My name is Karen Wain. I'm a genetic counselor at Geisinger and a member of
the ClinGen education workgroup. This presentation will provide a brief
introduction into how variant-spectrum evidence is incorporated into the ACMG
AMP sequence variant interpretation guidelines. Understanding the variant
spectrum of a gene is an important step in variant interpretation. It allows us
to become acquainted with the types of genetic variants that are known to cause
a disease within a specific gene, and what the mechanism of disease is. In
other words, why a variant causes a disease. We can also learn if there are
variant types that are often seen in the general population, in presumably
unaffected individuals, which can indicate that a variant is less likely
to cause disease. The location of a variant within a gene can be important
to consider. And finally, the types and locations of variants in a gene can be
associated with different phenotypes or even distinct disorders due to different
mechanisms of disease. So thinking about variant-spectrum is about knowing your
gene. It is important to be clear about the terms used to describe different
types of genetic variants and the expected consequences of each. Some types,
such as nonsense, frame shift, splice-site, and large deletions are often referred
to as "loss of function variants" since they are predicted to lead to complete
loss of gene product through nonsense mediated decay. Other variants such as in-
frame deletions or duplications and missense variants are generally expected
to result in an altered protein that may still be stable. Variants which are not
expected to change a protein include silent and intronic variants, though
these can impact splicing. Finally, whole gene duplications are associated with
disease i there is evidence of triplo- sensitivity for that gene, which is not
the same as gain of function. For reviews of variant nomenclature or
a general overview of variant spectrum data,
please view these specific webinars available in the ClinGen YouTube
channel. In 2015, the American College of Medical Genetics
and Genomics, ACMG, and the Association for Molecular Pathology, AMP, published a
joint consensus recommendation for standards and guidelines for the
interpretation of sequence variation. The overarching goal of these guidelines, in
addition to providing a consistent standard, was to create a framework to
determine if variant within a gene that has a definitive role in a Mendelian
disorder is pathogenic for that specific disorder. These guidelines are also
intended to reduce the number of variants that are reported as being
disease-causing, but have insufficient evidence. It's important to note that
this framework as written is not designed to consider variants in genes
with minimal evidence supporting their association to a Mendelian disease, which
are often referred to as "genes of uncertain significance." To accomplish
this, the guidelines defined 28 criteria that address various types and levels of
evidence that either support of a pathogenic classification for a given
variant, with levels ranging from supporting to very strong, or support of a
benign classification, with supporting, strong, or standalone evidence levels.
This figure provides a visual of the different criteria outlined as part of
this guideline, and how they are grouped into different types of evidence. For
this webinar, we will be focusing on criteria that relate to the type of
genetic variant or the location of the variant within the gene. As previously
mentioned, data can be used with different strengths of evidence which
are outlined at the top of the figure, and identified by color. The table to the
right shows how different combinations of evidence of different strengths are
used to determine a classification. For example, a variant can be classified
as pathogenic based on one very strong and two moderate criteria being met, or by
having two strong criteria being met. A variant can be classified as benign if
the standalone minor allele frequency criteria is met, or if two of strong
benign criteria are met.
This zoomed in view gives you a better look at the nine criteria that relate to
variant spectrum evidence. As we go through the different uses of variant
spectrum evidence in this framework, it is important to note that each criterion
we discuss will have a different level of strength.
These criteria outlined here are the starting point. Additional work is
underway to further refine this framework and provide guidance on when
to apply these criteria and how to modify the level of strength in
different circumstances. I would also like to point out how large-scale
population data is also incorporated into criteria relevant to variant-
spectrum by informing us of the types and locations of variants that are
common in the general population, and therefore are less likely to be
disease-causing. For additional review of population data and how it can be useful
in variant interpretation, please view the other webinars on the ClinGen
YouTube channel that specifically focus on this topic. There are three criteria
that specifically address variants that change the length of a protein. The first,
which we will consider is PVS1, which is the only criterion which has been
given a very strong level of evidence. This criterion can only be applied to
variants which are predicted cause a complete loss of gene function, and
absent protein product, often called "null variants." The variants that are usually
included in this category are nonsense, frame shift, canonical splice-site
variants, exonic deletions and duplications, and variants which
eliminate the initiation codon. When these variants result in complete loss
of gene expression or premature truncation that is expected to result in
nonsense mediated decay, they are predicted to cause loss of function.
However. it is critical to note that in order to apply PVS1, loss of function
must be the established mechanism of disease for a gene. A
predicted null variant would be expected to be pathogenic for a disorder caused
by loss of function, but not necessarily for other disease mechanisms. As
mentioned earlier, there are ongoing efforts to refine the use of individual
criterion. I would like to draw your attention to a recent publication in the
ClinGen ClinVar Special Edition of Human Mutation which specifically provides
guidance on how to modify the strength of PVS1 under certain circumstances.
As this table shows, it may be appropriate to downgrade the strength of
this evidence depending on how established the
mechanism of disease is for a gene- disease pair. There are also other
variant and gene-specific characteristics which may warrant
downgrading PVS1 to strong or moderate evidence. A
detailed flowchart of these circumstances is provided in the
manuscript. Next, we will discuss PM4, which is a moderate strength criterion.
This criterion applies to variants which change the protein lengths, but still
result in a stable protein.These could include the loss of a stop codon, leading
to an extended protein, or a truncating variant,
which occurs near the three prime end of the gene and escapes nonsense mediated
decay. In-frame deletions and duplications can also lead to stable
proteins of abnormal length. A moderate level of evidence is applied for these
types of variants because the impact that they have on protein function is
less certain.
In- frame deletions and duplications can
often occur within regions of highly repetitive DNA that naturally occur
within a coding region. However, these regions may or may not be relevant to
gene function, and they often are polymorphic within the general
population. Therefore, an in-frame deletion or duplication found within
such a repetitive region, if it is not known to be functionally relevant, would
be given the BP3 criterion, which is supporting evidence that the variant is
benign.
We will now focus on the four criteria which are relevant to missense variants.
A missense variant is a DNA nucleotide substitution which results in a new
codon, and therefore, a substitution of one amino acid for another within the
protein chain. Many missense variants are novel, making interpretation difficult.
There are two criterion which specifically addressed the use of
previously reported pathogenic variants, which occurr the same amino acid
residue as a novel variant. Of note, these should not be applied for variants
thought to cause disease due to to an impact on splicing. The first is PS1,
which has a strong level of evidence. This applies if a novel DNA change leads
to the same amino acid substitution as another previously described pathogenic
variant. Each DNA change is causing the same amino acid outcome, so we would
predict the same impact on protein function. The second criterion is PM5,
which is at the moderate level of evidence. In this situation, a novel
missense variant leads to a novel amino acid change that occurs at a residue
where a different amino acid change has been shown to be pathogenic. In this
situation, it is assumed that the amino acid residue is important, and so
changing it is likely problematic, but we have less certainty about whether the
novel amino acid change would have the same impact on protein function. There
are two criteria which consider the types of variants which typically are
pathogenic within a given gene and can be applied to novel missense variants as
supporting evidence toward either pathogenic or benign classifications.
These are PP2 and BP1.
If missense variants are known to commonly be pathogenic and if we do not
commonly see missense variants at a significant allele frequency in the
general population, then PP2 can be applied. If all known pathogenic variants
are truncating, then a missense change is less likely to be pathogenic, and BP1 can
be applied. Finally, there are two additional criteria which are relevant
to variant spectrum: PM1, a moderate level of evidence specifically, considers
the location of a variant within a gene. There can be hotspots for specific
regions of a gene, or domains of a protein, where disease-causing variants
tend to localize. This is often associated with specific disease
mechanisms such as gain of function and specific variant types. A novel variant
that occurs within a known hotspot or well studied functional domain that is
not known to contain benign variation would be appropriate for PM1. In BP7, a
supporting level of evidence toward a benign classification can pertain to
silent variants. Silent variants are DNA changes which are not predicted to
change an amino acid. However, they can sometimes impact splicing. In silico
splicing models can be used to evaluate if splicing is predicted to be impacted.
If not, BP7 can be applied.
To review, understanding the variant- spectrum of a gene is an important
step in variant interpretation. The types and locations of genetic variants that
are known to cause a disease within a specific gene, based on a known disease
mechanism, can inform the strength of evidence applied in classifying a novel
variant. Large population databases can be used to support whether a variant is
pathogenic or benign based on variant type and location. The ACMG AMP sequence
variant interpretation guidelines have operationalized the use of these types
of evidence, and refinement of individual criterion is ongoing through
collaborative ClinGen efforts. I hope this has been a useful introduction into
how variant-spectrum evidence is incorporated into the ACMG AMP
sequence variant interpretation guidelines. Please visit
www.clinicalgenome.org for additional educational resources, and to learn more about ClinGen initiatives.
Our funding sources are listed at the bottom. Please feel free to contact ClinGen by email at clingen@clinicalgenome.org
with questions or feedback. Thank you.