Interpretation of Sequence Variants

She was convicted of killing her four children. Could a gene mutation set her free?

Nature 09 November 2022

Each of these kinds of evidence can be used to score a variant using a five-tiered scale created by the American College of Medical Genetics and Genomics (ACMG), ranging from benign to pathogenic. In the middle of the scale are ‘variants of uncertain significance’, a messy classification that neither clears nor implicates a given mutation in causing disease.

Vinuesa and Arsov, who was involved in the Canberra team’s report, told the hearing that the classification of ‘likely pathogenic’ was based on several ACMG criteria: the variant seemed to be completely new because it was absent in population databases at the time (it has since turned up on CALM3 in one database). And computer simulations predicted it would be damaging to the function of the protein it encodes. The team also argued that there was a plausible pathway to explain how the variant triggers sudden deaths.

Vinuesa understood the significance - a new variant that results in an amino-acid change at the same location of a CALM gene is considered strong evidence in the ACMG guidelines. In his letter to Vinuesa, Schwartz wrote: “My conclusion is that the accusation of infanticide might have been premature and not correct.”

Guess Pathogenic or Benign

Gene HGVSc HGVSp HGVSp_Short Age Gender Diagnosis TumorType
BRCA2 c.10150C>T p.Arg3384Ter p.R3384* 63 M Adenocarcinoma, moderately differentiated Colon cancer
BRCA2 c.10150C>T p.Arg3384Ter p.R3384* 84 M Squamous cell carcinoma, keratinizing, poorly differentiated, with focal neuroendocrine differentiation Non-small cell lung cancer
BRCA2 c.10150C>T p.Arg3384Ter p.R3384* 74 M Non-small cell carcinoma, favor Adenocarcinoma with solid pattern, with focal neuroendocrine differentiation Non-small cell lung cancer

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

gnomAD

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 well established functional domain (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

Detailed Guidelines

ClinGen

Cancer Variant Interpretation Group UK (CanVIG-UK)

BRCA

BRCA1/BRCA2: CanVIG-UK Gene-Specific Guidance

It is predicted that truncating variants occurring at the 3’ end of the gene will not undergo nonsense-mediated RNA decay (NMD). The residues below demarcate the consensus boundary, 3’ of which protein truncating variants are not established to result in NMD and/or impairment of function of residual protein. BRCA1 (NM_007294.3): 1855 BRCA2 (NM_000059.3): 3309

BRCA1/BRCA2: CanVIG-UK Gene-Specific Guidance

PP4 (phenotypic specificity)

  • Patient’s phenotype or family history is highly specific for a disease with a single genetic aetiology

Cancer Variant Interpretation Group UK (CanVIG-UK)

Example (VUS)

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

Characteristics of BRCA1/2

  • LOF known mechanism of disease (for PVS1)
  • Mode of inheritance (for PM3/BP2)
    • AD/AR (BRCA2)
  • Missense pathogenic (for PP2/BP1)
    • BRCA2 1%
  • Hot spot or critical/well-established functional domain (for PM1/BP1)
    • BRCA1 RING (1-101), BRCT (1650-1863) COILEDCOIL DOMAIN (1391-1424) and BRCA2 DNA-binding domain (2481-3186)

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

  • ClinVar (Uncertain significance (Last evaluated: Nov 1, 2015)) (PP5, BP6)
  • Population AF: gnomAD 0.0016% (PM2, PM2_supporting, BA1, BS1)
  • Insilico SIFT 1.0, phyloP -0.72, PolyPhen-2 0.004
    • Use of meta-predictor REVEL (0.255) BP4
    • Use of multiple tools is no longer recommended
  • Missense pathogenic (for PP2/BP1)
    • BRCA2 1%
  • Hot spot or critical/well-established functional domain (for PM1/BP1)
    • DNA-binding domain (2481-3186)
    • \(\Delta\) score: 0 SpliceAI

Functional assay (PS3)

  • Guidugli et al, 2018
  • Hart et al, 2019
  • Richardson et al, 2021

Summary

  • Variant of unknown significance
  • 1 pathogenic supporting (PM2_supporting)
  • 2 benign supporting (BP1, BP4)

This sequence change replaces glutamic acid with lysine at codon 1895 of the BRCA2 protein (p.Glu1895Lys). The glutamic acid residue is weakly conserved and there is a small physicochemical difference between glutamic acid and lysine. This variant is present in population databases (rs146351301, ExAC 0.009%). This variant has been reported in individuals affected with breast cancer (PMID: 25682074, 20104584, 27257965, 28664449). In the literature, this variant is also known as 5911G>A. ClinVar contains an entry for this variant (Variation ID: 142307). Algorithms developed to predict the effect of missense changes on protein structure and function output the following: SIFT: “Tolerated”; PolyPhen-2: “Benign”; Align-GVGD: “Class C0”. The lysine amino acid residue is found in multiple mammalian species, suggesting that this missense change does not adversely affect protein function. These predictions have not been confirmed by published functional studies and their clinical significance is uncertain. In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance.

BRCA2 c.10150C>T p.Arf3384Ter

End truncation

BRCA1/BRCA2: CanVIG-UK Gene-Specific Guidance

  • BRCA2 c.10150C>T p.Arf3384Ter

It is predicted that truncating variants occurring at the 3’ end of the gene will not undergo nonsense-mediated RNA decay (NMD). The residues below demarcate the consensus boundary, 3’ of which protein truncating variants are not established to result in NMD and/or impairment of function of residual protein. BRCA1 (NM_007294.3): 1855 BRCA2 (NM_000059.3): 3309

Conclusions

  • ACMG guideline
  • CanVIG-UK Gene-Specific Guidance