Aims and objectives of the
workshop
David Moore
Aims
• Classification of variants is subjective and NEQAS results suggest
this is not a major problem
• To agree and recommend:
1.
2.
3.
4.
5.
6.
7.
Which class system to use
Which variants to report
Specific report wording for the different variant classes
Appropriate follow-up testing for different variant classes
What evidence to include in reports
Interpretation of frequency data
Interpretation of splice scores
1.
Which classification system to use
– 5 class
• 5 path, 4 likely path, 3UV, 2 unlikely path, 1 not path
– 4 class
• 4 path, 3 likely path, 2 unlikely path, 1 not path
– 3 class
• 3 path, 2 unknown, 1 not path
– A different system?
2. What classes of variants to report
– Yes: classes 3, 4, 5
• Clinically relevant or potential to be.
– ?: class 2
• Unlikely to be pathogenic
• Available on request?
– No: class 1
• Not pathogenic
3. Specific report wording for the different
variant classes
•
Class 5
–
•
Class 4
–
•
Uncertain pathogenicity, does not confirm or exclude diagnosis
Unsure about the pathogenicity and offer further work before offering further diagnostic or
carrier testing.
Class 2
–
–
•
Likely pathogenic, consistent with the diagnosis
Class 3
–
–
•
Predicted to be pathogenic, this result therefore confirms the diagnosis
Unlikely to be pathogenic, diagnosis not confirmed molecularly.
No evidence suggesting pathogenicity but not at a high enough frequency to say it’s not
pathogenic?
Class 1
–
–
–
Not pathogenic.
“Commom” polymorphism.
No evidence suggesting pathogenicity and at “high” frequency.
4. Appropriate follow-up testing for different
variant classes
•
•
Class 5
– Prenatal and predictive testing offered.
Class 4
– Should further work be offered prior to either prenatal or predictive
testing? If so, how much until deemed appropriate for PST/PND?
– Unlikely that any routine work available to diagnostic labs (bar RNA) will
lead to definitively calling it a 5.
– Should class 4 variants be treated as an almost 5 class and anything
that labs are unhappy to do further tests on be classed as a 3?
– Even if there is literature, the majority of missense variants are probably
still class 4’s, unless there is functional evidence?
– If it is a recessive disorder would you offer PND to a family where the
affected individual had:
• Class 5 and class 4 variant
• 2 class 4 variants
4. Appropriate follow-up testing for different
variant classes
• Class 3
– No to PST/PND
– Segregation analysis and any other further work available is warranted
– Is it always warranted, if evidence is weak, should you offer follow up?
• Class 2 or 1
– No further work required?
– If thought that there is a possibility of a class 2 being upgraded, then
should it be a class 3?
5. What evidence to include in reports
• Should labs list all of the lines of evidence used to classify variants
or just refer to Alamut and any relevant references?
–
This variant is likely to be pathogenic. The amino acid is highly conserved across mammals
and down to zebrafish, Alamut software (1) supports it as being pathogenic, the amino acid
change itself doesn’t have a large Grantham score but is from a basic to a polar uncharged
in a specific functional domain of the protein (EF-hand binding site) it is in the vicinity of
reported known pathogenic variants and has been reported by Waldmuller et al (2)
• Or
–
This variant is likely to be pathogenic (1,2)
–
(1)
Assessment of pathogenicity carried out using bioinformatics software Alamut v2.2 which includes tools;
AlignGVGD, SIFT, mutationTASTER polyphen and interrogation of the swissprot variation database and
utilises splicing algorithms: SpliceSiteFinder-like, MaxEntScan, NNSplice and GeneSplicer. (2) Waldmuller et
al in the Eur J Heart Fail 2011, vol13, p1185-1192.
• Should they only list evidence when justifying the reason for follow
up studies? i.e. class 3 variants.
6. Interpretation of frequency data
• Frequency to class a variant as a 1, issues– Frequency of the disease- known?
– Population specific
– “normal” population studied
• ESP uses individuals affected with heart, lung and blood disorders
– Modifying effect?
• c.131C>T (p.Ser44Leu) leads to early onset HSP if in-trans with pathogenic
variant.
• If freq of variant is “significantly higher” than that of
disease, then can it be a 1?
– What figure?
7. Interpretation of splice scores
CFTR c.1584G>A (p.Glu528Glu)
• Splicing– +/- 1, 2 (invariant sites): class 5?
– Other bases- drop of over 10% in at
least 3 programs- class 4?
– Some exceptions (CFTR exon 10).
– Degree of nt conservation an unreliable
indication (esp for variants in the coding
regions)