicable for any truncating variant distal of p.E643 and for single exon deletions that involve just non-coding exon 20. • SLC9A6: PVS1_Moderate is applicable for any truncating variant distal to p.Y602 and any frameshift variant that results in a read-through of the stop codon. • CDKL5: PVS1_Moderate is applicable for any truncating variant distal to p.R948 and canonical splice site variants that flank exon 17 (in-frame exon). |
Disease-Specific |
| PS4_Moderate | The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls. • 3–4 observations |
Strength |
| PP1_Moderate | Co-segregation with disease in multiple affected family members • 3–4 informative meiosis • Note: individuals must have disease consistent with reported phenotype (even if on the mild end of spectrum of the disease) |
Strength |
| SUPPORTING CRITERIA | ||
| PP1 | Co-segregation with disease in multiple affected family members • 2 informative meiosis • Note: individuals must have disease consistent with reported phenotype (even if on the mild end of spectrum of the disease) |
Strength |
| 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. • Do not use |
N/A |
| PP3 | Multiple lines of computational evidence support a deleterious effect on the gene or gene product • For missense variants use REVEL with a score ≥ 0.75 • For splice site variants use MaxEntScan, NNSPLICE and SpliceSiteFinder-like when all of the predictions program support splicing alteration |
None |
| PP4 | Phenotype specific for disease with single genetic etiology. • See gene specific clinical phenotype guidelines (Supp. Table S3) |
Disease-Specific |
| PP5 |
Reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation • Do not use |
N/A |
| PVS1_Supporting | Null variant in a gene where loss of function is a known mechanism of disease. • PVS1_Supporting is applicable for initiation codon variants in CDKL5, FOXG1, SLC9A6 and TCF4. |
Disease-Specific |
| PS3_Supporting | Well-established in vitro or in vivo functional studies supportive of a damaging effect • RNA studies that demonstrate abnormal splicing and an in-frame product (unless it affects an in-frame exon specified in the PVS1 section) • For FOXG1, MECP2, CDKL5, TCF4, UBE3A (Supp. Table S2) • Not to be used for SLC9A6 |
Disease-Specific |
| PS4_Supporting | The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls. • Use for 2nd independent occurrence |
Strength |
| PM2_Supporting | Absent/rare from controls in an ethnically-matched cohort population sample. • Use if absent, zero observations in control databases • If PVS1 is also applicable, variant can be classified as likely pathogenic |
Strength |
| PM4_Supporting | Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants. • Smaller in-frame events (< 3 amino acid residues) unless they occur in a functionally important region (see PM1 for functionally important domains for each gene). |
Strength |
| BENIGN CRITERIA | ||
| Criteria | Criteria Description | Specification |
| STAND ALONE CRITERIA | ||
| BA1 | Allele frequency • Use large population databases (i.e. gnomAD) • Use if variant is present at ≥0.0003 (0.03%) in any sub-population • Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles |
Disease-Specific |
| STRONG CRITERIA | ||
| BS1 | Allele frequency • Use large population databases (i.e. gnomAD) • Use if variant is present at ≥0.00008 (0.008%) and <0.0003 (0.03%) in any sub-population • Use if allele frequency is met in any general continental population dataset of at least 2,000 observed alleles |
Disease-Specific |
| BS2 | Observed in the heterozygous/hemizygous state in a healthy adult • 2 unaffected (related or unrelated) Het (FOXG1, TCF4), Hemi (SLC9A6), Het or Hemi (CDKL5, MECP2) • 4 unaffected (related and maternally inherited or unrelated) Het (UBE3A) |
Strength |
| BS3 | Well-established in vitro or in vivo functional studies shows no damaging effect on protein function • RNA functional studies that demonstrate no impact on splicing and transcript composition. It can be downgraded based on quality of data. • Not applicable for these genes for other functional studies (see tables for other accepted functional studies) |
Disease-Specific |
| BS4 | Lack of segregation in affected members of a family. • Absent in a similarly affected family member, when seen in two or more families • Need to confirm that the family member is ‘affected with a neurodevelopmental phenotype consistent with the gene’ at a minimum. |
Strength |
| BP5_Strong | Variant found in a case with an alternate molecular basis for disease • ≥3 cases with alternate molecular basis for disease |
Strength |
| SUPPORTING CRITERIA | ||
| BP1 | Missense variant in gene where only LOF causes disease • Do not use |
N/A |
| 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. • Applicable for MECP2, TCF4, FOXG1 for in trans state • Not applicable for SLC9A6, UBE3A and CDKL5 for in trans state |
Disease-Specific |
| BP3 | In-frame deletions/insertions in a repetitive region without a known function • Inframe expansions or deletions in FOXG1 repetitive regions: poly His (p.His47–p.His57), poly Gln (p.Gln70–p.Gln73) and poly Pro (p.Pro58–p.Pro61; p.Pro65–p.Pro69; p.Pro74–p.Pro80) |
Disease Specific |
| BP4 | Multiple lines of computational evidence suggest no impact on gene or gene product • For missense variants use REVEL with a score ≤ 0.15 • For splice site variants use MaxEntScan, NNSPLICE and SpliceSiteFinder-like when the majority of the predictions program support no splicing alteration |
None |
| BP5 | Variant found in a case with an alternate molecular basis for disease • UBE3A: variant should also be maternally inherited in the case with an alternate molecular basis for disease for this criteria to be used. • SLC9A6: the variant should be in the hemizygous state in the case with an alternate molecular basis for disease to be used. • Do not apply for any gene if variant is de novo |
Disease Specific |
| BP6 | Reputable source recently reports variant as benign but the evidence is not available to the laboratory to perform an independent evaluation • Do not use |
N/A |
| 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. • Defined “not highly conserved" regions as those with PhastCons score <1 and/or PhyloP score <0.1 and/or the variant is the reference nucleotide in one primate and/or three mammal species. |
None |
| BS2_Supporting | Observed in the heterozygous/hemizygous state in a healthy adult • 1 unaffected (related or unrelated) Het (FOXG1, TCF4), Hemi (SLC9A6), Het or Hemi (CDKL5, MECP2) • 2 unaffected (related and maternally inherited or unrelated) Het (UBE3A) |
Strength |
| BS4_Supporting | Lack of segregation in affected members of a family. • Absent in a similarly affected family member • Need to confirm that the family member is ‘affected with a neurodevelopmental phenotype consistent with the gene’ at a minimum. |
Strength |
CDKL5 (NM_001323289.2), FOXG1 (NM_005249.4), MECP2 (NM_004992.3), SLC9A6 (NM_006359.2), TCF4 (NM_001083962.1), UBE3A (NM_130838.2)
Key: Disease-Specific: Disease-specific modifications based on what is known about disorders; Strength: Increasing or decreasing strength of criteria based on the amount of evidence; N/A: not applicable for genes; None: no changes made to existing criteria definitions.