Criteria for the Interpretation of mRNA Analyses
The criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for
the evaluation of sequence variants with subsequent mRNA analysis are based on the
guidelines of the ENIGMA Consortium (see also [7 ]). The respective threshold values of potential splice variants for an empirical
predictive prognosis based on three commonly used predictive programmes are given
in Appendix A 1. Appendix A 4 ([Fig. 2 ]) gives a schematic representation of the areas considered by the VUS Task Force
when evaluating the splice variants. mRNA analysis is carried out using fresh blood
samples, cultured lymphocytes, cultured lymphoblastoid cell lines, etc. and compared
in parallel with at least 5 controls of the same type of material. A sequence variant
is described as pathogenic if it has the following effect on mRNA transcription: one
or more aberrant transcripts of the variant allele are detected, which lead to a stop
codon or an in-frame deletion and result in the destruction of known functional domains.
Sequencing of the full-length transcript of the variant allele or the presence of
an intronic variant of a cis-acting polymorphism is considered sufficient (evidence
of monoallelic expression) to determine the transcript amount (using semi-quantitative
or quantitative methods). Variants which show a transcript pattern comparable to the
mean value of controls are rated as neutral/not pathogenic due to the lack of aberrant
mRNA. As regards the cDNA primer design, the physiological splice variants/naturally
occurring isoforms must also be taken into account (see [8 ], [9 ]).
Fig. 2 Classification of variants which can affect splicing.
Caution: Certain BRCA1 and BRCA2 variants which are ± 1, 2 bp from the exon border and which are predicted or proven
to lead to at least 20 – 30% naturally occurring in-frame RNA isoforms per allele
could presumably result in some residual protein activity (see [9 ], [10 ], [11 ], [12 ]) and are therefore classified as VUS Class3, unless there is evidence to the contrary
(see Overview, Appendix 5, [Table 5 ])
Table 5 Excerpt from https://enigmaconsortium.org/wp-content/uploads/2018/10/ENIGMA_Rules_2017-06-29-v2.5.1.pdf .
Table 6: BRCA1 and BRCA2 exon boundary variants predicted or known to lead to naturally occurring in-frame
RNA isoforms that may rescue gene functionality. Variants at these positions should be considered Class3 Uncertain unless proven otherwise.*
Gene
Alternative splicing event
Variants implicated
Rationale
* This summary table does not yet capture the possibility of acceptor site changes
leading to small in-frame deletions > 3 bp, e.g. due to NAG (NNN)n NAG sites. It is recommended that bioinformatic prediction analysis is carried out
for variation in/near all donor and acceptor sites to assess the likelihood that a variant will or will not
cause alternative splicing.
Note: It could be argued that nonsense or frameshift variants in BRCA1 exon 9, BRCA1 exon 10, or BRCA2 exon 12 may not be associated with high risk of cancer due to rescue by the expression
of in-frame naturally occurring isoforms that bypass the premature termination codon
and thus encode a functional protein. A review of multiple clinical and control datasets
for the frequency of unique nonsense or frameshift variants – adjusted for exon size – does not provide strong support for this hypothesis at present (Spurdle, de la
Hoya, unpublished data). Additional research is underway to further investigate the
functional/clinical importance of germline nonsense or frameshift variants in these
exons.
Moreover, further work is planned within ENIGMA (led by Paolo Radice) to document
variants that have undergone splicing assays and are proven to be “leaky” variants,
to provide a record of all spliceogenic variants for which additional research is
necessary. This resource will identify variants that have already been classified
using clinical data, as positive and negative controls for future quantitative mRNA
studies.
BRCA1
Δ8p
c.442-1 (IVS7-1)
c.442-2 (IVS7-2)
BRCA1 exon 8 acceptor site is an experimentally validated tandem acceptor site (NAGNAG)
subject to alternative splicing (Colombo et al., 2014). c.442-1,-2 variants are predicted
to inactivate the 5′ acceptor site, but not the 3′ acceptor site, thus producing Δ8p
transcripts.
Δ9,10
c.548-1 (IVS8-1)
c.548-2 (IVS8-2)
c.593 to non-G
c.593+1 (IVS9+1)
c.593+2 (IVS9+2)
c.594-1 (IVS9-1)
c.594-2 (IVS9-2)
c.670 to non-G
c.670+1 (IVS10+1)
c.670+2 (IVS10+2)
Carriers of variants at these positions are predicted to produce normal (or increased)
levels of BRCA1 Δ(9,10), a major in-frame alternative splicing event (Colombo et al., 2014).
The BRCA1 variant c.594-2A>C (shown from ENIGMA research to co-occur in cis with c.641A>G),
has been reported to demonstrate clinical characteristics inconsistent with a high
risk of cancer expected for a pathogenic BRCA1 variant (Rosenthal et al., 2015). The haplotype of c.[594-2A>C; 641A>G] has been
shown from mRNA analysis in human samples to produce high levels of Δ10 transcripts
(70% of the overall expression, and has been designated as Class1 Not Pathogenic by the ENIGMA Consortium using multifactorial likelihood analysis that includes genetic
(segregation, case-control analysis) and pathology data (de la Hoya et al., 2016).
Δ11q, Δ11
c.4096 to non-G
c.4096+1 (IVS11+1)
c.4097+2 (IVS11+2)
Data collected by the ENIGMA consortium demonstrates that the BRCA1 c.4096+1G>A variant, proven to result in the production of naturally occurring in-frame
transcripts Δ11q (Bonatti et al., 2006) and also Δ11 (Radice, unpublished data), may
not exhibit the clinical characteristics of a standard high-risk pathogenic BRCA1 variant (Spurdle, unpublished data).
Δ13p
c.4186-1 (IVS12-1)
c.4186-2 (IVS12-2)
BRCA1 exon 13 acceptor site is an experimentally validated tandem acceptor site (NAGNAG)
subject to alternative splicing (Colombo et al., 2014). c.4186-1,-2 variants are predicted
to inactivate the 5′ acceptor site, but not the 3′ acceptor site, thus producing Δ13p
transcripts.
Δ14p
c.4358-1 (IVS13-1)
c.4358-2 (IVS13-2)
BRCA1 exon 14 acceptor site is an experimentally validated tandem acceptor site (NAGNAG)
subject to alternative splicing (Colombo et al., 2014). c.4358-1,-2 variants are predicted
to inactivate the 5′ acceptor site, but not the 3′ acceptor site, thus producing Δ14p
transcripts.
BRCA2
Δ12
c.6842-1 (IVS11-1)
c.6842-2 (IVS11-2)
c.6937 to non-G
c.6937+1 (IVS12+1)
c.6937+2 (IVS12+2)
Carriers of these variants are predicted to produce exon 12 skipping. BRCA2 Δ12 is
a naturally occurring in-frame splicing event (Fackenthal et al., 2016). BRCA2 exon
12 is functionally redundant (Li et al., 2009).
Approach of the VUS Task Force
The Consortium recommends routinely testing for 10 genes which are known (as per 8/19)
to be associated with breast and/or ovarian cancer: ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, PALB2, RAD51C, RAD51D and TP53 (http://www.konsortium-familiaerer-brustkrebs.de/ ).
The Consortiumʼs panel of experts (VUS Task Force) holds monthly telephone conferences
and, if necessary, meetings to reach a consensus on the classification of newly reported
sequence variants, discuss any new evidence available for the re-evaluation of already
known variants, and evaluate variants of unclear significance. In the event of a re-evaluation,
the central database of the Consortium will inform all centres about the reclassification
(recall system).
It should be expressly noted that new information can lead to changes in the classification
of variants and that these classifications are regularly reviewed by the panel of
experts. Similarly, new findings can lead to changes in the list of core genes for
which the German Consortium for Hereditary Breast and Ovarian Cancer recommends that
patients are tested. All such changes along with the inclusion of new findings into
the classification are published on the homepage of the German Consortium for Hereditary
Breast and Ovarian Cancer (
http://www.konsortium-familiaerer-brustkrebs.de/
).
Classification of Sequence Variants According to Their Functional Relevance
1. Class1 (functionally irrelevant/no loss of function) if one of the following
criteria is met:
1.1 Allele frequency of variants in large population groups (e.g. Caucasians, Africans,
or Asians) is ≥ 1% (minor allele frequency [MAF] ≥ 0.01). Caution: An allele frequency of ≥ 1% in subpopulations with a low-diversity gene pool (examples:
Finnish population, founder mutations!) is not sufficient.
1.2 Variants with a calculated multifactorial probability of < 0.001 of being pathogenic.
Caution: This currently only applies to the high-risk genes BRCA1/2 (for an exemplary calculation, see [13 ]).
1.3 Variants in high-risk genes which occur in at least 10 individuals in appropriate
cohorts of persons without disease ([Table 1 ]).
2. Class2 (probably no loss of function/functionally irrelevant) if one of the following
criteria are met:
2.1 Allele frequency of variants in large population groups (e.g. Caucasians, Africans,
or Asians) is 0.5 – 1% (MAF 0.005 – 0.01) Caution: An allele frequency of 0.5 – 1% in subpopulations with a low-diversity gene pool
(examples: Finnish population, founder mutations!) is not sufficient.
2.2 Exonic variants (A) which result in substitution of an amino acid (missense
variants) or small in-frame insertions/deletions (insertions/deletions of one or fewer
amino acid [s]) and whose a priori probability of pathogenicity is ≤ 2% (A-GVGD analysis,
http://priors.hci.utah.edu/PRIORS/ ); intronic variants (B) which are more than − 20 bp, + 10 bp from the exon border ; and synonymous variants (C) if these variants (A – C) will, according to bioinformatic
prediction programmes (see Appendix A 1), in all probability not change the splicing
mechanism. In non-BRCA1/2 genes, the above-mentioned variants must be present in large population groups with
an allele frequency of 0.001 ≤ MAF < 0.01.
2.3 Synonymous substitutions or intronic variants which show no mRNA aberrations
in the form of exon deletions/duplications or monoallelic expression of the wildtype
(wt) transcript in “in-vitro” laboratory tests even if, according to bioinformatic
prediction programmes (see Appendix A 1 for programmes and threshold values), in all
probability they change the splicing mechanism.
2.4 Variants which occur in the same gene with a clearly pathogenic variant in trans
(co-occurrence), if it has been verified that a homozygous or compound heterozygous
genotype is associated with a known, clinically unambiguous phenotype.
2.5 Variants with a calculated multifactorial probability of pathogenicity of 0.001 – 0.049.
Caution: This currently only applies to the high-risk genes BRCA1/2 (for an exemplary calculation, see: [13 ]).
2.6 Exon variants which code for the same amino acid exchange as a sequence variant
which has already been classified as Class1 but are based on a different nucleotide
exchange if no aberrant splicing is predicted.
2.7 Missense variants for which information from functional analyses, etc., is available
but not sufficient for multifactorial classification and which have been classified
as Class2 by a panel of experts (e.g. ENIGMA).
3. Class3 (unclear functional relevance) if one of the following criteria is met:
variants which cannot be unambiguously assigned to Class1, Class2, Class4, or Class5,
e.g.:
3.1 Special cases which could be assigned to one of the other classes based on the
evaluation criteria but are listed in Appendix A 5 among the characteristics of individual
core genes or in Table 5, Appendix of BRCA1/2 classification criteria, Version 2.5.1, July 2017 (ENIGMA) ([Table 5 ]).
3.2 Variants where the data used for their evaluation is contradictory and for which
further studies are still required.
3.3 Variants which are − 20 bp, + 10 bp from the exon border and which, based on bioinformatic predictive programmes (see Appendix A 1), probably
change the splicing mechanism as long as no in-vitro mRNA analysis has been done yet
([Fig. 2 ], Schematic representation of variants in the vicinity of splice sites).
3.4 Exon duplications which have not been analysed further (e.g. break point analysis,
cDNA analysis, etc.).
3.5 Variants with a calculated multifactorial probability of pathogenicity of 0.05 – 0.949.
Caution: This currently only applies to the high-risk genes BRCA1/2 (for an exemplary calculation, see [13 ]).
4. Class4 (probable loss of function/functionally relevant) if one of the following
criteria is met:
4.1 Variants with a calculated multifactorial probability of pathogenicity of 0.95 – 0.99.
Caution: This currently only applies to the high-risk genes BRCA1/2 (for an exemplary calculation, see Goldgar et al., 2004 [13 ]).
4.2 Variants which code for a premature termination of protein biosynthesis (nonsense
or frameshift variants) and do not necessitate the loss of known clinically relevant
functional protein domains as long as the location of the stop codon is not downstream
from the Nonsense-mediated decay-(NMD-)relevant site, 50 base pairs before the end
of the penultimate exon.
4.3 Intronic variants in position ± 1,2 or G > non-G in the last position of the
exon: if there is a positive splicing prediction (see Appendix A 1) and the first
6 bases in the intron are not GTRRGT and an aberrant in-vitro mRNA analysis is not yet available (i.e. has not [yet] been confirmed by a panel of experts or the pathomechanism
of loss of function has been confirmed to be exon skipping or allele-specific transcript
expression).
Exceptions:
A cryptic splice site (AG/GT) in the vicinity is activated and the (predicted) new
exon is spliced in-frame (→ Class3)
The (predicted) skipped exon (or exons) is alternatively spliced in significant quantities
(→ Class3) .
The (predicted) skipped exon (or exons) is spliced in-frame and contains no known
functional domain (→ Class3)
4.4 Variants which code for the same amino acid exchange as pathogenic missense variants
which have already been categorised as Class5 but are caused by another nucleotide
exchange and for which there is no positive splicing prediction (see Appendix A 1).
4.5 In-frame deletions (even for just one amino acid) which lead to the loss of a
missense variant already categorised as Class5 and which result in the interruption
of known, functionally important domains.
4.6 Extensive in-frame deletions which lead to the interruption/loss of known, functionally
important domains.
4.7 In-frame insertions verified by in-vitro mRNA analysis which result in the interruption
of functionally important domains.
4.8 Variants which lead to mutations of the translation initiation codon (AUG, methionine)
and for which there is no evidence (e.g. an alternative start codon in the immediate
vicinity) which would support an alternative classification.
4.9 Variants for which information from functional analyses, clinical data, etc.,
is available but insufficient for a multifactorial classification and which are categorised
as Class4 by a panel of experts (e.g. ENIGMA).
5. Class5 (loss of function/functionally relevant) if one of the following criteria
are met:
5.1 Variants which code for a premature termination of protein biosynthesis (nonsense
or frameshift variants) which prevents the expression of known, clinically relevant,
functional protein domains.
5.2 Variants with a calculated multifactorial probability of pathogenicity of > 0.99.
Caution: This currently only applies to the high-risk genes BRCA1/2 (for an exemplary calculation, see Goldgar et al., 2004 [13 ]).
5.3 Splice variants for which a frameshift effect was established by in-vitro mRNA
analysis, which leads to a premature termination of protein biosynthesis and prevents
the expression of known, clinically relevant, functional protein domains and for which
a wild-type transcript of the mutated allele has not been confirmed (monoallelic expression).
5.4 Splice variants for which in-vitro mRNA analysis detected an in-frame deletion/insertion
which leads to the interruption or loss of a known, clinically relevant domain or
functional inactivation through changes of the protein structure and for which a wild-type
transcript of the mutated allele has not been verified (monoallelic expression).
5.5 Copy-number deletion variants which result in the interruption or loss of one
or more exons with known, clinically relevant functional domains or lead to a reading
frameshift, which results, according to the prediction, in the inactivation of known,
clinically relevant, functional domains.
5.6 Copy-number duplication variants of any size, confirmed by laboratory analysis,
which duplicate one or more exons and lead to a reading frameshift, which results,
according to the prediction, in the inactivation of known, clinically relevant, functional
domains.
Appendix
A 1. Splicing prediction programmes and their threshold values
The splicing prediction programmes MaxEntScan (MES), Splice Site Finder (SSF), and
Human Splicing Finder (HSF) are considered relatively reliable and should therefore
be used to evaluate possible effects on the splicing process. MaxEntScan results are
considered non-normal for a deviation of delta of ≥ 15% [14 ], Human Splicing Finder for a delta of ≥ 4.1% [10 ] and Splice Site Finder for a deviation of delta of ≥ 5% [14 ]. An mRNA analysis should be done for evaluation in cases with non-normal prediction
(at least two of the three programmes mentioned below). The precondition is that the
physiological splice site is recognised by the respective prediction software based
on the following threshold values.
The threshold values (calculated for BRCA1/2 [14 ]) are:
MES > 3
SSF > 60
HSF > 80
An approximation of these threshold values can also be for the other genes. Once specific
threshold values have been defined, then the defined threshold values must be used.
A 2.
[Tables 1 ] and [2 ].
A 3. Evaluation Criteria Flow Chart
[Fig. 1 ].
A 4. Schematic Representation of Variants in the Vicinity of Splice Sites
[Fig. 2 ].
The use of predictive programmes to assess possible splicing effects is obligatory
for all new mutations including stop mutations as “rescue” effects can occur through
alternative transcripts.
A 5. Characteristics of Individual Genes
The above-mentioned general evaluation criteria should apply to all genes. However,
some exceptions, variations and special features are present in specific variants
and regions of individual genes, which, for the sake of clarity, are listed below.
A 5.1 BRCA1/2
The following should be categorised as Class3: truncating BRCA1 mutations after the amino acid position 1854 and truncating BRCA2 variants after amino acid position 3308 (they are not categorised as Class1, as structural
mutations cannot be excluded). Exception: truncating variants after the polymorphic
stop codon p.(Lys3326*) are classified as dispensable/neutral (Class1) [17 ] and ENIGMA: p.(Lys3326*) is a frequently detected polymorphism which is not associated
with a higher risk, OR 1.3 – 1.5 depending on breast or ovarian cancer. This means
that variants which lead to a stop downstream from p.(Lys3326*) will also not be associated
with an increased risk of developing disease.
The following should be categorised as Class5 in BRCA1/2 : all truncating BRCA1 variants up to the last mutation unequivocally identified as pathogenic at amino
acid position 1853 [18 ] and all truncating BRCA2 variants up to amino acid position 3308, c.9924C>G [19 ]. See ENIGMA BRCA1,2 functional domains, [Tables 3 ] and [4 ]. Caution: Be aware of the potential impact of NMD; the last 50 bp in the penultimate exon and
variants in the last exon usually are usually not subject to NMD. The predictive value
of RNA analysis of blood may be limited as it does not involve the target tissue.
Table 3 Excerpt from https://enigmaconsortium.org/wp-content/uploads/2018/10/ENIGMA_Rules_2017-06-29-v2.5.1.pdf .
Table 3: Catalogue of BRCA1 conserved domains/motifs and currently known, clinically
important amino acid residues, and relevance for classification of BRCA1 in-frame and terminal exon sequence variants.
Domain/Motif
AA start
AA end
AA alterations with demonstrated clinical importancea
Classification of in-frame deletions targeting domain/motifs
References and summary interpretationa
RING
1
101
L22S (c.65T>C [p.Leu22Ser])
T37K (c.110C>A [p.Thr37Lys])
C39R (c.115T>C [p.Cys39Arg])
H41R (c.122A>G [p.His41Arg])
C44S (c.130T>A [p.Cys44Ser])
C44Y (c.131G>A [p.Cys44Tyr])
C61G (c.181T>G [p.Cys61Gly])
Class5 if at least one clinically relevant residue is removed. Otherwise Class3.
http://www.ncbi.nlm.nih.gov/protein/15988069 ; http://hci-exlovd.hci.utah.edu ; multifactorial analysis for H41R (c.122A>G [p.His41Arg]) (Whiley et al., 2014).
NES
81
99
None reported
Class3
Domain location description (Rodriguez and Henderson, 2000).
NLS1
503
508
None reported
Class3
Domain location description (Chen et al., 1996, Thakur et al., 1997).
NLS2
607
614
None reported
Class3
Domain location description (Chen et al., 1996, Thakur et al., 1997).
NLS3
651
656
None reported
Class3
Domain location description (Chen et al., 1996).
COILED-COIL
1391
1424
None reported
Class3
Domain location description (Hu et al., 2000).
BRCT DOMAINS
1650
1863
T1685A (c.5053A>G [p.Thr1685Ala])
T1685I (c.5054C>T [p.Thr1685Ile])
V1688del (c.5062_5064del [p.Val1688del])
R1699W (c.5095C>T [p.Arg1699Trp])
G1706E (c.5117G>A [p.Gly1706Glu])
A1708E (c.5123C>A [p.Ala1708Glu])
S1715R (c.5143A>C [p.Ser1715Arg])
G1738R (c.5212G>A [p.Gly1738Arg])
L1764P (c.5291T>C [p.Leu1764Pro])
I1766S (c.5297T>G [p.Ile1766Ser])
M1775K (c.5324T>A [p.Met1775Lys])
M1775R (c.5324T>G [p.Met1775Arg])
C1787S (c.5359T>A [p.Cys1787Ser])
G1788V (c.5363G>T [p.Gly1788Val])
V1838E (c.5513T>A [p.Val1838Glu])
Class5 if at least one clinically relevant residue is removed. Otherwise Class3.
Domain boundaries derived from X-ray crystallography data are aa1646-1863 (1T15, http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=27907 ), and ENIGMA functional assay data (Monteiro, unpublished).
Digestion data indicate aa1860-1863 are dispensable based on susceptibility to digestion
(Lee et al., 2010), while pathogenic variant data indicate that 1855-1862 are dispensable
(Hayes et al., 2000). Position 1854 is implicated as clinically important by the observation
that Y1853X (c.5559C>G [p.Tyr1853Ter]) is a recognised high-risk pathogenic variant.
These combined data indicate that position 1854 or 1855 is the C-terminal border of
the BRCT/BRCA1 relevant for the clinical interpretation of sequence variants in exon
24 of BRCA1. That is, a variant predicted to disrupt expression of protein sequence
only downstream* of position 1855 would not be considered clinically important.
a Missense substitutions in specific functional domains that are designated as Class5
pathogenic based on multifactorial likelihood of the posterior probability of pathogenicity
> 0.99 (listed in http://hci-exlovd.hci.utah.edu or individual references), and which have no/little effect on the mRNA transcript
profile, unless the variant results in an aberrant transcript that encodes a discrete in-frame deletion
considered informative for the definition of clinically important domains.
* Typo was corrected in version 2.5.1.
Note: The following pathogenic exonic variants known to alter mRNA splicing have been
excluded from Table 3 above, as justified below:
Variant
mRNA Change
Predicted protein change
Reason for exclusion
BRCA1 R1495M (c.4484G>T [p.Arg1495Met])
r.[4358_4484del, 4358_4675del]
p.(Ala1453Glyfs
Predominant alternate transcript is out of frame. Loss of function is assumed due
to loss of full-length transcript from variant allele (Houdayer et al., 2012, Colombo
et al., 2013, Santos et al., 2014).
BRCA1 E1559K (c.4675G>A [p.Glu1559Lys])
r.[4665_4675del]
p.(Gln1366Alafs
Alternate transcript is out-of-frame. Level of full-length transcript not assessed
(Wappenschmidt et al., 2012).
BRCA1 A1623G (c.4868C>G [p.Ala1623Gly])
r.[4868_4986del]
p.(Ala1623Aspfs
Alternate transcript is out of frame. Variant allele produces some full-length transcripts
(Walker et al., 2010).
BRCA1 D1692N (c.5074G>A [p.Asp1692Asn])
r.[4987_5074del, 5074_5075ins5074+1_5074+153]
p.(Val1665Serfs
Predominant alternate transcript, based on minigene assay (Ahlborn et al., 2015),
is out of frame.
Table 4 Excerpt from https://enigmaconsortium.org/wp-content/uploads/2018/10/ENIGMA_Rules_2017-06-29-v2.5.1.pdf .
Table 4: Catalogue of BRCA2 conserved domains/motifs and currently known clinically
important amino acid residues, and relevance for classification of BRCA2 in-frame and terminal exon sequence variants.
Domain/Motif
AA start
AA end
AA alterations with demonstrated clinical importancea
Classification of in-frame deletions targeting domain/motifs
References and summary interpretationa
PALB2 Binding
10
40
None reported
Class3
Domain location description (Oliver et al., 2009, Xia et al., 2006)
BRC-1
1002
1036
None reported
Class3
http://www.ncbi.nlm.nih.gov/protein/NP_000050.2
BRC-2
1212
1246
None reported
Class3
http://www.ncbi.nlm.nih.gov/protein/NP_000050.2
BRC-3
1422
1453
None reported
Class3
http://www.ncbi.nlm.nih.gov/protein/NP_000050.2
BRC-4
1518
1549
None reported
Class3
http://www.ncbi.nlm.nih.gov/protein/NP_000050.2
BRC-5
1665
1696
None reported
Class3
http://www.ncbi.nlm.nih.gov/protein/NP_000050.2
BRC-6
1837
1871
None reported
Class3
http://www.ncbi.nlm.nih.gov/protein/NP_000050.2
BRC-7
1971
2005
None reported
Class3
http://www.ncbi.nlm.nih.gov/protein/NP_000050.2
BRC-8
2051
2085
None reported
Class3
http://www.ncbi.nlm.nih.gov/protein/NP_000050.2
DBD (DNA/DSS1 binding domain – helical, OB1, OB2, OB3)
2481
3186
W2626C (c.7878G>C [p.Trp2626Cys])
I2627F (c.7879A>T [p.Ile2627Phe])
E2663V (c.7988A>T [p.Glu2663Val])
T2722R (c.8165C>G [p.Thr2722Arg])
D2723G (c.8168A>G [p.Asp2723Gly])
D2723H (c.8167G>C [p.Asp2723His])
G2748D (c.8243G>A [p.Gly2748Asp])
I2778_Q2829del (c.8332_8487del [p.Ile2778_Gln2829del])
R3052W (c.9154C>T [p.Arg3052Trp])
Class5 if at least one clinically relevant residue (or all of AA2778-2829) is removed.
Otherwise Class3.
http://www.ncbi.nlm.nih.gov/protein/NP_000050.2 ; http://hci-exlovd.hci.utah.edu .
Pathogenic variant c.8486G>A (also recorded as Gln2829Arg) results in a transcript
encoding an in-frame exon 19 deletion only (Houdayer et al., 2012), indicating that
genetic variation encompassing loss of this entire exon (AA2778-2829) should be considered
clinically important. The clinical impact of alteration/deletion of individual amino
acids in exon 19 is not yet established.
NLS1
3263
3269
None reported
Class3
Domain local description (Guidugli et al., 2014)
BRC-9 or TR2
3265
3330
None reported
Class3
Note: although amino acids 3270-3305 within this fragment are reported to bind RAD51-DNA
filaments (Davies and Pellegrini, 2007), there is no sequence conservation with the
BRC repeats located between aa1002 and aa2014. Domain boundaries are derived from
x-ray crystallography data are aa3265-3330 (Esashi et al., 2005, Esashi et al., 2007).
Case-control and frequency data indicate that BRCA2 c.9976A>T (p.Lys3326Ter) does
not confer a high risk of cancer (OR 1.3–1.5, dependent on breast or ovarian cancer
subtype (Meeks et al., 2016), demonstrating that residues at and downstream of 3327
are likely dispensable.
Position 3308 is implicated as clinically important by the observation that a nonsense
variant c.9924C>G (p.Tyr3308Ter) is recognized as a high-risk pathogenic variant with
known functional relevance ([Vallee et al., 2016]; Bayes score 1122 : 1 from a single
large kConFab family, Spurdle unpublished data). There is currently no publicly available
clinical information to support pathogenicity of nonsense or frameshift variants located
between positions 3309 and 3325.
These data combined suggest that the C-terminal border of the BRC-9 relevant to the
clinical interpretation of sequence variants in exon 27 of BRCA2 lies between 3309
and 3325. That is, a variant predicted to disrupt expression only of protein sequence
downstream of position 3325 would be considered unlikely to be clinically important.
Further functional and clinical studies are underway to refine risk, if any, for predicted
nonsense or frameshift variants downstream of position 3326.
NLS2
3381
3385
No
Class3
Domain location description (Guidugli et al., 2014).
This domain is considered unlikely clinically relevant since it lies downstream of
position 3326.
a Missense substitutions in denoted functional domains that are designated as Class5
pathogenic based on multifactorial likelihood posterior probability of pathogenicity
> 0.99, and for which there is no/little effect on mRNA transcript profile –
unless
the variant results in an aberrant transcript that encodes a discrete in-frame deletion
considered informative to definition of clinically important domains. (Splicing aberrations
are reported for BRCA2 c.7988A>T [p.Glu2663Val] and c.8168A>G [p.Asp2723Gly] (Walker et al., 2010), but
these did not lead to complete loss of function of the full length transcript), and
missense alterations showed abrogation of functional activity using multiple assays
(Walker et al., 2010). An additional conserved region not commonly recognized as a
BRCA2 domain/motif is located AA 1110-1183, but no pathogenic missense substitutions
have been recorded for this region.
Note – The following pathogenic exonic variants known to alter mRNA splicing have
been excluded from Table 4 above, as justified below:
Variant
mRNA Change
Predicted protein change
Reason for exclusion
BRCA2 R2659K (c.7976G>A [p.Arg2659Lys])
r.[7806_7976del]
p.(Ala2603_
Alternate transcript is in-frame but level of full length transcript not assessed
(Farrugia et al., 2008)
BRCA2 R2659T (c.7976G>C [p.Arg2659Thr])
r.[7806_7976del]
p.(Ala2603_
Alternate transcript is in-frame but level of full length transcript not assessed
(Farrugia et al., 2008)
BRCA2 P3039P (c.9117G>A [p.Pro3039Pro])
r.[8954_9117del]
p.(Val2985
Allele-specific assay shows out-of-frame transcript (Houdayer et al., 2012)
Other special features are listed in the Appendix of the evaluation guidelines of
the ENIGMA Consortium, which can be accessed via the following link: https://enigmaconsortium.org/wp-content/uploads/2018/10/ENIGMA_Rules_2017-06-29-v2.5.1.pdf , Tables 3, 4 and 6 ([Tables 3 ] to [5 ]).
A 5.2 ATM
The evaluation criteria for ATM are based on a combination of the following criteria:
The 5-class IARC system for the assessment of the pathogenicity of BRCA1 and BRCA2 variants.
The 3-class system to evaluate the pathogenicity of ATM variants [20 ] which includes in silico analyses such as Align-GVGD.
The ACMG guidelines on the classification of variants [3 ], [21 ].
Additional literature: [22 ], [23 ], [24 ], [25 ], [26 ], [27 ], [28 ].
Class1:
If the allele frequency is ≥ 1% (MAF ≥ 0.01) in large population groups (e.g. Caucasians,
Africans, or Asians) or there is evidence of homozygous variant carriers in control
populations. If this is the case, then the variant is always categorised as Class1.
An allele frequency of ≥ 1% in subpopulations with a low-diversity gene pool (examples:
Finnish population, founder mutations!) is not sufficient.
Class2:
If the allele frequency is ≥ 0.5–< 1% (MAF ≥ 0.005 – 0.099) in large population groups
(e.g. Caucasians, Africans, or Asians), the variant is always categorised as Class2.
Missense variants which, according to in silico analysis (Align-GVGD, SIFT), are very
probably neutral and/or outside the functionally critical domain (FATKIN).
Class3:
Class4:
Variants with an in-frame deletion which are within the functionally critical domain
(FATKIN).
Missense variants which are within the functionally critical domain (FATKIN) and are,
according to in silico analysis (Align-GVGD, SIFT), very probably harmful and described
as functionally inactive.
Class5:
Truncating ATM variants up to the FATKIN domain.
Missense variants, in-frame deletion or splice mutations which reduce ATM protein
expression to < 20% for the mutated allele [28 ], [29 ].
Variants associated with classic AT.
Splice variants: see BRCA1 and BRCA2 .
Functional domains: FATKIN with FAT; PI3K-related kinase; FATC
[Table 6 ].
Table 6 ATM , functional domains and relevance for the interpretation of the clinical importance
of sequence variants – catalogue of clinically relevant functional domains and amino
acids.
Region
AA start
AA end
AA alterations with demonstrated clinical importance (AT) characterising known functional
domains
References and summary interpretation
Substrate binding
91
97
None reported
Domain location description [30 ]
Also contains p53- and BRCA1-binding domain
NLS
385
388
None reported
Domain location description [28 ], [31 ]
Leucine zipper
1218
1238
None reported
Domain location description [25 ], [28 ]
Proline rich
1373
1382
None reported
Domain location description [25 ], [28 ]
FATKIN
1893
3056
Yes, e.g.
p.(Val2424Gly)
p.(2546_2548del), in frame
p.(Asp2625Glu)
p.(Ala2626Pro)
p.(Val2716Ala)
p.(Ser2855_Val2856delinsArgIle)
AA alterations and in-frame deletions [26 ], [28 ], [29 ], [32 ], [33 ], [34 ], [35 ]
Domain location description [25 ], [27 ], [28 ], [36 ], [37 ]
Domains:
FAT: 1893-2612
KIN: 2612-3056 with ATP-binding: 2716-2730, substrate (nibrin and p53) binding: 2682-3012,
FATC with TIP60 binding: 3034-3056
Domain location description
Additional literature: [22 ], [23 ], [24 ], [25 ], [26 ], [28 ], [33 ], [35 ], [38 ].
A 5.3 PALB2
Additional literature: [35 ], [38 ], [40 ], [41 ], [42 ], [43 ], [44 ], [45 ], [46 ].
[Table 7 ].
Table 7 PALB2 , functional domains and relevance for the interpretation of the clinical importance
of sequence variants – catalogue of clinically relevant functional domains and amino
acids.
Region
AA start
AA end
AA alterations with potential clinical importance
References and summary interpretation
BRCA1 interaction domain
9
43
Yes, e.g.
p.(Leu35Pro)
Also covers oligomerisation domain/covers coiled-coiled motif
Domain location description [47 ], [48 ], [49 ];
Amino acid alteration in VUS and functional analysis [50 ].
DNA-binding site
1
200
None reported
Domain location description [51 ]
RAD51 binding site
101
184
None reported
Domain location description [51 ], [52 ]
DNA-binding site
372
561
None reported
Covers also chromatin association motif (ChAM, 395-446)
Domain location description [51 ], [53 ]
MRG15 (MORF4L1) interaction domain
611
764
None reported
Domain location description [48 ]
WD40 repeat
853
1186
Yes, e.g.
p.(Thr1030Ile)
p.(Leu1143Pro)
BRCA2 (1019-1098), RAD51C, XRCC3 and/or RAD51 complex formation
Domain location description [51 ], [52 ], [54 ], [55 ], [56 ], [57 ].
Amino acid alterations [48 ], [54 ], [58 ]
A 5.4 CHEK2
In exons 11 – 15, highly homologous, functionally inactive sequences (pseudogenes)
on various other chromosomes (2, 7, 10, 13, 15, 16, X, and Y) [59 ], [60 ] which can superimpose the relevant sequences > long-range PCR of exons 11 – 15 and
bioinformatic filtering of pseudogene reads, where possible.
Functional domains: SQ/TQ-rich domain*, forkhead-associated (FHA)** domain, kinase
domain***, nuclear localisation signal (NLS) [61 ], [62 ], [63 ].
Table 8 CHEK2 , functional domains and relevance for the interpretation of the clinical importance
of sequence variants – catalogue of clinically relevant functional domains and amino
acids.
Region
AA start
AA end
AA alterations with Potential Clinical Importance
References and summary interpretation
* Q/TQ consensus sites are sites phosphorylated by ATM/ATR [116 ]. e.g. phosphorylation of Thr-68 is important for CHEK2 activation and oligomerisation.
** The phosphorylated Thr-68 site of CHEK2 interacts with the FHA domain of another
CHEK2 molecule and thus leads to the formation of CHEK2 oligomers [66 ].
*** [67 ]
§ The CHEK2 c.470T>C p.Ile157Thr variant, although still present with various classifications
(VUS/likely pathogenic/pathogenic) in ClinVar and other databases has been reclassified
(date: 28.08.2018) as Class2/likely benign by the German Consortium on HBOC on the
following basis: It is frequently listed in large unaffected control cohorts (0.5%,
gnomAD V. 2.1.1, non-cancer). The population frequency in Finnish Europeans is 2.5%
(10 homozygous carriers). In addition, it is present in 47/7325 individuals (0.64%)
in the FLOSSIES database (non-cancer female controls of European descent aged > 70
years). Although showing functionally impaired dimerisation and autophosphorylation
[61 ], [62 ], [68 ], numerous large case-control studies show results indicating low or no increased
breast cancer risk [64 ], [69 ]: breast cancer OR = 1.58 (1.42 – 1.75), colon cancer OR = 1.67 (1.24 – 2.26) [70 ], [71 ]. Additionally, it has been observed with a frequency of 2% in controls [2 ]. However, it may act as a polygenic risk allele.
SQ/TQ-rich
19
69
e.g. c.85C>T,p.Gln29*
[62 ], [65 ]
FHA
92 [115]
205 [175]
p.Arg117Gly, p.Arg145Trp, p.Gly167Arg
[61 ], [62 ], [63 ]
Kinase
212
501
c.1040A>C, p.Asp347Ala #; c.1100del; c.1164dup; p.Thr476Met, c.1169A>C, p.Tyr390Ser;
c.1183G>T, p.Val395Phe#; c.1283C>T, p.Ser428Phe; c.1427C>T, p.Thr476Met
[61 ], [62 ], #ClinVar
NLS
515
538
e.g. c.1547delC, p.Ser516Leufs#; c.1555C>T, p.Arg519Ter#;
[63 ], #ClinVar
Numerous known missense variants in the FHA domain. Caution: Consult FLOSSIES database during evaluation! (e.g. c.470C>T; p.Ile157Thr: Class2
[see § footnote [Table 8 ]) or with unclear clinical relevance (e.g. c.434G>A, p.Arg145Gln; c.422A>C, p.Lys141Thr).
Missense variants in the kinase domain with unclear clinical relevance: e.g. c.1216C>T,
p.Arg406Cys.
Similarly, in the NLS domain, only truncating variants have been classified as pathogenic
to date ([Table 8 ]).
The investigation by Ow et al. [63 ] gives an overview of the identified mutations in the CHEK2 gene region. The publication by Roeb et al. (2012) includes a schematic overview
of the functional domains as well as the results of functional analyses of the missense
mutations located in these different CHEK2 domains [62 ].
A 5.5 TP53
IARC TP53 database; the functional analyses by Kato et al. (2003) and Monti et al. (2007, 2011)
are reliable [72 ], [73 ], [74 ], [75 ], [76 ], [77 ].
Functional domains: oligomerisation domain, core domain (DNA-binding).
Possible dominant negative effect of missense variants and stop variants which affect
the oligomerisation domain.
Mosaic mutations and clonal haematopoiesis are possible, therefore watch out for the
variant allele fraction when carrying out NGS analysis; if necessary, carry out further
analysis to confirm germline mutations.
[Table 9 ].
Table 9 p53 , functional domains and relevance for the interpretation of the clinical importance
of sequence variants – catalogue of clinically relevant functional domains and amino
acids.
Region
AA start
AA end
AA alterations with demonstrated clinical importance (including conflicting interpretations
of pathogenicity but criteria provided)
References and summary interpretation
Transcription activation
1
55
p.(Val10Ile)
p.(Val31Ile)
p.(Pro47Ser)
Amino acid alterations (ClinVar)
Domain location description [78 ]
Also binding site for numerous proteins including HDM2 (amino acids 15-29; IARC)
Proline-rich domain
61
94
p.(Pro82Leu)
p.(Ala83Val)
Amino acid alterations (ClinVar)
Domain location description [78 ]
DNA-binding region
102
292
p.(Gly105Asp)
p.(Lys120Glu)
p.(Thr125Met)
p.(Ser127Phe)
p.(Asn131Tyr)
p.(Cys141Tyr)
p.(Pro151Ser)
p.(Pro151Thr)
p.(Pro152Leu)
p.(Arg156His)
p.(Arg158Cys)
p.(Arg158His)
p.(Tyr163Asp)
p.(Tyr163Cys)
p.(Arg175Leu)
p.(Arg175His)
p.(Cys176Tyr)
p.(His179Tyr)
p.(Arg181Cys)
p.(Arg181His)
p.(Ala189Val)
p.(His193Arg)
p.(His193Leu)
p.(Leu194Phe)
p.(Ile195Thr)
p.(Arg213Gln)
p.(Val.216Met)
p.(Tyr220Cys)
p.(Tyr220Ser)
p.(Ile232Thr)
p.(Tyr234Cys)
p.(Asn235Ser)
p.(Tyr236Asp)
p.(Met237Val)
p.(Met237Ile)
p.(Cys238Tyr)
p.(Ser241Phe)
p.(Cys242Tyr)
p.(Gly245Asp)
p.(Gly245Ser)
p.(Gly245Cys)
p.(Met246Val)
p.(Met246Leu)
p.(Met246Arg)
p.(Arg248Gln)
p.(Arg248Trp)
p.(Ile251Leu)
p.(Ile251Ser)
p.(Thr256Ala)
p.(Leu257Arg)
p.(Glu258Lys)
p.(Arg267Trp)
p.(Arg267Gln)
p.(Val272Leu)
p.(Arg273Hisv
p.(Arg273Cys)
p.(Cys275Tyr)
p.(Cys277Tyr)
p.(Arg280Thr)
p.(Asp281Val)
p.(Asp281Gly)
p.(Arg282Gly)
p.(Arg282Leu)
p.(Arg282Trp)
p.(Arg283His)
p.(Arg283Lys)
p.(Glu286Lys)
Amino acid alterations (ClinVar)
Domain location description (IARC)
Also binding site for numerous proteins including 53BP1 (IARC) and RAD51 amino acids
94-160 and 264-315 [79 ]
Oligomerisation region
325
356
p.(Gly325Val)
p.(Arg337Leu)
p.(Arg337Cys)
p.(Glu339Lys)
p.(Arg342Pro)
Amino acid alterations (ClinVar)
Domain location description (IARC)
Also binding site for numerous proteins
Covering main nuclear localisation signal (amino acids 316-322) [80 ], [81 ]
Basic (repression of DNA-binding region)
369
388
None reported
Domain location description (IARC)
Also binding site for numerous proteins including RAD54 [82 ]
A 5.6 RAD51D
Functional domains: N-terminal domains and ATP-binding domain with the highly conserved
Walker A and B motifs [83 ], [84 ], [85 ].
[Table 10 ].
Table 10 RAD51D , functional domains and relevance for the interpretation of the clinical importance
of sequence variants – catalogue of clinically relevant functional domains and amino
acids.
Region
AA start
AA end
AA alterations with demonstrated clinical importance (including conflicting interpretations
of pathogenicity)
References and summary interpretation
N-terminal region
1
83
None reported
N-terminal domain required for ssDNA-specific binding function [86 ]
Linker
60
78
None reported
Proper interaction with RAD51C and XRCC2 [85 ]
ATPase domain and RAD51B, RAD51C, and XRCC2 binding
99
274
p.(G112A) (disrupts binding of RAD51D to RAD51C [87 ])
p.(S207L) (disrupts RAD51D-XRCC2 interaction [85 ])
p.(A210V) (predicted to be potentially pathogenic [88 ], [89 ])
p.(R266C) [90 ], Meindl et al. (unpublished)
ATPase, AAA+ type
Walker A and B motifs crucial for HR. These motifs are also implicated in binding
to RAD51C and XRCC2.
Additional literature: [91 ], [92 ], [93 ], [94 ]
A 5.7 RAD51C
References: [91 ], [95 ] – [101 ]
Functional domains: DNA repair/recombination protein RecA-like, ATP-binding domain
[Table 11 ].
Table 11 RAD51C , functional domains and relevance for the interpretation of the clinical importance
of sequence variants – catalogue of clinically relevant functional domains and amino
acids.
Region
AA start
AA end
AA alterations with demonstrated clinical importance (including conflicting interpretations
of pathogenicity)
References and summary interpretation
N-terminal region
1
66
None reported
Homology-derived putative DNA-binding domain[91 ]
ATPase domain and RAD51B, XRCC3, and RAD51D binding
79
376
p.(Gly125Val)
p.(Cys135Tyr)
p.(Leu138Phe)
p.(Gly153Asp)
p.(Asp159Asn)
p.(Val169Ala)
p.(Leu219Ser)
p.(Arg258His)
p.(Gly264Ser)
Amino acid alterations and functional consequences [95 ], [96 ], [97 ], [98 ] Domain location description [91 ] ATPase domain includes Walker A nucleotide binding motif (amino acids 125-132) and
Walker B nucleotide binding motif (amino acids 238-242) [91 ], [99 ]
Nuclear localisation signal
366
370
None reported
Domain location description [99 ]
A 5.8 BRIP1
[Table 12 ].
Table 12 BRIP1 , functional domains and relevance for the interpretation of the clinical importance
of sequence variants – catalogue of clinically relevant functional domains and amino
acids.
Region
AA start
AA end
AA alterations with demonstrated clinical importance (including conflicting interpretations
of pathogenicity)
References and summary interpretation
DEAD/DEAH box helicase domain
17
441
Domain location description [102 ]
Helicase superfamily c-terminal domain
697
851
Domain location description [102 ]
The BRCA1 interacting region of BRIP1
976
1006
Phosphorylation of FANCJ at Ser-990 is important for its interaction with BRCA1
[103 ]
MLH1 interaction
Lysines 141 and 142 are required for direct interaction of FANCJ with MLH1
[104 ]
Nuclear localisation signal
158
175
None reported
Domain location description [102 ]
Additional literature: [1 ], [102 ], [105 ], [106 ], [107 ], [108 ], [109 ], [110 ]
A 5.9 CDH1
References:
Focuses predominantly on molecular genetics: [111 ]
Review of functional analyses: [112 ]
Review of the HDGC Consortium: [113 ]
Review of lobular breast cancer: [114 ]
Distribution of pathogenic variants at the CDH1 locus [111 ]
Known pathogenic CDH1 variants are distributed across the entire locus; it is therefore
not possible to define a clinically relevant functional protein domain. The last known
truncating pathogenic variant in the last exon is c.2506G>T (p.Glu836*) [115 ]. All truncating variants upstream must therefore be categorised at least as Class4.
The proposal put forwards by the ClinGen Consortium to categorise variants with a
MAF > 0.2% as ACMG Class1, contrary to IARC guidelines, is currently being debated.
CDH1 Rule Specifications for the ACMG/AMP Variant Curation Guidelines ClinGen (https://www.clinicalgenome.org/site/assets/files/8816/clingen_cdh1_acmg_specifications_v1.pdf ).
