Geuess Pathogenic or Benign

Geuess Pathogenic or Benign

Question

• Can our lab classify novel missense variant into pathogenic or benign, not VUS?

Standards and Guidelines for the Interpretation of Sequence Variants

• To describe variants identified in Mendelian disorders
• American College of Medical Genetics and Genomics (ACMG)
• ENIGMA BRCA1/2 Gene Variant Classification Criteria
• International Agency for Research on Cancer (IARC)

Why is BRCA1/2 special?

• High prevalence in population
• Frequent benign variant

What about hereditary breast and ovarian cancer syndrome (HBOCS)

• BRCA1/2 and other genes
• Breast, ovarian cancer and other cancers
• Prevalence (between 1 in 400 to 1 in 800 people)
• Penetration rate (40-90%)

Categories of interpretation of variants

• Pathogenic
• Likely-pathogenic
• Uncertain (VUS)
• Likely-benign
• Benign

Let's guess the evidences

Famly pedigree

Segregation data (BS1, PP1)

• Caveat: linkage disequilibrium
• Penetration rate
• Difficult statistical evaluation

Population data

Population data

• 5%: benign stand alone (BA1)
• 0.5-5% (BS1)
• Wow! The first time observed variant! (Absent in population DB, PM2)

Null variant

• Frameshift, Nonsense, canonical +-1 or 2 splicing site, initiation codon
• Caveat: LOF variants at the extreme 3′ end of a gene
• Caveat: presence of multiple transcripts

Computational (in silico) data

• PolyPhen2, SIFT, MutationTaster, etc
• Mutational hot spot and/or critical and wellestablished (PM1)
• Protein length changes due to in-frame deletions/insertions and stop losses functional domain (PM4 BP3)
• Novel missense at the same position (PM5)

Other evidence

• de novo variants (PS2 PM6)
• Functional studies (PS3 BS3)
• Allelic data (BP2 PM3)

Evidences of interpretation

• Population data
• Computational data
• Functional data
• Segregation data
• De novo data
• Allele data
• Other databases
• Other data

27 variant attributes

Rules for combining criteria to classify sequence variants

Evaluation of ACMG-Guideline-Based Variant Classification of Cancer Susceptibility and Non-Cancer-Associated Genes in Families Affected by Breast Cancer[1]

• Whole-exome sequencing
• 180 medically relevant genes
• 404 individuals
• 253 families
• 1,640 variants

Example (VUS)

• 76/M, Lung adenocarcinoma
• NM_000059.3(BRCA2):c.5683G>A (p.Glu1895Lys)

Which evidences among 27 variants attributes are available in our lab?

Ion torrent

Characteristics of varant (NM_000059.3(BRCA2):c.5683G>A (p.Glu1895Lys))

• ClinVar (Uncertain significance (Last evaluated: Nov 1, 2015)) (PP5, BP6)
• Population AF: $8.29e^{-06}$ (PM2, BA1, BS1)
• Insilico SIFT 1.0, phyloP -0.72, PolyPhen-2 0.004 BP4

Functional assay (BS3)

• Findlay (2018) Nature 562: 217 PubMed: 30209399
• Guidugli (2013) Cancer Res 73: 265 PubMed: 23108138
• Biswas (2011) Blood 118: 2430 PubMed: 21719596
• Becker (2012) Breast Cancer Res Treat 135: 167 PubMed: 22729890

Summary

• Likely benign
• $\ge$ 2 supporting (BP1, BP4)

In Maxwell study [1]

Korean founder mutation BRCA1 p.Leu1780Pro

• 745 HBOC patients
• 86 VUS
• 64 missense

Korean founder mutation BRCA1 p.Leu1780Pro

• BRCA1 NM_007294.3:c.5339T>C Leu1780Pro
• No of patients: 11
• PS4: Odds ratio by Korean 41.2 (2.4-699.5)
• PS3: Functional study
• PP3: In silico test
• BP1: Missense

Question

• Can our lab classify novel missense variant into pathogenic or benign, not VUS?

  Answer

• Yes, we can but...

Unrobust result

• BP4 (How many in silico test)

HBOC cohort

Sherloc: a comprehensive refinement of the ACMG–AMP variant classification criteria [2]

• Iterative refinement of ACMG guideline
• 33 ACMG criteria to 108 criteria
• Combination of evidence to semiquantitative system

Challenges and Considerations in Sequence Variant Interpretation for Mendelian Disorders [3]

CMC pathology lab

• In house database
• https://brcaexchange.org/
• http://coda.nih.go.kr/coda/KRGDB/
• https://www.ncbi.nlm.nih.gov/clinvar/
• https://gnomad.broadinstitute.org/

BRCA2 c.10150C>T p.Arf3384Ter

KOBRA [4]

End truncation

HUMAN MUTATION Mutation in Brief 31: E 1200-E1240 (2010) Online

Finally, three sequence variants – BRCA2 c.9976A>T (BIC: K3326X), c.10095delCins11 (BIC: 10323delCins11) and c.10150C>T (BIC: R3384X) predicted to result in protein truncation were ruled as exceptions that could not be classified because of their location near the 3´-end and possibly dispensable part of the gene.

Clinvar

BRCA2 c.9976A>T (p.Lys3326*) variant, located upstream of this variant and also in the last exon of the gene, is a known benign variant.

Conclusions

• ACMG guideline
• Systemaic review system is requred
• Let's determine VUS to benign or even pathogenic