Bioinformatics and interpretation of variants identified using
ES.
In the PAGE study, sequenced data were assessed for candidate pathogenic
variants from a modified gene list of genes (DDG2P list (1628
genes)27) that were likely to be associated with
developmental disorders37. These were selected as they
were identified as rare, protein altering variants in which the
inheritance pattern of the variant matched that of the gene being
assessed for clinical review (by bioinformative filtering). These
‘candidate pathologic variants’ were then fed onto and reviewed by the
clinical review panel (CRP). The CRP (a multidisplinary group of
clinical geneticists, fetal medicine subspecialists, two clinical
scientists and a genetic bioinformaticians) reviewed anonymised variant
annotation data and clinical findings using the Sapientia software
(version 1.75; Congenica, Cambridge, UK). The CRP reached a consensus
view as to the variant classification (i.e. pathologic,
likely-pathogenic, variant of unknown significance), likely benign and
benign) and the likelihood that it was causative of the fetal phenotype.
Using this methodology, 0.4 variants were reviewed per proband (fetus
with structural anomaly)27.
The Petrovski paper used similar but not identical methodology for
identification of pathologic variants28. Again they
used trio analysis and a previously published frameworkto allow rapid and efficient identification of de-novo
and inherited variants38. This focused upon two
‘tiers’ of qualifying genotypes. Tier 1 were associated with the
assumption that a relevant genotype would be highly penetrant and be
absent from the parents (and controls). Tier 2 was a literature
motivated screen, which permitted genotypes to be observed at low
frequencies amongst controls (internal and external) and had to have
been previously classified a pathogenic on Clinvar or Human Gene
Mutation databases28,39. Again potential causative
variants were classified by a multidisplinary conference of specialists
to agree genotype/phenotype causation. This methods analysed all genes
and also incorporated ‘bioinformatic signatures’, assessing variants in
genes that were not yet linked to disease. This resulted in a ten-fold
increase in the variant interpretation burden compared to PAGE (4.8
variants per case versus 0.42 per case requiring manual interpretation
by PAGE), with a limited difference in the overall final pathogenic
variant yield28. This demonstrates the need to balance
a higher diagnostic yield versus higher interpretational burden in a
prenatal ES strategy, as well as considering the bioinformatics pipeline
adopted. It is also important to realise that causative variant
association with phenotype may alter with time and the ‘variant’ list
may be updated with time. This means that if the fetal trio ES were to
be periodically reanalysed (during childhood) every 1-2 years additional
pathologic variants may be identified. This has already been recognised
in the use of ES/WGS in paediatric datasets40.