EXOME SEQUENCING
ANALYSIS
Practical steps of clinical whole exome
sequencing
1. Exome sequencing
Coding genes 2. Gene panel analysis using bionformatics tools
20 000 The gene panel is selected according to the clinical
Disease-
picture of the patient
causing
4 000 genes 3. Molecular biology analysis, prediction of
pathogenicity of selected variants and clinical
assessment
Gene panel
of the disease 4. Possibility of data reanalysis based on newly
discovered disease genes
Clinical exome sequencing: how to identify
the variant of interest
Exome sequencing : ~25’000 variants
~500 rare missense
~150 LoF ; ~20 rares
~1-2 de novo variants
Scientific and medical challenges:
Interpretation & Classification
of rare genetic variants
1-2 variants of medical interest
TOOLS FOR VARIANT INTERPRETATION AND
CLASSIFICATION
Chr Start Gene AA change Zygosity AD Mol gnomAD_ALL sift pp2hvar ljb2_mt gerp++ dbscsnv11 clinvar
chr3 119'122'764 EXT1 NM_000127.2:c.521del:p.(Leu174*) het 108-43 AD 2X; 0.005 0 1 1 5.05 2xP
AD :
Gene accession number : Detection of 151 sequenced DNA fragments at that position.
coding sequence changes : heterozygote or 108 on the reference allele and 43 on the alternative allele
amino acid changes homozygote > 20 : good confidence in the accuracy of the detection of this
variant.
TOOLS FOR VARIANT INTERPRETATION AND
CLASSIFICATION (2)
Chr Start Gene AA change Zygosity AD Mol gnomAD_ALL sift pp2hvar ljb2_mt gerp++ dbscsnv11 clinvar
chr3 119'122'764 EXT1 NM_000127.2:c.521del:p.(Leu174*) het 108-43 AD 2X; 0.005 0 1 1 5.05 2xP
gnomAD_ALL:
gnomAD database
MAF=Minor allele frequency. Rare: MAF < 0.05.
sift, pp2hvar (polyphen 2) et mt (mutation taster):
Functional prediction of the DNA variant on the protein.
> Score sift <0.05 = pathogenic variant
> Score pp2hvar > 0.47 = pathogenic variant
> Score mt > 0.5 = pathogenic variant
TOOLS FOR VARIANT INTERPRETATION AND
CLASSIFICATION (3)
Chr Start Gene AA change Zygosity AD Mol gnomAD_ALL sift pp2hvar ljb2_mt gerp++ dbscsnv11 clinvar
chr3 119'122'764 EXT1 NM_000127.2:c.521del:p.(Leu174*) het 108-43 AD 2X; 0.005 0 1 1 5.05 2xP
gerp++
Score of the conservation of the nucleotide across different
species
Score ~5 = highly conserved; score ≤ 0 = not conserved
dbscsnv11
Functional prediction of the variant on the alternative splicing
score > 0.6, The splicing site is considered to be altered
Clinvar
A freely available public archive of human
genetic variants and interpretations of their
significance to disease
VARIANT INTERPRETATION AND
CLASSIFICATION
Exome = ~25’000 variants
Gene/type of variant/zygosity
Rare variant ? Variant
Known causal variant ? Annotation
Family variant testing ? &
Functional data
Bioinformatic predictions Classification
Likely Uncertain Likely
Benign Pathogenic
Benign Significance Pathogenic
Class 1 Class 5
Class 2 Class 3 Class 4
SOURCE
Laboratory Report 0-2 variants American College of Medical Genetics and Genomics (ACMG)
Association of Molecular Pathology (AMP)
