Approved ACMG-AMP RASopathy Spectrum Disease-Related Classification Criteria

These criteria should only be used to classify germline variants potentially associated with a
RASopathy phenotype. Please note that these adapted criteria are not currently designed to
classify variants relative to non-RASopathy phenotypes (e.g. loss of function variants in PTPN11
related to metachondromatosis); however, information about these other genotype:phenotype
correlations are noted within the supplemental material.

These criteria are also not designed to classify somatic variation in these genes. It is well-known
that information about known somatic mutations can be utilized as supporting evidence for
classifying variants relative to the RASopathy spectrum disorders given the disease mechanisms
are directly correlated. Future initiatives in conjunction with the ClinGen somatic working group
will aim to define this relationship in subsequent versions of this documentation. Currently,
specific phenotype:genotype correlations regarding somatic variants should not be used as
evidence to support germline pathogenicity.

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

RAS EP Commentary: LOF and/or haploinsufficiency has not been clearly identified as
disease mechanisms for these genes relative to the RASopathy spectrum phenotype,
therefore in general this rule is not applicable. Note that PTPN11 is currently the only
gene with a confirmed association to another non-RASopathy disorder due to LOF
alleles. Variants in PTPN11 with predicted LOF should not be evaluated by these
RASoathy specific criteria, but should defer to non-adjusted criteria. Given that some
historical LOF variants (e.g. canonical splice sites) could potentially result in a gain of
function, users should assess using these criteria and non-adjusted criteria to identify
the highest likelihood of pathogenicity for all associated diseases. We recommend that
the ClinGen Dosage Sensitivity Map Status
([Link] be reviewed for any
new apparently LOF disease associations prior to classification assessment.

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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
RAS EP Commentary: Previously established variant must be established as pathogenic
per these criteria for germline RASopathy variants. This evidence rule can also be
applied for the any observed analogous residue positions/regions throughout the gene
in highly analogous groupings below:
Group 1: HRAS, NRAS, KRAS
Group 2: MAP2K1, MAP2K2
Group 3: SOS1, SOS2

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

PS2_Very Strong: ≥2 independent occurrences of PS2 OR ≥2 independent occurrences

of PM6 and one occurrence of PS2. Evidence from literature must be fully evaluated to
support independent events. Also see PM6 definition.

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
RAS EP Commentary: Approved functional studies are available for each individual gene
in the supplemental material. Additional functional studies can be submitted to the
expert panel for approval.

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
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 unrelated patients with the same phenotype, and its absence in controls,
may be used as moderate level of evidence.
PS4: ≥5 independent occurrences
PS4_Moderate: 3-4 independent occurrences
PS4_Supporting: 1-2 independent occurrences

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
RAS EP Commentary: See supplemental material for approved functional domains and
residues. This evidence rule can also be applied for the same analogous residue
positions/regions in highly analogous groupings below:
Group 1: HRAS, NRAS, KRAS
Group 2: MAP2K1, MAP2K2
Group 3: SOS1, SOS2

PM2 Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing
Project, 1000 Genomes or ExAC
Caveat: Population data for indels may be poorly called by next generation sequencing
RAS EP Commentary: The variant must be completely absent from all population
databases.

PM3 For recessive disorders, detected in trans with a pathogenic variant

Note: This requires testing of parents (or offspring) to determine phase

RAS EP Commentary: This criterion is not applicable to the RASopathies.

PM4 Protein length changes due to in-frame deletions/insertions in a non-repeat region or

stop-loss variants

PM5 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
RAS EP Commentary: Previously established variant(s) must be established as
pathogenic per these criteria. Amino acid changes of variants should be concordant with
pathogenicity based on how conservative or non-conservative (within the context of
amino acid chain groupings) the residue change is relative to the known pathogenic
residue changes. This evidence rule can also be used for pathogenic missense variants
seen in the same analogous residue position in highly analogous groupings below:
Group 1: HRAS, NRAS, KRAS
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 Group 2: MAP2K1, MAP2K2
Group 3: SOS1, SOS2

This rule should not be used as independent criteria for calculating pathogenicity in
conjunction with PM1 if the amino acid residue being interrogated is explicitly
designated as a “mutational hot-spot”. For example, Gly12 in HRAS is listed as a hot-spot
for PM1 usage. In these situations, only PM1 should be used when combining criteria for
final variant classification in order to avoid premature designation of a likely pathogenic
classification in the absence of other evidence for pathogenicity.

PM5_Strong: ≥2 different pathogenic missense changes seen before at same residue of

missense change.

PM6 Assumed de novo, but without confirmation of paternity and maternity

PM6_Strong: ≥2 independent occurrences of PM6. Evidence from literature must be
fully evaluated to support independent events.
PM6_VeryStrong: ≥4 independent occurrences of PM6. Evidence from literature must
be fully evaluated to support independent events.
Also see PS2_VeryStrong: ≥2 independent occurrences of PS2 OR ≥2 independent
occurrences of PM6 and one occurrence of PS2. Evidence from literature must be fully
evaluated to support independent events.

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
RAS EP Commentary: Usage of PP1 requires 3-4 informative meioses. Segregation in
more than one family is recommended

PP1_Moderate: 5-6 informative meioses

PP1_Strong: ≥7 informative meioses

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
RAS EP Commentary: PP2 is applicable to all RASopathy genes described and curated
herein.

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.

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PP4 Patient’s phenotype or family history is highly specific for a disease with a single genetic
etiology.
RAS EP Commentary: This criterion is not applicable to the RASopathies. See PS4
criterion for proband counting options.

PP5 Reputable source recently reports variant as pathogenic but the evidence is not
available to the laboratory to perform an independent evaluation
RAS EP Commentary: Currently, there are no resources that are acceptable for this
criterion; however, additional groups are working on policies regarding use of somatic
variation for germline disorders. Once these policies are established, the RAS EP will
consider the use of other external resources (e.g. COSMIC database).

STAND ALONE EVIDENCE OF BENIGN IMPACT

BA1 Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes, or ExAC
RAS EP Commentary: An allele frequency ≥0.05% was approved. See supplemental
material for additional frequency information.

STRONG EVIDENCE OF BENIGN IMPACT

BS1 Allele frequency is greater than expected for disorder
RAS EP Commentary: An allele frequency ≥0.025% was approved. See supplemental
material for additional frequency information.

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.
RAS EP Commentary: Due to variable expressivity and severity, extensive clinical
workup for RASopathy spectrum features is warranted, thus general population data
should not be used for this criterion. Clinical laboratories are encouraged to accumulate
more than 3 instances of well phenotyped family members before applying this strong
criterion.

BS3 Well-established in vitro or in vivo functional studies shows no damaging effect on

protein function or splicing
RAS EP Commentary: Approved functional studies are available for each individual gene
in the supplemental material. Additional functional studies can be submitted to the
expert panel for approval.

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 an autosomal dominant disorder, further
confounding an apparent lack of segregation.

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ClinGen Steering Committee Approval Date: July 18, 2017 Page 5 of 10

 RAS EP Commentary: Requires only one informative meiosis and does not require an
additional piece of supporting evidence to classify variant as likely benign. Due to
variable expressivity and severity, individuals must be well-phenotyped.

SUPPORTING EVIDENCE FOR BENIGN IMPACT

BP1 Missense variant in a gene for which primarily truncating variants are known to cause
disease
RAS EP Commentary: This rule has contraindications for use with RASopathies. Given
the disease mechanism is gain-of-function for RASopathies, BP1 should be used for any
truncating variant (nonsense, frameshift, affects canonical splice sites, initiation codon,
entire gene or multi exon deletion) in genes without established LOF correlation to
disease. See the supplemental material regarding dosage sensitivity information for
each individual gene and potential association to disorders associated with LOF variants.

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

RAS EP Commentary: Currently, there are no resources that are acceptable for this
criterion; however, additional groups are working on policies regarding use of somatic
variation for germline disorders. Once these policies are established, the RAS EP will
consider the use of other external resources (e.g. COSMIC database).

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
RAS EP Commentary: This rule is also applicable for intronic positions (except canonical
splice sites) or non-coding variants and should be used in conjunction with BP4.

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ClinGen Steering Committee Approval Date: July 18, 2017 Page 6 of 10

RULES FOR COMBINING PATHOGENIC CRITERIA
Pathogenic
1. 1 Very Strong (PVS1) AND
a. ≥1 Strong (PS1‐PS4) OR
b. ≥2 Moderate (PM1‐PM6) OR
c. 1 Moderate (PM1‐PM6) and 1 Supporting (PP1‐PP5) OR
d. ≥2 Supporting (PP1‐PP5)
2. ≥2 Strong (PS1‐PS4) OR
3. 1 Strong (PS1‐PS4) AND
a. ≥3 Moderate (PM1‐PM6) OR
b. 2 Moderate (PM1‐PM6) AND ≥2 Supporting (PP1‐PP5) OR
c. 1 Moderate (PM1‐PM6) AND ≥4 Supporting (PP1‐PP5)

Likely Pathogenic
1. 1 Very Strong (PVS1) AND 1 Moderate (PM1‐PM6) OR
2. 1 Strong (PS1‐PS4) AND 1‐2 Moderate (PM1‐PM6) OR
3. 1 Strong (PS1‐PS4) AND ≥2 Supporting (PP1‐PP5) OR
4. ≥3 Moderate (PM1‐PM6) OR
5. 2 Moderate (PM1‐PM6) AND ≥2 Supporting (PP1‐PP5) OR
6. 1 Moderate (PM1‐PM6) AND ≥4 Supporting (PP1‐PP5)

RULES FOR COMBINING BENIGN CRITERIA

Benign
1. 1 Stand‐Alone (BA1) OR
2. ≥2 Strong (BS1‐BS4)

Likely Benign
1. 1 Strong (BS1‐BS4) and 1 Supporting (BP1‐BP7) OR
2. ≥2 Supporting (BP1–BP7)

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Summary of ACMG-AMP Criteria for the RASopathies

PATHOGENIC CRITERIA
Criteria Criteria Description Specification
VERY STRONG CRITERIA
PVS1 Null variant in a gene where loss of function is a N/A*
known mechanism of disease.
PS2_Very ≥2 independent occurrences of PS2 OR Strength
Strong ≥2 independent occurrences of PM6 plus 1 occurrence
of PS2
PM6_Very ≥4 independent occurrences of PM6 Strength
Strong
STRONG CRITERIA
PS1 Same amino acid change as a previously established Gene-
pathogenic variant regardless of nucleotide change. Specific
PS2 De novo (paternity confirmed) in a patient with the None
disease and no family history.
PS3 Well-established in vitro or in vivo functional studies Gene-
supportive of a damaging effect. Specific
PS4 The prevalence of the variant in affected individuals is Disease-
significantly increased compared with the prevalence Specific
in controls. Requires ≥5 independent occurrences/
probands.
PM5_Strong ≥2 different pathogenic missense changes at residue Strength
PM6_Strong 2-3 independent occurrences of PM6 Strength
PP1_Strong ≥7 segregations with disease Strength
MODERATE CRITERIA
PM1 Located in a mutational hot spot and/or critical and Gene-
well-established functional domain. specific
PM2 Absent from controls. Variant must be absent in large Disease-
control population cohorts. specific
PM3 For recessive disorders, detected in trans with a N/A
pathogenic variant.
PM4 Protein length changes due to in-frame None
deletions/insertions in a non-repeat region or stop-
loss variants.
PM5 Missense change at an amino acid residue where a Gene-
different missense change determined to be specific
pathogenic has been seen before.
PM6 Confirmed de novo without confirmation of paternity None
and maternity.
PS4_Moderate 3-4 independent occurrences/probands. Strength
PP1_Moderate 5-6 segregations with disease Strength
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ClinGen Steering Committee Approval Date: July 18, 2017 Page 8 of 10

 SUPPORTING CRITERIA
PP1 Co-segregation with disease in 3-4 affected family Disease-
members. specific
PP2 Missense variant in a gene that has a low rate of benign Gene-
missense variation and where missense variants are a specific
common mechanism of disease. Note: Applicable to all
RASopathy genes.
PP3 Multiple lines of computational evidence support a None
deleterious effect on the gene or gene product
PP4 Phenotype specific for disease with single genetic N/A
etiology.
PP5 Reputable source recently reports variant as N/A
pathogenic but the evidence is not available to the
laboratory to perform an independent evaluation
PS4_ 1-2 independent occurrence/proband. Strength
Supporting
* PTPN11 is the only gene with sufficient evidence to support haploinsufficiency associated with
autosomal dominant metachondromatosis. It is recommended that predicted loss-of-function or null
alleles in PTPN11 be assessed using unmodified ACMG-AMP criteria.

BENIGN CRITERIA
Criteria Criteria Description Specification
STAND ALONE CRITERIA
BA1 Allele frequency is ≥ 0.0005 based on the filtering allele Disease-
frequency (FAF) in ExAC specific
STRONG CRITERIA
BS1 Allele frequency is ≥ 0.00025 based on the filtering Disease-
allele frequency (FAF) in ExAC specific
BS2 Observed in ≥3 well-phenotyped unaffected individuals. Disease-
specific
BS3 Well-established in vitro or in vivo functional studies Gene-
shows no damaging effect on protein function or specific
splicing
BS4 Lack of segregation in affected members of a family. Disease-
Requires only one informative meiosis. specific
SUPPORTING CRITERIA
BP1 Loss of function or truncating variant (nonsense, Disease-
frameshift, affects canonical splice sites, initiation specific
codon, entire gene or multi exon deletion). (Note this is
a contraindication of original criteria)

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ClinGen Steering Committee Approval Date: July 18, 2017 Page 9 of 10

 BP2 Observed in trans with a pathogenic variant for a fully None
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 None
without a known function
BP4 Multiple lines of computational evidence suggest no None
impact on gene or gene product
BP5 Variant found in a case with an alternate molecular None
basis for disease
BP6 Reputable source recently reports variant as benign but N/A
the evidence is not available to the laboratory to
perform an independent evaluation
BP7 A synonymous (silent) variant for which splicing Disease-
prediction algorithms predict no impact to the splice specific
consensus sequence nor the creation of a new splice
site AND the nucleotide is not highly conserved. Also
applicable to intronic (except canonical splice sites) and
non-coding variants.

Key: Gene-specific: Specifications that are specified at the gene level; Disease-Specific:
Disease-specific modifications based on what is known about the RASopathies; Strength:
Increasing or decreasing strength of criteria based on the amount of evidence; N/A: not
applicable to the RASopathies; None: no changes made to existing criteria definitions.

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