Issues arising from UKNEQAS schemes. Ottie O Brien, Northern Genetics Service, Newcastle, UK 15 th May 2014

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1 Issues arising from UKNEQAS schemes Ottie O Brien, Northern Genetics Service, Newcastle, UK 15 th May 2014

2 2013 schemes There was great variation in the way HGVS nomenclature was applied Scheme would like to promote a universal system and encourages labs to follow HGVS guidelines Marks were deducted when HGVS was not used except for dups/dels and triplet repeat expansions Brackets were a common problem, especially at the protein level, but marks were not deducted Most variation was in reporting of dels/dups

3 Duplications / deletions 5 disease schemes: BRCA, FH, LS, Rett, VHL A minority of labs attempted to use HGVS Up to 6 different versions in one scheme Do not want to insist on HGVS for this reason But if dup/del is described in words, the report must say what is meant by the numbering of the exons

4 Exon numbering GenBank ref seqs do not include reference to exon numbers May have exons missing e.g. BRCA1 NM_ has no exon 4 The report must explain the numbering used The MLPA kit ref no. is not sufficient to indicate the exon number LRG sequences

5 LRG Locus Reference Genomic sequences LRG sequences provide a stable genomic DNA framework for reporting mutations with a permanent ID and core content that never changes. The Scheme has agreed to promote the use of LRGs and will include all published LRGs in future documentation.

6 BRCA1 & 2 scheme Heterozygous deletion of exon 3 in BRCA1 Most labs described it in words Only 6/24 attempted HGVS

7 Summary of the HGVS nomenclature used in BRCA 2013 EQA Q1. Nomenclature No. of labs BRCA1 ref seq c.81-?_134+?del p.(cys27*) 2 NM_ c.81-?_134+?del c.[81-?_134+?];[=] c.[81-?_134+?del];[=]; p.[?];[=] c.[81-?_134+?del];[=] p.[(cys27*)];[=] c.[81-?_134+?del](;)[=] p.([cys27*];[=]) 1 U

8 HGVS feedback comments Should not give a protein description without RNA analysis having been performed Should use p.? or put in brackets to indicate it s a prediction

9 Summary of the HGVS nomenclature used in BRCA 2013 EQA Q1. Nomenclature c.81-?_134+?del p.(cys27*) No. of labs BRCA1 ref seq 2 NM_ HGVS feedback comment c.81-?_134+?del c.[81-?_134+?];[=] c.[81-?_134+?del];[=]; p.[?];[=] c.[81-?_134+?del];[=] p.[(cys27*)];[=] c.[81-?_134+?del](;)[=] p.([cys27*];[=]) 1 U

10 Summary of the HGVS nomenclature used in BRCA 2013 EQA Q1. Nomenclature c.81-?_134+?del p.(cys27*) No. of labs BRCA1 ref seq 2 NM_ HGVS feedback comment c.81-?_134+?del c.[81-?_134+?];[=] c.[81-?_134+?del];[=]; p.[?];[=] c.[81-?_134+?del];[=] p.[(cys27*)];[=] c.[81-?_134+?del](;)[=] p.([cys27*];[=]) 1 U

11 Summary of the HGVS nomenclature used in BRCA 2013 EQA Q1. Nomenclature c.81-?_134+?del p.(cys27*) No. of labs BRCA1 ref seq 2 NM_ c.81-?_134+?del HGVS feedback comment c.[81-?_134+?];[=] c.[81-?_134+?del];[=]; p.[?];[=] c.[81-?_134+?del];[=] p.[(cys27*)];[=] c.[81-?_134+?del](;)[=] p.([cys27*];[=]) 1 U

12 Summary of the HGVS nomenclature used in BRCA 2013 EQA Q1. Nomenclature c.81-?_134+?del p.(cys27*) No. of labs BRCA1 ref seq 2 NM_ c.81-?_134+?del c.[81-?_134+?];[=] c.[81-?_134+?del];[=]; p.[?];[=] c.[81-?_134+?del];[=] p.[(cys27*)];[=] HGVS feedback comment Incorrect nucleotide description as does not indicate del. c.[81-?_134+?del](;)[=] p.([cys27*];[=]) 1 U

13 Summary of the HGVS nomenclature used in BRCA 2013 EQA Q1. Nomenclature c.81-?_134+?del p.(cys27*) No. of labs BRCA1 ref seq 2 NM_ c.81-?_134+?del c.[81-?_134+?];[=] c.[81-?_134+?del];[=]; p.[?];[=] c.[81-?_134+?del];[=] p.[(cys27*)];[=] HGVS feedback comment Incorrect nucleotide description as does not indicate del. c.[81-?_134+?del](;)[=] p.([cys27*];[=]) 1 U

14 Summary of the HGVS nomenclature used in BRCA 2013 EQA Q1. Nomenclature c.81-?_134+?del p.(cys27*) No. of labs BRCA1 ref seq 2 NM_ c.81-?_134+?del c.[81-?_134+?];[=] c.[81-?_134+?del];[=]; p.[?];[=] c.[81-?_134+?del];[=] p.[(cys27*)];[=] HGVS feedback comment Incorrect nucleotide description as does not indicate del. c.[81-?_134+?del](;)[=] p.([cys27*];[=]) 1 U

15 Summary of the HGVS nomenclature used in BRCA 2013 EQA Q1. Nomenclature c.81-?_134+?del p.(cys27*) No. of labs BRCA1 ref seq 2 NM_ c.81-?_134+?del c.[81-?_134+?];[=] c.[81-?_134+?del];[=]; p.[?];[=] c.[81-?_134+?del];[=] p.[(cys27*)];[=] c.[81-?_134+?del](;)[=] p.([cys27*];[=]) 1 U HGVS feedback comment Incorrect nucleotide description as does not indicate del. Incorrect nucleotide The semicolon should not be in brackets.

16 Familial Hypercholesterolaemia Heterozygous duplication of exons 3-8 in LDLR 4/9 labs used HGVS Nomenclature Number of labs LDLR reference sequence quoted c.[191-?_c.1186+?(2)];[=] 1 NM_ c.[191-?_1186+?dup];[=] 1 NM_ c.191-?_1186+?dup 2 NM_ dup should only be used if the duplication has been shown to be in tandem 2 should be used if no experimental evidence as the second copy could be anywhere in the genome

17 Lynch syndrome Heterozygous deletion of exons 9-16 in MSH2 6 labs (29%) used HGVS Summary of the HGVS nomenclature used in Lynch syndrome 2013 EQA Question 1. Nomenclature No. of labs MSH2 reference sequence quoted c.1387-?_2805+?del and c.[1387-?_2805+?del];[=], p.[?];[=] 1 NM_ c.[1387-?_c.2805+?del];[=] 1 NM_ c.1387-?_2805+? 1 NM_ c.1387-?_(*279_?)del 2 NM_ Nomenclature submitted described a different deletion, so not reproduced here 1 N/A

18 Heterozygous deletion of exons 9-16 in MSH2 GenBank MSH2 ref seq recently updated: NM_ c.1387-?_(*272_?)del NM_ c.1387-?_(*279_?)del Exon 16 is last exon LRG sequence for MSH2 now available

19 Rett syndrome Heterozygous for a partial deletion of exon 4 of MECP2 5/11 labs used HGVS Summary of the HGVS nomenclature used in Rett syndrome 2013 EQA Question 1. Nomenclature c.[378-?_704+?del];[=] p.[?];[=] No. of labs MECP2 reference sequence quoted 1 NM_ c.[378-?_1029+?del];[=] 1 NM_ c.378-?_1051+?del 1 NM_ c ?_1051+?del c ?_1087+?del 1 NM_ (MECP2_e2) NM_ (MECP2_e1) c ?_1029+?del 1 NM_ Following HGVS, best answer is the most precise description by taking into account the MLPA probe sequences

20 Von Hippel Lindau Heterozygous deletion of exons 1-3 of VHL 3/8 used HGVS Summary of the HGVS nomenclature used in VHL 2013 EQA Question 2. Nomenclature Number of labs VHL reference sequence quoted c.1-?_642+?del (correct) 1 NM_ c.1-?_642+?del (correct) 1 Not stated c.(?_-213) (*3705_?)del (incorrect) 1 NM_

21 BRCA dup /del nomenclature summary Need HGVS nomenclature (especially when submitting the information to the mutation databases) Need to be comprehensible to all recipients, so advisable to describe the exonic deletion in words. DMD best practice guidelines: dup / dels should be described in words rather than HGVS. Most labs provided a ref seq but only 5 gave the MLPA kit and version no. in the BRCA scheme. In future EQA schemes a ref seq will be required.

22 Nomenclature (all) What is clearest for the clinician? What is clearest for other labs reading the report to carry out e.g. predictives? What is clearest for the disease-specific mutation databases and does this need to be on the report? Could all these be included in one report and should they be?

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24 Recommendations? Could have het del exon 3 in words and then other descriptions in the footnotes Do we need square bracket versions? To indicate a termination codon either (*) or Ter is acceptable. Is it clear though? All variants listed on report (e.g. table of those tested for in CF screen) should be in HGVS, but for templates only need to set this up once. CF clinical team is always asking for extra copies of this indication it s confusing?.

25 Recommendations? Will insisting on full HGVS with brackets become too onerous for busy labs? Is writing and checking every variant in HGVS creating unnecessary work? What will we expect in the QA returns as a minimum? (>1 correct answer so difficult to assess) Will labs only do it for QA?

26 Mutation nomenclature examples To describe a no mutation detected result: To describe a heterozygous result: To describe the presence of two different mutations when the phase is not known: To describe the presence of two different mutations shown to be in trans: To describe the presence of two different heterozygous mutations shown to be in cis: To describe a confirmed homozygous result (e.g. confirmed by parental testing): To describe an apparent homozygous result: To describe a mutation when also using accompanying words to state heterozygous/homozygous/etc.: c.[=];[=] p.[(=)];[(=)] c.[1521_1523del];[=] p.[(phe508del)];[(=)] c.[1521_1523del(;)1657c>t] p.[(phe508del(;)arg553*)] c.[1521_1523del];[1657c>t] p.[(phe508del)];[(arg553*)] c.[1521_1523del;1657c>t];[=] p.[(phe508del;arg553*)];[(=)] c.[1521_1523del];[1521_1523del] p.[(phe508del)];[(phe508del)] c.[1521_1523del];[(1521_1523del)] p.[(phe508del)];[(phe508del)] c.1521_1523del p.(phe508del)

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