NODAL variants are associated with a continuum of laterality defects from simple D-transposition of the great arteries to heterotaxy

Abstract

Background: NODAL signaling plays a critical role in embryonic patterning and heart development in vertebrates. Genetic variants resulting in perturbations of the TGF-β/NODAL signaling pathway have reproducibly been shown to cause laterality defects in humans. To further explore this association and improve genetic diagnosis, the study aims to identify and characterize a broader range of NODAL variants in a large number of individuals with laterality defects.

Methods: We re-analyzed a cohort of 321 proband-only exomes of individuals with clinically diagnosed laterality congenital heart disease (CHD) using family-based, rare variant genomic analyses. To this cohort we added 12 affected subjects with known NODAL variants and CHD from institutional research and clinical cohorts to investigate an allelic series. For those with candidate contributory variants, variant allele confirmation and segregation analysis were studied by Sanger sequencing in available family members. Array comparative genomic hybridization and droplet digital PCR were utilized for copy number variants (CNV) validation and characterization. We performed Human Phenotype Ontology (HPO)-based quantitative phenotypic analyses to dissect allele-specific phenotypic differences.

Results: Missense, nonsense, splice site, indels, and/or structural variants of NODAL were identified as potential causes of heterotaxy and other laterality defects in 33 CHD cases. We describe a recurrent complex indel variant for which the nucleic acid secondary structure predictions implicate secondary structure mutagenesis as a possible mechanism for formation. We identified two CNV deletion alleles spanning NODAL in two unrelated CHD cases. Furthermore, 17 CHD individuals were found (16/17 with known Hispanic ancestry) to have the c.778G > A:p.G260R NODAL missense variant which we propose reclassification from variant of uncertain significance (VUS) to likely pathogenic. Quantitative HPO-based analyses of the observed clinical phenotype for all cases with p.G260R variation, including heterozygous, homozygous, and compound heterozygous cases, reveal clustering of individuals with biallelic variation. This finding provides evidence for a genotypic-phenotypic correlation and an allele-specific gene dosage model.

Conclusion: Our data further support a role for rare deleterious variants in NODAL as a cause for sporadic human laterality defects, expand the repertoire of observed anatomical complexity of potential cardiovascular anomalies, and implicate an allele specific gene dosage model.

Keywords: NODAL; Congenital heart disease; Genetic diagnosis; Heterotaxy; Laterality defects; Single ventricle; Structural variation; Transposition.

Fig. 1

Comprehensive pedigrees and their genotypes for families with NODAL variants. Standard pedigree structures are utilized—filled circles and squares denote clinically affected individuals, and probands are indicated by black arrows. A Pedigrees of probands harboring heterozygous NODAL variants. B Pedigrees of probands harboring biallelic NODAL variants

Fig. 2

Schematic diagram of NODAL protein structure from conceptual translation of transcript NM_018055 with mapping location (vertical lollipops) of the exact map position of amino acid variants observed in this study. In total, we identified twelve different SNV alleles and indel that are distributed among the NODAL domains. The yellow horizontal rectangle represents the TGF-beta propeptide (amino acid 29-166) and the orange horizontal rectangle represents the mature transforming growth TGF-beta domain (amino acid 247-347). Blue circles denote missense variants, green circles denote splice site variants, red circles denote nonsense variants, and brown circles denote a complex indel variant. Numbers inside the circles refer to the variant frequency in our cohort

Fig. 3

Heart depictions illustrating anatomy, blood flow (yellow arrows) and oxygenation (color coded red for oxygenated, blue for unoxygenated, and purple for poorly oxygenated) for different heart defects compared to A the normal heart anatomy. These include B dextro-transposition of the great arteries (DTGA), C double outlet right ventricle with D-malposed great arteries, D tricuspid atresia with D-malposed great arteries, E congenitally corrected transposition of the great arteries (CCTGA) with left ventricular looping and L-transposition of the great arteries, F double inlet left ventricle (DILV) with L-looped ventricles. CCTGA and DILV are illustrated with dextrocardia, but may be levocardic or dextrocardic. G Heterotaxy, asplenia syndrome/right atrial isomerism type

Fig. 4

Complex NODAL indel variants in two probands with congenital heart disease. A Pedigree structure for Family 6 (top) with integrative genomics viewer (IGV) view of the heterozygous complex indel variant in NODAL (below). Under the IGV view are Sanger dideoxy sequence traces for the proband, LAT0191 (left) and mother, LAT0192, (right). The Sanger sequence trace panels at top represent PCR amplification products of the variant region from genomic DNA. Individual alleles are not discernable for the proband genomic DNA trace at left. Beneath the genomic DNA results are Sanger sequence traces from cloning the amplified variant region using TA cloning kits as described in “Methods”. Top and bottom panels are representative of different populations of clones for each allele. At left, the complex c.700_723delinsTTGACTTCC, p.R234_P241delinsLTS variant is apparent in the Allele 2 (Mut) Sanger trace for the proband, LAT0191. B Pedigree, IGV view, and Sanger traces for Family 7. In addition to the c.700_723delins, p.R234_P241delinsLTS variant, Sanger traces for the nearby c.778G > A:p.G260R variant are shown. Allele 1 in LAT1763 is shown to be WT for the c.700_723 locus, while containing the c.778G > A variant. Allele 2 was shown to contain the c.700_723delins, p.R234_P241delinsLTS variant while being WT at the c.778G > A locus, confirming these variant alleles are in a trans configuration. C, D Secondary structure predictions for WT and mutant RNA using the RNAfold Server (http://rna.tbi.univie.ac.at/) using the minimum free energy (C) and thermodynamic ensemble (D) functions. Base pair probability is shown using color coding; cooler colors (blue) represent lower probability and warmer colors (red) represent higher probability. Variant RNA has more stable secondary structure as shown at right in C and D. E UCSC genome browser view of the 12 clones sequenced for LAT0191 and LAT1763, showing populations with the deletion, and with WT sequence. All variant data shown is for NODAL transcript NM_018055.5

Fig. 5

NODAL proband phenotypic similarity heatmap—A heatmap was generated using proband phenotypic similarity scores and ordered based on Hierarchical Agglomerative Clustering of proband phenotypic similarity. Dendrograms showing clusters are present at the left and top sides of the heatmap. Proband IDs and variants are shown at right and bottom, and color coded based on cluster. Five clusters are highlighted by color-coded boxes on the heatmap, from top left diagonally to bottom right: red, olive, green, teal, and purple. The 4 probands with biallelic variation group in one cluster (purple box). A key for phenotypic similarity score based on color (blue corresponding to a lower score and red corresponding to a higher score) is shown at bottom right. An asterisk denotes probands with biallelic variants in NODAL

Fig. 6

NODAL heterozygous copy number variant (CNV) deletion alleles in laterality defect cases. A Proband LAT1415 pedigree (Family 30) (delCNV/+ heterozygous, +/+ WT homozygous). B HMZDelFinder analyses detected a CNV deletion at the 5′ start of NODAL using ES read count data (RPKM); red vertical dashed lines align to each gene exon, horizontal jagged lines (black: controls; red: deletion CNV subject) show distribution of individual read depth values for that given exon. C The Alu-Alu mediated rearrangement (AAMR) risk score for NODAL and EIF4EBP2 using AluAluCNVpredictor tool. D Family 30 aCGH showed a 46-kb deletion in proband and father. E The 1% agarose gel electrophoresis of PCR products showing the recombinant junction. The junction primer pair was designed to produce an amplicon size of 465 base pairs for deleted alleles resulting from AAMR with the formation of a chimeric Alu. F Family 31 pedigree of proband (CVG007). G Confirmation by aCGH showed a ~4 kb deletion CNV spanning NODAL exon 3. H The gel electrophoresis of PCR products showing the recombinant junction with lighter 700 base pair bands representing heterozygous deleted alleles and more intense bands (~4 kb) representing WT allele. I A schematic representation of NODAL and EIF4EBP2. Note convergent transcripts for NODAL and EIF4EBP2 (black arrow heads representing gene’s orientation). Breakpoint sequences for NODAL deletions are also shown. The proximal reference sequence and its matching proband breakpoint sequences are shown in green for LAT1415 and orange for CVG007, the distal reference sequence and its matching proband breakpoint sequences are in blue for LAT1415 and purple for CVG007, and microhomology at the junction is shown in red. The 46 kb deletion in LAT1415 presumably results from AAMR between AluSx1/AluSq2, whereas the 4 kb deletion in CVG007 proposed to result from AAMR between AluSg/AluSq2