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.