Variant filtering, digenic variants, and other challenges in clinical sequencing: a lesson from fibrillinopathies

Abstract

Genome-scale high-throughput sequencing enables the detection of unprecedented numbers of sequence variants. Variant filtering and interpretation are facilitated by mutation databases, in silico tools, and population-based reference datasets such as ExAC/gnomAD, while variants are classified using the ACMG/AMP guidelines. These methods, however, pose clinically relevant challenges. We queried the gnomAD dataset for (likely) pathogenic variants in genes causing autosomal-dominant disorders. Furthermore, focusing on the fibrillinopathies Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), we screened 500 genomes of our patients for co-occurring variants in FBN1 and FBN2. In gnomAD, we detected 2653 (likely) pathogenic variants in 253 genes associated with autosomal-dominant disorders, enabling the estimation of variant-filtering thresholds and disease predisposition/prevalence rates. In our database, we discovered two families with hitherto unreported co-occurrence of FBN1/FBN2 variants causing phenotypes with mixed or modified MFS/CCA clinical features. We show that (likely) pathogenic gnomAD variants may be more frequent than expected and are challenging to classify according to the ACMG/AMP guidelines as well as that fibrillinopathies are likely underdiagnosed and may co-occur. Consequently, selection of appropriate frequency cutoffs, recognition of digenic variants, and variant classification represent considerable challenges in variant interpretation. Neglecting these challenges may lead to incomplete or missed diagnoses.

Keywords: Marfan syndrome; congenital contractural arachnodactyly; digenic variants; genome sequencing; variant interpretation.

Figure 1

Overview of used workflow. Categories I and II were applied to all genes associated with autosomal‐dominant disorders in gnomAD, whereas categories III‐VI were only applied to FBN1 and FBN2. *,† Data obtained from the Tables “wgEncodeCrgMapabilityAlign75mer” (*) and “knownCanonical” (†) from the UCSC Table browser; http://genome.ucsc.edu/cgi-bin/hgTables. ‡ Data obtained from the Online Mendelian Inheritance in Man (OMIM) dataset, 05.2018; http://omim.org. § According to Quinodoz et al. (2017).13 ¶ According to Lek et al. (2016).2 ** According to Karczewski et al. (2019).3 †† In FBN2 the prevalence calculation was limited to the CCA‐mutation‐hotspot region (exons 23‐34) for categories II‐VI, while for category I (nonsense and frameshift) all exons were considered. ‡‡ Sequence variants predicted “damaging” or “deleterious” by all six used in silico prediction tools (FATHMM, FATHMM‐MKL, MutationAssessor, MutationTaster, Polyphen2, SIFT; see also Supporting Information Table S1). CCA, congenital contractural arachnodactyly; gnomAD, Genome Aggregation Consortium; HGMD, Human Gene Mutation Database; indel, insertion/deletion; MFS, Marfan syndrome

Figure 2

FBN1 and FBN2 a priori (likely) pathogenic variants in gnomAD and FBN1/FBN2 dual variants in Family 1 and Family 2. Lollipops show the type and position of variants in relation to protein domain structure. Red boxes indicate the severe/neonatal region in FBN1 (exons 23‐34) and the comparable congenital contractural arachnodactyly (CCA)‐mutation‐hotspot region in FBN2 (exons 23‐34). cbEGF, calcium‐binding epidermal growth factor; EGF, epidermal growth factor; HGMD, Human Gene Mutation Database; indel, small insertion/deletion; TB, transforming growth factor β binding. Information on protein domains was obtained from umd.be/FBN1 and umd.be/FBN2. Lollipop diagrams were generated using the R package “trackViewer,” available from http://bioconductor.org/packages/release/bioc/html/trackViewer.html [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 3

Pedigrees of Family 1 (A) and Family 2 (B). Arrows denote index patients harboring FBN1/FBN2 dual variants. The vertical line in the symbols (circle, female; square, male) denotes molecular genetic testing for the respective variants. Black halves (left) represent an FBN1 variant, striped halves (right) represent an FBN2 variant, and white halves represent absence of variant. The diagonal line through a symbol indicates deceased family members. The age at examination (y, years) or the year of birth and, where applicable, death is given in parentheses