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Pathogenicity of sequence variants interpretation pilot EQA

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Title: Pathogenicity of sequence variants interpretation pilot EQA


1
Pathogenicity of sequence variants interpretation
pilot EQA
  • David Moore

2
Aims of the scheme
  • Determine whether assessment is consistent.
  • Determine which methods are being applied to
    variant assessment.
  • Determine some of the thresholds being used to
    determine pathogenicity
  • Create a generic scheme to allow assessment of
    labs that dont participate in NEQAS

3
Reasons to assess
  • NGS is going to reveal variants in genes that
    have had little or no work carried out on them,
    so published data and LSDB info may be limited.
  • Less confusion for clinicians if all labs are
    reporting variants in the same way and offering
    to do follow up on the same types of variant.
  • Clearer message on reports of what is pathogenic
    and what is worth following up.
  • Avoid adverse clinical impact
  • Inappropriately confirming diagnosis if the cause
    is due to another gene.
  • Doing inappropriate presymptomatic testing on
    family members.
  • Doing unwitting presymptomatic testing on
    unaffected family members during segregation
    analysis for late onset disorders
  • Not offering/suggesting follow up studies when
    they are indicated
  • Doing costly follow up studies that are not
    warranted, including bleeding patients
    unnecessarily.

4
Cases and results supplied
  • Question 1
  • Helen McMillan (dob 02/08/2004) has been referred
    by a Metabolic Consultant as she is presenting
    with Glycogen Storage Disease type 12. She has a
    reduction in Aldose levels and is presenting with
    myopathic symptoms, including muscle weakness and
    premature muscle fatigue. Helen has been found to
    have a large deletion by array CGH which has been
    shown to include exons 1 to 6 of the aldolase A
    (ALDOA) gene (NM_00034.3). There is no other
    history of this disease in the family. Variants
    in this gene act in a recessive manner, so your
    laboratory has been asked to screen the gene by
    Sanger sequencing and has detected the
    heterozygous variant c.363CgtT p.().
  • Key points
  • Aldolase A deficiency and a clinical presentation
    that matches the expected phenotype of GSDXII.
  • As a deletion has been detected and the c.363CgtT
    variant introduces a cryptic donor splice site
    that could be pathogenic, does this confirm
    diagnosis?
  • The points to cause some concern are that exons
    1-6 are non-coding and it is purely a splicing
    prediction.

5
Cases and results supplied
  • Indicators to establish the pathogenicity of the
    variant were decided to be whether the variants
    occurred in-cis or in-trans and to suggest RNA
    studies to establish whether aberrant splicing
    was occurring.
  • 17 (59) labs scored the variant as a class 4, 1
    as a 3.5 and the remaining 11 (38) as a class 3-
    giving the variant an average scoring of 3.6.

6
Cases and results supplied
  • Question 2
  • Steven Lawson (dob 06/09/1954) is presenting with
    hereditary spastic paraplegia (HSP). He has a
    strong family history of HSP and a Consultant
    Neurologist has requested testing of the HSP
    genes. Your laboratory has recently set up a next
    generation sequencing panel to cover all the
    known dominant HSP genes and only the
    heterozygous variant c.1361AgtC p.(Glu454Ala) was
    detected in the spastin (SPAST) gene
    (NM_014946.3). The technology being used has not
    been validated to robustly detect exonic copy
    number variants.

7
Cases and results supplied
  • The variant c.1361AgtC p.(Glu454Ala) occurs in the
    highly conserved functional AAA domain in the
    vicinity of reported pathogenic missense
    variants.

8
Cases and results supplied
  • 16 (55) of labs score the variant as a class 4,
    2 (6) as 3.5, 10 (34) scored as a class 3. One
    lab classed it as a 2- an average score of 3.5.
  • The key points to consider when trying to
    establish pathogenicity were determined to be to
    rule out any copy number variants and to offer
    segregation analysis of the disease and the
    variant.
  • Copy number could potentially be done by
    validating this on the NGS, or by a combination
    of commercial and in-house designed MLPA kits, or
    other methods.
  • Individual 3iii was the best member of the family
    to test for co-segregation, as she is the
    furthest removed from the individual tested.
  • As HSP has a variable age of onset (but in the
    majority of cases late onset), then it is
    inappropriate to test unaffected family members
    for segregation as they may go on to develop
    symptoms which could not only confuse
    co-segregation results but also act as an
    unwitting predictive test. There are also six
    living affected family members to test.

9
Cases and results supplied
  • Question 3
  • Sarah Archibald (dob 06/02/1979) is Caucasian and
    has presented with multiple melanomas. She has an
    affected brother and unaffected sister. A
    Consultant Dermatologist has requested screening
    the XPC gene (NM_004628.4) and your laboratory
    has carried out Sanger sequencing of the coding
    regions of the XPC gene including 50 bp of the
    intronic regions flanking the exons and only
    found the heterozygous sequence variant c.658CgtT
    p.(Arg220). XPC variants have been shown to act
    in an autosomal recessive manner.

10
Cases and results supplied
  • A premature stop codon in exon 6 out of 16 and
    has been reported in the literature.
  • class 5 variant.
  • XPC is autosomal recessive and only the XPC gene
    has been screened by Sanger sequencing, so there
    remains two possibilities
  • There is a second pathogenic XPC variant that has
    not yet been detected.
  • It is an incidental finding and the patient is
    harbouring pathogenic variants in another gene
    causing XP.
  • In order to determine between the two
  • Dosage of XPC
  • Linkage analysis could be done to establish
    whether two affected individuals shared the same
    haplotype and whether this differs to the
    unaffected sibling.
  • Promoter and intronic regions of the XPC gene
    could be sequenced.
  • Complementation group studies.
  • Ruling out XPC as the cause in the first instance
    before doing further screening was thought to be
    the best course of action.
  • 28 (97) of labs scored the variant as a class 5,
    with only one classing the variant as a 4. Some
    labs stated that the variant was a class 5 but
    was a class 3 for this actual patient until a
    second XPC variant was found. However the markers
    thought that this could cause confusion.

11
Cases and results supplied
  • Question 4
  • Zachary Sampson is a 7 year old boy (dob
    10/02/2005) presenting with albinism and bleeding
    diathesis. His sister is presenting with the same
    symptoms. A Consultant Clinical Geneticist has
    made a diagnosis of Hermansky Pudlak syndrome.
    Your laboratory has carried out next generation
    sequencing of genes involved with this disorder
    on a sample from Zachary and detected two clearly
    pathogenic variants in the HPS3 gene NM_032383.3
    (c.13031GgtA and 18312TgtG, reported in Huizing
    et al (2001) Am J Hum Genet vol69, p1022-1032).
    Your laboratory has also detected a heterozygous
    variant in the HPS1 gene (NM_00195.3) c.12CgtT.

12
Cases and results supplied
  • The HPS3 variants are causative.
  • No evidence to suggest that the variant is
    pathogenic
  • HPS is recessive and no other variants have been
    detected.
  • The implication is that the variant is very
    unlikely to be pathogenic.
  • 21 (72) labs scoring it as a class 2
  • 1 scoring it as a 1.5
  • 6 (20) scoring it as a 1.
  • The average score for the variant was 1.8.
  • Classification made by frequency of the variant
    compared to frequency of the disease, or that two
    pathogenic changes in the other gene rules this
    variant out as being pathogenic.
  • 1 lab classed the variant as a 3 and recommended
    to do both RNA studies and dosage analysis which
    the markers deemed inappropriate.

13
Other schemes- FH Question 2
  • Question
  • Irene Lambert (dob 14/08/1958) has probable
    familial Hypercholesterolaemia based on elevated
    LDL cholesterol levels and a family history of
    cardiovascular disease (reported, not
    documented). Irene has 3 children. The Consultant
    Lipidologist has requested molecular analysis to
    confirm the diagnosis.
  • Results
  • Found to be heterozygous for c.-188CgtT in LDLR.
  • No evidence of any other sequence variants other
    than known neutral polymorphisms.

14
Other schemes- FH Question 2
  • Interpretation
  • Reported on LDLR _at_ www.ucl.ac.uk/ldlr/LOVDv.1.1.0/
    Fouchier et al Hum Mutat 2005 vol 26, p550-556.
  • Variant is located in the promoter region of the
    LDLR gene at the Sp1 binding site and is reported
    to lead to reduction in promoter activity.
    However LDLR expression studies for the c.-188CgtT
    mutation have not been published.
  • 5 out of 9 labs classified it as Variant of
    Uncertain Significance and the remaining 4 as
    pathogenic (3 as class 4, 1 as class 5).
  • 6 offered co-segregation analysis.

15
Other schemes- FH Question 3
  • Question
  • Brian Bedford (dob 09/01/1973) was diagnosed with
    familial hypercholesterolaemia based on a
    moderately increased concentration of plasma
    cholesterol and history of cardiovascular disease
    in the family. The Consultant Lipidologist has
    requested molecular genetic testing to confirm
    the diagnosis.
  • Results
  • Heterozygous for c.148GgtT and heterozygous for
    c.2282CgtT in LDLR. No evidence of any other
    sequence variants other than known neutral
    polymorphisms.
  • Interpretation
  • c.148GgtT p.(Ala50Ser) has been reported in FH
    mutation databases and in several publications as
    a (relatively) rare, benign polymorphism.
  • c. 2282CgtT p.(Thr761Met)
  • Align GVGD- C0
  • SIFT- deleterious
  • Mutation Taster- disease causing
  • Polyphen probably pathogenic
  • However data in FH mutation databases and
    literature is inconsistent.

16
Other schemes- FH Question 3
  • 3 labs scored the c. 2282CgtT p.(Thr761Met)
    variant as a VUS (class 3), 6 as unlikely
    pathogenic (class 2).
  • 1 lab didnt report the variants- presumably
    because local practice is to not report class 1
    or 2 variants.
  • Some suggested co-segregation analysis (2 labs)
  • 1 used the class system on a report
  • 1 said that no mutations were found but that
    two variants had been detected
  • 1 said unlikely to be clinically significant,
    but unknown effect so suggested segregation
    analysis

17
Other schemes- FAP Question 2
  • Question
  • A request was received for Joshua NAYLOR to be
    tested for the familial APC variant c.295CgtT
    p.(Arg99Trp).
  • Affected with bowel cancer at 36.
  • Joshua was found to be heterozygous for c.295CgtT
    p.(Arg99Trp).

18
Other schemes- FAP Question 2
  • A summary of evidence used in the reports
  • Result indicates the variant may be segregating
    with disease in this family.
  • Reported in the literature as possibly pathogenic
    based on its segregation in a family.
  • In silico protein prediction software indicates
    it may affect protein function.
  • An unpublished report of this variant occurring
    together with a known pathogenic truncating
    mutation, c.646CgtT, in an affected individual
  • May be a rare neutral variant (freq of 0.1) in
    cohorts of varying ethnicity.
  • No functional assays.
  • Consensus
  • It was thought not appropriate to offer
    pre-symptomatic testing
  • Further testing suggested included segregation
    studies of other affected relatives in this
    family and full screening of APC and MUTYH in
    Joshua.
  • Responses included
  • 2 of 13 reports specifically said no to PST.
  • Variant was referred to as poss-path, likely
    path, UV with segregation analysis, UV with no
    suggested segregation analysis, unlikely path,
    VUS1.
  • 9 of 13 essentially classed it as a 3, 2 as a
    possible/likely path and 2 as unlikely path
  • One lab stated that it was consistent with FAP
    but could be a benign polymorphism or possibly
    pathogenic.

19
Other schemes- Rett syndrome Question 1
  • Paul Steward (dob 26/09/2008) has developmental
    delay, hypotonia, seizures and apnoea and has
    been referred by a Consultant Paediatric
    Neurologist. ?Rett syndrome.
  • Hemizygous for c.452AgtG No evidence of any other
    sequence variants other than known neutral
    polymorphisms.
  • Several lines of evidence suggest pathogenic
    nature of this variant
  • Reported in atypical Rett patients
  • In silico prediction algorithms support it as
    possible pathogenic.
  • Codon 151 is conserved and located in functional
    MECP2 Methyl-binding domain (MBD)
  • Not present in ESP
  • Taken together, these results suggest, but do not
    formally prove, that the variant is causative.
  • In addition this variant was found de novo in a
    patient in one of the participating laboratories
    underscoring the pathogenic nature of this
    variant.

20
Other schemes- Rett syndrome Question 1
  • As some laboratories are more cautious than
    others, two types of answers were deemed
    acceptable for this question
  • Class 3- clinical diagnosis can neither be
    confirmed nor excluded- 8 labs.
  • Class 4- makes diagnosis of MECP2 related
    syndrome highly likely- 5 labs.
  • It was thought necessary that regardless of class
    that the mother of the patient should be tested
    for carrier status and possible de novo testing
  • One offered PND for a UV
  • All participating laboratories provided multiple
    lines of evidence suggesting clinical relevance
    of the variant.
  • One laboratory only mentioned in silico
    prediction algorithms (SIFT and PPH-2) with no
    reference to further analysis.

21
Other schemes- Rett syndrome Question 3
  • ?Rett syndrome. John Benedict (dob 28/09/2008)
    has been referred by a local Consultant
    Paediatric Neurologist.
  • Hemizygous for c.499CgtT No evidence of any other
    sequence variants other than known neutral
    polymorphisms.

22
Other schemes- Rett syndrome Question 3
  • Several lines of evidence suggest pathogenic
    nature of this variant
  • Reported to be associated with non-specific
    X-linked mental retardation in males in a four
    generation family.
  • Reported de novo in affected mother of 3-year old
    female patient
  • In silico prediction algorithms support it being
    possibly pathogenic.
  • Codon167 is conserved and situated in region
    between MBD and TRD. Grantham score 101.
  • Not present in 6500 exomes
  • These results strongly suggest that the variant
    is clinically relevant but presents with a
    slightly different phenotype than Rett syndrome.
  • Two types of answers were deemed acceptable for
    this question.
  • Class 3- 8 labs
  • Class 4 or 5- 5 labs
  • It was thought necessary that regardless of class
    labs would suggest testing of maternal DNA for
    carrier status and possible de novo testing
  • One laboratory stated pathogenic mutation
    identified, confirmed diagnosis. In view of the
    results provided by the other laboratories, this
    could suggest a slight over interpretation of the
    available data.
  • Not all laboratories that opted for class 4 in
    Question 1 also opted for class 4 in Question 3.

23
Classifying variants
  • Assessment is down to interpretation which is
    unique to each variant and can vary even within a
    laboratory with some investigators being more
    cautious.
  • An attempt to standardise the scoring of variants
    can be attempted and while some tightening could
    be achieved, looking at the results of the
    variant scheme this is unnecessary and could be
    hard to accomplish.
  • 13 labs (45) scored the variants 4, 4, 5.
  • 6 labs (21) scored the variants 3, 4, 5
  • 6 labs (21) scored the variants 4, 3, 5.
  • 4 labs (13) scored the variants 3, 3, 5

24
After a classification has been made
  • Although the classifications were similar across
    the labs the work offered after classifications
    varied e.g.
  • For class 4 changes some laboratories would offer
    presymptomatic and prenatal testing, while others
    wouldnt.
  • Class 4 variants seemed to be falling into 2
    categories-
  • labs that used class 4 as almost a class 5
    variant- probable pathogenic but not sufficient
    evidence to push it into the class 5 category,
  • labs used class 4 almost as an extension of class
    3- unknown significance, requiring further work
    before any conclusions were made and prenatal or
    presymptomatic testing was offered.
  • Some labs seemed inconsistent in their approach
    to the classes of variants.
  • In question 2 some labs offered to do
    confirmatory testing for the SPAST variant but
    not presymptomatic testing.
  • Some labs would not offer prenatal for the
    variants in question 1, but would offer
    presymptomatic for the SPAST variant in question
    2 even though both were classed as 4s.
  • One lab would offer prenatal diagnosis for the
    variants in question 1 but wouldnt offer
    presymptomatic testing for the SPAST variant
    until further work had been done, even though
    they classed both variants as class 4.
  • So clearly some attempt to unify laboratories
    approaches to variants is required, especially if
    a clinical genetics department is receiving
    reports from multiple testing centres.

25
Classification system used
  • Some of the variation could be down to the
    familiarity with the 5 class system used in the
    scheme.
  • 12 labs (42) said they used this system, with
    another saying they used the concept. (5 path,
    4 likely path, 3UV, 2 unlikely path, 1 not path)
  • 4 used the 4 class system (4 path, 3 likely path,
    2 unlikely path, 1 not path)
  • 3 used the three class system (3 path, 2 unknown,
    1 not path)
  • 5 had in-house methods.
  • 2 stated they did it case by case
  • 1 did not state what class system they used.
  • No labs said that they included a numerical score
    on reports but used the descriptions of the
    classes instead, in line with the current best
    practice guidelines.

26
Report wording used
  • Some labs still used mutation to indicate a
    pathogenic variant.
  • Some reports seemed contradictory referring to
    variants in different ways within the same report
    which led to them being unclear
  • Report wording varied
  • Variant of unknown significance/variant of
    unclear significance
  • Possibly pathogenic/likely pathogenic
  • Benign polymorphism/unlikely pathogenic
  • Presentation and amount of evidence extremely
    variable

27
Other collected information
  • The frequencies used to classify something as a
    class 1 varied from 1-10 but most labs stated
    that it depended on the frequency of disease,
    mode of inheritance and the population studied,
    which was deemed the best approach.
  • 22 labs (76) classed the -2, -1, 1, 2 variants
    as class 5 (some with the caveat of
    classification after literature searches), 3 as
    class 4 or 5 and 4 labs would class them as
    class 4.
  • The drop in splice score necessary to deem a
    variant into class 4 or 5 varied from 2.4 to
    100, with one lab stating that they didnt
    look- presumably because they do not come across
    this type of variant, however they classed
    question 1 as a class 4.The most common response
    was over 10 in three programs which 6 labs
    (21) supplied as their answer, but the response
    to this question varied the most.
  • All laboratories that participated use Alamut to
    assess pathogenicity. As all labs are utilising
    the same software this provides a good platform
    to attempt to attain some parity in assessment.
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