Discussion
Clinical interpretation of genomic duplications identified by CMA is a challenge faced by clinical genetic laboratories. As exemplified by this report, clarifying the genotype-phenotype correlations of genomic duplications requires molecular breakpoint characterization since duplications can be pathogenic by more than one mechanism, including gene dosage alterations, gene disruption at breakpoints, creation of pathogenic gene fusions, and even the re-wiring of the local and distal regulatory landscape of chromatin organization [6, 17].
For the presented prenatal case, the 145 Kbp duplication was found to be in inverted tandem orientation to the normal SMAD2 allele, disrupting the coding sequence of this gene and reducing SMAD2expression to about half its normal value. The characterization of this duplication and its associated functional impact could not have been achieved without a molecular method, such as MPseq, to determine rearrangement positional information. Similar to our case, MPseq has been applied to the analysis of structural variants in the prenatal setting, and shown to resolve complex chromosome rearrangements with important consequences to prenatal diagnosis and perinatal management [18]. To our knowledge, this is the first report of a partialSMAD2 duplication in inverted orientation associated with heterotaxy. In a murine model, Smad2 has been shown to act in a dosage-dependent manner in the Nodal signaling pathway to regulate left-right developmental patterning [13, 19]. Similarly in humans,SMAD2 has been classified as a haploinsufficient gene as evidenced by reports of deletions of SMAD2 in association with congenital heart defects, including dextrocardia [14].
It remains uncertain how the presence of the duplication in proband’s mother was not tied to any major clinical concerns despite the inheritance of the duplication in affected offspring. While duplications are often parentally inherited, the functional outcome and interpretation can be obscured by mosaicism, incomplete penetrance, and/or variable expressivity. For example, a subject with a parentally inherited 1.68 Mb deletion encompassing SMAD2 reported in the Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DECIPHER)[21] does not present with congenital heart defects, but pulmonary stenosis was detected, a feature which can also be seen in heterotaxy (Supplementary Table 2). In contrast, ClinVar [22] entries of SMAD2 mutations include four cases with reported cardiac anomalies (Supplementary Table 3) [14]. For M1 we were unable to determine if the partial SMAD2 duplication was mosaic, or if additional heterotaxy-related pathogenic variants were present in her genome or that of her partners.
With more cases being published and variants made accessible to the genetics community through collective efforts such as the DECIPHER and ClinVar databases, prediction of clinical outcomes of prenatal duplications may be more accurate. Nucleotide-level resolution studies will, ultimately, provide the final answer to the complex problem of clinical duplication interpretation in routine genetic testing.