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 don’t 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.363C>T 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.363C>T 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.1361A>C p.(Glu454Ala) wasdetected 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.1361A>C 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.658C>T 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.1303+1G>A and 1831+2T>G, 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.*12C>T.

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.-188C>T in LDLR. • No evidence of any other sequence variants other than known neutral polymorphisms.

14. Other schemes- FH Question 2 Interpretation • Reported on LDLR @ 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.-188C>T 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.148G>T and heterozygous for c.2282C>T in LDLR. No evidence of any other sequence variants other than known neutral polymorphisms. Interpretation • c.148G>T p.(Ala50Ser) has been reported in FH mutation databases and in several publications as a (relatively) rare, benign polymorphism. • c. 2282C>T 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. 2282C>T p.(Thr761Met) variant as a VUS (class 3), 6 as unlikely pathogenic (class 2). • 1 lab didn’t 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.295C>T p.(Arg99Trp). • Affected with bowel cancer at 36. • Joshua was found to be heterozygous for c.295C>T 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.646C>T, 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.452A>G 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.499C>T 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 wouldn’t. • 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 4’s. • One lab would offer prenatal diagnosis for the variants in question 1 but wouldn’t 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 “didn’t 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.